MtDNA Sequencing

NIST SPECIAL PUBLICATION 260-155
U. S. DEPARTMENT OF COMMERCE/Technology Administration

National Institute of Standards and Technology
Standard Reference Materials®
Human Mitochondrial DNA—Amplification and

Sequencing Standard Reference Materials—
SRM 2392 and SRM 2392-I
B. C. Levin, D. K. Hancock, K. A. Holland, H. Cheng and K. L. Richie

NIST Special Publication 260-155
Standard Reference Materials®
Human Mitochondrial DNA—Amplification and

Sequencing - Standard Reference Materials—
SRM 2392 and SRM 2392-I
B. C. Levin, D. K. Hancock, K. A. Holland, H. Cheng and K. L. Richie
Biotechnology Division
Chemical Science and Technology Laboratory
Gaithersburg, MD 20899-8311
U.S. DEPARTMENT OF COMMERCE, Donald L. Evans, Secretary

TECHNOLOGY ADMINISTRATION, Phillip J. Bond, Under Secretary of Commerce for Technology
NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY, Arden L. Bement, Jr., Director
Issued September 2003

Certain commercial equipment, instruments, or materials are identified in this paper in
order to specify the experimental procedure adequately. Such identification is not intended
to imply recommendation or endorsement by the National Institute of Standards and
Technology, nor is it intended to imply that the materials or equipment identified are
necessarily the best available for the purpose.
National Institute of Standards and Technology Special Publication 260-155

Natl. Inst. Stand. Technol. Spec. Publ. 260-155, 89 pages (September 2003)
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Contents
Abstract......................................................................................................................................... vi
1. Introduction................................................................................................................................1
2. Materials and Methods..............................................................................................................1
2.1. Origin of Extracted DNA...................................................................................................1
2.2. Isolation and Cloning of mtDNA Containing the C-Stretch in SRM 2392 ...................2
2.3 Bacteriophage DNA Isolation and Sequencing................................................................3
2.4. mtDNA Primers used in Both SRM 2392 and 2392-I .....................................................4
2.5. Polymerase Chain Reaction (PCR) SRM 2392 Completed in 1999...............................4
2.6. Polymerase Chain Reaction (PCR) SRM 2392-I Completed in 2002 ............................4
2.7. Sequencing SRM 2392 Completed in 1999.......................................................................4
2.8. Sequencing SRM 2392-I Completed in 2002 ...................................................................5
2.9. Interlaboratory Evaluation for SRM 2392 Completed in 1999 ....................................5
2.10. Differences in Methodology used by Laboratories in Interlaboratory Evaluation of

SRM 2392, 1999 ................................................................................................................6
2.11. Interlaboratory Evaluation for SRM 2392-I, 2002 .......................................................6
2.12. Differences in Methodology used by the Laboratories in the Interlaboratory

Evaluation for SRM 2392-I, 2002 ...................................................................................7
2.12.1. Armed Forces DNA Identification Laboratory (AFDIL), SRM 2392-I .........7
2.12.1.1. AFDIL PCR Amplification, SRM 2391-I ...........................................7
2.12.1.2. AFDIL Sequencing, SRM 2392-I ........................................................7
2.12.2. Georgia Bureau of Investigation (GBI), Interlaboratory Evaluation, SRM

2392-1 ...................................................................................................................8
2.12.3. Federal Bureau of Investigation (FBI), Interlaboratory Evaluation, SRM

2392-I ...................................................................................................................9
iii
2.12.3.1. FBI Polymerase Chain Reaction (PCR), Interlaboratory
Evaluation, SRM 2392-I .......................................................................9
2.12.3.2. FBI Sequencing, Interlaboratory Evaluation, SRM 2391-I .............9
2.13. Permissions .....................................................................................................................10
2.14. Final Preparation of DNA for SRM 2392 and SRM 2392-I ......................................10
2.14.1. CHR DNA Preparation and Confirmation, SRM 2392 .................................10
2.14.2. CHR Cloned DNA Production, SRM 2392 .....................................................10
2.14.3. 9947A DNA Production, SRM 2392 ................................................................10
2.14.4. HL-60 Production, SRM 2392-I .......................................................................11
3. Results and Discussion ...........................................................................................................12
3.1. SRM Templates ............................................................................................................12
3.2. Primers ..........................................................................................................................12
3.3. Differences between the SRM DNAs and the Cambridge Reference Sequence ......13
3.4. Meaning of the Differences from the Cambridge Reference Sequence ..................13
3.5. The Interlaboratory Evaluation of the CHR and HL-60 Templates ......................14
4. Conclusions .............................................................................................................................15
5. Acknowledgments ..................................................................................................................16
6. References ...............................................................................................................................16
Table 1. Sequences for Primer Sets Used for PCR Amplification of Human DNA ..............19
Table 2. Certified Human mtDNA Sequence Differences from the Cambridge Reference
Sequence (CRS) found in the Two Templates (CHR and 9947A) in NIST SRM 2392,
One Template (HL-60) in NIST SRM 2392-I and in GM03798 and GM10742A .........22
Table 3. Universal Genetic Code and (Human mtDNA Differences) ....................................29
iv
Figure 1. Schematic of Human mtDNA Showing all the Differences from the Cambridge
Reference Sequence in CHR, 9947A, HL-60, GM03798 and GM10742A ....................30
Appendixes....................................................................................................................................33
Appendix A. Certificate of Analysis, SRM 2392
Appendix B. Certificate of Analysis, SRM 2392-I
Appendix C. “A Human Mitochondrial DNA Standard Reference Material for Quality
Control in Forensic Identification, Medical Diagnosis, and Mutation Detection,”
B. C. Levin, H. Cheng, and D. J. Reeder [Genomics 55, 135-146 (1999)].
Appendix D. “Comparison of the Complete mtDNA Genome Sequences of Human Cell
Lines - HL-60 and GM10742A - From Individuals with Pro-Myelocytic Leukemia and
Leber Hereditary Optic Neuropathy, Respectively, and the Inclusion of HL-60 in the
NIST Human Mitochondrial DNA Standard Reference Material - SRM 2392-I,”
B. C. Levin, K. A. Holland, D. K. Hancock, M. Coble, T. J. Parsons, L. J. Kienker, D.
W. Williams, MP. Jones, and K. L. Richie [Mitochondrion 2, 387-400 (2003)].
v
NIST Special Publication 260-155
Standard Reference Materials:

Human Mitochondrial DNA – Amplification and Sequencing – SRM 2392 and SRM 2392-I
Barbara C. Levin, Diane K. Hancock, Koren A. Holland, Haiyan Cheng and Kristy L. Richie
Chemical Science and Technology Laboratory
Gaithersburg, MD 20899-8311
Abstract
Standard Reference Materials SRM 2392 and 2392-I are intended to provide quality control when
performing the polymerase chain reaction (PCR) and sequencing of human mitochondrial DNA
(mtDNA) for forensic identification, medical diagnosis, or mutation detection. They may also serve
as controls when amplifying (PCR) and sequencing any DNA. These SRMs can also be used for
quality assurance when determining the sequence of in-house DNA controls. SRM 2392 is certified
for the sequences of the entire human mtDNA (16,569 base pairs) from two lymphoblastoid cell
culture lines (CHR and 9947A) from apparently normal individuals and the cloned HV1 region of
CHR containing a C-stretch through which it is difficult to sequence. SRM 2392-I is certified for the
mtDNA sequence from HL-60, a promyelocytic cell line prepared from the peripheral blood
leukocytes from an individual with acute promyelocytic leukemia. The mtDNA sequence
information (but not the DNA) of two additional DNA templates (GM03798 and GM10742A) that
were amplified and sequenced in their entirety multiple times at NIST are provided in this Special
Publication for comparison purposes. The sequences of fifty-eight unique primer sets that allow any
area or the entire mtDNA (16,569 base pairs) to be amplified and sequenced are also provided. We
found during the sequencing of these five mtDNA templates that some of the single nucleotide
polymorphisms (SNPs) did result in amino acid changes when compared with the Cambridge
Reference Sequence (Anderson et al.,1981; Andrews et al.,1999). Two interlaboratory evaluations,
one for the amplification, sequencing, and data analysis of the CHR template and one for the HL-60
template were each conducted by three different laboratories and NIST. Corroboration of the results
in SRMs 2392 and 2392-I will provide quality assurance that any unknown mtDNA is also being
amplified and sequenced correctly.
Keywords: CHR; 9947A; HL-60; GM03798; GM10742A; Forensic Identification; Medical

Diagnosis; Mitochondrial DNA sequence; Mutation Detection; Single Nucleotide Polymorphism
(SNP); Standard Reference Material (SRM);
vi
1. Introduction
Each human cell can have a few dozen to several thousand molecules of mtDNA (Bogenhagen and
Clayton, 1974; King and Attardi, 1989). Sequence analysis of mtDNA is used by the forensic
community for human identification especially in those cases where the genomic DNA is highly
degraded or non-existent (Holland et al., 1993; Holland et al., 1995). Forensic analysis to
distinguish between non-maternally related individuals is primarily based on the considerable
sequence variation found in the two hypervariable regions (HV1, HV2) located in the non-coding
displacement loop (D-loop). The medical community is also using sequence analysis of mtDNA for
diagnoses of diseases associated with specific mutations and deletions in the coding regions
(Wallace et al., 1997). A third area of research which is largely unexplored and which needs
sequence analysis is the examination of the mutagenic effects of chemical and physical agents on
mtDNA (Grossman, 1995; Ballinger et al., 1996).
Human mitochondrial DNA (mtDNA) has been completely sequenced and found to be circular
double-stranded molecules containing 16,569 base pairs (Fig. 1) (Anderson et al., 1981). The
sequence determined by Anderson et al. (1981) is referred to as the Cambridge Reference Sequence
and was a consensus sequence based primarily on the results from a human placenta, partly on the
sequence from HeLa cells, and in five areas where the results were ambiguous, the authors inserted
the results from the bovine mtDNA that they had also sequenced. In 1999, Andrews et al.
resequenced the original placenta and found that the 1981 sequence contained a number of errors
and rare polymorphisms that were specific for that placenta. In SRM 2392, which became available
in 1999, we compared the mtDNA sequence of three apparently normal individuals with the 1981
Cambridge Reference Sequence. We also found a number of places that appeared to be errors and
which were later confirmed by Andrews et al. (1999). In this NIST Special Publication on the
development of the mtDNA amplification and sequencing SRM 2392 (Levin et al., 1999) and SRM
2392-I (Levin et al., 2003, in press), we compare the sequencing results from three apparently
normal individuals and two individuals with diseases with both the 1981 and 1999 Cambridge
Reference Sequences. Human mtDNA SRMs 2392 and 2392-I can be used for quality control in
amplification, sequencing, forensic identifications, medical diagnostics, and mutation detection.
2. Materials and Methods
2.1. Origin of Extracted DNA
In SRM 2392, the DNA template designated CHR came from human white blood cells that were
transformed with the Epstein-Barr virus and immortalized as a cell culture line (CHR cells) by the
American Type Culture Collection (ATCC, Manassas, VA). After transformation, the cells were
grown in Iscove’s Modified Dulbecco’s Media or RPMI 1640 media with L-glutamine, sodium
bicarbonate, penicillin, streptomycin and 20% fetal calf serum (Life Technologies, Inc., Grand
Island, NY). The cell cultures were grown at 37 oC in an humidified atmosphere containing 5% CO2
and 95% air. The DNA was extracted from 2 x 108 CHR cells by the Qiagen Plasmid/Cosmid
Purification Protocol (Qiagen, Inc., Chatsworth, CA). This procedure enhanced the concentration of
mtDNA and reduced, but did not eliminate, nuclear DNA.
1
The CHR data presented in this paper were obtained primarily with the above mentioned
immortalized CHR cell culture line. However, before starting production of the final SRM 2392, it
was necessary to obtain fresh blood from CHR and to reestablish the cell line. This second CHR cell
line was established by ATCC as above. The sequence of the mtDNA of this second CHR cell line
was examined and found to be identical to that of the first CHR cell line, with the single exception
that no heterplasmy was noted at nucleotide position (np) 68491; the mtDNA of the second CHR cell
line agreed with the Cambridge Reference Sequence at np 6849. It is the DNA from the second
CHR cell line that is included in SRM 2392.
Also in SRM 2392, the DNA template 9947A was obtained from Life Technologies, Inc. (Grand
Island, NY) who prepared it from a Epstein-Barr virus immortalized human lymphoid cell line.
DNA from 9947A is also used in the PCR-based DNA profiling standard (SRM 23912) designed for
forensic and paternity testing, law enforcement training, and research.
SRM 2392-I contains the extracted DNA from HL-60; this DNA was prepared by the Professional
Services Department of ATCC (Manassas, VA).
The entire mtDNA of two additional cell lines (GM03798 and GM10742A) were amplified and
sequenced multiple times at NIST and these sequences are provided in this NIST Special Publication
for information and comparison purposes. Although the extracted DNA from GM03798 and
GM10742A are not part of SRM 2392 or 2392-I, the cell cultures can be obtained from the National
Institute of General Medical Sciences (NIGMS) Human Genetic Mutant Cell Repository, Coriell
Institute for Medical Research, Camden, NJ. GM03798, an apparently normal human
lymphoblastoid cell culture, was grown in the same manner as the CHR cells and the DNA was
extracted using DNA NOW™, a phenol-free DNA isolation reagent (BIOGENTEX, Seabrook, TX).
The lymphoblast cell line GM10742A came from a patient with Leber Hereditary Optic Neuropathy
(LHON), a disease that causes blindness in young adults and that has been associated with a number
of mtDNA primary, intermediate and secondary mutations (Wallace et al., 1997). This cell line
(GM10742A) was purchased from NIGMS. The cells were grown at NIST in RPMI 1640 plus L-
glutamine and sodium bicarbonate growth media (Sigma, St. Louis, MO), fetal calf serum (20%)
(Sigma), and the antibiotics streptomycin and penicillin (final concentration: 100 U/mL) (Sigma).
DNA was extracted from GM10742A using the QIAGEN Plasmid Kit following the Plasmid Mini
Purification Protocol.
2.2. Isolation and Cloning of mtDNA Containing the C-Stretch in SRM 2392
Confluent CHR cells were harvested by centrifugation at 1500 rpm for 5 min. The mtDNA was
isolated using the Qiagen Plasmid/Cosmid Purification Protocol (Qiagen Inc., Chatsworth, CA).
Following isolation, the mtDNA was digested with restriction enzymes SacI and KpnI (New
England Biolabs, Inc., Beverly, MA) into five fragments which were separated on a 0.7% low
All nucleotide numbers referred to in this paper are based on the numbering system of the
1
Cambridge Reference Sequence (Anderson et al., 1981 and Andrews et al., 1999).
2
SRM 2391 may be obtained from the Standard Reference Material Program, NIST, Gaithersburg,
MD 20899.
2
melting agarose gel. Bands of the size of the fragment containing the HV1 region were cut from the
gel and melted at 65 oC. DNA was extracted with phenol twice and precipitated by adding sodium
acetate (150 mmol/L) and two volumes of 100% ethanol. The final product was resuspended in 10
mmol/LTris-1mmol/L EDTA (TE) buffer. The cloning vector, M13mp18, was also digested with
SacI and KpnI, treated with Calf Intestinal Alkaline Phosphatase (New England Biolabs, Inc.,
Beverly, MA) extracted with phenol and precipitated with sodium acetate and ethanol as described
above. The vector was incubated with the mtDNA product and T4 DNA ligase (Life Technologies,
Inc., Grand Island, NY) at 4 oC overnight. An overnight culture of E. coli host TG-1 cells was
diluted and grown at 37 oC in LB media (Sambrook et al., 1989) until the OD650 reached 0.4 - 0.5.
The cells were harvested by centrifugation at 1500 rpm for 5 min. The cell pellet was resuspended in
10 mL calcium chloride (50 mmol/L) and incubated for one hour on ice, centrifuged, and
resuspended in 1 mL calcium chloride (50 mmol/L) and incubated for 30 min on ice. The treated
TG-1 cell suspension (0.3 mL) was incubated for 30 min on ice with 20 µL of a ligation mixture
containing the isolated mtDNA fragment, the cloning vector that had been treated overnight, T4
Ligase and ligation buffer (Life Technologies, Inc., Gaithersburg, MD). This mixture was heat
shocked at 42 oC for 2 min, mixed with 0.2 mL of untreated TG-1 cells (from the overnight culture),
4 µL of a 1mol/L isopropylthio-β-D-galactoside (IPTG), 40 µL of a 20 mg/mL 5-bromo-4-chloro-3-
indolyl-β-D-galactopyranoside (X-gal) and 3 mL of melted (55 oC) top agar and spread on the
surface of freshly prepared LB agarose plates (Sambrook et al., 1989). The plates were incubated at
37 oC overnight. Both colorless and blue plaques were visible in the morning. The colorless plaques
indicate that insertion of the mtDNA fragment into the vector has occurred; whereas, the blue
plaques have no insertion.
2.3. Bacteriophage DNA Isolation and Sequencing
Single, well isolated colorless plaques from the above LB plates were each placed in a sterile tube
with 1.5 mL of a TG-1/ LB cell suspension which contained TG-1 cells that were grown overnight
and diluted 1/100 in LB media and grown for 1 hour at 37 oC. The plaques and the TG-1/ LB cell
suspension were grown at 37 oC for 5 hours. Cells were removed by centrifugation at 15000 rpm for
5 min. The supernatant containing the bacteriophage was incubated with 0.2 mL polyethylene
glycol (20% PEG in 2.5 mol/L NaCl) overnight at 4 oC and the resultant precipitate containing the
DNA phage particles was pelleted by centrifugation at 15000 rpm for 15 min. The bacteriophage
DNA was isolated by phenol extraction and sodium acetate/ethanol precipitation as described above
and then dissolved in 25 µL of TE buffer. The bacteriophage DNA was cycle sequenced with
AmpliTaq DNA polymerase, FS and the -21M13 primer: 5'-TGTAAAACGACGGCCAGT-3'
according to the protocol in the ABI PRISMTM Dye Terminator Cycle Sequencing Ready Reaction
Kit (PerkinElmer, Foster city, CA). The cycle sequencing was conducted in a PerkinElmer Model
9600 thermocycler by first heating the DNA reaction mixture at 96 oC for 1 min and then subjecting
the mixture to 25 cycles of 96 oC for 10 sec, 50 oC for 5 sec and 60 oC for 2 min. The cycle
sequencing product was purified using a Centri-Sep spin column (Princeton Separations, Inc.,
Adelphia, NJ). The DNA pellet was rinsed with 70% ethanol, vacuum dried, resuspended in loading
buffer prepared by combining deionized formamide and 25 mmol/L EDTA (pH 8.0) in a ratio of 5:1,
loaded onto a 4.75% acrylamide gel and electrophoresed on an ABI 373A DNA sequencer. One of
the clones containing the C-stretch sequence was used as the source of the cloned DNA for the
SRM.
3
2.4. mtDNA Primers used in both SRM 2392 and 2392-I
Fifty-eight sets of unique primers (19-28 bp) for sequencing the entire human mtDNA (16,569 bp)
were computer-designed using GENE RUNNER FOR WINDOWS (Hastings Software, Inc.,
Hastings, NY). The -21M13 primer was used to sequence the cloned HV1 region of the DNA from
the CHR template. The sequences of all the primers are shown in Table 1 and are the same as those
used for SRM 2392 (Levin et al., 1999; Appendix C) with the exception of the reverse primer of set
51. During the course of the development of SRM 2392-I, this primer was changed since it
contained a C at np 14368. Since this is the reverse primer, it would bind to a G at np 14368.
Andrews et al. (1999) in their reevaluation of the placenta originally used to sequence human
mtDNA in 1981 (Anderson et al., 1981) found that np 14368 should have a C at that position.
Therefore, the primer should have a G at np 14368. The new reverse primer 51 is
5’-TTAGCGATGGAGGTAGGATTGG-3’ (np 14368 is in bold and underlined) which binds to the
C at position 14368. The 5’ end is np 14388 and the 3’ end is 14367. The 58 sets of primers were
custom-made by Bio-Synthesis, Inc. (Lewisville, TX); the new reverse primer 51 was obtained from
Invitrogen (Carlsbad, CA).
2.5. Polymerase Chain Reaction (PCR) SRM 2392 completed in 1999
Extracted DNA was resuspended in TE buffer (pH 7.5) containing 10 mmol/L Tris and 1 mmol/L
EDTA. The PCR reaction mixture contained: DNA (1µL), Taq DNA polymerase (0.5 µL or 2.5
units)((Boehringer Mannheim, Indianapolis IN) and 10x buffer (5 µL) (Boehringer Mannheim),
dNTP’s (0.2 mmol/L each) (Life Technologies, Inc., Gaithersburg, MD), forward and reverse
primers (0.4 µmol/L each) plus H2O to a final volume of 50 µL. The 10x buffer (pH 8.3) contained
Tris-HCl (100 mmol/L), MgCl2 (15 mmol/L), and KCl (500 mmol/L). Thermal cycling was
conducted in a PerkinElmer Model 9600 thermocycler and consisted of 1 min at 96 oC, followed by
32 cycles of 15 sec at 94 oC (denaturation), 30 sec at 56 oC (annealing), and 15 sec at 72 oC
(extension), and ending with a final extension of 7 min at 72 oC. A sample of the amplified DNA
was electrophoresed in 0.7% agarose and stained with ethidium bromide to assess the purity and size
of the PCR product. Before sequencing, extraneous materials were removed from the PCR product
with a QIAquick PCR Purification Kit (QIAGEN, Inc., Chatsworth, CA).
2.6. Polymerase Chain Reaction (PCR) SRM 2392-I completed in 2002
The PCR mixture contained: DNA (1µL; 1.4 ng), AmpliTaq Gold® DNA polymerase, (0.5 µL; 2.5
units) (Applied Biosystems), 10x buffer (5 µL) containing 100 mmol/L Tris-HCl, pH 8.3, 500
mmol/L KCl, 15 mmol/L MgCl2 and 0.01% (w/v) gelatin (Applied Biosystems), 10 mmol/L dNTP
mix (1 µL) (Invitrogen), 10 µmol/L forward and reverse primers (1 or 2 µL), plus water to make a
final volume of 50 µL. Thermal cycling was conducted in a PerkinElmer thermocycler Model 9700
and started with 10 minutes at 95 ºC, followed by 35 cycles of 20 sec at 94 ºC (denaturation), 10 sec
at 56 ºC (annealing), 30 sec at 72 ºC (extension) and ended with a final extension of 7 min at 72 ºC.
Amplified DNA was purified with a QIAquick PCR Purification Kit (Qiagen) and the purity and size
of the PCR product was determined by electrophoresis in 2% agarose gels in 1x TBE buffer
containing 0.5 µg/mL ethidium bromide (Sigma).
2.7. Sequencing SRM 2392 completed in 1999
4
Cycle sequencing using fluorescent dye-labeled terminators was performed with an ABI PRISMTM
Dye Terminator Cycle Sequencing Kit with AmpliTaq® DNA Polymerase, FS (PerkinElmer).
Thermal cycling was conducted in a PerkinElmer Model 9600 thermocycler and started with one
minute at 96 oC. The reaction then underwent 25 cycles of 96 oC for 15 sec (denaturation), 50 oC
for 5 sec (annealing), and 60 oC for 2 min (extension). The DNA products were purified by passage
through a Centri-Sep spin column (Princeton Separations, Inc.). Electrophoresis and sequencing of
the fluorescently-labeled purified DNA were performed with a 373A ABI Sequencer (PerkinElmer)
using a 4.75% acrylamide gel. Data analysis was executed with the Sequence Navigator software
package (PerkinElmer).
2.8. Sequencing SRM 2392-I completed in 2002
Cycle sequencing using fluorescent dye-labeled terminators was performed with an ABI PRISM®
BigDye® Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA Polymerase, FS
(Applied Biosystems). Cycle sequencing reactions in both the forward and reverse modes were
conducted with a 9700 PerkinElmer thermal cycler and started with one minute at 96 ºC. The
reaction then underwent 25 cycles of 96 ºC for 15 sec (denaturation), 50 ºC for 5 sec (annealing),
and 60 ºC for 2 min (extension). The DNA products were purified using Edge Gel Filtration
Cartridges (Edge BioSystems, Gaithersburg, MD). Sequencing and data analysis of the purified
DNA were performed using an Applied Biosystems PRISM® Model 310 Genetic Analyzer with
POP-6™ polymer system and 47 cm x 50 µm capillaries (Applied Biosystems). Sequence data
were analyzed with Sequencing Analysis Software 3.3, and comparisons to the Cambridge
Reference Sequence (Anderson et al.,1981) were performed with Sequence Navigator Software
1.01.
2.9. Interlaboratory Evaluation for SRM 2392 completed in 1999
Three laboratories in addition to NIST participated in an interlaboratory evaluation of the CHR

template. These laboratories were The Bode Technology Group, Inc., 21515 Ridgetop Circle, Suite
140, Sterling, VA 20166; IIT Research Institute, Virginia Technology Center, 8510 Cinderbed Road,
Suite 300, P.O. Box 899, Newington, VA 22122; and Lark Technologies, Inc., 9545 Katy Fwy, Suite
465, Houston, TX 77024.
Each laboratory was sent:

1. Two tubes of DNA from the first CHR cell culture line. One tube contained extracted DNA
ready for PCR amplification of the entire mtDNA. The other contained the cloned DNA ready for
cycle sequencing of the HV1 region (this DNA did not need to be PCR amplified).
2. Fifty-eight sets of primers labeled with either F# (forward primer) or R# (reverse primer).
Forward and reverse primers with the same number were paired and numbered from the 5' end.
Primers were diluted to 10 µL and ready for use. Also enclosed was the -21M13 primer to do the
sequencing of the cloned HV1 region of the CHR DNA, which covered base pairs 16133 through
16569 and 1 through 40.
3. The protocol used at NIST to amplify and sequence the DNA. The laboratories, however, were
5
free to use any protocol with which they were familiar and felt comfortable.
4. A form table to record the results. This table provided the number of the Primer Set, the region
that each Primer Set amplified, and the length of the amplified region. We requested that the
laboratory fill in the differences found when they compared the sequence that they determined for
the mtDNA from CHR with that of the Cambridge Reference Sequence (Anderson et al., 1981).
5. All the laboratories received the following cautionary note:

WARNING: The DNA and cells were derived from a cell culture line from an apparently
healthy human subject. The cell culture line has been tested and found to be nonreactive for
hepatitis B surface antigen and HIV. However, no test method can ensure that a product derived
from human blood does not contain HIV, hepatitis or other infectious agents. HANDLE AS IF
CAPABLE OF TRANSMITTING DISEASE. (The second CHR cell culture line generated from the
same individual was not tested again for hepatitis or HIV. Normal precautions should be used.)
2.10. Differences in Methodology used by Laboratories in Interlaboratory Evaluation of SRM 2392,

