CN106566827B - dye marking method - Google Patents
dye marking method Download PDFInfo
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- CN106566827B CN106566827B CN201610988458.1A CN201610988458A CN106566827B CN 106566827 B CN106566827 B CN 106566827B CN 201610988458 A CN201610988458 A CN 201610988458A CN 106566827 B CN106566827 B CN 106566827B
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- 238000000034 method Methods 0.000 title abstract description 13
- 238000002372 labelling Methods 0.000 claims abstract description 12
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 claims abstract 5
- 230000003321 amplification Effects 0.000 claims description 15
- 239000012634 fragment Substances 0.000 claims description 15
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 15
- 239000013612 plasmid Substances 0.000 claims description 7
- 238000001514 detection method Methods 0.000 abstract description 28
- 230000000694 effects Effects 0.000 abstract description 7
- 238000012546 transfer Methods 0.000 abstract description 7
- PWZJEXGKUHVUFP-UHFFFAOYSA-N ATTO 590 meta-isomer Chemical compound [O-]Cl(=O)(=O)=O.C1=2C=C3C(C)=CC(C)(C)N(CC)C3=CC=2OC2=CC3=[N+](CC)C(C)(C)C=C(C)