1999.
The Bode Technology Group, Inc. essentially followed the NIST protocol except used a 6%
acrylamide/8.3 mol/L urea gel for the sequencing electrophoresis instead of a 4.75% acrylamide.
IIT Research Institute also followed the NIST protocol except used Taq Gold (PerkinElmer) for the
amplification reaction which was modified to include a hot start of 95 oC for 11 minutes. Microcon
100 microconcentrators (Amicon, Inc., Beverly, MA) were used to purify the PCR products. The
quantities of DNA were determined by capillary electrophoresis (CE) with a Beckman P/ACE 5010
System (Beckman Instruments, Inc., Fullerton, CA) as follows: 1 µL of the amplified product was
mixed with 25 µL of sterile deionized H2O containing 0.52 ng/µL of a 200 bp internal standard
(GenSura Laboratories, Inc., Del Mar, CA) and run on the CE. One determines the quantity of the
amplified product from the ratio of the PCR product peak area to the internal standard peak area
multiplied by a migration standard. A 6% acrylamide gel was used for the sequencing
electrophoresis instead of a 4.75% acrylamide.
Lark Technologies, Inc., followed the NIST protocol with the following differences: Ampli-Taq
DNA polymerase (PerkinElmer) was used to amplify the DNA; the dNTP’s were purchased from
Pharmacia Biotech, Inc. (Piscataway,NJ); the products were purified with Qiaquick PCR purification
kit (Qiagen); in the sequencing reactions, the amount of PCR product used varied from 1 to 3 µL
based on the concentration estimated from agarose gels; cycling conditions were 95 oC for one
minute, followed by 25 cycles of 96 oC for 15 seconds, 50 oC for 15 seconds, and 60 oC for 4
minutes; the sequence reactions were cleaned up by ethanol precipitation; a 4.25% polyacrylamide
gel was used for the sequencing electrophoresis instead of a 4.75% acrylamide; and a ABI 377
automated sequencer was used instead of the ABI 373. Electropherograms were printed for each
reaction and the sequences were manually edited based on the electropherogram patterns. Printed
electropherograms and a floppy disc with the sequence data were sent to NIST where the data were
compared to the Cambridge Reference Sequence (Anderson et al.,1981).
2.11. Interlaboratory Evaluation for SRM 2392-I, 2002
6
Three laboratories, in addition to NIST, participated in an interlaboratory evaluation of the mtDNA
sequence of HL-60. These laboratories included the FBI Laboratory, FBI Academy, Quantico, VA
22135; Armed Forces DNA Identification Laboratory (AFDIL), Armed Forces Institute of
Pathology, Rockville, MD 20850; and the Georgia Bureau of Investigation (GBI), Decatur, GA
30034. Each laboratory was asked to amplify and sequence the entire mtDNA of HL-60. NIST
provided: 1. A tube of DNA containing the extracted DNA ready for PCR amplification, 2. The 58
sets of primers labeled with either F# (forward primer) or R# (reverse primer), 3. A table to record
the results, and 4. Any other supplies that were needed and requested by the participants. They were
allowed to use any protocol for amplification or sequencing that they wished, but were requested to
provide a copy of that protocol to NIST. NIST also requested copies of the electropherograms to
enable us to resolve any discrepancies; although, as it turned out, there were no discrepancies.
2.12. Differences in Methodology Used by the Laboratories in the Interlaboratory Evaluation for
SRM 2392-I
2.12.1. Armed Forces DNA Identification Laboratory (AFDIL), SRM 2392-I
AFDIL has developed a high-throughput, automated sequencing procedure using 12 primer sets that
produce overlapping PCR products ranging from 825 to 1886 bp. The primers used to amplify the
first 11 products are based on those published in Levin et al. (1999). They are designated: Amp01 –
F361/R2216; Amp02 – F1993/R3557; Amp03 – F3441/R4983; Amp04 – F4797/R6526; Amp05 –
F6426/R8311; Amp06 – F8164/R9848; Amp07 – F9754/R11600; Amp08 – F11403/R13123;
Amp09 – F12793/R14388; Amp10 – F14189/R15396; Amp11 – F15260/R16084. The primers used
to amplify the control region were developed at AFDIL and are Amp12 – F15878/R649 (F15878 is
TTAACTCCACCATTAGCACC and R649 is TTTGTTTATGGGGTGATGTGA).
2.12.1.1. AFDIL PCR Amplification, SRM 2392-I:
The PCR mixture contained HL-60 DNA (1 µL), AmpliTaqGold® DNA polymerase (1 µL)
(Applied Biosystems), 10x PCR buffer (5 µL) (Applied Biosystems), dNTP’s (0.2 mmol/L)
(Invitrogen), 2 µL of forward and reverse primers (10 µmol/L) (MWG Biotech, High Point, NC)
plus dH2O to a final volume of 50 µL. The 10x PCR buffer was the same as that used by NIST.
Thermal cycling was conducted in a PerkinElmer 9700 thermocycler with the following conditions:
10 min at 96 oC (activation of AmpliTaq Gold®), plus 40 cycles of 94 oC for 15 sec, 56 oC for 30 sec,
and 72 oC for 1 min. The purity and size of the PCR products were assessed by electrophoresis in a
0.7% agarose gel containing 0.3 µg/mL of ethidium bromide. The PCR products were purified with
Shrimp Alkaline Phosphatase/Exonuclease I (Amersham Pharmacia, Piscataway, NJ). 5 µL of
exonuclease I (10 U/µL) and 10 µL of Shrimp Alkaline Phosphatase (1 U/µL) was added to each
tube containing PCR product. The tubes were heated at 37oC for 15 min followed by 94oC for 15
min in a PerkinElmer 9700 thermocycler.
2.12.1.2. AFDIL Sequencing, SRM 2392-I :
Cycle sequencing was performed with the ABI PRISM® BigDye® terminators (original version)
cycle sequencing kit (Applied Biosystems). The sequencing mixture contained 9 µL dH2O, 6 µL
7
BigDye® dilution buffer (400 mmol/L TRIS, 10 mmol/L MgCl2, pH 9.0), 2 µL BigDye®
terminator reaction mixture, 1 µL of forward or reverse primer (10 µmol/L each) and 2 µL of HL-60
PCR product for a total volume of 20 µL. A few of the sequencing primers (e.g., R649) required the
use of the ABI PRISM® dGTP BigDye® terminator kit (Applied Biosystems). Thermal cycling was
conducted in a PerkinElmer 9700 thermocycler at the following conditions: an initial 1 min
denaturation at 96 oC, followed by 25 cycles of 94 oC for 15 sec, 50 oC for 5 sec, and 60 oC for 2 min.
The DNA product was purified by filtration through a spin column matrix (Edge BioSystems,
Gaithersburg, MD). Electrophoresis and sequencing were performed with an ABI PRISM® 3100
Genetic Analyzer (Applied Biosystems) using POP-6™ polymer (Applied Biosystems) with a 50 cm
capillary. Data analysis was executed using Sequencher Plus 4.0.5b11 (Gene Codes, Ann Arbor,
MI). The HL-60 sequence differences were identified by comparison to the Cambridge Reference
Sequence as revised by Andrews et al. (1999). In most cases, sequence information was acquired for
both the forward and reverse directions. In some regions, two separate reactions using the same
primer were conducted (indicated by 2X in the following list of primers). A total of 95 sequencing
reactions plus 1 pGEM reaction were conducted in a 96 well format. If the primer failed the first
trial, the reaction was repeated. The finding of a heteroplasmy at np 12071 was also confirmed by an
additional PCR and sequencing reaction. The following primers from Levin et al. (1999) were used
to sequence the 12 PCR amplicons:
Amp01 (F361/R2216): F361, R921, F1234, R1425, F873, R2216, F1657, R1769
Amp02 (F1993/R3557): F1993, R2660, R2834, R3557, F2417, R3006, F3234
Amp03 (F3441/R4983): F3441, R3940, F3931 (2X), R4982, F4392, R4162
Amp04 (F4797/R6526): F4797 (2X), R6526, F5700 (2X), F5318, R5882, F6242
Amp05 (F6426/R8311): F6426 (2X), R7255, F7645 (2X), R8311, F7075, R7792
Amp06 (F8164/R9848): F8164 (2X), R9059, F8903, R9848, F8539, R9403, F9309
Amp07 (F9754/R11600): F9754 (2X), R10556, F11001 (2X), R11600, F10386, R11267
Amp08 (F11403/R13123): F11403 (2X), F12357, R13123, F11901 (2X), F12601, R12876 Amp09
(F12793/R14388): F12793, R13611, F13518 (2X), R14388, F13188, R13343,
F13899, R13935
Amp10 (F14189/R15396): F14189 (2X), R15396, F14909, R14996, F14470
Amp11 (F15260/R16084): F15260, R16084, F15574, R15774
Amp12 (F15878/R649): F15971, R16175 (2X), F16450 (2X), R274, F314 (2X), R649 (2X),
F16190, R16400
In Amp12, F15971 came from Levin et al. (1999). The other primers were designed by AFDIL and
were as follows:
R16175: TGGATTGGGTTTTTATGTA
F16450: GCTCCGGGCCCATAACACTTG
R274: TGTGTGGAAAGTGGCTGTGC
F314: CCGCTTCTGGCCACAGCACT
R649: TTTGTTTATGGGGTGATGTGA
F16190: CCCCATGCTTACAAGCAAGT
R16400: GTCAAGGGACCCCTATCTGA
2.12.2. Georgia Bureau of Investigation (GBI), Interlaboratory Evaluation, SRM 2392-I
The Georgia Bureau of Investigation used the same protocol as that used by NIST with the
8
following exceptions: 1. A PerkinElmer model 9600 was used for thermal cycling; 2. Amplified
DNA was electrophoresed in 2.75% agarose gels; 3. The cycle sequencing was performed with a
PerkinElmer model 9600 using the program provided with the BigDye® kit (25 cycles of 96 oC for
10 sec, 50 oC for 5 sec, and 60 oC for 4 min); 4. Some samples that needed to be cycle sequenced
again using more amplicon were not purified before precipitation; 5. The samples were precipitated
using isopropanol precipitation as per the BigDye® instructions; and 6. Comparison of the sequence
data was performed with Sequencer 3.1.1 software (Gene Codes, Ann Arbor, MI).
2.12.3. Federal Bureau of Investigation (FBI), Interlaboratory Evaluation, SRM 2392-I
2.12.3.1. FBI Polymerase Chain Reaction (PCR):
The PCR mixture contained from 0.1 to 1.4 ng HL-60, AmpliTaq Gold® Polymerase (2.5 units)
(Applied Biosystems), 10x PCR buffer (5 µL) (Applied Biosystems), GeneAmp® dNTPs (0.2
mmol/L each) (Applied Biosystems), forward and reverse primers (0.4 µmol/L each), plus dH2O to a
final volume of 50 µL. The 10x buffer (pH 8.3) was the same as used by NIST. Thermal cycling was
conducted in a GeneAmp® PCR System 9700 (PerkinElmer) and consisted of 10 min at 95 oC
followed by 35 cycles of 94 oC for 20 sec, annealing temperatures of 50 oC (primer set 49), 51 oC
(primer sets 1, 7, 44, 57), 52 oC (primer sets 6, 8, 30, 45), 53 oC (primer set 53) and 56 oC (all other
primer sets) for 10 sec, and 72 oC for 30 sec and ending with a final extension of 7 min at 72 oC.
Amplified products were purified by treatment with Exo-SAP-IT (5 µL for every 25 µL of PCR
product) (USB Corp., Cleveland, OH). Samples of the PCR products were electrophoresed in 1.2%
agarose gels containing ethidium bromide to assess the purity, size, and quantity of the PCR
products.
2.12.3.2. FBI Sequencing:
Cycle sequencing using fluorescent dye–labeled terminators was performed with an ABI PRISM®
dRhodamine Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA Polymerase,
FS (Applied Biosystems). Thermal cycling was conducted in a GeneAmp® PCR System 9700
(Applied Biosystems) and started with 1 min at 96 oC followed by 25 cycles of 96 oC for 15 sec, 50
o
C for 1 sec and 60 oC for 1 min and ended with a final incubation at 15 oC for 10 min. These
products were purified with Centri-Sep™ spin columns (Princeton Separation, Inc., Adelphia, NJ)
and electrophoresed on an ABI PRISM® 310 Genetic Analyzer (Applied Biosystems) using POP-
6™ and either a 47 or 61 cm capillary. Comparison of the sequence data was performed with
Sequencer 4.1 software (Gene Codes).
The FBI also discovered the problem with original primer set 51 (see Materials and Methods). The
reverse primer incorporated a G at position 14368 [an error in the original Cambridge Reference
Sequence (Anderson et al., 1981) that was found and corrected by Andrews et al. (1999)]. However,
sequencing with primer set 52 showed a C at position 14368. They prepared a new set of primers
(primer set 51.5) to amplify approximately a 400 bp fragment that encompassed np 14368. The new
primers were:
F14217: 5’-CTAATCAACGCCCATAATCATAC-3’ and
R14620: 5’-GTTTTCTTCTAAGCCTTCTCC-3’.
The new primer set confirmed that the correct base at position 14368 was a C.
9
2.13. Permissions
The development of SRM 2392-I was deemed exempt from the policy of Part 27 of Title 15 of the
Code of Federal Regulations by the NIST Institutional Review Board and the Director of the
Chemical Science and Technology Laboratory. This work fit into the exemption category described
in 15 CFR 27.101(b)(4) which states: “Research, involving the collection or study of existing data,
documents, pathological specimens, or diagnostic specimens, if, these sources are publicly available
or if the information is recorded by the investigator in such a manner that subjects cannot be
identified, directly or through identifiers linked to the subjects.”
ATCC also waived condition 3(c) in their Material Transfer Agreement which states that the
“purchaser shall not sell, lend, distribute or otherwise transfer the material or replicates to any
others” for the use of HL-60 in the NIST mtDNA SRM. They stated that, in their view, “as a
government agency, NIST will not be providing this material as a commercial product despite the
collection of fees for the SRM.”
2.14. Final Preparation of DNA for SRM 2392 and SRM 2392-I
2.14.1. CHR DNA Preparation and Confirmation SRM 2392:
CHR cells were grown in RPMI-1640 media supplemented with L-glutamine, sodium bicarbonate,
penicillin, streptomycin and 10% fetal calf serum (Sigma Chemical Co.), centrifuged and
resuspended in 10 mL of phosphate buffered saline (PBS) to a final cell count of 1.2 x 106 cells/mL.
DNA was extracted using the Qiagen Maxi Kit according to the Qiagen protocol (Qiagen Inc.). The
concentration of the DNA in the extract was determined with an UV spectrophotometer to be 82.3
ng/µL.
PCR amplification was performed on the extract using primer set 57 (HV1 region) and primer set 1
(HV2 region) in order to verify the sequence. The extract was diluted 1/10 (8.23 ng/µL), and 1 µL
was used in PCR reaction. The PCR reaction mixture was the same as that described in the
polymerase chain reaction section of Materials and Methods, with the exception that the forward and
reverse primers were used at a concentration of 0.2 µM.
Thermal cycling was conducted in a PerkinElmer 9600 (HV1) or 9700 (HV2) and consisted of 30
sec at 95 oC, followed by 32 cycles of 20 sec at 94 oC (denaturation), 10 sec at 56 oC (annealing), and
30 sec at 72 oC (extension), and ending with a final extension of 7 minutes at 72 oC. A sample of the
amplified DNA was electrophoresed and stained with ethidium bromide and purified as described in
the polymerase chain reaction section of Materials and Methods except that a 2% agarose gel was
used. Cycle sequencing, electrophoresis of the products, and the analysis of the sequence data were
performed as described in the sequencing section of Materials and Methods. The polymorphisms of
the HV1 and HV2 regions and the C-stretch (HV1 region) seen in the pre-production mode samples
were confirmed in the production mode samples.
The DNA extract was diluted to 1 ng/µL with TE buffer and PCR amplified using Primer Set 1
(HV2) to verify that this concentration was appropriate to use for PCR. Sixty µL of the CHR DNA
extract at a concentration of 1 ng/µL was aliquotted into 260 tubes.
10
2.14.2. CHR Cloned DNA Production, SRM 2392:
DNA from one of the C-stretch clones (containing 12 C’s) prepared as described earlier was used to
transform TG-1 cells. Following the transformation and growth of the clones, the bacteriophage
DNA was isolated and sequenced to verify the presence of the 12 C region. Following this
verification, a large scale transformation was performed for SRM production. The cloned DNA that
was obtained following this transformation was quantitated by UV spectrophotometry and diluted to
100 ng/µL with TE buffer. The cloned DNA was sequenced and analyzed as described in section
entitled Bacteriophage DNA Isolation and Sequencing in Materials and Methods to verify that it
contained the C-stretch of 12 C’s plus all the additional polymorphisms previously found in the HV1
region of the CHR cells. For the final SRM, 260 tubes containing 10 µL at a concentration of 100
ng/µL were prepared.
2.14.3. 9947A DNA Production, SRM 2392:
Cell culture line 9947A was grown by Life Technologies, Inc. (Gaithersburg, MD) who extracted the
DNA and submitted a sample (at a concentration of 1 ng/µL) to NIST for verification of the
sequence. The HV1 (primer set 57) and HV2 (primer set 1) regions as well as the region amplified
by primer set 30 were checked by PCR and sequencing to verify that the DNA was the same as
sequenced earlier. One µL of DNA (1 ng) was used as the template in the PCR reaction mixture,
which was performed as described in the polymerase chain reaction section of the Materials and
Methods section, with the exception that the forward and reverse primers were used at a
concentration of 0.2 µmol/L. Thermal cycling was conducted in a PerkinElmer 9700 and consisted
of 30 sec at 95oC, followed by 32 cycles of 20 sec at 94 oC (denaturation), 10 sec at 56 oC
(annealing), and 30 sec at 72 oC (extension), and ending with a final extension of 7 minutes at 72 oC.
A sample of the amplified DNA was electrophoresed and stained with ethidium bromide and
purified as described in the section entitled polymerase chain reaction except that a 1% agarose gel
was used. Cycle sequencing, electrophoresis of products, and analysis of sequence data were
performed as described earlier. Examination of the results from primer sets 57 (HV1), 1 (HV2), and
30 indicated that the DNA supplied by Life Technologies was the same as that examined earlier in
the pre-production mode.
After NIST verified that the sequence was okay, Life Technologies, Inc. prepared 250 tubes
containing 60 µL of 9947A DNA at a concentration of 1 ng/µL.
2.14.4.HL-60 Production, SRM 2392-I:
DNA from the HL-60 cell culture was extracted, isolated, and quantified by the Professional
Services Department of the American Type Culture Collection (ATCC, Manassas, VA). DNA was
isolated from the HL-60 cell culture using the QIAamp DNA Blood Mini Kit (Qiagen, Inc.).
Quantification was determined by the Quantiblot Human DNA Quantification Kit (Applied
Biosystems). The final concentration, 1.4 ng/µL, is based on 12 replicate tests. The DNA purity
(A260:A280 = 1.9) was determined spectrophotometrically. The integrity of DNA was determined
electrophoretically using 0.4% agarose gels.
11
Sixty-five µL of HL-60 DNA were dispensed into 260 tubes. Three randomly chosen tubes were
used to send to the laboratories that agreed to participate in the Interlaboratory Evaluation. Each of
the three laboratories plus NIST used all 58 primer sets to sequence the entire 16,569 base pairs of
the HL-60 mtDNA. All four laboratories found the identical sequence, thereby confirming the
accuracy of the sequence determined at NIST.
3. Results And Discussion
3.1. SRM Templates:
Two DNA templates, CHR and 9947A, are included in the NIST human mtDNA amplification and
sequencing SRM 2392. The CHR and 9947A DNA samples came from human cell culture lines that
were developed from apparently normal individuals. The DNA template in SRM 2392-I, HL-60,
came from a promyelocytic cell line prepared from the peripheral blood leukocytes from an
individual with acute promyelocytic leukemia. The DNA extracted from 9947A and HL-60 is the
total DNA including nuclear DNA. The DNA from CHR was isolated in a manner which enhanced
the concentration of the mtDNA, but did not totally eliminate the nuclear DNA. However, both types
of extracted DNA amplified and sequenced accurately. SRM 2392 also provides cloned DNA from
the HV1 region of the CHR template which contains a C-stretch. In most people, the HV1 region
has a string of cytosine (C) residues interrupted by a thymine (T) at np 16189. In some individuals,
however, a transition occurs which changes the T to a C producing a long string of C’s called the C-
stretch. When this happens, sequencing beyond the C-stretch becomes very difficult, if not
impossible. Clones of the HV1 region containing the C-stretch indicated that the number of C’s
differed among the different clones and the difficulty in sequencing was due to frameshifts that
resulted from the simultaneous sequencing of templates with differing numbers of C’s (Bendall and
Sykes, 1995; Levin et al., 1995; Levin et al., 1997). We found, however, that one could sequence
through the entire HV1 region including the C-stretch without problems if one used the clone of the
area. Therefore, we included the cloned HV1 region of the CHR DNA template in SRM 2392.
In addition to templates CHR and 9947A in SRM 2392 and HL-60 in SRM 2392-I, we have included
all the mtDNA sequence information from two additional templates in Table 2: 1. GM03798, a
normal lymphoblastoid cell line and 2. GM10742A from an individual with the mtDNA disease,
Leber Hereditary Optic Neuropathy (LHON), which causes blindness in early adulthood. Both of
these cell lines were obtained from the NIGMS Human Genetic Mutant Cell Repository. The DNA
from these cell lines are not part of SRMs 2392 or 2392-I and the data are included for information
and comparison purposes only.
3.2. Primers:
The 58 sets of unique primers were designed to allow the amplification and sequencing of any
region or the entire 16,569 base pairs that comprise human mtDNA. The sequence of both the
forward and reverse primers that are in each set are shown in Table 1. The numbers indicate the 5'
end of the primer. They are all between 19 and 28 base pairs long and the criteria that were used to
choose these primers were primer melting temperatures (Tm) between 50 and 65 oC, primer length
between 15 and 30 base pairs, and PCR product length between 220 and 1000 base pairs. In
actuality, these primers gave PCR product lengths between 224 and 976 base pairs. The primers
12
were designed to produce sequences that overlapped with both the previous and following regions to
allow those areas in the beginning and end of electropherograms which are difficult to sequence to
become readable and to prevent any gaps in the sequence. The readable region is always smaller
than that of the amplified region. In the process of developing SRM 2392-I, we found that reverse
primer of primer set 51 needed to be changed (see explanation in section entitled “mtDNA primers”
in the Materials and Methods section). The new reverse primer 51 is shown in Table 1. The change
is in bold and underlined.
In addition to the designed primers, we also used the -21M13 primer (Table 1) to sequence the
cloned DNA from the HV1 region of the CHR template which contained the C-stretch. The PCR
products were produced under the same conditions for all 58 primer sets and generated single,
distinct bands in all cases (Levin et al., 1999).
3.3. Differences between the SRM DNAs and the Cambridge Reference Sequence:
Human mtDNA was completely sequenced in 1981 and is referred to as the Cambridge Reference
Sequence (Anderson et al., 1981). This sequence is a consensus sequence based primarily on a
placenta, a few areas sequenced from HeLa cells, and five ambiguous areas where the sequence from
bovine mtDNA was used. All investigators, who subsequently examined human mtDNA, have used
the numbering system of the Cambridge Reference Sequence and have compared their sequence
findings to those found in 1981. In 1999, Andrews et al. resequenced the original placenta used in
1981. They found that the 1981 sequence had a number of errors and that the placenta had a number
of rare polymorphisms. At the present time, investigators compare their sequences to the 1999
revised sequence. However, the DNA from this placenta is not available for use as a positive control
during actual experiments; whereas, NIST SRMs 2392 and 2392-I are available. Table 2 shows the
mtDNA differences compared to the Cambridge Reference Sequence that were found at NIST with
the three templates - CHR and 9947A (in SRM 2392) and HL-60 (in SRM 2392-I) and the two
additional templates - GM03798, and GM10742A - that are included for information and
comparison purposes. In all five templates, all 58 areas comprising the entire mtDNA were
completely amplified and sequenced at least twice. There are 13, 9, 11, 4, and 12 differences in the
non-coding regions of templates CHR, 9947A, HL-60, GM03798, and GM10742A, respectively and
33, 23, 33, 19, and 31 differences in the coding regions of templates CHR, 9947A, HL-60,
GM03798, and GM10742A, respectively. All of the differences from the Cambridge Reference
Sequence found in CHR, 9947A, HL-60, GM03798, and GM10742A are shown in Figure 1 along
with many of the mutations associated with Leber Hereditary Optic Neuropathy (LHON) that have
been noted in the literature (Wallace et al., 1997). With one exception, none of the base pair changes
found in the coding regions of the apparently normal templates (CHR, 9947A, GM03798) sequenced
at NIST correlate with any of the changes found associated with the published disease states found
in the MitoMap web site: http://www.mitomap.org The one exception was a secondary LHON
mutation found in CHR. HL-60 and GM10742A, however, did have some mutations that have been
associated with LHON.
3.4. Meaning of the Differences from Cambridge Reference Sequence:
Since three templates came from apparently normal individuals, one from an individual with acute
promyelocytic leukemia, and one from an individual with LHON, it was of interest to determine if
13
the differences in the coding regions would actually cause amino acid changes in the resultant
protein structures. The genetic code for human mtDNA is slightly different from the universal
genetic code. Table 3 shows the differences in the universal genetic code that one needs to consider
when determining the amino acid sequence designated by the three base pair codons in mtDNA.
Many of the differences from the Cambridge Reference Sequence were in the third position wobble
and did not affect the amino acid sequence. However, CHR, 9947A, HL-60, GM03798, and
GM10742A had 11, 12, 15, 8, and 14 different base pairs, respectively, that would result in a
different amino acid from that designated by the Cambridge Reference Sequence of 1981. However,
comparison with the revised Cambridge Reference Sequence of 1999, indicate that only 3, 4, 7 plus
2 in tRNAs, 0, and 7 would result in amino acid changes in CHR, 9947A, HL-60, GM03798, and
GM10742A, respectively. These structural changes, however, do not necessarily mean a functional
change has occurred in the protein. To determine if a functional change has occurred, one still needs
to decipher whether the amino acid change is in an active site on the protein. We have developed an
interactive web site that will easily and quickly determine if the DNA change causes an amino acid
change (Lee and Levin, 2002). The changes that result in amino acid changes are shown in Table 2.
3.5. The Interlaboratory Evaluation of the CHR and HL-60 Templates:
The interlaboratory evaluation of CHR was conducted by four laboratories including NIST. The
interlaboratory evaluation of HL-60 was conducted by four laboratories including NIST. Any
changes made by the various laboratories to the NIST protocol are listed in the section on Materials
and Methods. Each laboratory was instructed to amplify and sequence the 58 areas designated by
the 58 primer sets and those laboratories that sequenced CHR were also to sequence the cloned DNA
from the HV1 region. Those sequencing CHR were designated Labs 1, 2, 3, and 4. Labs 1, 2 and 3
found essentially the same polymorphisms. Laboratory 4 had less experience with sequencing
mtDNA and did find differences that the other laboratories did not observe. Interlaboratory data was
considered ambiguous and excluded if the following problems were observed: 1. If the computer
results were ambiguous as indicated by calling a peak “N” rather than A, C, G or T. 2. If the
differences from the Cambridge Reference Sequence were not consistently seen within a laboratory,
i.e., if the laboratory sequenced in both the forward and reverse directions and one direction agreed
with the Cambridge Reference Sequence and the other direction did not agree, we assumed the
results that agreed with the Cambridge Reference Sequence were correct. 3. If, within any one
laboratory, the difference from Cambridge Reference Sequence was seen with one primer set, but
not in the overlapping sequences seen in the previous or subsequent primer sets, the results agreeing
with the Cambridge Reference Sequence were considered correct. Even with these exclusions,
Laboratory 4 had many differences that were not seen by the other labs. One problem was that they
did not provide data from primer sets 29, 39, and 41 and we were unable to check the overlapping
sequences. Laboratory 2 was unable to sequence the clone, which was not a problem for the other
laboratories. Laboratory 3 was missing data from primer sets 36 and 48. Laboratories 1 and 3 noted
a heteroplasmy3 at base number 6849 (the Cambridge Reference Sequence found an A at this site).
In final preparation of SRM 2392, a new blood sample was obtained from CHR and a new cell
3
culture line was established. The sequence analysis of the new CHR was identical to the first cell
line except no heteroplasmy was found at bp 6849. Therefore, the cell line supplied with SRM
2392 does not have this heteroplasmy. The data on the first cell line are included in the text to
indicate the agreement in the interlaboratory evaluation which was done with the first cell line.
14
Laboratory 1 found a G at this site, but closer examination of the electropherogram showed that an A
peak existed under the G peak. Laboratory 3 also noted the A/G heteroplasmy at this site.
Laboratory 4 did not note the heteroplasmy, but when their electropherograms were examined at
NIST, the A/G heteroplasmy was noted. NIST did not have the electropherograms of Laboratory 2,
but on questioning them, they agreed that the heteroplasmy was there, but that they had missed it.
One of the problems with finding heteroplasmic sites is that if the computer call is the same as the
Cambridge Reference Sequence, one would not necessarily examine that site more closely. If the
computer call is different from the Cambridge Reference Sequence, one would look more closely at
the electropherogram and then note the presence of a smaller peak under the main peak. The cell
line CHR was reestablished with fresh blood and the entire 16,569 base pairs were amplified and
sequenced again. The heteroplasmy at 6849 was not present in the new cell line. The new cell line
agreed with the Cambridge Reference Sequence at this site. It is the DNA from the new cell line that
is in SRM 2392.
With the exceptions of the differences noted here, the interlaboratory evaluation of CHR was
successful in that most of the laboratories found the same results. The many differences noted by
Laboratory 4, who was less experienced at sequencing mtDNA, confirms and emphasizes the need
for Standard Reference Materials for sequencing mtDNA. If Laboratory 4 had the NIST mtDNA
SRM 2392 or SRM 2392-I and had run it along side of their unknown sample, they would have
realized that they were finding an undue number of differences and could have reexamined their
procedures to try to determine the reason for these excessive differences.
The Interlaboratory Evaluation of the mtDNA sequence of HL-60 was conducted by four
laboratories (see Materials and Methods section) including NIST. All the laboratories produced
identical results ).
4. Conclusions
Two NIST Standard Reference Materials (SRM 2392 and 2392-I) have been prepared. These SRMs
allow one to amplify and sequence any region or the entire 16,569 base pairs which comprise human
mtDNA. Fifty-eight pairs of unique primers have been designed, tested and shown to work well in
the amplification and sequencing procedures (note: the reverse primer for primer set 51 has been
changed from that originally listed in SRM 2392, in Levin et al., 1999, and in Appendix C). The
three DNA templates (CHR and 9947A in SRM 2392, and HL-60 in SRM 2392-I) have
characteristic polymorphisms throughout the non-coding and coding regions of the DNA and
therefore, can serve as positive controls during PCR amplification and sequencing. With one
exception, none of the polymorphisms found in CHR and 9947A correspond to any of the published
base pair changes that have been correlated with specific diseases. The one exception is that CHR
has one mutation that has been associated with a secondary mutation associated with LHON. HL-
60, however, has 4 mutations that have been associated with the mitochondrial disease LHON.
Compared to the 1981 Cambridge Reference Sequence, CHR mtDNA had 13 differences in the non-
coding regions and 33 differences in the coding regions, 9947A mtDNA had 9 differences in the
non-coding regions and 23 differences in the coding regions, HL-60 had 11 differences in the non-
coding regions and 33 differences in the coding regions. GM03798 and GM 10742A, whose data
are included for comparison and information, had 4 and 12 differences in the non-coding regions,
15
respectively and 19 and 14 differences in the coding regions, respectively. All of these differences
are shown in Fig. 1 and Table 2. These differences in the coding regions do result in some amino
acid changes in the proteins coded for by mtDNA. Four laboratories participated in an
interlaboratory evaluation of the CHR template and four laboratories participated in the
interlaboratory evaluation of HL-60; some differences between laboratories were noted in the CHR
evaluation, but, in general, agreement was good. No differences were noted in the HL-60
interlaboratory evaluation. The use of NIST SRM 2392 and SRM 2392-I will provide quality control
to the scientific and medical communities when they amplify and sequence human mtDNA.
5. Acknowledgments
The authors are grateful to The Bode Technology Group, Inc., Sterling, VA; IIT Research Institute,
Newington, VA; and Lark Technologies, Inc., Houston, TX for their participation in the
Interlaboratory Evaluation of the CHR template and to the FBI Laboratory, FBI Academy, Quantico,
VA; the Armed Forces DNA Identification Laboratory (AFDIL), Armed Forces Institute of
Pathology, Rockville, MD; and the Georgia Bureau of Investigation (GBI), Decatur, GA for their
participation in the Interlaboratory Evaluation of HL-60. The research to prepare SRM 2392-I
containing HL-60 was supported by Interagency Agreement number 1999-IJ-R-094 from the
National Institute of Justice. The authors also acknowledge the funding received from the National
Institute of Justice through the NIST Office of Law Enforcement Standards and the support from the
Advanced Technology Program at NIST to develop SRM 2392. Points of view are those of the
authors and do not necessarily represent the position of the U.S. Department of Justice. The authors
thank Prasad Reddy for his help and advice on cloning, Lois A. Tully (when serving as a post-
doctoral research associate at NIST) and Julie T. Chen for checking selected sequence areas of DNA
samples from CHR, the CHR clone, and 9947A before the final assembly of SRM 2392 that became
available in 1999. We also thank MaryPat Jones for confirming the sequences of some areas of the
second generation of the CHR cell line. The authors are grateful to Gettysburg College for the partial
funding of Koren A. Holland during her sabbatical year at NIST. We also acknowledge and thank
Mary Pat Jones and Kathryn Kapfer for help in sequencing GM10742A at NIST.
6. References
Anderson, S., Bankier, A. T., Barrell, B. G., deBrujin, M. H. L., Coulson, A. R., Drouin, J., Eperon,
I. C., Nierlich, D. P., Roe, B. A., Sanger, F., Schreier, P. H., Smith, A. J. H., Staden, R., and Young,
I. G. (1981). Sequence and organization of the human mitochondrial genome. Nature 290: 457-465.
Andrews, R.M., Kubacka, I., Chinnery, P.F., Lightowlers, R.N., Turnbull, D.M., Howell, N. (1999).
Reanalysis and revision of the Cambridge Reference Sequence for human mitochondrial DNA.
Nature Genetics 23:147.
Ballinger, S. W., Bouder, T. G., Davis, G. S., Judice, S. A., Nicklas, J. A., and Albertini, R. J.
(1996). Mitochondrial genome damage associated with cigarette smoking. Cancer Research 56:
5692-5697.
Bendall, K. E., and Sykes, B. C. (1995). Length heteroplasmy in the first hypervariable segment of
the human mtDNA control region. Am. J. Hum. Genet. 57: 248-256.
16
Bogenhagen, D., and Clayton, D. A. (1974). The number of mitochondrial deoxyribonucleic acid
genomes in mouse L and human HeLa cells. J. Biol. Chem. 249: 7991-7995.
Grossman, L. I. (1995). Mitochondrial mutations and human disease. Env. & Mol. Mutag. 25:
(supplement 26) 30-37.
Holland, M. M., Fisher, D. L., Mitchell, L. G., Rodriquez, W. C., Canik, J. J., Merril, C. R., and
Weedn, V. W. (1993) Mitochondrial DNA sequence analysis of human skeletal remains:
Identification of remains from the Vietnam War. J. Forensic Sciences 38: 542-553.
Holland, M. M., Fisher, D. L., Roby, R. K., Ruderman, J., Bryson, C., and Weedn, V. W. (1995)
Mitochondrial DNA sequence analysis of human remains. Crime Laboratory Digest 22: 109-115.
Howell, N., McCullough, D. A., Kubacka, I., Halvorson, S., and Mackey, D. (1992). The sequence
of human mtDNA: the question of errors versus polymorphisms. Am. J. Hum. Genet. 50: 1333-1337.
King, M. P., and Attardi, G. (1989). Human cells lacking mtDNA: Repopulation with exogenous
mitochondria by complementation. Science 246: 500-503.
Lee, M.S. and Levin, B.C. (2002). MitoAnalyzer, a computer program and interactive web site to
determine the effects of single nucleotide polymorphisms and mutations in human mitochondrial
DNA. Mitochondrion 1: 321-326.
Levin, B. C., Cheng, H., O'Connell, C., Reeder, D. J., and Holland, M. (1995). Evaluation of
alternative enzymes and additives to improve sequencing of mitochondrial DNA. Proceedings of the
Fifth International Symposium on Human Identification, Scottsdale, AZ, October 8-12, 1994, p.
170.
Levin, B. C., Cheng, H., Holland, M., and Reeder, D. J. (1997). Heteroplasmy responsible for
difficulty experienced in sequencing the human mitochondrial DNA HV1 region containing the C-
stretch. Proceedings of the Seventh International Symposium on Human Identification, Scottsdale,
AZ, September 19-21, 1996, p. 166.
Levin, B. C., Cheng, H., and Reeder, D. J. (1999). A human mitochondrial DNA Standard Reference
Material for quality control in forensic identification, medical diagnosis, and mutation detection.
Genomics 55: 135-146.
Levin, B.C., Holland, K.A., Hancock, D.K., Coble, M., Parsons, T.J., Kienker, L.J., Williams, D.W.,
Jones, MP., and Richie, K.L., (2003). Comparison of the complete mtDNA genome sequences of
human cell lines - HL-60 and GM10742A - from individuals with promyelocytic leukemia and leber
hereditary optic neuropathy, respectively, and the inclusion of HL-60 in the NIST human
mitochondrial DNA Standard Reference Material - SRM 2392-I. Mitochondrion (in press, 2003).
Marzuki, S., Lertrit, P., Noer, A. S., Kapsa, R. M. I., Sudoyo, H., Byrne, E., and Thyagarajan, D.
(1992). Reply to Howell et al.: the need for a joint effort in the construction of a reference data base
17
for normal sequence variants of human mtDNA. Am. J. Hum. Genet. 50: 1337-1340.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning, A Laboratory Manual,
Second Edition, Cold Spring Harbor Laboratory Press, NY.
Wallace, D. C. (1992). Mitochondrial genetics: a paradigm for aging and degenerative diseases.
Science 256: 628-632.
Wallace, D. C., Brown, M. D., and Lott, M. T. (1997). Mitochondrial genetics. In “Emery and
Rimoin’s Principles and Practice of Medical Genetics” (D. L. Rimoin, J. M. Connor, and R. E.
Pyeritz RE, Eds.), Vol. 1, Third Edition, pp. 277-332, Churchill Livingstone, NY.
18
Table 1. Sequences for Primer Sets Used for PCR Amplification of Human mtDNA
Primer
Set Primer Sequence
Number
F15 CACCCTATTAACCACTCACG
1(HV2)
R484 TGAGATTAGTAGTATGGGAG
F361 ACAAAGAACCCTAACACCAGC
2
R921 ACTTGGGTTAATCGTGTGACC
F756 CATCAAGCACGCAGCAATG
3
R1425 AATCCACCTTCGACCCTTAAG
F873 GGTTGGTCAATTTCGTGCCAG
4
F1234 CTCACCACCTCTTGCTCAGC
5
R1769 GCCAGGTTTCAATTTCTATCG
F1587 TGCACTTGGACGAACCAGAG
6
R2216 TGTTGAGCTTGAACGCTTTC
F1657 CTTGACCGCTCTGAGCTAAAC
7
F1993 AAACCTACCGAGCCTGGTG
8
F2105 GAGGAACAGCTCTTTGGACAC
9
R2660 AGAGACAGCTGAACCCTCGTG
F2417 CACTGTCAACCCAACACAGG
10
R3006 ATGTCCTGATCCAACATCGAG
F2834 CCCAACCTCCGAGCAGTACATG
11
R3557 AGAAGAGCGATGGTGAGAGC
F2972 ATAGGGTTTACGACCTCGATG
12
F3234 AGATGGCAGAGCCCGGTAATC
13
F3441 ACTACAACCCTTCGCTGACG
14
R3940 TGAAGCCTGAGACTAGTTCGG
F3635 GCCTAGCCGTTTACTCAATCC
15
R4162 TGAGTTGGTCGTAGCGGAATC
F3931 TCAGGCTTCAACATCGAATACG
16
R4728 TTATGGTTCATTGTCCGGAGAG
F4183 TTTCTACCACTCACCCTAGCATTAC
17
F4392 CCCATCCTAAAGTAAGGTCAGC
18
R4983 GGTTTAATCCACCTCAACTGCC
F4447 TTGGTTATACCCTTCCCGTAC
19
R4982 GTTTAATCCACCTCAACTGCC
F4797 CCCTTTCACTTCTGAGTCCCAG
20
R5553 AGGGCTTTGAAGGCTCTTG
F4976 ATTAAACCAGACCCAGCTACG
21
19
F5318 CACCATCACCCTCCTTAACC
22
R5882 GCTGAGTGAAGCATTGGACTG
F5700 TAAGCACCCTAATCAACTGGC
23
R6262 GCCTCCACTATAGCAGATGCG
F5999 TCTAAGCCTCCTTATTCGAGC
24
R6526 ATAGTGATGCCAGCAGCTAGG
F6242 CGCATCTGCTATAGTGGAGG
25
F6426 GCCATAACCCAATACCAAACG
26
R7030 TGGGCTACAACGTAGTACGTG
F6744 GGCTTCCTAGGGTTTATCGTG
27
R7255 TTTCATGTGGTGTATGCATCG
F7075 GAGGCTTCATTCACTGATTTCC
28
R7792 GGGCAGGATAGTTCAGACGG
F7215 CGACGTTACTCGGACTACCC
29
F7645 TATCACCTTTCATGATCACGC
30
R8215 GACGATGGGCATGAAACTG
F7901 TGAACCTACGAGTACACCGACTAC
31
R8311 AAGTTAGCTTTACAGTGGGCTCTAG
F8164 CGGTCAATGCTCTGAAATCTGTG
32
R8669 CATTGTTGGGTGGTGATTAGTCG
F8539 CTGTTCGCTTCATTCATTGCC
33
R9059 GTGGCGCTTCCAATTAGGTG
F8903 CCCACTTCTTACCACAAGGC
34
R9403 GTGCTTTCTCGTGTTACATCG
F9309 TTTCACTTCCACTCCATAACGC
35
R9848 GAAAGTTGAGCCAATAATGACG
F9449 CGGGATAATCCTATTTATTACCTCAG
36
R9995 AGAGTAAGACCCTCATCAATAGATGG
F9754 AGTCTCCCTTCACCATTTCCG
37
R10275 AAAGGAGGGCAATTTCTAGATC
F10127 ACTACCACAACTCAACGGCTAC
38
R10556 GGAGGATATGAGGTGTGAGCG
F10386 GGATTAGACTGAACCGAATTGG
39
R11166 CATCGGGTGATGATAGCCAAG
F10704 GTCTCAATCTCCAACACATATGG
40
R11267 TGTTGTGAGTGTAAATTAGTGCG
F11001 AACGCCACTTATCCAGTGAACC
41
R11600 CTGTTTGTCGTAGGCAGATGG
F11403 GACTCCCTAAAGCCCATGTCG
42
R11927 TTGATCAGGAGAACGTGGTTAC
F11760 ACGAACGCACTCACAGTCG
43
R12189 AAGCCTCTGTTGTCAGATTCAC
F11901 TGCTAGTAACCACGTTCTGGTG
44
R12876 GATATCGCCGATACGGTTG
45 F12357 AACCACCCTAACCCTGACTTCC
20
F12601 TTCATCCCTGTAGCATTGTTCG
46
R13123 AGCGGATGAGTAAGAAGATTCC
F12793 TTGCTCATCAGTTGATGATACG
47
R13343 TTGAAGAAGGCGTGGGTACAG
F13188 CACTCTGTTCGCAGCAGTATG
48
R13611 TCGAGTGCTATAGGCGCTTGTC
F13518 CATCATCGAAACCGCAAAC
49
R13935 TGTGATGCTAGGGTAGAATCCG
F13715 GAAGCCTATTCGCAGGATTTC
50
R14118 TGGGAAGAAGAAAGAGAGGAAG
F13899 TTTCTCCAACATACTCGGATTC
51 R14388 TTAGCGATGGAGGTAGGATTGG (New Primer)
R14388 TTAGCGATGGAGGTAGGATTCG (Old Primer)
F14189 ACAAACAATGGTCAACCAGTAAC
52
R14926 TGAGGCGTCTGGTGAGTAGTGC
F14470 TCCAAAGACAACCATCATTCC
53
R14996 CGTGAAGGTAGCGGATGATTC
F14909 TACTCACCAGACGCCTCAACCG
54
R15396 TTATCGGAATGGGAGGTGATTC
F15260 AGTCCCACCCTCACACGATTC
55
R15774 ACTGGTTGTCCTCCGATTCAGG
F15574 CGCCTACACAATTCTCCGATC
56
R16084 CGGTTGTTGATGGGTGAGTC
F15971 TTAACTCCACCATTAGCACC
57 (HV1)
R16451 GCGAGGAGAGTAGCACTCTTG
F16097 TACATTACTGCCAGCCACCATG
58
R336 TTAAGTGCTGTGGCCAGAAG
-21M13 F TGTAAAACGACGGCCAGT
HV2: Hypervariable region 2

F: forward primer
R: reverse primer
These are the same primers used for SRM 2392, Levin et al., 1999, and Appendix C except the
reverse primer of set 51 has been changed to: TTAGCGATGGAGGTAGGATTGG. The change (C
to G) occurs at np 14368 and is in bold and underlined. Those using SRM 2392 or SRM 2392-I
should use the new reverse primer 51.
21
Table 2. Certified Human mtDNA Sequence Differences from the Cambridge Reference Sequence (CRS) Found in the Two
Templates (CHR and 9947A) in NIST SRM 2392, One Template (HL-60) in NIST SRM 2392-I and in GM03798 and GM10742A.
Comparison with the Cambridge Reference Sequence (CRS)

CRS
Template Template Template Template Template Amino acid

Baseb Region
CHRd 9947Ad HL-60 GM03798 GM10742A change
# a 1981/1999
73 A G - G - G HV2
93 A - G - - - HV2
150 C - - T - - HV2
152 T - - C - - HV2
185 G - - - - A HV2
195 T C C - - - HV2
204 T C - - - - HV2
207 G A - - - - HV2
214 A - G - - - HV2
228 G - - - - A HV2
263*R A G G G G G HV2
295 C - - T - T HV2
303-309 - C (ins) CC (ins) - - - HV2
311-315*R - C (ins) C (ins) C (ins) C (ins) C (ins) HV2
462 C - - - - T HV2
22
482 T - - - - C HV2
489 T - - C - C HV2
709 G A - - A - 12sRNA
750 *R A G G G G G
12sRNA
1438*R A G G G G G 12sRNA
1719 G A - - - - 16sRNA
2706 A G - G - G 16sRNA
2841 T - - - - A 16sRNA
3010 G - - - A A 16sRNA
3106-3107*E C/del del C del C del C del C del C 16sRNA
3394 T - - - - C Tyr → His ND1 LHON
3423*E G/T T T T T T Silent ND1
4135 T - C - - - Tyr → His ND1
4216 T - - C - C Tyr → His ND1 LHON
4769*R A G G G G G Silent ND2
4985*E G/A A A A A A Silent ND2
5186 A G - - - - Silent ND2
5228 C - - G - - Silent ND2
-
5633 C - - T - tRNA Ala
6221 T C - - - - Silent COI

6371 C T - - - - Silent COI
23
6791 A G - - - - Silent COI
6849h A G(0.3A)h - - - - Thr → Alah COI
7028 C T - T - T Silent COI
7476 C - - T - - tRNA Ser
7645 T - C - - - Silent COII
7861 T - C - - - Silent COII
8020 G - - - - A Silent COII
8448 T - C - - - Met → Thr ATPase 8
8503 T C - - - - Silent ATPase 8
8860*R A G G G G nd Thr → Ala ATPase6
9315 T - C - - - Phe → Leu COIII
9559*E G/C C C C C C Arg → Pro COIII
10172 G - - A - - Silent ND3
10398 A - - G - G Thr → Ala ND3
11251 A - - G - G Silent ND4
11287 T - - - - C Silent ND4
11335*E T/C C C C C C Silent ND4
11719 G A - A - A Silent ND4
11778 G - - - - A Arg → His ND4 LHON
11878 T C - - - - Silent ND4
12071het T - - C/Thet - - Phe→Leuhet ND4
12612 A G - G - G Silent ND5
12705 C T - - - - Silent ND5
13572 T - C - - - Silent ND5
24
13702*E G/C C C C C C Gly → Arg ND5
13708 G A - A - A Ala → Thr ND5 LHON
13759 G - A - - - Ala → Thr ND5
13966 A G - - - - Thr → Ala ND5
14199*E G/T T T T T T Pro → Thr ND6
14272*E G/C C C C C C Phe → Leu ND6
14365*E G/C C C C C C Silent ND6
14368*E G/C C C C C Phe → Leu ND6

C
14470 T C - - - - Silent ND6
14569 G - - A - - Silent ND6
14766*E T/C T C T C T Ile → Thr ND6
14798 T - - - - C Phe → Leu CYT B
15257 G - - A - - Asp→ Asn CYT B LHON
15326*R A G G G G G Thr → Ala CYT B

15452 C - - A - A Leu → Ile CYT B
15646 C - - - T - Silent CYT B

15812 G - - A - - Val → Met CYT B LHON
16069 C - - T - T HV1
16126 T - - - - C HV1
25
16183 A C - - - - HV1
16184-93 - C (ins) - - - - HV1
16189 T C - - - - HV1
16193 C - - T - -
HV1
16223 C T - - - -
HV1
16278 C T - T - -
HV1
16292 C - - - - T
16311 - - - HV1
T C -
16357 - C - HV1
T - -
16362 T - - C - - HV1
16519 T C C - C nd HV1
a: Numbers correspond to Cambridge Reference Sequence (Anderson et al., 1981).

b: Base found in 1981 (Anderson et al., 1981)/ Base found in 1999 (Andrews et al., 1999).
d: SRM 2392 (Levin et al., 1999).
e: SRM 2392-I (Levin et al., 2003 in press).
-: Base pair same as in 1981 Cambridge Reference Sequence (Anderson et al., 1981).
h: Possible heteroplasmic site. This heteroplasmy seen in the mtDNA from the first CHR cell culture line is not seen in the mtDNA
from the second CHR cell culture line. The second CHR cell culture line agrees with the CRS at np 6849. It is DNA from the
second CHR cell culture line that is supplied in NIST SRM 2392.
*R: Rare polymorphisms in Cambridge Reference Sequence discovered by reanalysis of original placenta (Andrews et al., 1999).
*E: Error in Cambridge Reference Sequence discovered by reanalysis of original placenta (Andrews et al., 1999).
del: Deletion
ins: Insertion
het: Heteroplasmy found in HL-60 at np 12071.
HV1: Non-coding region found from 16024 and 16569.
HV2: Non-coding region found from 1 and 576.
CHR DNA: Sequence based on two amplifications and cycle sequencing procedures with DNA from the first cell culture line and at
least one amplification and cycle sequencing procedure with DNA from the second cell culture line.
9947A DNA: Sequence based on two amplifications and cycle sequencing procedures.
26
HL-60 DNA: Sequence based on two amplifications and cycle sequencing procedures in both the forward and reverse directions for a
total of 4 sequences
ATPase 6: ATP synthase 6
ATPase 8: ATP synthase 8
CYTB: Cytochrome B
COI: Cytochrome C Oxidase I
COII: Cytochrome C Oxidase II
COIII: Cytochrome C Oxidase III
ND1: NADH dehydrogenase 1
27
Table 3. Universal Genetic Code and (Human mtDNA Differences)
2nd Position
5’ End 3’ End
T C A G
Phe Ser Tyr Cys T
Phe Ser Tyr Cys C
T
Leu Ser TER1 TER1 (Trp) A
Leu Ser TER1 Trp G
Leu Pro His Arg T
Leu Pro His Arg C
C
Leu Pro Gln Arg A
Leu Pro Gln Arg G
Ile (INT)2 Thr Asn Ser T
Ile (INT)2 Thr Asn Ser C
A
Ile (Met) (INT)2 Thr Lys Arg (TER)1 A
Met (INT)2 Thr Lys Arg (TER)1 G
Val Ala Asp Gly T
Val Ala Asp Gly C
G
Val Ala Glu Gly A
Val Ala Glu Gly G
1. TER: Termination codon

2. INT: Initiation codon
29
30
Figure 1. Schematic of human mtDNA showing its circular double-stranded DNA and all the
differences from the Cambridge Reference Sequence (Anderson et al., 1981) found in CHR
(red), 9947A (green), HL-60 (black), GM03798 (blue), and GM10742A (purple) as numbers
along the outside of the color-coded circle. Locations of the control region, rRNAs and genes
(see legend to Table 2 for abbreviations) coded by human mtDNA are shown. The locations
of the 22 tRNAs are noted by white areas in the circle and designated by their single letter
amino acid code. Since a number of mutations found in GM10742A and HL-60 and one
change in CHR have been associated with primary, intermediate or secondary mutations
linked to the disease LHON, the position of these mutations plus other LHON mutations are
shown on the inside of the circle (Wallace et al., 1997). The question mark following the np
of the LHON mutations indicates the assignment is not confirmed. One of the primary
mutations that have been associated with LHON, G11778A, was found in GM10742A (Table
2) but not found in the other DNA templates examined in this research. (Modified from
Levin et al., 1999).
31
32
Appendix A
SRM 2392 Certificate
33
National Institute of Standards & Technology
Certificate of Analysis
Standard Reference Material 2392
Mitochondrial DNA Sequencing
(Human)
This Standard Reference Material (SRM) is intended to provide quality control when performing the polymerase chain
reaction (PCR) and sequencing of human mitochondrial DNA (mtDNA) for forensic identifications, medical diagnosis,
or mutation detection. It may also be used as a control when amplifying (PCR) and sequencing any DNA. This SRM
can also be used for quality assurance when assigning values to in-house control materials. It is certified for the
sequences of the entire human mtDNA (16 569 base pairs) from two lymphoblastoid cell culture lines (CHR and
GM09947A) from apparently normal individuals, plus the cloned HV1 region of CHR containing a C-stretch which is
difficult to sequence. The SRM is packaged in a single box containing three components: (1) extracted DNA from cell
culture line CHR (tube contains 60 µL of DNA at a concentration of 1 ng/µL); (2) extracted DNA from cell culture line
GM09947A (tube contains 60 µL of DNA at a concentration of 1 ng/µL); and (3) cloned DNA from the CHR HV1 region
containing the C-stretch (tube contains 10 µL of DNA at a concentration of 100 ng/µL).
This SRM is composed of well-characterized extracted human DNA from CHR and GM09947A and cloned DNA from
the HV1 region of CHR. Table 1 contains the certified sequence information of two entire mtDNA templates (CHR and
GM09947A). Table 2 contains the reference sequences of 58 unique primer sets which were designed to amplify any
portion or the entire human mtDNA. The sequence information of a third DNA template (GM03798) that was amplified
and sequenced in its entirety three to four times at NIST is provided in reference 1. Although the extracted DNA from
GM03798 is not provided, the cell culture line can be obtained from NIGMS Human Genetic Mutant Cell Repository,
Coriell Institute for Medical Research, Camden, NJ.
Expiration of Certification: The certification of this SRM is valid until 31 May 2008, provided the SRM is handled
and stored in accordance with the instructions given in this certificate. This certification is nullified if the SRM is
damaged, contaminated, or modified.
Maintenance of SRM Certification: NIST will monitor this SRM over the period of its certification. If substantive
technical changes occur that affect the certification before the expiration of this certificate, NIST will notify the
purchaser. Return of the attached registration card will facilitate notification.
The overall direction and coordination of the technical measurements leading to the certification were performed by
B.C. Levin and D.J. Reeder of the NIST DNA Technologies Group, Biotechnology Division.
The analytical determination and technical measurements for the certification of this SRM were performed by
B.C. Levin, H. Cheng, L.A. Tully, M.P. Jones, and D.J. Reeder of the NIST DNA Technologies Group,
Biotechnology Division.
The support aspects involved in the preparation, certification, and issuance of this SRM were coordinated through the
NIST Standard Reference Materials Program by J.C. Colbert and B.S. MacDonald of the NIST Measurement Services
Division.
Vincent L. Viker, Chief

Gaithersburg, MD 20899 John Rumble, Jr., Chief

Certificate Issue Date: 17 June 2003 Measurement Services Division
See Certificate Revision History on Last Page
SRM 2392 Page 1 of 14

NOTICE AND WARNING TO USER
Warning: SRM 2392 IS A HUMAN SOURCE MATERIAL. SINCE THERE IS NO CONSENSUS ON THE
INFECTIOUS STATUS OF EXTRACTED DNA, HANDLE PRODUCT AS A BIOHAZARDOUS MATERIAL
CAPABLE OF TRANSMITTING INFECTIOUS DISEASE.
Storage: Store frozen at a temperature of -20 oC. DO NOT use a self-defrosting freezer because periodic cycling of
temperatures may cause shortened shelf life of this SRM.
INSTRUCTIONS FOR USE
It is recommended that once thawed, each SRM component should be used in its entirety. Repeated freezing and thawing
is NOT recommended as this might shorten the shelf life of the SRM. If it is necessary to perform repeated analyses,
thaw the SRM and divide the tube contents into aliquots that will be kept frozen until use. Thawing can be conducted at
refrigerator temperatures, room temperature, or at 37 oC. Once thawed, the sample should be processed without delay.
SOURCE AND ANALYSIS1
Source of Material: CHR DNA, both extracted and cloned, was prepared in the NIST DNA Technologies Group,
Biotechnology Division. DNA for GM09947A was prepared by Life Technologies, Inc., Gaithersburg, MD.
NIST Analysis: NIST extracted DNA from the CHR cell culture, PCR was used to amplify both the CHR DNA and
GM09947A DNA with all 58 primer sets multiple times, and sequenced the PCR products with a Perkin-Elmer Applied
Biosystems, Inc. (ABI) 373 automated sequencer or an ABI 310 sequencer. The cloned DNA was prepared at NIST as
described in reference 1. The sequence of the CHR clone and of representative PCR products of the final CHR and
GM09947A DNA included in SRM 2392 was reanalyzed to ensure sequence accuracy.
Interlaboratory Analysis: An interlaboratory evaluation of the amplification, sequencing, and data analysis of the CHR
template was conducted by four laboratories, including NIST. These laboratories were: The Bode Technology Group,
Inc., Sterling, VA; IIT Research Institute, Virginia Technology Center, Newington, VA; and Lark Technologies, Inc.,
Houston, TX. Description of the interlaboratory analyses is described in reference 1.
Description of Components: Three components are included in each unit; all components must be stored at -20 ºC.
# 1 Extracted DNA from cell culture line CHR (tube contains 60 µL of DNA at a concentration of 1 ng/µL)
# 2 Extracted DNA from cell culture line GM09947A (tube contains 60 µL of DNA at a concentration of
1 ng/µL)
# 3 Cloned DNA from the CHR HV1 region containing the C-stretch (tube contains 10 µL of DNA at a
concentration of 100 ng/µL)
NOTE: DNA concentrations given are nominal values and are not intended for use as concentration standards.
1
Certain commercial equipment, instruments, materials, or companies are identified in this paper to specify the experimental
procedure. Such identification does not imply recommendation or endorsement by NIST, nor does it imply that the materials or
equipment identified are the best available for this purpose.

Table 1. Certified Human mtDNA Sequence Differences from the
Cambridge Reference Sequence [2] Found in Two Templates at NIST
Primer Set Amplified Regiona Length of Comparison with Cambridge Reference Sequence (CRS) Amino Acid
Amplified
Region CRS # bp Template Template
Change
CHR GM09947A
1 15 - 484 470 Start 39 Start 39

(HV2) 73 A G -
93 A - G
195 T C C
204 T C -
207 G A -
214 A - G
263 A G G
309.1 C(ins) C(ins)
309.2 - C(ins)
315.1 C(ins) C(ins)
End 436 End 473
2 361 - 921 561 Start 429 Start 421

709 G A -
750 A G G
End 891 End 846
3 756 - 1425 670 NONE Start 778 Start 778

End 1197 End 1278
4 873 - 1425 553 NONE Start 931 Start 928

End 1335 End 1377
5 1234 - 1769 536 Start 1279 Start 1275

1438 A G G
1719 G A -
End 1738 End 1741

Table 1. Continued
6 1587 - 2216 630 Start 1632 Start 1632

1719d G A -
End 2106 End 2106
7 1657 - 2216 560 Start 1691 Start 1686

1719d G A -
End 2170 End 2173
8 1993 - 2216 224 NONE Start 2036 Start 2018

End 2213 End 2217
9 2105 - 2660 556 NONE Start 2157 Start 2150

End 2636 End 2586
10 2417 - 3006 590 Start 2465 Start 2458

2706 A G -
End 2920 End 2956
11 2834 - 3557 724 Start 2861 Start 2869

3106/3107 C Del Del
End 3350 End 3373
12 2972 - 3557 586 Start 2999 Start 2999

3106/3107d C Del Del
3423 G E T Silent
End 3422 End 3460
13 3234 - 3557 324 Start 3265 Start 3258

3423d G T T Silentd
End 3548 End 3545
14 3441 - 3940 500 NONE Start 3487 Start 3491

End 3916 End 3920
15 3635 - 4162 528 NONE Start 3667 Start 3662

End 4126 End 4061
16 3931 - 4728 798 Start 3964 Start 3968

4135 T - C Try➜His
End 4399 End 4427
Table 1. Continued
17 4183 - 4728 546 NONE Start 4208 Start 4249

End 4657 End 4657
18 4392 - 4982 591 Start 4449 Start 4453

4769 A G G Silent
End 4860 End 4935
19 4447 - 4982 536 Start 4492 Start 4492

4769d A G G Silentd
End 4958 End 4921
20 4797 - 5553 757 Start 4838 Start 4845

4985 G A A Silent
5186 A G - Silent
End 5327 End 5324
21 4976 - 5553 578 Start 5000 Start 5007

5186d A G - Silentd
End 5516 End 5521
22 5318 - 5882 565 NONE Start 5361 Start 5360

End 5754 End 5758
23 5700 - 6262 563 NONE Start 5741 Start 5744

End 6149 End 6163
24 5999 - 6526 528 Start 6043 Start 6058

6221 T C - Silent
6371 C T - Silent
End 6442 End 6503
25 6242 - 6526 285 Start 6271 Start 6302

6371d C T - Silentd
End 6520 End 6520
26 6426 - 7030 605 Start 6451 Start 6474

6791 A G - Silent
6849* A G(0.3A)h* - Thr➜Ala*
End 6916 End 6930

Table 1. Continued
27 6744 - 7255 512 Start 6775 Start 6782

6849d* A G(0.3A)h* - Thr➜Alad*
7028 C T - Silent
End 7215 End 7221
28 7075 - 7792 718 NONE Start 7123 Start 7123

End 7602 End 7601
29 7215 - 7792 578 Start 7263 Start 7280

7645 T - C Silent
End 7722 End 7769
30 7645 - 8215 571 Start 7671 Start 7666

7861 T - C Silent
End 8149 End 8155
31 7901 - 8311 411 NONE Start 7960 Start 7959

End 8289 End 8288
32 8164 - 8669 506 Start 8211 Start 8212

8448 T - C Met➜Thr
8503 T C - Silent
End 8646 End 8641
33 8539 - 9059 521 Start 8581 Start 8582

8860 A G G Thr➜Ala
End 9019 End 8999
34 8903 - 9403 501 Start 8947 Start 8944

9315 T - C Phe➜Leu
End 9380 End 9381
35 9309 - 9848 540 Start 9334 Start 9333

9559 G C C Arg➜Pro
End 9823 End 9827
36 9449 - 9995 547 Start 9476 Start 9485

9559d G C C Arg➜Prod
End 9964 End 9940

Table 1. Continued
37 9754 - 10275 522 NONE Start 9777 Start 9781

End 10225 End 10251
38 10127 - 10556 430 NONE Start 10168 Start 10166

End 10534 End 10536
39 10386 - 11166 781 NONE Start 10410 Start 10416

End 10899 End 10916
40 10704 - 11267 564 NONE Start 10734 Start 10742

End 11223 End 11197
41 11001 - 11600 600 Start 11026 Start 11040

11335 T C C Silent
End 11461 End 11517
42 11403 - 11927 525 Start 11428 Start 11432

11719 G A - Silent
End 11795 End 11853
43 11760 - 12189 430 Start 11784 Start 11802

11878 T C - Silent
End 12159 End 12164
44 11901 - 12876 976 NONE Start 11926 Start 11926

End 12404 End 12443
45 12357 - 12876 520 Start 12404 Start 12391

12612 A G - Silent
12705 C T - Silent
End 12769 End 12849
46 12601 - 13123 523 Start 12627 Start 12645

12705d C T - Silentd
End 13102 End 13045
47 12793 - 13343 551 NONE Start 12817 Start 12807

End 13295 End 13307

Table 1. Continued
48 13188 - 13611 424 Start 13238 Start 13238

13572 T - C Silent
End 13587 End 13593
49 13518 - 13935 418 Start 13541 Start 13541

13572d T - C Silentd
13702 G C C Gly➜Arg
13708 G A - Ala➜Thr
13759 G - A Ala➜Thr
End 13910 End 13921
50 13715 - 14118 404 Start 13775 Start 13760

13966 A G - Thr➜Ala
End 14094 End 14110
51 13899 - 14388 490 Start 13926 Start 13927

13966d A G - Thr➜Alad
14199 G T T Pro➜Thr
14272 G C C Phe➜Leu
14365 G C C Silent
End 14369 End 14374
52 14189 - 14926 738 Start 14216 Start 14216

14272d G C C Phe➜Leud
14365d G C C Silentd
14368 G C C Phe➜Leu
14470 T C - Silent
14766 T E C Ile➜Thr
End 14699 End 14806
53 14470 - 14996 527 Start 14502 Start 14513

14766d T - C Ile➜hrd
End 14957 End 14972
54 14909 - 15396 488 Start 14941 Start 14933

15326 A G G Thr➜Ala
End 15380 End 15373

Table 1. Continued
55 15260 - 15774 515 Start 15305 Start 15293

15326d A G G Thr➜Alad
End 15754 End15950
56 15574 - 16084 511 NONE Start 15637 Start 15599

End 16056 End 16058
57 15971 - 16451 481 Start 16014 Start 16011

(HV1) 16183 A C -
16189 T C -
16311 T E C
End 16193 End 16430
58 16097 - 336 809 Start 16125 Start 16130

16183d A C -
16189d T C -
16311d T E C
16519 T E C
End 16193 End 59
-21M13c 16133 - 40 477 Start 16131

cloned DNA 16183d A C ND
16189d T C
16193.1 C(ins)
16223 C T
16278 C T
16519d T C
End 40

Table 1. Continued
a
Numbers correspond to Cambridge Reference Sequence [2].
E Base pair change came after the readable sequence.
- Base pair same as in Cambridge Reference Sequence [2].
h* Possible heteroplasmic site
* This heteroplasmy seen in the first CHR cell culture line was not seen with the second CHR cell culture line. It is the second CHR cell culture line
that is supplied in NIST SRM 2392.
c
This primer is used for sequencing the cloned DNA of the HV1 region.
d
Change also seen in previous primer set.
Start Start of readable sequence.
End End of readable sequence.
CHR cells Sequence based on two amplifications and cycle sequencing procedures in first cell culture line and at least one amplification and cycle sequencing
procedure with the second cell culture line.
GM09947A cells Sequence based on two amplifications and cycle sequencing procedures.
Ins Insertion
Del Deletion
ND Not done

Table 2. Reference Sequences for Primer Sets Used for PCR
Amplification of Human mtDNA
PRIMER SET
PRIMER SEQUENCE
NUMBER
1 F15 CACCCTATTAACCACTCACG
(HV2) R484 TGAGATTAGTAGTATGGGAG
2 F361 ACAAAGAACCCTAACACCAGC
3 F756 CATCAAGCACGCAGCAATG
4 F873 GGTTGGTCAATTTCGTGCCAG
5 F1234 CTCACCACCTCTTGCTCAGC
6 F1587 TGCACTTGGACGAACCAGAG
7 F1657 CTTGACCGCTCTGAGCTAAAC
8 F1993 AAACCTACCGAGCCTGGTG
9 F2105 GAGGAACAGCTCTTTGGACAC
10 F2417 CACTGTCAACCCAACACAGG
11 F2834 CCCAACCTCCGAGCAGTACATG
12 F2972 ATAGGGTTTACGACCTCGATG
13 F3234 AGATGGCAGAGCCCGGTAATC
14 F3441 ACTACAACCCTTCGCTGACG
15 F3635 GCCTAGCCGTTTACTCAATCC
16 F3931 TCAGGCTTCAACATCGAATACG

Table 2. Continued
17 F4183 TTTCTACCACTCACCCTAGCATTAC
18 F4392 CCCATCCTAAAGTAAGGTCAGC
19 F4447 TTGGTTATACCCTTCCCGTAC
20 F4797 CCCTTTCACTTCTGAGTCCCAG
21 F4976 ATTAAACCAGACCCAGCTACG
22 F5318 CACCATCACCCTCCTTAACC
23 F5700 TAAGCACCCTAATCAACTGGC
24 F5999 TCTAAGCCTCCTTATTCGAGC
25 F6242 CGCATCTGCTATAGTGGAGG
26 F6426 GCCATAACCCAATACCAAACG
27 F6744 GGCTTCCTAGGGTTTATCGTG
28 F7075 GAGGCTTCATTCACTGATTTCC
29 F7215 CGACGTTACTCGGACTACCC
30 F7645 TATCACCTTTCATGATCACGC
31 F7901 TGAACCTACGAGTACACCGACTAC
32 F8164 CGGTCAATGCTCTGAAATCTGTG
33 F8539 CTGTTCGCTTCATTCATTGCC
34 F8903 CCCACTTCTTACCACAAGGC

Table 2. Continued
35 F9309 TTTCACTTCCACTCCATAACGC
36 F9449 CGGGATAATCCTATTTATTACCTCAG
37 F9754 AGTCTCCCTTCACCATTTCCG
38 F10127 ACTACCACAACTCAACGGCTAC
39 F10386 GGATTAGACTGAACCGAATTGG
40 F10704 GTCTCAATCTCCAACACATATGG
41 F11001 AACGCCACTTATCCAGTGAACC
42 F11403 GACTCCCTAAAGCCCATGTCG
43 F11760 ACGAACGCACTCACAGTCG
44 F11901 TGCTAGTAACCACGTTCTGGTG
45 F12357 AACCACCCTAACCCTGACTTCC
46 F12601 TTCATCCCTGTAGCATTGTTCG
47 F12793 TTGCTCATCAGTTGATGATACG
48 F13188 CACTCTGTTCGCAGCAGTATG
49 F13518 CATCATCGAAACCGCAAAC
50 F13715 GAAGCCTATTCGCAGGATTTC
51a F13899 TTTCTCCAACATACTCGGATTC

R14388 TTAGCGATGGAGGTAGGATTGG (New Primer)
52 F14189 ACAAACAATGGTCAACCAGTAAC

Table 2. Continued
53 F14470 TCCAAAGACAACCATCATTCC
54 F14909 TACTCACCAGACGCCTCAACCG
55 F15260 AGTCCCACCCTCACACGATTC
56 F15574 CGCCTACACAATTCTCCGATC
57 (HV1) F15971 TTAACTCCACCATTAGCACC

58 F16097 TACATTACTGCCAGCCACCATG
a
The reverse primer of set 51 has been changed. One should use the new primer.
REFERENCES
[1] Levin, B.C.; Cheng, H.; Reeder, D.J.; Human Mitochondrial DNA Standard Reference Material for Quality
Control in Forensic Identification, Medical Diagnosis, and Mutation Detection; Genomics, Vol. 55, pp. 135-146
(1999).
[2] Anderson, S.; Bankier, A.T.; Barrell, B.G.; deBrujin, M.H.L.; Coulson, A.R.; Drouin, J.; Eperon, I.C.;
Nierlich, D.P.; Roe, B.A.; Sanger, F.; Schreier, P.H.; Smith, A.J.H.; Staden, R.; Young, I.G.; Sequence and
Organization of the Human Mitochondrial Genome; Nature, Vol. 290, pp. 457-465 (1981).
Certificate Revision History: 17 June 2003 (This revision reports an extension in expiration date and replacement of reverse primer 51); 29
December 1999 (Original certificate date).
Users of this SRM should ensure that the certificate in their possession is current. This can be accomplished by
contacting the SRM Program at: telephone (301) 975-6776; fax (301) 926-4751; e-mail srminfo@nist.gov; or via the
Internet http://www.nist.gov/srm.

Appendix B
SRM 2392-I Certificate

National Institute of Standards & Technology
Certificate of Analysis
Standard Reference Material 2392-I
Mitochondrial DNA Sequencing
(Human HL-60 DNA)
This Standard Reference Material (SRM) is intended to provide quality control when performing the polymerase
chain reaction (PCR) and sequencing of human mitochondrial DNA (mtDNA) for forensic identification, medical
diagnosis, or mutation detection. It may also serve as a control when amplifying (PCR) and sequencing any DNA.
This SRM can also be used for quality assurance when assigning values to in-house control materials. It is certified
for the sequences of the entire human mtDNA (16 569 base pairs) from a promyelocytic cell line (HL-60) prepared
from the peripheral blood leukocytes from an individual with acute promyelocytic leukemia. A unit of SRM 2392-I
consists of 65 µL of extracted DNA from cell culture line HL-60 at a nominal concentration of 1.4 ng/µL, which is
contained in a vial packaged in a protective plastic box.
Certified Values: The certified sequence information of extracted human DNA from HL-60 is provided in Table 1.
Also provided in Table 1 is the certified sequence information for two additional entire mtDNA templates, CHR and
GM09947A, which are provided in SRM 2392. SRM 2392-I only contains the HL-60 template. Table 2 contains
the sequences of 58 unique primer sets that were designed to amplify any portion or the entire human mtDNA [1].
Supplemental Information: The sequence information of an additional two DNA templates, (GM03798 [1] and
GM10742A [2]), that were amplified and sequenced in their entirety multiple times at NIST are provided in
references 1 or 2. Although the extracted DNA from GM03798 and GM10742A are not provided, the cell cultures
can be obtained from NIGMS Human Genetic Mutant Cell Repository, Coriell Institute for Medical Research,
Camden, NJ. A schematic of the differences from the Cambridge Reference Sequence [3] found in the mtDNA
from all five templates is shown in Figure 1.
Expiration of Certification: The certification of this SRM is valid until 31 March 2008, provided the SRM is
handled and stored in accordance with the instructions given in this certificate. This certification is nullified if the
SRM is damaged, contaminated, or modified.
Maintenance of SRM Certification: NIST will monitor this SRM over the period of its certification. If
substantive technical changes occur that affect the certification before the expiration of this certificate, NIST will
notify the purchaser. Return of the attached registration card will facilitate notification.
The analytical determination, technical measurements and analysis of data for the certification of this SRM were
performed by D.K. Hancock, K.L. Richie, K.A. Holland (on sabbatical from Gettysburg College, Gettysburg, PA),
and B.C. Levin of the NIST DNA Technologies Group, Biotechnology Division.
The overall direction and coordination of the technical measurements leading to the certification was performed by
B.C. Levin of the NIST DNA Technologies Group, Biotechnology Division.
The support aspects involved in the issuance of this SRM were coordinated through the NIST Standard Reference
Materials Program by B.S. MacDonald of the NIST Measurement Services Division.
Vincent L. Vilker, Chief

Gaithersburg, MD 20899 John Rumble, Jr., Chief

Certificate Issue Date: 13 June 2003 Measurement Services Division
SRM 2392-I Page 1 of 9

Support for the preparation and certification of this SRM was provided by the National Institute of Justice through
the NIST Office of Law Enforcement Standards.
NOTICE AND WARNINGS TO USER
Warning: SRM 2392-I IS A HUMAN SOURCE MATERIAL. SINCE THERE IS NO CONSENSUS ON THE
INFECTIOUS STATUS OF EXTRACTED DNA, HANDLE PRODUCT AS A BIOHAZARDOUS MATERIAL
CAPABLE OF TRANSMITTING INFECTIOUS DISEASE.
Permissions: The research to use HL-60 DNA in SRM 2392-I was deemed exempt from the policy of Part 27 of
Title 15 of the Code of Federal Regulations by the NIST Institutional Review Board and the Director of the
Chemical Science and Technology Laboratory. This work fit into the exemption category described in 15 CFR
27.101(b)(4) which states: “Research, involving the collection or study of existing data, documents, pathological
specimens, or diagnostic specimens, if, these sources are publicly available or if the information is recorded by the
investigator in such a manner that subjects cannot be identified, directly or through identifiers linked to the
subjects.”
ATCC also waived condition 3(c) in their Material Transfer Agreement which states that the “purchaser shall not
sell, lend, distribute or otherwise transfer the material or replicates to any others” for the use of HL-60 in the NIST
mitochondrial DNA SRM. They stated that, in their view, “as a government agency, NIST will not be providing this
material as a commercial product despite the collection of fees for the SRM.”
Storage: Store frozen at a temperature of –20 oC. DO NOT use a self-defrosting freezer because of periodic
cycling of temperatures may shorten the shelf life of this SRM.
INSTRUCTIONS FOR USE
It is recommended that once thawed, each SRM component should be used in its entirety. Repeated freezing and
thawing is NOT recommended as this might shorten the shelf life of the SRM. If it is necessary to perform repeated
analyses, thaw the SRM and divide the tube contents into aliquots that will be kept frozen until use. Thawing can be
conducted at refrigerator temperatures, room temperature, or at 37 oC. Once thawed, the sample should be
processed without delay. DNA concentrations given are nominal values and are NOT intended for use as
concentration standards.
SOURCE AND ANALYSIS1
Source of Material: DNA from HL-60 was prepared by the Professional Services Department of the American
Type Culture Collection (ATCC), Manassas, VA. This material was subsequently vialed at NIST into 65 µL
portions (nominal DNA concentration of 1.4 ng/µL) and labeled SRM 2392-I Component D (Components A, B, and
C are available in SRM 2392).
NIST Analysis: PCR was used to amplify the HL-60 DNA in its entirety multiple times using all 58 primer sets.
The PCR products were sequenced with an Applied Biosystems, Inc. 310 automated sequencer. The sequences of
representative PCR products of the final HL-60 DNA included in SRM 2392-I were reanalyzed to ensure sequence
accuracy.
Interlaboratory Analyses: An interlaboratory evaluation of the amplification, sequencing and data analysis of the
HL-60 template was conducted by four laboratories, including NIST. These laboratories were: The Armed Forces
DNA Identification Laboratory (AFDIL), Rockville, MD; Federal Bureau of Investigation Laboratory (FBI),
Quantico, VA; and The Georgia Bureau of Investigation (GBI), Decatur, GA. The sequences obtained by all of the
laboratories were identical. Description of the interlaboratory analysis of HL-60 is described in reference 2.
1
Certain commercial equipment, instruments, or materials are identified in this certificate in order to specify adequately
the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of
Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the
purpose.

Table 1. Certified Human mtDNA Sequence Differences from the Cambridge Reference Sequence
(CRS) [3,4] Found in the Two Templates (CHR and GM09947A) in NIST SRM 2392
and One Template (HL-60) in NIST SRM 2392-I
CRS
Baseb 1981/ Template Template Amino acid

Template
#a 1999 CHRd 9947Ad HL-60e change Region
73 A G - G HV2
93 A - G - HV2
150 C - - T HV2
152 T - - C HV2
195 T C C - HV2
204 T C - - HV2
207 G A - - HV2
214 A - G - HV2
263*R A G G G HV2
295 C - - T HV2
303-309 - C (ins) CC (ins) - HV2
311-315*R - C (ins) C (ins) C (ins) HV2
489 T - - C HV2
709 G A - - 12sRNA
750 *R A G G G 12sRNA
1438*R A G G G 12sRNA
1719 G A - - 16sRNA
2706 A G - G 16sRNA
3106-3107*E CC/del del C del C del C 16sRNA

3423*E G/T T T T Silent ND1
4135 T - C - Tyr → His ND1
4216 T - - C Tyr → His ND1 LHON
4769*R A G G G Silent ND2
4985*E G/A A A A Silent ND2
5186 A G - - Silent ND2
5228 C - - G Silent ND2
5633 C - - T tRNA Ala
6221 T C - - Silent COI

6371 C T - - Silent COI

CRS

Template
6791 A G - - Silent COI

h h h
6849 A G(0.3A) - - Thr → Ala COI
7028 C T - T Silent COI

7476 C - - T tRNA Ser
7645 T - C - Silent COII
7861 T - C - Silent COII
8448 T - C - Met → Thr ATPase 8
8503 T C - - Silent ATPase 8
8860*R A G G G Thr → Ala ATPase 6

9315 T - C - Phe → Leu COIII
9559*E G/C C C C Arg → Pro COIII
10172 G - - A Silent ND3
10398 A - - G Thr → Ala ND3
11251 A - - G Silent ND4
11335*E T/C C C C Silent ND4
11719 G A - A Silent ND4
11878 T C - - Silent ND4
het het het
12071 T - - C/T Phe→Leu ND4
12612 A G - G Silent ND5
12705 C T - - Silent ND5
13572 T - C - Silent ND5

13702*E G/C C C C Gly → Arg ND5
13708 G A - A Ala → Thr ND5 LHON
13759 G - A - Ala → Thr ND5
13966 A G - - Thr → Ala ND5
14199*E G/T T T T Pro → Thr ND6

14272*E G/C C C C Phe → Leu ND6
14365*E G/C C C C Silent ND6
14368*E G/C C C C Phe → Leu ND6

14470 T C - - Silent ND6
14569 G - - A Silent ND6
14766*E T/C T C T Ile → Thr ND6
15257 G - - A Asp→ Asn CYT B LHON

CRS

Template
15326*R A G G G Thr → Ala CYT B

15452 C - - A Leu → Ile CYT B
15812 G - - A Val → Met CYT B LHON

16069 C - - T HV1
16183 A C - - HV1
16184-93 - C (ins) - - HV1
16189 T C - - HV1
16193 C - - T HV1
16223 C T - - HV1
16278 C T - T HV1
16311 T - C - HV1
16362 T - - C HV1
16519 T C C - HV1
a
Numbers correspond to Cambridge Reference Sequence [3]
b
Base found in 1981[3] Base found in 1999 [4]
d
SRM 2392, reference 1
e
SRM 2392-I, reference 2
-
Base pair same as in 1981 Cambridge Reference Sequence [3]
h
Possible heteroplasmic site. This heteroplasmy seen in the mtDNA from the first CHR cell culture line is not seen in the
mtDNA from the second CHR cell culture line. The second CHR cell culture line agrees with the CRS at np 6849. It is
DNA from the second CHR cell culture line that is supplied in NIST SRM 2392.
*R Rare polymorphisms in Cambridge Reference Sequence discovered by reanalysis of original placenta by Andrews et al.,
1999 [4].
*E Error in Cambridge Reference Sequence discovered by reanalysis of original placenta by Andrews et al., 1999 [4].
del Deletion
ins Insertion
het Heteroplasmy found in HL-60 at np 12071
HV1 Non-coding region found from 16024 and 16569
HV2 Non-coding region found from 1 and 576
CHR DNA Sequence based on two amplifications and cycle sequencing procedures with DNA from the first cell culture line
and at least one amplification and cycle sequencing procedure with DNA from the second cell culture line.
GM09947A DNA Sequence based on two amplifications and cycle sequencing procedures.
HL-60 DNA Sequence based on two amplifications and cycle sequencing procedures in both the forward and reverse
directions for a total of 4 sequences.
ATPase 6 ATP synthase 6
ATPase 8 ATP synthase 8
CYTB Cytochrome B
COI Cytochrome C Oxidase I
COII Cytochrome C Oxidase II
COIII Cytochrome C Oxidase III
LHON Leber Hereditary Optic Neuropathy
ND1 NADH dehydrogenase 1

Table 2. Reference Sequences for Primer Sets Used for PCR Amplification of Human mtDNA
Primer Set
Primer Sequence
Number
F15 CACCCTATTAACCACTCACG
1(HV2)
R484 TGAGATTAGTAGTATGGGAG
F361 ACAAAGAACCCTAACACCAGC
2
F756 CATCAAGCACGCAGCAATG
3
F873 GGTTGGTCAATTTCGTGCCAG
4
F1234 CTCACCACCTCTTGCTCAGC
5
F1587 TGCACTTGGACGAACCAGAG
6
F1657 CTTGACCGCTCTGAGCTAAAC
7
F1993 AAACCTACCGAGCCTGGTG
8
F2105 GAGGAACAGCTCTTTGGACAC
9
F2417 CACTGTCAACCCAACACAGG
10
F2834 CCCAACCTCCGAGCAGTACATG
11
F2972 ATAGGGTTTACGACCTCGATG
12
F3234 AGATGGCAGAGCCCGGTAATC
13
F3441 ACTACAACCCTTCGCTGACG
14
F3635 GCCTAGCCGTTTACTCAATCC
15
F3931 TCAGGCTTCAACATCGAATACG
16
F4183 TTTCTACCACTCACCCTAGCATTAC
17
F4392 CCCATCCTAAAGTAAGGTCAGC
18
F4447 TTGGTTATACCCTTCCCGTAC
19
F4797 CCCTTTCACTTCTGAGTCCCAG
20
F4976 ATTAAACCAGACCCAGCTACG
21
F5318 CACCATCACCCTCCTTAACC
22
F5700 TAAGCACCCTAATCAACTGGC
23
F5999 TCTAAGCCTCCTTATTCGAGC
24
F6242 CGCATCTGCTATAGTGGAGG
25
F6426 GCCATAACCCAATACCAAACG
26
F6744 GGCTTCCTAGGGTTTATCGTG
27

Primer Set
Primer Sequence
Number
F7075 GAGGCTTCATTCACTGATTTCC
28
F7215 CGACGTTACTCGGACTACCC
29
F7645 TATCACCTTTCATGATCACGC
30
F7901 TGAACCTACGAGTACACCGACTAC
31
F8164 CGGTCAATGCTCTGAAATCTGTG
32
F8539 CTGTTCGCTTCATTCATTGCC
33
F8903 CCCACTTCTTACCACAAGGC
34
F9309 TTTCACTTCCACTCCATAACGC
35
F9449 CGGGATAATCCTATTTATTACCTCAG
36
F9754 AGTCTCCCTTCACCATTTCCG
37
F10127 ACTACCACAACTCAACGGCTAC
38
F10386 GGATTAGACTGAACCGAATTGG
39
F10704 GTCTCAATCTCCAACACATATGG
40
F11001 AACGCCACTTATCCAGTGAACC
41
F11403 GACTCCCTAAAGCCCATGTCG
42
F11760 ACGAACGCACTCACAGTCG
43
F11901 TGCTAGTAACCACGTTCTGGTG
44
F12357 AACCACCCTAACCCTGACTTCC
45
F12601 TTCATCCCTGTAGCATTGTTCG
46
F12793 TTGCTCATCAGTTGATGATACG
47
F13188 CACTCTGTTCGCAGCAGTATG
48
F13518 CATCATCGAAACCGCAAAC
49
F13715 GAAGCCTATTCGCAGGATTTC
50
F13899 TTTCTCCAACATACTCGGATTC
51 R14388 TTAGCGATGGAGGTAGGATTGG (New Primer)
F14189 ACAAACAATGGTCAACCAGTAAC
52
F14470 TCCAAAGACAACCATCATTCC
53
F14909 TACTCACCAGACGCCTCAACCG
54
F15260 AGTCCCACCCTCACACGATTC
55

Primer Set
Primer Sequence
Number
F15574 CGCCTACACAATTCTCCGATC
56
F15971 TTAACTCCACCATTAGCACC
57 (HV1)
F16097 TACATTACTGCCAGCCACCATG
58

F: forward primer
R: reverse primer
These are the same primers used for SRM 2392 and reference 1 except the reverse primer of set 51 has been
changed to: TTAGCGATGGAGGTAGGATTGG. The change (C to G) occurs at np 14368 and is in bold and
underlined. Those using SRM 2392 should also use the new reverse primer 51.
REFERENCES
[1] Levin, B.C.; Cheng, H.; Reeder, D.J.; A Human Mitochondrial DNA Standard Reference Material for Quality
Control in Forensic Identification, Medical Diagnosis, and Mutation Detection; Genomics, Vol. 55, pp. 135-
146 (1999).
[2] Levin, B.C.; Holland, K.A.; Hancock, D.K.; Coble, M.; Parsons, T.J.; Kienker, L.J.; Williams, D.W.; Jones,
MP.; Richie, K.L.; Comparison of the Complete mtDNA Genome Sequences of Human Cell Lines - HL-60 and
GM10742A - from Individuals with Promyelocytic Leukemia and Leber Hereditary Optic Neuropathy,
Respectively, and the Inclusion of HL-60 in the NIST Human Mitochondrial DNA Standard Reference Material
- SRM 2392-I; Mitochondrion, Vol. 2, pp. 386-399 (2003).
[3] Anderson, S.; Bankier, A.T.; Barrell, B.G.; deBrujin, M.H.L.; Coulson, A.R.; Drouin, J.; Eperon, I.C.; Nierlich,
D.P.; Roe, B.A.; Sanger, F.; Schreier, P.H.; Smith, A.J.H.; Staden, R.; Young, I.G.; Sequence and Organization
of the Human Mitochondrial Genome; Nature, Vol. 290, pp. 457-465 (1981).
[4] Andrews, R.M.; Kubacka, I.; Chinnery, P.F.; Lightowlers, R.N.; Turnbull, D.M.; Howell, N.; Reanalysis and
Revision of the Cambridge Reference Sequence for Human Mitochondrial DNA; Nature Genetics, Vol. 23,
p. 147 (1999).
Users of this SRM should ensure that the certificate in their possession is current. This can be accomplished by
contacting the SRM Program at: telephone (301) 975-6776; fax (301) 926-4751; e-mail srminfo@nist.gov; or via
the Internet http://www.nist.gov/srm.

Figure 1. Schematic of human mtDNA showing its circular double-stranded DNA and all the differences from
Cambridge Reference Sequence (1981) found in CHR (red), 9947A (green), HL-60 (black), GM03798 (blue), and
GM10742A (purple) as numbers along the outside of the color-coded circle. Locations of the control region, rRNAs
and genes coded by human mtDNA are shown. The locations of the 22 tRNAs are noted by white areas in the circle
and designated by their single letter amino acid code. Since a number of mutations found in GM10742A and HL-60
and one change in CHR have been associated with primary, intermediate or secondary mutations linked to the
disease Leber Hereditary Optic Neuropathy (LHON), the position of these mutations plus other LHON mutations
are shown on the inside of the circle (Wallace et al., 1997). The question mark following the np of the LHON
mutations indicates the assignment is not confirmed. One of the primary mutations that have been associated with
LHON, G11778A, was found in GM10742A [2] but not found in the other DNA templates examined in this
research. (Modified from Levin et al., 1999).

Appendix C
Journal Article:
B. C. Levin, H. Cheng, and D. J. Reeder, “A Human Mitochondrial
DNA Standard Reference Material for Quality Control in Forensic
Identification, Medical Diagnosis, and Mutation Detection,”
Genomics 55, 135-146 (1999).
(See end of Appendix C for color representation of Figure 2.)
, *.
Genomics55, 135- 146 (1999)

Article ID geno. 1998. 5513 , available online at http://www.idealibrary. com on IDE ~ LQ!)
Human Mitochondrial DNA Standard Reference Material

for Quality Control in Forensic Identifi. cation,
Medical Diagnosis, and Mutation Detection
Barbara C. Levin 1 Haiyan Cheng, t and Dennis J. Reeder*
*Biotechnology Division, Chemical Science and Technology Laboratory, National Institute of Standards and Technology,
Gaithersburg, Maryland 20899; and tGEO- CENTERS, Inc. , Newton Centre, Massachusetts 02159
Received May 8 . 1998; accepted August 4 . 1998
INTRODUCTION
A human mitochondrial DNA (mtDNA) standard ref-
erence material (SRM 2392) will provide quality con- Human mitochondrial DNA (mtDNA) has been com-
trol when mtDNA is sequenced for forensic identifica- pletely sequenced and found to be circular double-
tions, medical diagnosis, or mutation detection. SRM stranded molecules containing 16, 569 bp (Anderson
2392 includes DNA from two lymphoblast cell cultures al. 1981). Each human cell can have a few dozen to
(CRR and 9947A) and cloned DNA from the CRR HV1 several thousand molecules of mtDNA (Bogenhagen
region, which contains a C stretch and is difficult to and Clayton , 1974; King and Attardi , 1989). Sequence
sequence. The mtDNA sequence (but not the DNA) of a
third human template GM03798 is provided analysis of mtDNA is being used by the forensic com-
for com-
parison. Fifty-eight unique primer sets allow any area munity for human identification , especially in those
Or the entire mtDNA (16, 569 bp) to be amplified and cases in which genomic DNA is highly degraded or
sequenced. While none of the differences in these nonexistent (Holland et al. 1993 , 1995). Forensic anal-
three templates correspond to published mutations ysis to determine the distinction between individuals is
associated with specific diseases, some of these differ- primarily based on the considerable sequence variation
ences did result in animo acid changes compared with found in the two hypervariable regions (lIVl , lIV2)
that published by S. Anderson et al. (1981, Nature 290: located in the noncoding displacement loop (D loop).
457- 465). An interlaboratory evaluation of theampli- The medical community is also using sequence analy-
fication, sequencing, and data analysis of the CRR sis of mtDNA for diagnoses of diseases associated with
template was conducted by four laboratories. Corrob- specific mutations and deletions (Wallace et al. 1997).
oration of the SRM results will provide quality assur- A third area of research , which is largely unexplored
ance that any unknown mtDNA is also being amplified and which needs sequence analysis , is the examination
and sequenced correctly. ~ 1999 Academic Press of the mutagenic effects of chemical and physical
agents on mtDNA (Grossman , 1995; Ballinger et al.
1996). The objective of this research was to develop a
This paper is a contribution of the U. S. National Institute of human mtDNA standard reference material (SRM) for
Standards and Technology (NIST) and is not subject to copyright. quality control in sequencing, forensic identifications
Certain commercial equipment , instruments , materials , or compa" medical diagnostics , and mutation detection.
nies are identified in this paper to specify the experimental proce-
dure. Such identification does not imply recommendation or endorse-
ment by NIST, nor does it imply that the materials or equipment MATERIALS AND METHODS
identified are the best available for this purpose. Some of these data
have been presented at the following meetings: Environmental Mu- Origin of extracted DNA. The DNA template designated CHR
tagen Society 27th Annual Meeting, Victoria, British Columbia, Can- came from human white blood cells that were transformed with the
ada, March 1996; Seventh International Symposium on Human Epstein-Barr virus and immortalized as a cell culture line (CHR
Identification , Scottsdale , Arizona , September 1996; Human Identi- cells) by the American Type Culture Collection (ATCC; Rockville
fication User s Meeting, Rockville , Maryland, November 1996; The MD). After transformation , the cells were grown in Iscove s modified
American Association of Clinical Chemistry, Inc. , San Diego , Cali- Dulbecco s media or RPMI 1640 media with L- glutarnine , sodium
fornia , November 1996; The Society of Toxicology Annual Meeting, bicarbonate , penicillin , streptomycin , and 20% fetal calf serum (Life
Cincinnati , Ohio , March 1997; Genetic Toxicology Gordon Confer- Technologies, Inc. , Grand Island, NY). The cell cultures were grown
ence, New London, New Hampshire , June 1997; and the 47th Annual at 37O C in humidified atmosphere containing 5% CO2 and 95% air.
Meeting of The American Society of Human Genetics, Baltimore The DNA was extracted from 2 X 108 CRR cells by the Qiagen
Maryland , October 1997. Plasmid/Cosmid Purification Protocol (Qiagen , Inc. , Chatsworth
1 To whom correspondence should
be addressed at 100 Bureau CA). This procedure enhanced the concentration of mtDNA and
Drive , Stop 8311 , National Institute of Standards and Technology, reduced , but did not eliminate , nuclear DNA.
Gaithersburg, Maryland 20899- 8311. Telephone: (301) 975- 6682. The CHR data presented in this paper were obtained primarily
Fax: (301) 330- 3447. E-mail: barbara.levin~st. gov. with the above-mentioned immortalized CRR cell culture line. How-
135
0888- 7543/99 $30.
Copyright ~ 1999 by Academic Press
All rights of reproduction in any form reserved.
136 LEVIN, CHENG, AND REEDER
ever , before production of the final SRM began , it was necessary to (20% PEG in 2. 5 M NaC!) overnight at 4O , and the resultant
obtain fresh blood from CHR and to reestablish the cell line. This precipitate containing the DNA was pelleted by centrifugation at
second CHR cell line was established by the ATCC as above. The 000 rpm for 15 min. The bacteriophage DNA was isolated by
sequence of this second CHR cell line was examined and found to be phenol extraction and NaClIethanol precipitation as described above
identical to that of the first CHR cell line , with the single exception and then dissolved in 25 p.l ofTE buffer. The bacteriophage DNA was
that no heteroplasmy was noted at bp6849; the second CHR cell line cycle sequenced with AmpliTaq DNA polymerase , FS , and the ~
agreed with Anderson at bp 6849. It is the second CHR cell line that M13 primer: 5' TGTAAAACGACGGCCAGT- 3' according to the pro-
is included in SRM 2392. tocol in the ABI PRISM Dye TerIninator Cycle Sequencing Ready
The DNA template 9947A was obtained from Life Technologies Reaction Kit (Perkin-Elmer , Foster City, CA). The cycle sequencing
Inc. , who prepared it from a Epstein-Barr virus-immortalized hu- was conducted in a Perkin~Elmer Model .9600 thermocycler by first
man lymphoid cell line. DNA from 9947A is also used in the PCR- heating the DNA reaction mixture at 96O C for 1 min and then
based DNA profiling standard (SRM 2391)2 designed for forensic and subjecting the mixture to 25 cycles of 96O C for 10 s , 500 C for 5 s , and
paternity testing, law enforcement training, and research. 600 Cfor 2 min. The cycle sequencing product was purified using a
A third DNA template was extracted from an apparently normal Centri-Sep spin column (Princeton Separations , Inc. , Adelphia , NJ).
human lymphoblastoid cell culture (GM03798) obtained from The DNA pellet was rinsed with 70% ethanol , vacuum dried , resus-
NIGMS3 and grown in the same manner as the CHR cells. The DNA pendedin loading buffer prepared by combining deionized form-
was extracted using DNA NOW, a phenol-free DNA isolation reagent amide and 25 mM EDTA (pH 8. 0) in a ratio of 5:1 , loaded onto a
(BIOGENTEX, Seabrook , TX). The information on this template is 75% acrylamide gel , and electrophoresed on an ABI 373 DNA
included for informational purposes only; the DNA is not included as sequencer. One of the clones containing the C stretch sequence was
part of this SRM. used as the source of the cloned DNA for the SRM.
Isolation and cloning of mtDNA containing the C stretch.
Conflu- mtDNA primers. Fifty-eight sets of unique primers (19-28 bp)
ent CHR cells were harvested by centrifugation at 1500 rpm for 5
for sequencing the entire mtDNA (16 569 bp) were computer- de-
min. The mtDNA was isolated using the Qiagen Plasmid/Cosmid signed using Gene Runner for Windows (Hastings Software, Inc.
Purification Protocol (Qiagen , Inc. ). Following isolation , the mtDNA Hastings , NY) and custom made by Bio-Synthesis, Inc. (Lewisville
was digested with restriction enzymes Sac! and KpnI (New England TX). The - 21M13 primer was used to sequence the cloned HV1
Biolabs, Inc. , Beverly, MA) into fivefragments which were separated
region of the DNA from the CHR template. The sequences of all the
on a 0. 7% low-melting-agarose gel. Bands of the size of the fragment
primers are shown in Table 1.
containing the HV1 region were cut from the gel and melted at 65O
DNA was extracted with phenol twice and precipitated by adding Polymerase chain reaction (PCR). Extracted DNA WaS resus-
sodium chloride (150 mM) and 2 vol of 100% ethanol. The final pended in TE buffer (pH 7. 5) containing 10 mM Tris and 1 mM
product was resuspended in Tris~EDTA (TE) buffer. EDTA. The PCR mixture contained DNA (1 p.!), Taq DNA polymer-
The cloning vector, M13mp18 , was also digested with Sac! and ase (0. 5 p.l or 2. 5 units) (Boehringer Mannheim), and lOX buffer (5
KpnI treated with calf intestinal alkaline phosphatase (New En- p.!) (Boehringer Mannheim), dNTP' s (0. 2 mM each) (Life Technolo-
gland Biolabs , Inc. ), extracted with phenol , and precipitated with gies , Inc. ), forward and reverse primers (0.4 p.M each), plus H2 O to a
NaCl and ethanol as described above. The vector was incubated with final volume of 50 p.l. The lOxbuffer (pH 8. 3) contained Tris-HCl
the mtDNA product and T. DNA ligase (Life Technologies, Inc. ) at (100 mM), MgCl2 (15 mM), and KCl (500 mM).
C overnight. An overnight culture of Escherichia coli host TG- Thermal cycling Was conducted in a Perkin-Elmer Model 9600
cells was diluted and grown at 37O C in LB media (Sambrook et al. thermocycler and consisted of 1 min at 96O C; followed by 32 cycles of
1989) until the ODs5o reached 0.4~ 5. The cells were harvested by 15 s at 94O C (denaturation), 30 s at 56O C (annealing), and 15 s at
centrifugation at 1500 rpm for 5 min. The cell pellet was resus- 72O C (extension); and ending with a final extension of 7 rnin at 72O
pended in 10 ml calcium chloride (50 mM) and incubated for 1 h on A sample of the amplified DNA was electrophoresed in 0. 7% aga-
ice , centrifuged , and resuspended in 1 ml calcium chloride (50 rnM) rose and stained with ethidium bromide to assess the purity and size
and incubated for 30 min on ice. The treated TG- 1 cell suspension of the PCR product. Before sequencing, extraneous materials were
(0. 3 m!) was incubated for 30min on ice with 20 p.l of a ligation removed from the PCRproduct with a QIAquick PCR Purification
mixture containing the isolated mtDNA fragment , the cloning vector Kit (QIAGEN, Inc.
that had been treated overnight , T. ligase , and ligation buffer (Life
Technologies, Inc. ). This mixt1lI'e was then exposed to a heat shock of Sequencing. Cycle sequencing using fluorescent dye-labeled ter-
42O C for 2 min; mixed with 0. 2 ml of untreated TG- 1 cells (from the minators was performed with an ABI PRISM Dye Terminator Cycle
overnight culture), 4 p.l of 1 M isopropylthio-(3-D-galactoside, 40 p.lof Sequencing Kit with AmpliTaq DNA polymerase, FS (Perkin-Elmer
20 mg/ml 5-bromo- 4-chloro- indolyl-(3-D-galactopyranoside , and 3 Foster City, CA). Thermal cycling was conducted ip. a Perkin~Elmer
ml of melted (55O C) top agar; and spread on the surface of freshly Model 9600 thermocycler and started with 1 min at 96O C. The reac-
tion then underwent 25 cycles of 96O C for 15 s (denaturation), 500
prepared LB agarose plates (Sambrook et al. 1989). The plates were
incubated at 37O C overnight. Both colorless and blue plaques were for 5 s (annealing), and 600 C for 2 min(extension). The DNA product
visible in the morning. The colorless plaques indicate that insertion was purified by passage through a Centri- Sep spin column (Prince-
of the vector has occurred , whereas the blue plaques have no inser- ton Separations , Inc.
tion of the vector. Electrophoresis and sequencing of the fluorescently labeled puri-
fied DNA were performed with a 373 ABI Sequencer (Perkin-Elmer)
Bacteriophage DNA isolation and sequencing. Single, well-iso- using a 4. 75% acrylamide gel. Data analysis was executed with the
lated colorless plaques from the above LB plates were each placed in Sequence Navigator software package (Perkin-Elmer).
a sterile tube with 1.5 rnl of a TG- l/LB cell suspension that contained
TG- 1 cells that were grown overnight and diluted 1/100 in LB media Interlaboratory evaluation. Three laboratories in addition to
and grown for 1 h at 37O C. The plaques and the TG- l/LB cell sus- NIST participated in an interlaboratory evaluation of the CRR tem-
pension were grown at 37O C for 5 h. Cell debris WaS removed by plate. These laboratories were The Bode Technology Group, Inc.
centrifugation at 15
000 rpm for 5 min. The supernatant containing (21515 Ridgetop Circle , Suite 140 , Sterling, VA 20166), IIT Research
the bacteriophage was incubated with 0. 2 ml polyethylene glycol Institute (Virginia Technology Center , 8510 Cinderbed Road , Suite
300 , P. O. Box 899 , Newington , VA 22122), and Lark Technologies
Inc. (9545 Katy Fwy, Suite 465 , Houston, TX 77024).
2 SRM 2391 may be obtained from the Standard Reference Mate- Each laboratory was sent:
rial Program , NIST , Gaithersburg, Maryland 20899. 1. Two tubes of DNA from the first CHR cell culture line. One tube
3 NIGMS Human Genetic Mutant Cell Repository, Coriell Institute
contained extracted DNA ready for PCR amplification of the entire
for Medical Research , 401 Haddon Avenue , Camden , New Jersey mtDNA. The other contained the cloned DNA ready for cycle sequenc-
08103. ing of the HV1 region (this DNA didnot need to be PCR amplified).
HUMAN mtDNA STANDARD REFERENCE MATERIAL 137
2. Fifty-eight sets of primers labeled with either F# (forward not totally eliminate the nuclear DNA. The SRM also
primer) or R# (reverse primer). Forward and reverse primers with
the same number were paired and numbered from the 5' end. Prim-
provides cloned DNA from the HVI region of the CHR
ers were diluted to 10 J.Ll and ready for use. Also enclosed was the template , which contains a C stretch. In most people,
-21M13 primer for the sequencing of the cloned HV1 region of the the HVI region has a string of cytosine (C) residues
CHR DNA, which covered basepairs 16133 to 40. interrupted by a thymine(T) at nucleotide position
3. The protocol used at NIST to amplify and sequence the DNA. 16189. 4 In some individuals , however , a transition that
The laboratories , however, were free to use any protocol with which changes the T to a C occurs , producing a long string of
they were familiar and felt comfortable. Cs called the C stretch. When this happens , sequencing
4. A form table to record the results. This table provided the
number of the primer set , the region that each primer set amplified,
beyond the C stretch becomes very difficult, if not im-
and the length of the amplified region. We requested that the labo- possible. Clones of the HVI region containing the C
ratory fill in the differences found when they compared the sequence stretch indicated that the number of Cs differed among
that they determined for the SRM with that of Anderson et al. (1981). the different clones and the difficulty in sequencing
5. The following cautionary note: was due to frameshifts that resulted from the simulta-
WARNING: The DNA and cells were derived from a cell culture neous sequencing of templates with differing numbers
line from an apparently healthy human subject. The cell culture of Cs (Bendall and Sykes , 1995; Levin et al. 1995
line has been tested and found to be nonreactive for hepatitis B 1997). We found , however , that one could sequence
surface antigen and HIV. However, no test method can enSure through the entire HVI region including the C stretch
that a product derived from human blood does not contain HIV
hepatitis or other infectious agents. HANDLE AS IF CAPABLE
without problems if one used the clone of the area.
OF TRANSMITTING DISEASE. (The second CHR cell culture Therefore , we have included the cloned HVI region of
line generated from the same individual was not tested again for the CRR DNA template in the SRM.
hepatitis or HIV. Normal precautions should be used. In addition to templates CHR and 9947 A, we have
Differences in methodology used by laboratories in interlaboratory included all the information regarding a third template
evaluation. The Bode Technology Group, Inc. , essentially followed from a lymphoblastoid cell line (GM03798) that was
the NIST protocol except that they used a 6% acrylamide/8. 3 M urea
gel for the sequencing electrophoresis instead of a 4. 75% acrylamide.
obtained from the NIGMS Human Genetic Mutant Cell
IIT Research Institute also followed the NIST protocol except that Repository and that was completely sequenced at NIST
they used Taq Gold (Perkin-Elmer) for the amplification reaction three times. The DNA from this . cellline is not part of
which was modified to include a hot start of 95O C for 11 min. Micro- the SRM , and the data are included for information
con 100 rnicroconcentrators (Arnicon , Inc. , Beverly, MA) were used to
only.
purify the PCR products. The quantities of DNA were detemined by
capillary electrophoresis (CE) with a Beckman PlACE 5010 System Primers. The 58 sets of unique primers were de-
(Beckman Instruments , Inc. , Fullerton , CA) as follows: 1 J.Ll of the signed to allow the amplification and sequencing of any
amplified product was mixed with 25 J.Ll of sterile deionized H2 region or the . entire 16 569 bp that comprise human
containing 0. 52 ng/pJ of a 200-bp internal standard (GenSura Lab- mitochondrial DNA. The sequences of both the forward
oratories , Inc. , Del Mar, CA) and run on the CEo One determines the
quantity of the amplified product from the ratio of the PCR product and the reverse primers that are in each set are shown
peak area to the internal standard peak area multiplied by a migra- in Table 1. The numbers indicate the 5' end of the
tion standard. A 6% acrylamide gel was used for the sequencing primer. They . are all between 19 and 28 bp long, and
electrophoresis instead of a 4. 75% acrylamide. the criteria that were used to choose these primers
Lark Technologies, Inc. , followed the NIST protocol with the fol-
lowing differences: AmpliTaq DNA polymerase (Perkin-Elmer) was
were primer m = 50- 65O , primer length = 15-30 bp,
used to amplify the DNA; the dNTP' s were purchased from Pharma" and PCR product length = 400- 850 bp. The primers
cia Biotech , Inc. (Piscataway, NJ); the products were purified with were designed to produce sequences that overlapped
Qiaquick PCR purification kit (Qiagen); in the sequencing reactions with both the previous and the following regions to
the amount of PCR product used varied from 1 to 3 J.Ll based on the allow those areas in the beginning and end of electro-
concentration estimated from agarose gels; cycling conditions were
95O C for 1 rnin followed by 25 cycles of 96O C for 15 s , 50o C for 15 s
pherograms , which are difficult to sequence , to become
and 60o C for 4 min; the sequence reactions were cleaned up by readable. Table 2 shows the number of the base where
ethanol precipitation; a 4. 25% polyacrylamide gel was used for the the readable sequence begins and ends (indicated in
sequencing electrophoresis instead of 4. 75% acrylamide; and an ABI the table as " start" and "end" ). The readable region is
377 was used instead of the ABI 373. Electropherograms were always smaller than the amplified region.
printed for each reaction , and the sequences were manually edited
based on the electropherogram patterns. Printed electropherograms
In addition to the designed primers , we also used
and a floppy disk with the sequence data were sent to NIST where the -21M13 primer (Table 1) to sequence the cloned
the data were compared to the Anderson sequence. DNA from the HVI region of the CHR template that
contained the C stretch. The PCR products produced
RESULTS AND DISCUSSION single, distinct bands for all 58 primer sets (Figs. lA
and IB).
SRM templates. Two DNA templates CHR and Differences between the SRM templates and the Ander-
9947 A, are included in the NIST human mtDNA se-
son sequence. Anderson and his co-workers completely
quencing SRM 2392. Both of these DNA samples come sequenced human mtDNA in 1981 (Anderson
from human cell culture lines that were developed from et al.
1981). All investigators who subsequently examined hu-
apparently normal individuals. The DNA from 9947 A
man mtDNA have used the numbering system of Ander-
is the total extracted DNA, which also includes nuclear
DNA. The DNA from CHR was isolated in a manner 4 All nucleotide numbers referred to in this paper are based
on the
that enhanced the concentration of the mtDNA, but did numbering system of Anderson et al. (1981).
TABLE 1
Primer Sets Used for PCR Amplification of Human mtDNA
Primer set Primer sequence Primer set Primer sequence
1 (HV2) F15 CACCCTATTAACCACTCACG R8215 GACGA TGGGCATGAAACTG

R484 TGAGATTAGTAGTATGGGAG F7901 TGAACCT ACGAGTACACCGACT AC
F361 A C AAAGAAC C CT AA CAC C A GC R8311 AA GTT A GC TTT A CAG TGGGCTCT A G
R921 A CTTGGG TTAA TC G TG TGA C C F8164 C GG TCAATGCTCTGAAA TCTG TG
F756 CATCAAGCACGCAGCAATG R8669 CA TTG TTGGGTGGTGATT AGTCG
R1425 AA TCCACCTTCGACCCTT AAG F8539 CTGTTCGCTTCA TTCA TTGCC
F873 GG TTGG TC AA TTTC GTGC CA G R9059 GTGGCGCTTCCAATTAGGTG
R1425 AATCCA CCTTC G ACCCTTAA G F8903 CCCACTTCTTACCACAAGGC
F1234 CTCA C C A C CTCTTGCTCAGC R9403 GTGCTTTCTCGTGTT ACA TCG
R1769 GCCAGGTTTCAATTTCT A TCG F9309 TTTCACTTCCACTCCA T AACGC
F1587 TGCACTTGGACGAACCAGAG R9848 GAAAGTTGAGCCAA T AA TGACG
R2216 TG TTGA GCTTG AA CGCTTTC F9449 CGGGA T AA TCCT A TTT A TTACCTCAG
F1657 CTTG A CC GCTCTG A GCT AAA C R9995 AGAGT AAGACCCTCA TCAA T AGATGG
R2216 TG TTG A GCTTGAA C GCTTTC F9754 A G TCTC C CTTCA CC A TTTCCG
F1993 AAACCTACCGAGCCTGGTG R10275 AAAGGAGGGCAA TTTCTAGATC
R2216 TG TTG AGCTTGAA CGCTTTC F10:!.27 ACT ACCACAACTCAACGGCT AC
F2105 G A GG AAC A GCTCTTTGG ACA C R10556 GGAGGA T A TGAGGTGTGAGCG
R2660 A GAGA CA GCTGAA CCCTC G TG F10386 GGATTAGACTGAACCGAATTGG
F2417 CACTGTCAACCCAACACAGG Rl1166 CATCGGGTGATGATAGCCAAG '
R3006 A TGTCCTGA TCCAACA TCGAG F1O704 GTCTCAATCTCCAACACA T A TGG
F2834 CCCAACCTCCGAGCAGT ACA TG R11267 TG TTGTG A G TGT AAA TT A G TGCG
R3557 AGAAGAGCGATGGTGAGAGC F11001 AACGCCACTT A TCCAGTGAACC
F2972 AT AGGGTTT ACGACCTCGA TG R11600 CTG TTTG TC GT AGGC A GATGG
R3557 AGAAGAGCGATGGTGAGAGC F11403 GACTCCCT AAAGCCCA TGTCG
F3234 AGA TGGCAGAGCCCGGT AA TC R11927 TTGA TCAGGAGAACGTGGTT AC
R3557 A GAAG A GCGA TGGTGA GA GC F11760 ACGAACGCACTCACAGTCG
F3441 ACTACAACCCTTCGCTGACG R12189 AA GCCTCTGTTG TCAGA TTC A C
R3940 TGAA GC CTG AGA CT A G TTC GG F11901 TGCT AGT AACCACGTTCTGGTG
F3635 GCCT AGCCGTTT ACTCAA TCC R12876 GATATCGCCGATACGGTTG
R4162 TGAGTTGGTCGT AGCGGAA TC F12357 AA C CA CC CT AA CC CTGA CTTCC
F3931 TCAGGCTTCAACATCGAA T ACG R12876 GATATCGCCGATACGGTTG
R4728 TT A TGGTTCA TTGTCCGGAGAG F12601 TTCA TCCCTGT AGCA TTGTTCG
F4183 TTTCT ACCACTCACCCT AGCA TT AC R13123 AGCGGATGAGT AAGAAGA TTCC
R4728 TT ATGGTTCA TTGTCCGGAGAG F12793 TTGCTCA TCAGTTGATGA T ACG
F4392 CCCA TCCT AAAGT AAGGTCAGC R13343 TTGAA GAAGGC GTGGG T A CA
R4983 GG TTT AA TC CACCTCAACTGCC F13188 CACTCTGTTCGCAGCAGT A TG
F4447 TTGGTTA T ACCCTTCCCGT AC R13611 TCGAGTGCT AT AGGCGCTTGTC
R4982 G TTT AA TC CA C CTC AA CTGC C F13518 CATCATCGAAACCGCAAAC
F4797 C C CTTTCA CTTCTG A G TC C CAG R13935 TGTGA TGCT AGGGT AGAA TCCG
R5553 AGGGCTTTGAAGGCTCTTG F13715 GAAGCCT A TTCGCAGGA TTTC
F4976 A TT AAACCAGACCCAGCT ACG R14118 TGGGAAGAAGAAAGAGAGGAAG
R5553 AGGGCTTTGAAGGCTCTTG F13899 TTTCTC CAA CAT A CTCGG A TTC
F5318 CACCATCACCCTCCTTAACC R14388 TTAGCGA TGGAGGT AGGA TTCG
R5882 GCTGA G TG AA GC A TTGGACTG F14189 ACAAACAA TGGTCAACCAG T AAC
F5700 T AAGCACCCT AA TC AA CTGGC R14926 TG AGGC GTCTGG TG AG TAG TGC
R6262 GCCTCCACT A T AGCAGA TGCG F14470 TC CAAA GA CAAC C ATCA TTCC
F5999 TCT AAGCCTCCTTA TTCGAGC R14996 CGTGAAGGT AGCGGATGA TTC
R6526 AT AGTGA TGCCAGCAGCT AGG F14909 T A CTCA CCA G A C GCCTCAA C CG
F6242 CGCATCTGCTATAGTGGAGG R15396 TT A TCGGAA TGGGAGGTGA TTC
R6526 AT AG TGATGCCAGCAGCTAGG F15260 AGTCCCACCCTCACACGA TTC
F6426 GCCA T AACCCAA T ACCAAACG R15774 A CTGG TTG TCCTC CG A TTCAGG
R7030 TGGGCT ACAACGT AGT ACGTG F15574 CGCCT ACACAATTCTCCGA TC
F6744 GGCTTCCT AGGGTTT A TCGTG R16084 CGGTTGTTGATGGGTGAGTC
R7255 TTTCATGTGGTG T A TGCA TCG 57 (HV1) F15971 TTAACTCCACCATTAGCACC
F7075 G A GGCTTC A TTCACTGA TTTC C R16451 GCG A GGA GAG T A GCA CTCTTG
R7792 GGGCAGGATAGTTCAGACGG F16097 T ACATT ACTGCCAGCCACCA TG
F7215 CGACGTTACTCGGACTACCC R336 TTAAGTGCTGTGGCCAGAAG
R7792 GGGCAGGATAGTTCAGACGG 21M13 TGTAAAACGACGGCCAGT
F7645 TATCACCTTTCA TGA TCACGC
son .and have compared their sequence findings to those would be available. Table 2 shows the mtDNA differences
described by Anderson. However , the DNA sequenced by compared to the Anderson sequence that were found at
Anderson is not available for use as a positive control NlST with all three template&-CHR , 9947 A , and
during actual experiments , whereas NlST SRM 2392 GMO3798. In all three templates , all 58 areas comprising
TABLE 2
Primer Sets Used for PCR Amplification of Human mtDNA and Differences with the Anderson Sequence
Found in Three Templates at NIST
Comparison with Anderson
Length of
Amplified amplified Anderson Anderson Template Template Template Amino acid
Primer set region region No. CHR 9947A GM03798 change
1 (HV2) 15-484 470 Start 39 Start 39 Start 55
195
204
207
214
263
309. C(ins) C(ins)
309. C(ins)
315. C(ins) C(ins) C(ins)
End 436 End 473 End 454
361-921 561 Start 429 Start 421 Start 415
709
750
End 891 End 846 End 834
756-1425 670 None Start 778 Start 778 Start 818
End 1197 End 1278 End 1146
873- 1425 553 None Start 931 Start 928 Start 938
End 1335 End 1377 End 1323
1438
1719
End 1738 End 1741 End 1654
1719b
End 2106 End 2106 End 2031
1719b
End 2170 End 2173 End 2097
End 2213 End 2217 End 2212
End 2636 End 2586 End 2560
2706
End 2920 End 2956 End 2915
3010
3106/3107 Del Del Del
End 3350 End 3373 End 3243
31O6/31O7b Del Del Del
3423 Silent
End 3422 End 3460 End 3425
3423b Silentb
End 3548 End 3545 End 3541
End 3916 End 3920 End 3847
End 4126 End 4061 End 4044
4135 Try ... His
End 4399 End 4427 End 4436
End 4657 End 4657 End 4642
4769 Silent
End 4860 End 4935 End 4877
4769b Silent
TABLE Continued
Length of
End 4958 End 4921 End 4931

4985 Silent
5186 Silent
End 5327 End 5324 End 5215
5186. Silentb
End 5516 End 5521 End 5400
End 5754 End 5758 End 5800
End 6149 End 6163 End 6136
6221 Silent
6371 Silent
End 6442 End 6503 End 6456
6371. Silent.
End 6520 End 6520 End 6520
6791 Silent
6849* G (0. 3A)h* Thr .. Ala
End 6916 End 6930 End 6885
6849b. G (0. 3A)h* Thr .. Ala
7028 Silent
End 7215 End 7221 End 7177
End 7602 End 7601 End 7547
7645 Silent
End 7722 End 7769 End 7706
7861 Silent
End 8149 End 8155 End 8156
End 8289 End 8288 End 8258
8448 Met.. Thr
8503 Silent
End 8646 End 8641 End 8637
8860 Thr .. Ala
End 9019 End 8999 End 8991
9315 Phe .. Leu
End 9380 End 9381 End 9370
9559 Arg.. Pro
End 9823 End 9827 End 9800
9559b Arg .. Pro
End 9964 End 9940 End 9911
End 10225 End 10251 End 10184
End 10534 End 10536 End 10524
End 10899 End 10916 End 10865
End 11223 End 11197 End 11167
11001- 11600 600 Start 11026 Start 11040 Start 11059
11335 Silent
End 11461 End 11517 End 11497
TABLE Continued
Length of
11719 Silent
End 11795 End 11853 End 11855
11878 Silent
End 12159 End 12164 End 12163
End 12404 End 12443 End 12397
12612 Silent
12705 Silent
End 12769 End 12849 End 12775
12705b Silentb
End 13102 End 13045 End 13024
End 13295 End 13307 End 13266
13572 Silent
End 13587 End 13593 End 13590
13572b Silentb
13702 Gly ... Arg
13708 Ala ... Thr
13759 Ala ... Thr
End 13910 End 13921 End 13900
13966 Thr ... Ala
End 14094 End 14110 End 14104
13966b Thr ... Ala
14199 Pro'" Thr
14272 Phe ... Leu
14365 Silent
End 14369 End 14374 End 14342
14272b Phe ... Leu
14365b Silentb
14368 Phe ... Leu
14470 Silent
14766 lIe ... Thr
End 14699 End 14806 End 14698
14766b lIe ... Thrb
End 14957 End 14972 End 14956
15326 Thr ... Ala
End 15380 End 15373 End 15359
15326b Thr ... Ala.
15646 Silent
End 15754 End 15950 End 15723
15646b Silentb
End 16056 End 16058 End 16030
57 (HV1) 15971-16451 481 Start 16014 Start 16011 Start 16004
16183
16189
16311
16357
End 16193 End 16430 End 16403
16183b
16189b
142 LEVIN , CHENG, AND REEDER
TABLE Continued
Length of
1631lb
16357b
16519
End 16193 End 59 End 103
-21M13C
cloned DNA 16133-40 477 Start 16131
16183b
16189b
16193. C(ins)
16223
16278
16519b
End 40
Note. , basepair change came before the readable sequence; E., basepair change came af'kr the readable sequence; - , basepair same as
in Anderson sequence; h* , possible heteroplasmic site. * This heteroplasmy seen in the first CHR cell culture line was not seen with the
second CHR cell culture line. It is the second CHR cell culture line that is supplied in NIST SRM 2392; Start , start of readable sequence; End,
end of readable sequence; CHR cells, sequence based on two amplifications and cycle sequencing procedures in first cell culture line and at
least one amplification and cycle sequencing procedure with the second cell culture line; 9947A cells , sequence based on two amplifications
and cycle sequencing procedures; GM03798 , sequence based on three to four amplifications and cycle sequencing procedures; ins , insertion;
Del , deletion; ND, not done.
" Numbers correspond to Anderson sequence (Anderson et al. 1981).
Change also seen in previous primer set.
This primer is used for sequencing the cloned DNA of the HV1 region.
the entire mtDNA were completely amplified and sequence (Table 2). However , data from the literature
quenced at least twice (GM03798 was done three indicate that perhaps 14 basepair designations in the
times). There were 13 , 9 , and 4 differences in the non- consensus sequence of Anderson may not be the se-
coding regions of templates CHR , 9947A, and quence found in the majority of normal individuals
GM03798 , respectively, and 33, 23 , and 19 differences (Howellet al. 1992; Marzuki et al. 1992). Our results
in the coding regions of templates CHR , 9947 A , and agree with 11 of these 14 new designations (Table 3).
GM03798 , respectively. All of the differences from Examination of our results using these new designa-
Anderson found in these three templates are shown in tions indicates that only 4 (3 without the heteroplasmy
Fig. 2 along with many of the diseases that have been at bp 6849), 6, and 2 differences for CHR , 9947A, and
noted in the literature (Wallace et al. 1997). None of GM03798, respectively, would result in amino acid
the basepair changes found in the coding regions of the changes. These structural changes , however , do not
three templates sequenced at NIST correlate with any necessarily mean a functional change has occurred in
of the changes found associated with these published the protein. To determine if a functional change has
disease states. occurred , one still needs to decipher whether the amino
Meaning of the differences from Anderson. Since all acid change is in an active site on the protein.
three templates had come from apparently normal in- The interlaboratory evaluation of the CRR template.
dividuals , it was of interest to determine if the differ- An interlaboratory evaluation was conducted by four
ences in the coding regions would actually cause amino laboratories including NIST. All of the laboratories
acid changes in the resultant protein structures. The essentially followed the NIST protocol sent with the
genetic code for human mtDNA is slightly different DNA from the first CHR cell culture line and the prim~
from the universal genetic code (Anderson et al. 1981). ers. Any changes to the protocol are listed under Ma-
One needs to consider these . differences in the univer-
terials and Methods. Each laboratory was instructed to
sal genetic code when determining the amino acid se- amplify and sequence the 58 areas designated by the
quence designated by the 3-bp codons in mtDNA. Many 58 primer sets and also to sequence the cloned DNA for
of the differences from the Anderson sequence were in the HVI region. Laboratory 1 amplified and sequenced
the third position wobble and did not affect the amino each area at least twice. The other labs amplified and
acid sequence (silent changes) (Table 2). However sequenced the areas from one to six times. Laborato-
CHR , 9947A, and GM03798 had 10 (9 without the ries 1 , 2 , and 3 found essentially the same polymor-
heteroplasmyat bp 6849), 12 , and 8 different base- phisms. Laboratory 4 had less experience with se-
pairs , respectively, that would result in a different quencing mtDNA and did find differences that the
amino acid from that designated by the Anderson se- other laboratories did not observe. Data were . excluded
FIG. 1. Agarose gel electrophoresis of PCR products from 58 primer sets designed for human mitochondrial DNA. (A) Lane L, 123-
ladder from Gibco BRL; lane c1 , negative control with no primers; lane c2, negative control with no DNA; lanes 1-
28, PCR products from
primer sets 1-28. (B) Lane L, 123-bp ladder from Gibco BRL; lanes 29-58, PCR products from primer sets 29-
58.
from the analysis of this interlaboratoy evaluation if at base number 6849 (Anderson found an A at this
the following conditions were observed: (1) The com- site). Laboratory 1 found a G at this site , but closer
puter results were ambiguous as indicated by calling a examination of the electropherogram showed that an A
peak "N" rather than A , C , G, or T. (2) The differences peak existed under the G peak. Laboratory 3 also noted
from Anderson were not consistently seen within a the A/G heteroplasmy at this site. Laboratory 4 did not
laboratory, i.e. , if the laboratory sequenced in both the note the heteroplasmy, but when their electrophero-
forward and the reverse directions and one direction
grams were .examined at NIST , the A/G heteroplasmy
agreed with Anderson and the other direction did not was noted. NIST did not have the electropherograms of
agree , we assumed the results that agreed with Ander- Laboratory 2 , but on questioning them , they agreed
son were correct. (3) Within anyone laboratory, the that the heteroplasmy was there, but that they had
difference from Anderson was seen with one primer missed it. One of the problems with finding
heteroplas.
set, but not in the overlapping sequences seen in the mic sites is that if the computer call is the
previous or subsequent primer sets. Even with these Anderson , one would not necessarily examine same as
that site
exclusions , Laboratory 4 had many differences that more closely. If the
were not seen by the other labs. One problem was that Anderson computer call is different from
, one would look more closely at the electro-
they did not provide data from primer sets 29 , 39 , and
, and we were unable to check those overlapping pherogram and then note the presence of a smaller
peak under the main peak.
sequences. Laboratory 2 was unable to sequence the
clone , which was not a problem for the other laborato-
With the exceptions of the differences noted here
ries. Laboratory 3 was missing data from primer sets the interlaboratory evaluation was successful in that
36 and 48. Laboratories 1 and 3 noted a heteroplasmy most of the laboratories found the same results. The
many differences noted by Laboratory 4 , which was
5 In final preparation of this SRM less experienced at sequencing mtDNA , confirm and
, anew blood sample was ob-
tained from CHR and a new cell culture line was established. The emphasize the need for a standard reference mate-
sequence analysis of the new CHR was identical to the first cell line
except no heteroplasmy was found at bp 6849. Therefore , the cell line
supplied with this SRM does not have this heteroplasmy. The data in the interlaboratory evaluation that was done with the first cell
on the first cell line are included in the text to indicate the agreement line.
4135
:1 512
10. kb deletion
Complex III genes

Complex I genes
(NADH dehydrogenase)
(ubiquinoJ: cytochrome c DTransfer RNA genes
oxidoreductase)
Complex IV genes Complex IV genes

(cytochrome c oxidase) (ATP synthase) Ribosomal RNA genes
FIG. 2. Human mitochondrial DNA (color-coded circle) showing positions of genes , diseases , and deletions (Wallace, 1992; Wallace et al. 1997)
and the polymorphisms found in three apparently normal individuals examined for this study. The pink (CHR), green (9947A), and blue (GMO3798)
numbers and lines along the outside of the color-coded circle indicate the locations of differences from Anderson found in this study. Locations of
disease base substitutions are shown on the inside of the circle. Abbreviations: ADPD , Alzheimer disease and/or Parkinson disease; DEAF
neurosensory hearing loss; LDYT, LHON plus dystonia;LHON, Leber hereditary optic neuropathy; MELAS , mitochondrial encephalomyopathy,
lactic acidosis, and stroke-like episodes; MERRF, myoclonic epilepsY and ragged-red fiber disease; MMC, myopathy and cardiomyopathy; NARP
neurogenic muscle weakness , ataxia , and retinitus pigrnentosum. Large deletions are shown by the concentric black semicircles along the outside
of the colored circle. tRNA genes (white . areas in color-coded circle) are indicated by their amino acid single-letter abbreviation. Modified with
permission of the publisher from Wallace (1992) where additional information on this figure may be found. 6849* , see legend to Table 2.
TABLE 3
Errors vs Polymorphisms in mtDNA Sequence Determined by Anderson
Anderson designation ... Anderson designation ...
literature designation NIST mt SRM designation
Basepair (No. foundINo. examined) (No. foundINo. examined) Change
1438 A... Ga A"'G(3/3) 12 s rRNA
3423 G... T (87/87)b G-+T (3/3) SILENT
4769 A... G (28/3W A"'G (3/3) SILENT
4985 G... A (9/W G"'A (3/3) SILENT
8860 A... Ga A"'G (3/3) Thr ... Ala
11335 T ... C (8/8)b T"'C (3/3) SILENT
11719 G'" A (26/37)b G...A (1/3) SILENT
12308 A ... G (3/W Change not found tRNA'eu
13702 G ... C (105/105)b G"'C (3/3) Gly ... Arg
14199 G... T(9/W G"'T (3/3) Pro'" Thr
14272 G ... C (9/W G"'C (3/3) Phe ... Leu
14365 G ... C (9/W G"'C (3/3) SILENT
14368 G ... C (9/W G"'C (3/3) Phe ... Leu
15326 A... G (6/6)b A"'G (3/3) Thr ... Ala
Note. Anderson et at. (1981) seqllenced mtDNA mainly from a single h1lIIlan placenta , although some regions were from HeLa cells. Five
ambiguous regions (bp 10 , 934 , 935 , 14272 , 14365) were assumed by Anderson to be same as that found in bovine mtDNA.
a Marzllki
et al. 1992.
Howell et al.,1992.
rial for sequencing mtDNA. If Laboratory 4 had the use of NIST SRM 2392 will provide quality control to
NIST mtDNA SRM 2392 and had run it alongside the scientific and medical communities when they se-
their unknown sample , they would have realized quence human mtDNA.
that they were finding an undue number of differ-
ences and could have reexamined their procedures to
try to determine the reason for these excessive ACKNOWLEDGMENTS
changes.
The allthors are grateful to The Bode Technology Group, Inc.
(Sterling, VA), lIT Research Institute (Newington, VA), and .Lark
CONCLUSIONS Technologies , Inc. (Houston, TX) for their participation in the inter-
laboratory evaluation. The authors also acknowl~dge the funding
received from the National Institute of Justice throllgh the NIST
A NIST standard reference material (SRM 2392) Office of Law Enforcement Standards and the support from the
that allows one to sequence any region or the . entire Advanced Technology Program at NIST. Th~ authors thank Prasad
569 bp that comprise human mtDNA has been pre- Reddy for his help and advice on cloning, MaryPat Jones for con-
pared. Fifty-eight pairs of unique primers have been firming the sequences of some areas of the second CHR cells, and
Catherine O' Connell for the use of her compllter with the Gene
designed , tested , and shown to work well in the ampli- Runner Program and her advice in the use of the program.
fication and sequencing procedures. The two DNA tem-
plates (CHR and 9947A) included in the SRM have
characteristic polymorphisms throughout the noncod- REFERENCES
ing and coding regions of the DNA and , therefore , can
serve as positive controls during PCR amplification Anderson, S. , Bankier, A. T. , Barrell , B. G. , deBrujin , M. H. L.
Coulson , A. R. , Drouin, J. , Eperon, 1. C. , Nierlich , D. P. , Roe, B. A.,
and sequencing. None of thesepolymorphisms corre- Sanger , F. , Schreier, P. H. , Smith , A. J. H. , Staden , R, and Young,
spond to any of the published basepair changes that 1. G. (1981). Sequence and organization of the human mitochon-
have been correlated with specific diseases. drial genome. Nature 290: 457- 465.
Compared to the Anderson sequence , CRR mtDNA Ballinger, S. W. , Bouder , T. G. , Davis , G. S. , Judice , S. A. , Nicklas
had 13 differences in the noncoding regions and 33 J. A. , and Albertini , R. J. (1996). Mitochondrial genome damage
differences in the coding regions , and the 9947

associated with cigarette smoking. Cancer Res. 56: 5692-5697.
mtDNA had 9 differences in the noncoding regions and Bendall , K. E. , and Sykes, B. C. (1995). Length heteroplasmy in the
first hypervariable segment of the human mtDNA control region.
23 differences in the coding regions. GMO3798 , whose Am. J. Hum. Genet. 57: 248-256.
data are included for comparison and information , had Bogenhagen , D. , and Clayton , D. A. (1974). The number ofmitochon-
4 differences in the noncoding regions and 19 differ- drial deoxyribonucleic acid genomes in mouse L and human HeLa
ences in the coding regions. These differences in the cells. J. Biol. Chem. 249: 7991-7995.
coding regions do result in some amino acid changes in Grossman, L. 1. (1995). Mitochondrial mutations and human disease.
the proteins coded for by mtDNA. Four laboratories Environ. Mol. Mutagen. 25(Suppl. 26): 30-37.
participated in an interlaboratory evaluation of the Holland , M. M. , Fisher, D. L., Mitchell , .L. G. , Rodriquez , W. C.,
Canik, J. J. , Merril , C. R. , and Weedn, V. W. (1993). Mitochondrial
CHR template; some differences between laboratories DNA sequence analysis of h1lIIlan skeletal remains: Identification
were noted , but , in general , agreement was good. The of remains from the Vietnam War. J. Forensic Sci. 38: 542-553.
, pp.
146 LEVIN , CHENG, AND REEDER
Holland , M. M. , Fisher , D. L. , Roby, R. K Ruderman , J. , Bryson , C. stretch. Proc. Seventh Int. Symp. on Human Identification, Scotts-
and Weedn , V. W. (1995). Mitochondrial DNA sequence analysis of dale , Arizona , September 19- , 1996, p. 166.
human remains. Crime Lab. Dig. 22: 109- 115. Marzuki , S. , Lertrit , P. , Noer , A. S. , Kapsa, R. M. 1. , Sudoyo , H.
Howell , N. , McCullough , D. A, Kubacka , 1., Halvorson , S. , and Byrne , E. , and Thyagarajan , D. (1992). Reply to Howell et at. The
Mackey, D. (1992). The sequence of human mtDNA: The question need .for a joint effort in the construction of a reference data base
of errors versus polymorphisms. Am. J. Hum. Genet. 50: 1333- for normal sequence variants of human mtDNA Am. J. Hum.
1337. 50: 1337- 1340.
Genet.
King, M. P. , and Attardi , G. (1989). Human cells lacking mtDNA: Sambrook, J. Fritsch , E. F. , and Maniatis , T. (1989). "Molecular
Repopulation with exogenous mitochondria by complementation. Cloning: A Laboratory Manual " 2nd ed. , Cold Spring Harbor Lab-
Science 246: 500-503. oratory Press , Cold Spring Harbor , NY.
Levin, B. C. , Cheng, H. , O' Connell , C. , Reeder, D. J. , and Holland, M. Wallace, D. C. (1992). Mitochondrial genetics: A paradigm for aging
(1995). Evaluation of alternative enzymes and additives to improve and degenerative . diseases. Science 256: 628- 632.
sequencing of mitochondrial DNA Proc. Fifth Int. Symp. on Human Wallace , D. C. , Brown , M. D. , and Lott , M. T. (1997). Mitochon"
Identification, Scottsdale , Arizona , October 8- 1994, p. 170. drial genetics. In Emery and Rimoin s Principles and Practice
Levin, B. C. , Cheng, H. , Holland , M. , and Reeder , D. J. (1997). of Medical Genetics " (D. L. Rimoin , J. M. Connor , and R. E.
Heteroplasmy responsible for difficulty experienced in sequencing Pyeritz , Eds. ), 3rd ed. , Vol. 1 277- 332 , Churchill Living-
the human mitochondrial DNA HV1 region containing the C- stone , New York.
Appendix D
Journal Article:
B. C. Levin, K. A. Holland, D. K. Hancock, M. Coble, T. J. Parsons, L.
J. Kienker, D. W. Williams, MP. Jones, and K. L. Richie,
“Comparison of the Complete mtDNA Genome Sequences of
Human Cell Lines - HL-60 and GM10742A - From Individuals with
Pro-Myelocytic Leukemia and Leber Hereditary Optic Neuropathy,
Respectively, and the Inclusion of HL-60 in the NIST Human
Mitochondrial DNA Standard Reference Material - SRM 2392-I,”
Mitochondrion 2, 387-400, (2003).
(See end of Appendix D for color representation of Figure 1.)
lIitochondrion
ELSEVIER Mitochondrion 2 (2003) 387-.400
www. elsevier.comllocate/mito
Comparison of the complete mtDNA genome sequences of human

cell lines - HL- 60 and GMIO742A - from individuals with
pro-myelocytic leukemia and leber hereditary optic neuropathy,
respectively, and the inclusion of HL- 60 in the NIST
human mitochondrial DNA standard reference
material - SRM 2392-
Barbara C. Levin , Koren A: Hollanda , Diane K. Hancocka

Michael Coble , Thomas J. Parsons , Laura J. Kienker
Diana W. Williams , MaryPat Jones , Kristy L. Richie
NationalInstitute of Standards and Technology, lOa Bureau Drive, Mail Stop 83Jl, Gaithersburg, MD 20899- 83JI. USA
Gettysburg, FA , USA
Gettysburg College ,
Armed Forces DNA Identification Laboratory, Rockville, MD, USA
The George Washington University, Washington, DC, USA
Federal Bureau of Investigation Laboratory, Quantico, VA, USA
Georgia Bureau of Investigation, Decatur, GA , USA
gNational Human Genome Research Institute, National Institutes of Health, Bethesda, MD , USA
Received 12 December 2002; received in revised form 26 January 2003; accepted 28 January 2003
* This paper is a contribution of the U. S. National Institute of Standards and Technology (NIST), Armed Forces DNA Identification
Laboratory (AFDIL), Federal Bureau of Investigation (FBI), the Georgia Bureau of Investigation (GBI) and the National Human Genome
Research Institute (NHGRI), National Institutes of Health (NIH) and is not subject to copyright Certain commercial equipment , instruments,
materials, or companies may be identified in this paper to specify the experimental procedure. Such identification does not imply
recommendation or endorsement by the authors, nor does it imply that the materials or equipment identified are the best available for this
purpose.
** Orders and requests for information concerning this SRM should be directed to the Standard Reference Materials Program, National
Institute of Standards and Technology, 100 Bureau Drive, Stop 3222 , Gaithersburg, MD 20899- 2322 , USA. Tel.: + 1- 301 "975- 6776; fax: + I"
301-948-3730. E-mail: srminfoC1Ynistgov , web address http://www. nistgov/srm.
* Some of the data in this paper has been presented at the following meetings: The 12th International Symposium on Human Identification
Biloxi, MS, October, 2001; the American Academy of Forensic Sciences Annual Meeting, Atlanta, GA, February, 2002; the Third Annual
Biotechnology Retreat , Shepherdstown, WV, May, 2002; the United Mitochondrial Disease Foundation Milo Dallas 2002 Symposium and
Mitochondrial Standards Workshop, Dallas , TX , June , 2002; the Third Annual DNA Grantees ' Workshop, National Institute of Justice, June
2002; and the 13th International Symposium on Human Identification, Phoenix, AZ , October, 2002.
* Corresponding author. Tel.: + 1-301"975- 6682; fax: + 1- 301-975- 8505.
E-mail address: barbara.levinC1Ynist. gov (B. c. Levin).
1567- 7249103/$20. 00 ~ 2003 Published by Elsevier Science B. V. on behalf of Mitochondria Research Society.
16/S 1567- 7249(03)000 I 0-
doi: 10. 10
388 c. Levin et at. Mitochondrion (2003) 387-400
Abstract
Forensic and clinical laboratories benefit from DNA standard reference materials (SRMs) that provide the quality control and
assurance that their results from sequencing unknown samples are correct. Therefore , the mitochondrial DNA (mtDNA)
genome of HL- 6Q, a promyelocytic leukemia cell line, has been completely sequenced by four laboratories and will be available
to the forensic and medical communities in the spring of 2003; it will be called National Institute of Standards and Technology
(NIST) SRM 2392- 1. NIST human mtDNA SRM 2392 will continue to be available and include the DNA from two apparently
healthy individuals. Both SRM 2392 and 2392-1 contain all the information (e. g. the sequences of 58 unique primer sets) needed
to use these SRMs as positive controls for the amplification and sequencing any DNA. Compared to the templates in SRM 2392
the HL- 60 mtDNA in SRM 2392- 1 has two tRNA differences and IIlore polymorphisms resulting in amino acid changes. Four of
these HL- 60 mtDNA polymorphisms have been associated with Leber Hereditary Optic Neuropathy (LHON), one as an
intermediate mutation and three as secondary mutations. The mtDNA from a cell line (GM1O742A) from an individual with
LHON was also completely sequenced for comparison and contained some of the same LHON mutations. The combination of
these particular LHON associated mutations is also found in phylogenetic haplogroup J and its subset , J2, and may only be
indicative that HL- 60 belongs to haplogroup J, one of nine haplogroups that characterize Caucasian individuals of European
descent or may mean that haplogroup J is mOre prone to LHON. Both these mtDNA SRMs will provide enhanced quality
control in forensic identification , medical diagnosis , and single nucleotide polymorphism detection.
~ 2003 Published by Elsevier Science B. V. on behalf of Mitochondria Research Society.
Keywords: Forensic identification; GMI0742A; Haplogroup J; HL- 60; Leber hereditary optic neuropathy (LHON); Medical diagnosis;
Mitochondrial DNA sequence; Single nucleotide polymorphism (SNP); Standard reference material (SRM)
1. Introduction polymerase chain reaction (PCR) amplification pro-

cess , cycle sequencing steps , gel separation , and data
On July 15, 1998, the Federal13ureau of Investi- analysis to obtain the final DNA sequence is provided
gation (FBI) Director signed Standard 9.5 , which as well as the information on the sequence of
stated " The laboratory shall check its DNA pro- unique primer sets that allow the sequencing of any
cedures annually or whenever substantial changes are specific portion or the entire mtDNA (16, 569 bp)
made to the protocol(s) against an appropriate and without any gaps. Following an interlaboratory evalu-
available National Institute of Standards and Tech~ ation , SRM 2392 became available to the public in
nology (NIST) standard reference material or standard December 1999.
traceable to a NIST standard" . At the present time The FBI acknowledges the utility of DNA SRMs to
there are a number of human DNA standard reference provide the quality control and quality assurance that
materials (SRMs) available from NIST (Levin et aI. the results from forensic laboratories that are sequenc-
2001). One of these SRMs on mitochondrial DNA ing unknown samples are correct. One of the FBI's
(mtDNA SRM 2392) is used by the forensic com- Combined DNA Index Systems now includes mtDNA
munity for human identification . and by the medical from unidentified remains, as well as from relatives of
community for diagnoses of a number of human missing persons. In order for authorized laboratories
diseases now known to be associated with specific to contribute to these indices, certain quality standards
mutations, insertions and deletions of mtDNA (Levin must be met. These include the use of DNA from the
et aI. , 1999). This SRM was prepared by NIST to human cell line HL- 60 as a positive control to be run
provide quality control to the scientific community in conjunction with the unknown samples. HL- 60 was
when they amplify and sequence human mtDNA or chosen as the positive control because of several
any DNA and includes DNA from two apparently features present in HL- 60 but not in the cell lines
healthy individuals (CHR and 9947A) plus cloned currently available in NIST SRM 2392. Some of the
DNA of an area in CRR containing a C- Stretch advantageous features of HL- 60 are well-spaced
beyond which sequencing becomes difficult. All the polymorphisms throughout the non-coding hypervari-
information necessary to successfully conduct the able regions, HV1 and HV2 , of the mtDNA control
13. C. Levin et at. Mitochondrion (2003) 387-400 389
region , and no insertions at the HV2 C-stretch area considered either intermediate or secondary mutations
(positions 303~ 309). One of the current templates in associated with leber hereditary optic neuropathy
SRM 2392 , the CHR DNA has a C-stretch in the HV1 (LHON) (Wallace et aI. , 1997). We, therefore,
region caused by . a T to Cchange at position 16189 sequenced the mtDNA from cell line GM1O742A
that produces a length heteroplasmy (Bendall and that was prepared from a patient with LHON for
Sykes, 1995; Butler and Levin, 1998). Sequencing comparison with the HL- 60 mtDNA sequence. Two
through this C-stretch is difficult and time-consuming of the four LHON-associated mutations in HL-
and results in the need to perform additional were also found in GM10742A. Two additional
sequencing reactions to resolve this region and the mutations associated with LHON were found in
area following the C-stretch. The CHR template was GM10742A , one primary and one secondary muta-
chosen by NIST specifically for the C-stretch region tion. Many of the polymorphisms common to both
since some laboratories wanted the opportunity to HL- 60 and GM10742A (some of which are associated
address this difficult sequencing problem and try to with LHON) characterize the phylogenetic haplo-
resolve it. The DNA from the other . current SRM group J and sub- haplogroup Jz, a subset of J (FinniHi
DNA template , 9947 A , has only two polymorphisms et aI., 2001; Helgason et aI., 2001; Herrnstadt et aI.
in the HV1 region with respect to the Cambridge 2002). Haplogroup J is one of nine haplogroups .that
Reference Sequence (Anderson etaI., 1981; Andrews characterize Caucasian populations of European
et aI. , 1999) and those polymorphisms are at common decent. A number of papers in the literature indicate
sites. For the work that the forensic laboratories are that individuals with LHON usually belong to haplo-
doing on human identification, several evenly spaced group J and suggest that those in haplogroup J may
polymorphisms within. the HV1 region are more therefore be mC?re prone to this mtDNA disease than
desirable to differentiate the positive control from the those in other haplogroups (Brown et aI., 2002;
test sample. Thus , the FBI suggested to NIST that the Herrnstadt et aI. , 2002; Torroni et aI. , 1997).
development of a SRM containing the HL- 60 DNA
template would be of great utility to the forensic
community. All of the research and the interlaboratory 2. Materials and methods
evaluation of theHL- 60 template necessary to pro-
duce this new SRM containing HL- 60 have been 1. HL- 60 DNA
completed and are described in this paper. Both SRM
2392 (containing two DNA templates) and SRM DNA from the HL- 60 cell culture was extracted,
2392- 1 (containing one DNA template) should fulfill isolated, and quantified by the Professional Services
the needs of forensic laboratories by providing Department of the American Type Culture Collection
additional quality control when sequencing human (A TCC, Manassas , VA).
mtDNA. Corroboration of the SRM results will DNA was isolated from the HL- 60 cell culture
provide assurance that the techniques being used for using the QIAamp DNA Blood Mini Kit (Qiagen
amplification and sequencing unknown DNA are Inc. , Valencia, CA). Quantification was determined
being conducted correctly. by the Quantiblot Human DNA Quantification Kit
The DNA templates in SRM 2392 came from (Applied Biosystems , Foster City, CA). The final
apparently healthy individuals; however , HL- 60 in concentration , 1.4 ng/lJ.l, is based on 12 replicate tests.
SRM 2392- 1 is from a promyelocytic cell line from The DNA purity (A260:A280 = 1.9) was deter-
peripheral blood leukocytes provided by a 36- year-old mined spectrophotometrically. The integrity of DNA
Caucasian female with acute promyelocytic leukemia. was determined electrophoretically using 0.4%
Therefore, it was of interest to determine if the HL- agarose gels.
DNA contained specific mutations that may be
characteristic of this disease. Although more DNA 1. GM1O742A DNA
samples from leukemic patients would have to be The lymphoblast cell line GM1O742A was pur-
examined to associate specific mutations with this chased from NIGMS Human Genetic Mutant Cell
disease , we did find four polymorphisms that are Repository, Coriell Institute of Medical Research
390 c. Levin et at. Mit9chondrion (2003) 387-400
Camden , NJ. The cells were grown at NIST in RPMI (extension) and ended with a final extension of 7 min
1640 plus L- glutamine and sodium bicarbonate. at no
growth media (Sigma , St. Louis , MO) plus fetal calf Amplified DNA was purified with a QlAquick
serum (20%) (Sigma) plus the antibiotics Strepto" PCR Purification Kit (Qiagen) and the purity and size
U/ml)
mycin and Penicillin (final concentration: 100 of the PCR product was determined by electrophoresis
(Sigma). DNA was extracted using the QIAGEN in 2% agarose gels in 1 X TBE buffer containing
Plasmid Kit following the Plasmid Mini Purification 5 j.Lg/ml ethidium bromide (Sigma).
Protocol.
1.4. Sequencing at NIST
1.2. mtDNA primers Cycle sequencing using fluorescent dye- labeled
Fifty-eight sets of unique primers (19- 28 bp) terminators was performed with an ABI PRISM
for sequencing any portion or the entire human BigDye Terminator Cycle Sequencing Ready Reac"
mtDNA (16 569 bp), including both the HV1 and tion Kit with AmpliTaq (jj) DNA Polymerase , FS
ffV2 regions , were computer- designed using GENE (Applied Biosystems).
RUNNER FOR WINDOWS (Hastings Software , Inc. Cycle sequencing reactions in both the forward
Hastings, NY) and were the same as those used for and reverse modes were conducted with a 9700
SRM 2392 (Levin et aI. , 1999) with the exception of PerkinElmer thermal cycler and started with 1 min at
the reverse primer of set 51. During the course of this 96O e. The reaction then underwent 25 cycles of 96O
study, this primer was changed since it contained a C for 15 s (denaturation), 50oC for 5 s (annealing), and
at nucleotide position (np) 14 368. Since this is the 60oC for 2 min (extension). The DNA products were
reverse primer, it would bind to a G at np 14 368. purified using Edge Gel Filtration Cartridges (Edge
Andrews et al. (1999) in their reevaluation of the BioSystems, Gaithersburg, MD).
placenta originally used to sequence human mtDNA Sequencing and data analysis of the purified
in 1981 (Anderson eta1. , 1981) found that np 14 368 DNA were performed using an Applied Biosystems
should have a C at that position. The new reverse PRISM Model 310 Genetic Analyzer with POP- 6TM
primer 51 is 5' - TT AGCGA TGGAGGT AGGATf polymer system and 47 cm X 50 j.Lm capillaries
3' (np 14 368 is in bold and underlined) which binds (Applied Biosystems). Sequence data were analyzed
to the C at position 14 368. The 5' end is np 14, 388 with Sequencing Analysis Software 3. , and com~
and the 3' end is 14 367. The 58 sets of primers were parisons to the Cambridge Reference Sequence
custom-made by Bio- Synthesis, Inc. (Lewisville (1981) were performed with Sequence Navigator
TX); the new reverse primer 51 was obtained from Software 1.01.
Invitrogen (Carlsbad , CA). Those laboratories using
either SRM 2392 or SRM 2392- 1 should use the new 2. Interlaboratory evaluation of HL-
reverse primer 51.
Three laboratories, in addition to NIST , partici-
1.3.Polymerase chain reaction (PCR) at NIST pated in an interlaboratory evaluation of the sequence
The PCR mixture contained: HL- 60 DNA (1 j.Ll; of HL-60 DNA. These laboratories included the FBI
1.4 ng), AmpliTaq Gold(jj) DNA polymerase, (0. 5j.Ll; Laboratory, FBI Academy, Quantico, V A 22135;
5 units) (Applied Biosystems), 10 buffer (5 j.Ll) Armed Forces DNA Identification Laboratory
containing 100 mmolll Tris- HCl , pH 8. 3, 500 mmol/l (AFDIL), Armed Forces Institute of Pathology,
KCl , 15 mmolll MgCl2 and 0. 01 % (w/v) gelatin Rockville , MD 20850; and the Georgia Bureau of
(Applied Biosystems), 10 mmolll dNTP mix (1 j.Ll) Investigation (GBI), Decatur, GA 30034. Each
(Invitrogen), 10 j.Lmolll forward and reverse primers laboratory was asked to amplify and sequence the
(lor 2 j.Ll), plus water to make a final volume of 50 j.L1. entire mtDNA ofHL- 60. NIST provided: (1) a tube of
Thermal cycling was conducted in a PerkinElmer DNA containing the extracted DNA ready for PCR
thermocycler Model 9700 and started with 10 min amplification; (2) the 58 sets of primers labeled with
at 95O C, followed by 35 cycles of 20 s at 94O either F# (forward primer) or R# (reverse primer); (3)
(denaturation), 10 s at 56O C (annealing), 30 sat no a table to record the results; and (4) any other supplies
Mitochondrion (2003) 387-400 391
c. Levin et at.
that were needed and requested by the participants. followed by 94O C for 15min in a perkinElmer 9700
They were allowed to use any protocol for amplifi- thermocycler.
1.1. 2. Cycle sequencing
cation or sequencing that they wished , but were AFD1L sequencing.
i!9 BigDye i!9
requested to provide a copy of that protocol to NIST. was performed with the ABI PRISM
NIST also requested copies of the electropherograms terminators (original version) cycle sequencing kit
to enable us to resolve any discrepancies; although as (Applied Biosystems). The sequencing mixture con-
i!9 dilution buffer
it turned out , there were no discrepancies. tained 9 j.Ll dHz O, 6 j.Ll BigDye
(400 mmolll TRIS, 10 mmol/l MgClz, pH 9. 0), 2 j.Ll
1. Differences in methodology used by the BigDye i!9 terminator reaction mixture, 1 j.Ll of forward
laboratories in the interlaboratory evaluation or reverse primer (10 j.Lmolll each) and 2 j.Ll of HL-
PCR product for a total vol of 20 j.Ll. A few of the
Armed forces DNA identification laboratory

1. sequencing primers (e. g. R649) required the use of the
i!9
(AFD1L). AFDIL has developed a high- throughput Am PRISMi!9 dGTP BigDye terminator kit (Applied
automated sequencing procedure using 12 primer sets Biosystems). Thermal cycling was conducted in a
that produce overlapping PCR products ranging from PerkinElmer 9700 thermocycler at the following
825 to 1886 bp. The primers used to amplify the first conditions: an initial 1 min denaturation at 96O
11 products were based on those published in Levin followed by 25 cycles of 94OC for 15 s , 50oC for 5 s,
et al. (1999) and are: AmpOl - F361/R2216; Amp02 and 60o C for 2 min. The DNA product was purified by
- F1993/R3557; Amp03 - F3441/R4983; Amp04 - filtration through a spin column matrix (Edge
F4797/R6526; Amp05 - F6426/R83 11; Amp06 BioSystems, Gaithersburg, MD). Electrophoresis
F8164/R9848; Amp07- F9754/R11600; Amp08 - and sequencing were performed with an Am PRISMi!9
Fl1403/R13123; Amp09 - F12793/R14388; Amp10 3100 Genetic Analyzer (Applied Biosystems) using
- F14189/R15396; Amp11 - F15260/R16084. The POP- 6TM polymer (Applied Biosystems) with a 50
primers used to amplify the control region were capillary. Data analysis was executed using
developed at AFDIL and are Amp12 - F15878/R649 Sequencher Plus 4. 5b11 (Gene Codes , Ann Arbor,
(Fl5878 is TTAACTCCACCATTAGCACC and MI). The HL- 60 sequence differences were identified
R649 is TTTGTTT A TGGGGTGATGTGA). by comparison to the Cambridge Reference Sequence
2.1.1. 1. AFD1L PCR amplification. The PCR as revised by Andrews et aI. (1999). In most cases
mixture contained HL- 60 DNA (1 j.Ll), AmpliTaq- sequence information was acquired for both the
Goldi!9 DNA polymerase (1 j.Ll) (Applied Biosystems), forward and reverse directions. In some regions, two
10 X PCR buffer (5 j.L1) (Applied Biosystems), separate reactions using the same primer were
dNTP' s (0. 2 nunol/l) (Invitrogen), 2 j.Ll of forward routinely conducted (indicated by ' 2 X ' in the
and reverse primers (10 j.Lmol/l) (MWG Biotech following list of primers) to achieve full sequence
High Point , NC) plus dHzO to a final volume of confirmation. A total of 95 sequencing reactions plus
50 . j.Ll. The lOx PCR buffer was the same as that used one pGEM reaction were conducted in a 96 well
by NIST. Thermal cycling was conducted in a format. If the primer failed the first trial, the reaction
PerkinElmer 9700 thermocycler with the following was repeated. The finding of a heteroplasmy at
conditions: 10 min at 96O C (activation of AmpliTaq nucleotide position (np) 12, 071 was also confirmed
Goldi!9 ), plus 40 cycles of 94OC for 15 s, 56OC for 30 s, by an additional PCR and sequencing reaction.
and noc for 1 min. The purity and size of the PCR The following primers from Levin et al. (1999)
products were assessed by electrophoresis in a 0. were used to sequence the 12 PCR amplicons:
agarose gel containing 0. 3 j.Lg/ml of ethidium bro-
mide. The PCR products' were purified with Shrimp AmpOl (F3611R2216): F361 , R921, F1234,
Alkaline Phosphatase/Exonuclease I (Amersham R1425, F873, R2216, F1657, R1769
Pharmacia , Piscataway, NJ). Five j.Ll of exonuclease Amp02 (F1993/R3557): F1993 , R2660, R2834,
1(10 U/j.Ll) and 10 j.Ll of Shrimp Alkaline Phosphatase R3557, F2417, R3006 , F3234
(1 U/j.Ll) was added to each tube containing PCR Amp03 (F3441/R4983): F3441, R3940 , F3931
product. The tubes were heated at 37O C for 15 min (2 x), R4982 , F4392, R4162
392 B.c. Levin et al. Mitochondrion (2003) 387-400
Amp04 (F4797/R6526): F4797 (2 X), R6526, with Sequencer 3. 1.1 software (Gene Codes, Ann
F5700 (2 X), F5318 , R5882 , F6242 Arbor, MI).
Amp05 (F6426/R83 11): F6426 (2 X), R7255,
F7645 (2X), R8311, F7075, R7792 1.3. Federal Bureau of Investigation (FBI)
Amp06 (F8164/R9848): F8164 (2 X), R9059, 1.3. 1. FBI Polymerase chain reaction (PCR).
F8903, R9848 , F8539, R9403, F9309 The PCR mixture contained from 0. 1 to 1.4 ng HL- 60,
Amp07 (F9754/Rl1600): F9754 (2 X), R1O556 AmpliTaq Gold4\) Polymerase (2. 5 units) (Applied
F11001 (2 X ), R11600, F1O386, R11267 Biosystems), 10 X PCR buffer (5 fJ-l) (Applied
Amp08 (F11403/R13123): F11403 (2 X), F12357 Biosystems), GeneAmp 4\) dNTPs (0. 2 mmol/l each)
R13123 , F11901 (2 X ), F12601, R12876 (Applied Biosystems), forward and reverse primers
Amp09 (F12793/RI4388): F12793 , R13611 (0.4 fJ-mol/l each), plus dH2O to a final volume of
F13518 (2 X), R14388, F13188 , R13343, 50 fJ-l. The 10 X buffer (pH 8. 3) was the same as used
F13899, R13935 by NIST. Thermal cycling was conducted in a
Amp 10 (F14189/R15396): F14189 (2 X), R15396, GeneAmp 4\) PCR System
97QO (PerkinElmer) and
F14909, R14996, F14470 consistedof lO min at 95O C followed by 35 cycles of
Amp11 (F15260/R16084): F15260, R16084 94O C for 20 s , annealing temperatures of 50oC (primer
F15574, R15774 set 49), 51O C (primer sets 1 , 7 , 44 , 57), 52O C (primer
Amp12 (F15878/R649): F15971 , R16175 (2 X), sets 6, 8 , 30, 45), 53O C (primer set 53) and 56OC (all
F16450 (2 X), R274 , F314 (2 X), R649 (2 X), other primer sets) for 10 s, and 72O C for 30 sand
F16190, Rl6400. ending with a final extension of 7 min at 72O
Amplified products were purified by treatment with
In Amp12 , F15971 came from Levin et al. (1999). Exo- SAP- IT (5 fJ-l for every 25 fJ-I of PCR product)
The other primers were designed by AFDIL and were (USB Corp. , Cleveland , OH). Samples of the PCR
as follows: products were electrophoresed in 1.2% agarose gels
containing ethidium bromide to assess the purity, size
R16175: TGGATTGGGTTTTTATGTA and quantity of the PCR products.
F16450: GCTCCGGGCCCATAACACTTG 1.3. 2. FBI sequencing. Cycle sequencing using
R274: TGTGTGGAAAGTGGCTGTGC fluorescent dye~ labeled terminators was performed

F314: CCGCTTCTGGCCACAGCACT irith an ABI PRISM4\) dRhodamine Terminator Cycle
4\)
R649: TTTGTTTA TGGGGTGA TGTGA Sequencing Ready Reaction Kit with AmpliTaq
F16190: CCCCATGCTTACAAGCAAGT DNA Polymerase, FS (Applied Biosystems). Thermal
R16400: GTCAAGGGACCCCT A TCTGA. cycling was conducted in a GeneAmp 4\) PCR System
9700 (Applied Biosystems) and started with 1 min at
96O C followed by 25 cycles of 96O C for 15 s, 50o C for
1.2. Georgia Bureau of Investigation (GBI). The 1 s and 60o C for 1 min and ended with a final
GBI used the same protocol as that used by NIST with incubation at 15OC for 10 min. These products were
the following exceptions: (1) a PerkinElmer model TM spin columns
purified with Centri- Sep (Princeton
9600 was used for thermal cycling; (2) amplified Separation , Inc., Adelphia, NJ) and electrophoresed
DNA was electrophoresed in 2. 75% agarose gels; (3) on an ABI PRISM 4\) 310 Genetic Analyzer (Applied
the cycle sequencing was performed with a Perkin~ Biosystems) using POP- 6TM and either a 47 or 61
Elmer model 9600 using the program provided with capillary. Comparison of the sequence data was
the BigDye 4\) kit (25 cycles of 96O C for 10 s, 50o C
for performed with Sequencer 4. 1 software (Gene
5 s, and . 60oC for 4 min); (4) some samples that Codes).
needed 'to be cycle sequenced again using more The FBI also discovered the problem with original
amplicon were not purified before precipitation; (5) primer set 51 (see Section 2). The reverse primer
the samples were precipitated u.sing isopropanol incorporated a G at position 14, 368 (an error in the
precipitation as per the BigDye 4\) instructions; and original Anderson et al. (1981) sequence that was
(6) comparison of the sequence data was performed found and corrected by Andrews et aI. (1999)).
B.c. Levin et at. Mitochondrion (2003) 387-400 393
However, sequencing with primer set 52 showed a C previous publication (Levin et aI., 1999), and is shown
at position 14 368. They prepared a new set of primers in Fig. 1 in this paper; however , that DNA is not
(primer set 51. 5) to amplify approximately a 400 bp included in SRM 2392 or SRM 2392- 1. Another
fragment that encompassed np 14 368. The new mtDNA template (GM1O742A) from a 30- year-old
primers were: F14217: 5' CTAATCAACGCCCA- Caucasian male patient with LHON was also
TAATCATAC- 3' and R14620: 5' GTTTTCTTC- sequenced at NIST; that sequence is shown in
TAAGCCTTCTCC- . The new primer set Table 1 and Fig. 1, but the DNA is not included in
confirmed that the correct base at position 14 368 SRM 2392 or 2392- 1. Both GM03798 and
was a C. GM1O742A can be obtained from the National
Institute of General Medical Sciences Human Genetic
2.3. Permissions Mutant Cell Repository, Coriell Institute for Medical
Research , Camden , NJ. At NIST , 58 primer sets
The research to prepare SRM 2392- 1 containing (Levin et al, 1999) were used to amplify the entire
HL- 60 DNA was deemed exempt from the policy of HL- 60 mtDNA and GMI0742A at least twice. In the
Part 27 of Title 15 of the Code of Federal Regulations case of HL- 60, all the PCR products were sequenced
by the NIST Institutional Review Board and the in both the forward and reverse directions for a total of
Director of the Chemical Science and Technology four sequences for each amplicon. During this study,
Laboratory. This work fit into the exemption category reverse primer 51, which contained np 14 368, was
described in 15 CFR 27. 1O1(b)(4) which states: changed (see Section 2) to accommodate the differ-
Research , involving the collection or study of ence found at that position by Andrews et aI. (1999)
documents , pathological specimens, or
existing data , when they resequenced the original placenta used by
diagnostic specimens , if, these sources are publicly Anderson et aI. (1981). Any sequence ambiguities
available or if the information is recorded by the between experiments at NIST were resolved by
investigator in such a manner that subjects cannot be additional PCR reactions and sequencing.
identified, directly or through identifiers linked to the The NIST sequencing results from the mtDNA of
subjects . ATCC also waived condition 3(c) in their HL- 60, CHR, 9947A and GM1O742A are shown in
Material Transfer Agreement which states that .the Table 1; all of these sequences plus GMO3798 are
purchaser shall not sell , lend , distribute or otherwise shown in Fig. 1. Table 1 also compares the CHR,
transfer the material or replicates to any others " for 9947A, HL- 60 and GMI0742A templates to the
the use of HL- 60 in the NIST mitochondrial DNA original Cambridge Reference Sequence (Anderson
SRM 2392- 1. They stated that , in their view as a et al., 1981) and the revised sequence (Andrews et aI.
government agency, NIST will not be providing this 1999). In Fig. 1, the HL- 60 results are shown in black
material as a commercial product despite the collec- numbers surrounding the circular depiction of human
tion of fees for the SRM" mtDNA; the CHR results are shown in red; 9947A
results are shown in green; GMO3798 results are
shown in blue; and GM1O742A are in purple. At the
3. Results and discussion present time, GM03798 and GM1O742A are not part
of SRM 2392 or SRM 2392- , and the results are
HL- 60 is a promyelocytic cell line from the peri- presented for information and comparison only.
pheral blood leukocytes of a Caucasian female (age The numbering system in Table 1 and Fig. 1 is that
36) with acute promyelocytic leukemia. HL- 60 DNA of the original Cambridge Reference Sequence
will be available in SRM 2392~ 1. The two DNA (1981). The Cambridge Reference Sequence (1981)
templates available in SRM 2392 are from apparently was based on a consensus analysis of a placenta, the
healthy individuals (both the DNA from CHR and HeLa cell line and the bovine sequence (the Dovine
9947 A are from Caucasian females in their twenties sequence was used in five ambiguous human sites).
and thirties). The entire mtDNA of GM03798 (a The 1981 results were reexamined in 1999 by
lO- year-old apparently healthy Caucasian male) was Andrews et aI. who resequenced the original placenta.
amplified and sequenced at NIST, discussed in a The original Cambridge Reference Sequence was
~-- __.
394 B.C. Levin et at. Mitochondrion (2003) 387-400
'216--
4216
ATPase 8
Fig. I. HL-60 DNA polymorphic differences from the Cambridge Reference Sequence and positions of LHON mutations are added to the figure
from Levin et al., 1999. This figure is a schematic of human mtDNA showing its circular double-stranded DNA and all the differences from
Cambridge Reference Sequence found in CHR (red), 9947A (green), GM03798 (blue), HL-60 (black), and GM1O742A (purple) as numbers
along the outside of the color- coded circle. Locations of the control region , rRNAs and genes (see footnote to Table I for abbreviations) coded
by human mtDNA are shown. The locations of the 22 tRNAs are noted by white areas in the circle and designated by their single letter amino
acid code. (Modified from Levin et aI. , 1999).
found to have a number of rare polymorphisms and acid changes), HL- 60 has two polymorphisms (one
errors , which are noted in Table 1 with an asterisk. producing an amino acid change and is hetero-
Table 1 also shows the unique (D) coding region plasmic), and GM1O742A has two (one in the
polymorphisms (either silent with no amino acid 16sRNA and one that is silent) that were not found
change or resulting in an amino acid change) in in the MITOMAP database.
CRR, 9947 A , HL- 60 and GM1O742A that were Table 2 shows the number of differences compared
not found in the MITOMAP web site database to the Cambridge Reference Sequence found in CRR,
(http://www. rnitornap. org). This is a very extensive 9947 A , HL- 60 and GM10742A. There are 13, 9,
internet database on the mitochondrial DNA poly- and 12 differences in the non-coding regions of CRR
morphisms found in the literature. CRR has five 9947A, HL- 60, and GM10742A, respectively. The
polymorphisms (one produces an amino acid change), non-coding region is the primary area used by the
9947 A has four polymorphisms (two producing amino forensic community for human identification. There
B.C. Levin et al. Mitochondrion (2003) 387-400 395
are 33, 33 and 31 differences ~n the coding regions in tRNA for leucine have been associated with LHON
of CHR, 9947 A , HL- 60, and GM1O742A , respect- (http://www. mitomap. org). Some of these mutations
ively. These differences would have resulted in 11 , 12 are shown inside the inner circle of Fig. 1. The four
15 and 14 amino acid changes in their respective mutations considered ' primary ' in causing LHON are
proteins if one compares the results to the original G14459A, Gl1778A , G3460A , and T14484C and are
Cambridge Reference Sequence published by presented in order of decreasing severity (Wallace
Anderson et aI. (1981). If compared to the revised et aI., 1997). The mutations associated with LHON
Cambridge Reference Sequence (Andrews et aI. found in this study are considered ' primary
1999), the actual amino acid changes would be (Gl1778A is in GM1O742A), ' intermediate
reduced to 3 in CHR, 4 in 9947 A , and 7 in both (G 15257 A is in HL- 60) or ' secondary ' (T3394C in
HL- 60 and GMI0472A (the non-normal cell lines GMI0742A; T4216C in HL- 60 and GMI0742A;
sequenced). HL- 60 also has a change in the tRNA for Gl3708A in HL- , CHR and GMI0742A;
alanine and the tRNA for serine. It would be of G15812A in HL- 60). According to Wallace et aI.
interest to examine other cell lines from patients with (1997), the intermediate or secondary mutations may
acute promyelocytic leukemia or LHON to see if they increase the probability of having LHON or may be
have any of these same mutations. linked to one of the primary mutations. It is interesting
Table 3 shows the HL- 60 and GMI0742A poly- that GMI0742A has two of the secondary mutations
morphisms in the coding regions that (with the also found in HL- , namely T4216C and G13708A.
exception of one CHR mutation included because CHR also has the G 13708A mutation. Torroni et aI.
of its association with LHON) were not found in (1997) found that the combination of the np 4216 and
the other templates in SRM 2392. This table also 708 mutations were more frequent among the
indicates if the mutation has been associated with a LHON patients containing the mutation at 11 778 than
disease according to the MITOMAP web site among the controls.
database. At least four HL- 60 mutations (T4216C in Characteristic sets of polymorphisms in human
NADH dehydrogenase 1 , G13708A in NADH dehy- mtDNA are being used to distinguish various groups
drogenase 5 , and G15257A and G15812A in Cyto- and to trace their maternal genealogy (Macaulay et aI.
chrome B) were found to be associated with LHON 1999). Haplogroup J and sub-haplogroup Jz have
(Wallace et aI., 1997). Four mutations associated with specific polymorphisms that distinguish this group of
LHON were also found in GM1O742A; these are Caucasians with European ancestry from the other
T3394C and T4216C in NADH dehydrogenase 1 eight haplogroups (H , I , K , T , U , V, Wand X) that
G 11778A in NADH dehydrogenase 4 , and G 13708A have also been found to characterize those with a
in NADH dehydrogenase 5. Two of these mutations European background (Torroni et aI. , 1997). Both
T4216C and G13708A , were found in both HL- 60 and HL- 60 and GM1O742A have a number of identical
GM1O742A. The mutation G13708A was also seen in polymorphisms that place them in Haplogroup J and
the CHR template. LHON causes central vision loss in Sub- haplogroup Jz (Table 4).
patients in their twenties or thirties although the onset In the interlaboratory evaluation of the entire
of symptoms can occur both earlier and later. The sequence of HL- 60 that was conducted by three
disease is inherited like other mitochondrial DNA laboratories plus NIST , all four laboratories found the
diseases through the maternal lineage. LHON was same sequence with the exception of the Georgia
first associated with a G 11778A mtDNA mutation Bureau of Investigation who had the problem noted
(Wallace et aI. , 1988) that results in an arginine to in Section 2 with primer set 51 and was unable to
histidine change in NADH dehydrogenase 4 at posi- examine that amplicon and therefore , did not find the
tion 340 in a protein that contains 460 amino acids polymorphism at 14 199.
(Lee and Levin , 2002). G 11778A is considered a In conclusion , NIST has sequenced the entire
primary mutation causing LHON and was seen in mtDNA 06 569 bp) from the HL- 60 cell line multiple
GMI0742A , a cell line from a patient who experi- times and compared these results with those from
enced the sudden onset of blindness at the age of 24. AFDIL , FBI , and GBI who participated in the
Since 1988, 27 missense mutations and one mutation interlaboratory evaluation. All four laboratories
396 B.c. Levin et al. Mitochondrion (2003) 387-400
Table 1
Cambridge Reference Sequence nucleotide differences (some unique) found at NIST in the two DNA templates , CHR and 9947 A included in
SRM 2392; HL-60 included in SRM 2392- 1 and GM1O742A DNA"
Comparison with Cambridge Reference Sequence (CRS)
CRS Base Template Template Template Template Amino acid Region

19811199ge CHR 9947Ab HL, 60c GMI0472A change
HV2
HV2
150 HV2
152 HV2
185 HV2
195 HV2
204 HV2
207 HV2
214 HV2
228 HV2
263*Rg HV2
295 HV2
303- 309 C (ins) CC (ins) HV2
311- 315*R C (ins) C (ins) C (ins) C (ins) HV2
462 HV2
482 HV2
489 HV2
709 12sRNA
750*R 12sRNA
1438*R 12sRNA
1719 16sRNA
2706 16sRNA
2841 A(U) 16sRNA
3010 16sRNA
31O6- 31O7*Eh C/del del del del del 16sRNA
3394 Tyr -+ His NDI LHON
3423*E GIT Silent NDI
4135 C(U) Tyr -+ His NDI
4216 Tyr -+ His NDI LHON
4769*R Silent ND2
4985*E G/A Silent ND2
5186 G (U) Silent ND2
5228 G (U) Silent ND2
5633 tRNA Ala
6221 Silent cor
6371 T(U) Silent cor
6791 Silent cor
6849i G(0. Thr -+ Ala cor
7028 Silent cor
7476 tRNA Ser
7645 Silent COIl
7861 C (U) Silent COIl
8020 Silent COIl
8448 Met -+ Thr ATPase 8
8503 Silent ATPase 8
8860*R Thr -+ Ala ATPase 6
9315 C (U) Phe -+ Leu COllI
9559*E G/C Arg -+ Pro COllI
172 Silent ND3
10, 398 Thr -+ Ala ND3
11, 251 Silent ND4
287 C (U) Silent ND4
c. Levin et at. Mitochondrion (2003) 387-400 397
Table I (continued)
Comparison with Cambridge Reference Sequence (CRS)
11, 335*E TIC Silent ND4

719 Silent ND4
11, 778 Arg..... His ND4 LHON
II , 878 C (U) Silent ND4
071 het ClThet (u) Phe ..... Leu het ND4
612 Silent ND5
705 Silent ND5
13, 572 C (U) Silent ND5
13, 702*E G/C Gly -+ Arg ND5
708 Ala -+ Thr ND5 LHON
13, 759 Ala -+ Thr ND5
966 Thr -+ Ala ND5
14, 199*E GIT Pro -+ Thr ND6
14, 272*E G/C Phe -+ Leu ND6
365*E G/C Silent ND6
14, 368*E G/C Phe -+ Leu ND6
470 Silent ND6
569 Silent ND6
766*E TIC Ile -+ Thr ND6
798 Phe -+ Leu CYTB
15, 257 Asp -+ Asn CYT B LHON
15, 326*R Thr -+ Ala CYTB
15,452 Leu -+ Ile CYTB
15, 812 Val..... Met CYT B LHON
16, 069 HVI
16, 126 HVI
16, 183 HVI
16, 184- C (ins) HVI
16, 189 HVI
16, 193 HVI
16, 223 HVI
16, 278 HVI
16, 292 HVI
16, 311 HVI
16, 362 HVI
16, 519 HVI
" ATPase 6, ATP synthase 6; ATPase 8, ATP synthase 8; CYTB , Cytochrome B; CO!, Cytochrome C Oxidase I; COIl, Cytochrome C
Oxidase II; com , Cytochrome C Oxidase III; del, deletion; het, heteroplasmy found in HL- 60 at np 12 071; HVl , non-coding region found
from 16, 024 and 16, 569; HV2, non-coding region found from I and 576; ins, insertion; nd , area not amplified or sequenced; NDl, NADH
dehydrogenase I; ND2 , NADH dehydrogenase 2; ND3, NADH dehydrogenase 3; ND4, NADH dehydrogenase 4; ND5, NADH dehydrogenase
5; ND6, NADH dehydrogenase 6; (U), Unique polymorphisms found in coding regions of CHR, 9947 A, fIL- 60and GM1O742A determined by
comparison with the MITOMAP database (http://www. mitomap. org).
b Levin et al. (1999). CHR DNA: Sequence based on two amplifications and cycle sequencing procedures with DNA from the first cell culture
line and at least one amplification and cycle sequencing proc'edure with DNA from the second cell culture line. 9947 A DNA: Sequence based on
two amplifications and cycle sequencing procedures.
c HL-
60 DNA: Sequence based on two amplifications and cycle sequencing procedures in both the forward and reverse directions for a total of
four sequences.
d Numbers correspond to Cambridge Reference Sequence (Anderson et aI. , 1981).
e Base found in 1981 (Anderson
et aI., 1981)/base found in 1999 (Andrews et al., 1999).
f Base pair same as in 1981 Cambridge Reference Sequence.
g *R: Rare polymorphisrns in Cambridge Reference Sequence discovered by reanalysis of original placenta by Andrews et al. (1999).
h *E: Error in Cambridge Reference Sequence discovered by reanalysis of original placenta by Andrews et at (1999).
i Possible heteroplasmic site. This heteroplasmy seen in the mtDNA from the first CHR ceUculture line is not seen in the mtDNA from
the second CHR cell culture line. It is DNA from the second CHR cell culture line that is supplied in NIST SRM 2392.
398 c. Levin et at. Mitochondrion (2003) 387-400
Table 2 found identical results in all amplified regions. SRM

Number of differences from the Cambridge Reference Sequence 2392- 1 containing HL- 60 will be available in the
DNA template Non-coding Coding Amino Real change spring of 2003. SRM 2392 that currently includes the
regions region acid CHR and 9947A templates also will continue to be
changes available. The CHR and 9947A DNAs come from
apparently normal individuals, but the HL- 60 DNA
CHR
9947Ad
comes from an individual who has acute promyelo-
HL- 7 + 2 tRNA cytic leukemia. Since this is the first time that the
GMlO742A entire mtDNA from HL- 60 has been sequenced, it
" The non-coding regions cover the areas from 16, 024
would be interesting to determine if the differences
to 16, 569
and I to 576. found in HL- 60 are also found in other leukemia
b Amino acid changes compared to original 1981 Cambridge patients. Another interesting finding is the four
Reference Sequence. differences in HL- 60 that have been associated with
C Amino acid and tRNA changes compared to revised 1999 Cam-
LHON disease. For comparison , we also sequenced
bridge Reference Sequence and not counting those changes
considered a rare polymorphism. GM1O472A , a cell line from a patient with LHON
d Levin et al. (1999). and found many of the same polymorphisms and
mutations. Analysis of these polymorphisms places
both HL- 60 and GM1O742A in Haplogroup J. These
results support the premise that individuals in Haplo-
group J may be more prone to LHON. At this time,
Table 3
HL- 60 and GMI0472A Polymorphisms in Coding Regions and not found in original SRM 2392"
Nucleotide position Nucleotide change Region Amino acid difference Associated disease Reference
2841GM T-+A 16sRNA

30lOGM G-+A 16sRNA MM,
3394 T-+C NDI Tyr -+ His LHON , NIDDM
4216HL60;GM T-+C NDI Tyr -+ His LHON
5228HL60 C-+G ND2 Silent
5633 HL60 C-+T tRNA ala MM,
7476HL60 C-+T tRNA ser MM,
8020 G-+A COIl Silent
HL6o
G-+A ND3 Silent MM,
1O, 398HL60;GM A-+G ND3 Thr -+ Ala MM, MA , Mt-
HL60;GM
251 A-+G ND4 Silent MM,
1l, 287 T-+C ND4 Silent
778 G-+A ND4 Arg -+ His LHON MM,
I 2, 071 HL60 T-+ CIT ND4 Phe -+ Leu
13, 708HL60;GMC G-+A ND5 Ala -+ Thr LHON
14, 569HL60 G-+A ND6 Silent MM,
14, 798GM T-+C CYTB Phe -+ Leu
257 HL6o G-+A CYTB Asp -+ Asn LHON MM,
15,452HL60;GM C-+A CYTB Leu -+ Ile MM,
812HL6o G-+A CYTB Val-+ Met LHON MM,
" GM, GMIO742A;NIDDM , non-insulin dependent diabetes mellitus; MM http:llwww. mitomap. org; MA , http://www. cstI. nistgovlbiotechl
strbaselmitoanalyzer.html; Mt- , Levin, BC, Sekiguchi, K, Tully, LA, Chen, JT , and Gropman , A. A patient with chronic progressive external
ophthalmoplegia reexamined 30 years later. (Manuscript In preparation, 2003).
b See footnote to Table 1.
C Also seen in CHR as well as HL- 60 and GMI0742A.
B.c. Levin et at. Mitochondrion (2003) 387~OO 399
Table 4
Polymorphisms common to haplogroup J and sub-haplogroup Jz found in the mtDNA of HL- 60 and GM1O742A"
Polymorphisms Polymorphisms Found Found in Also found in Disease associated

characteristic of characteristic of in HL- GM1O742A
haplogroup J sub-haplogroup Jz
A73G Yes Yes CHR

CI50T Yes
Tl52C Yes
GI85A Yes
Tl95C CHR 9947A
G228A Yes
C462T Yes
T489C Yes Yes
G3010A Yes
T4216C Yes Yes LHON (secondary)
A1O398G Yes Yes
A1l251G Yes Yes
Gll719A Yes Yes CHR
A12612G Yes Yes CHR
Gl3708A Yes Yes CHR LHON (secondary)
Tl4798C Yes
CI5452A Yes Yes
CI6069T Yes Yes
TI6126C Yes
Tl6189C CHR
CI6278T Yes CHR
Tl631lC 9947A
C295T Yes Yes
C5633T Yes
C7476T Yes
GI0172A Yes
GI5257A Yes LHON (intermediate)
Gl5812A Yes LHON (secondary)
CI6193T Yes
" References: Torroni et al. (1997); and FinniHi et al. (2001).
since the linkage of HL- 60 to the actual donor has NIST. We also acknowledge and thank Kathryn
been broken , there is no way to determine if this Kapfer for help in sequencing GMI0742A at NIST.
patient actually had LHON.
References
Acknowledgements
Anderson, S., Bankier, AT., Barrell, B. G., et aI., 1981. Sequence
and organization of the human mitochondrial genome. Nature

This research was supported by Interagency 290, 457 - 465.
Agreement number 1999- IJ- O94 from the National Andrews, R.M. , Kubacka, I., Chinnery, P. F., Lightowlers, R.N.
Institute of Justice. Points of view are those of the Turnbull, D. M., Howell, N., 1999. Reanalysis and revision of
the Cambridge Reference Sequence for human mitochondrial
authors and do not necessarily represent the position
DNA. Nat Genet 23, 147.
of the U. S. Department of Justice. The authors are Bendall , K.E., Sykes, B. C., 1995. Length heteroplasmy in the first
grateful to Gettysburg College for the partial funding hypervariable segment of the human mtDNA control region.
of Koren A. Holland during her sabbatical year at Am. J. Hum. Genet 57 , 248- 256.
400 G. Levin et al. Mitochondrion (2003) 387-400
Brown , M. , Starikovskaya , E. , Derbeneva, 0. , et aI., 2002. The forensic identification, medical diagnosis , and mutation detec"
role of mtDNA background in disease expression: a new tion. Genomics 55, 135- 146.
primary LHON mutation associated with Western Eurasian Levin , B. , Cheng, H., Kline , M.c., Redman , J. W., Richie, KL
haplogroup. J. Hum. Genet 110, 130~ 138. 2001. A review of the DNA standard reference materials
Butler, J. , Levin, B. , 1998. Forensic applications of mitochon- developed by the National Institute of Standards and Tech-
drial DNA Trends Biotechnol. 16 , 158- 162. nology. Fresenius J. Anal. Chem. 370, 213- 219.
Finnilii, A., Lehtonen, M. , Majamaa, K , 2001. Phylogenetic Macaulay, V., Richards, M. , Hickey, E. , et aI., 1999. The emerging
network for European mtDNA Am. J. Hum. Genet 68, tree of West Eurasian mtDNAs: a synthesis of control region
1475~ 1484. sequences and RFLPs. Am. J. Hum. Genet 64, 232- 249.
Helgason, A., Hickey, E. , Goodacre , S. , et ai., 2001. MtDNA and Torroni, A, Petrozzi, M. , D' Urbano, L , et aI., 1997. Haplotype and
the islands of the North Atlantic: estimating the proportions phylogenetic analyses suggest that one European-specific
of Norse and Gaelic Ancestry. Am. J. Hum. Genet 68, mtDNA background plays a role in the expression of Leber
723- 737. Hereditary Optic Neuropathy by increasing the penetrance of
Herrnstadt, c., Elson , J. , Fahy, E. , et aI. , 2002. Reduced-median- the primary mutations 11, 778 and 14,484. Am. J. Hum. Genet
network analysis of complete mitochondrial DNA coding- , 1107- 1121.
region sequences for the major African , Asian, and European Wallace, D., Singh, G. , Lott, M. T., etal., 1988. Mitochondrial
haplogroups. Am. J. Hum. Genet 70, 1152~ 1171. DNA mutation associated with Leber Hereditary Optic Neuro-
Lee, M. , Levin , B. C., 2002. MitoAnalyzer , a computer program pathy. Science 242, 1427~ 1430.
and interactive web site to determine the effects of single Wallace , D, c., Brown, M. D., Lott , M. T., 1997. Mitochondrial
nucleotide polymorphisms and mutations in human mitochon- genetics. In: Rimoin , D. , Connor , J. M., Pyeritz, RE. (Eds.
drial DNA Mitochondrion I, 321~ 326. Chapter 13 in Emery and Rimoin s Principles and Practice of
Levin, B. c., Cheng, H., Reeder, DJ., 1999. A human mitochondrial Medical Genetics, Churchill Livingstone, New York, Edin-
DNA Standard Reference Material for quality control in burgh , London, pp. 277- 332.

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NIST SPECIAL PUBLICATION 260-155

U. S. DEPARTMENT OF COMMERCE/Technology Administration

Standard Reference Materials®

Human Mitochondrial DNA—Amplification and

B. C. Levin, D. K. Hancock, K. A. Holland, H. Cheng and K. L. Richie

Human Mitochondrial DNA—Amplification and

B. C. Levin, D. K. Hancock, K. A. Holland, H. Cheng and K. L. Richie

U.S. DEPARTMENT OF COMMERCE, Donald L. Evans, Secretary

Issued September 2003

National Institute of Standards and Technology Special Publication 260-155

U.S. GOVERNMENT PRINTING OFFICE

For sale by the Superintendent of Documents, U.S. Government Printing Office

2. Materials and Methods..............................................................................................................1

2.1. Origin of Extracted DNA...................................................................................................1

2.3 Bacteriophage DNA Isolation and Sequencing................................................................3

2.5. Polymerase Chain Reaction (PCR) SRM 2392 Completed in 1999...............................4

2.7. Sequencing SRM 2392 Completed in 1999.......................................................................4

2.8. Sequencing SRM 2392-I Completed in 2002 ...................................................................5

2.9. Interlaboratory Evaluation for SRM 2392 Completed in 1999 ....................................5

2.10. Differences in Methodology used by Laboratories in Interlaboratory Evaluation of

2.11. Interlaboratory Evaluation for SRM 2392-I, 2002 .......................................................6

2.12. Differences in Methodology used by the Laboratories in the Interlaboratory

2.12.1.1. AFDIL PCR Amplification, SRM 2391-I ...........................................7

2.12.1.2. AFDIL Sequencing, SRM 2392-I ........................................................7

2.12.2. Georgia Bureau of Investigation (GBI), Interlaboratory Evaluation, SRM

2.12.3. Federal Bureau of Investigation (FBI), Interlaboratory Evaluation, SRM

2.12.3.2. FBI Sequencing, Interlaboratory Evaluation, SRM 2391-I .............9

2.13. Permissions .....................................................................................................................10

2.14.1. CHR DNA Preparation and Confirmation, SRM 2392 .................................10

2.14.2. CHR Cloned DNA Production, SRM 2392 .....................................................10

2.14.3. 9947A DNA Production, SRM 2392 ................................................................10

2.14.4. HL-60 Production, SRM 2392-I .......................................................................11

3. Results and Discussion ...........................................................................................................12

3.1. SRM Templates ............................................................................................................12

3.2. Primers ..........................................................................................................................12

Table 3. Universal Genetic Code and (Human mtDNA Differences) ....................................29

Standard Reference Materials:

Keywords: CHR; 9947A; HL-60; GM03798; GM10742A; Forensic Identification; Medical

2. Materials and Methods

2.1. Origin of Extracted DNA

2.3. Bacteriophage DNA Isolation and Sequencing

2.5. Polymerase Chain Reaction (PCR) SRM 2392 completed in 1999

2.6. Polymerase Chain Reaction (PCR) SRM 2392-I completed in 2002

2.7. Sequencing SRM 2392 completed in 1999

2.8. Sequencing SRM 2392-I completed in 2002

2.9. Interlaboratory Evaluation for SRM 2392 completed in 1999

Three laboratories in addition to NIST participated in an interlaboratory evaluation of the CHR

Each laboratory was sent:

5. All the laboratories received the following cautionary note:

2.10. Differences in Methodology used by Laboratories in Interlaboratory Evaluation of SRM 2392,

2.11. Interlaboratory Evaluation for SRM 2392-I, 2002

2.12.1. Armed Forces DNA Identification Laboratory (AFDIL), SRM 2392-I

2.12.1.1. AFDIL PCR Amplification, SRM 2392-I:

2.12.1.2. AFDIL Sequencing, SRM 2392-I :

2.12.2. Georgia Bureau of Investigation (GBI), Interlaboratory Evaluation, SRM 2392-I

2.12.3. Federal Bureau of Investigation (FBI), Interlaboratory Evaluation, SRM 2392-I

2.12.3.1. FBI Polymerase Chain Reaction (PCR):

2.12.3.2. FBI Sequencing: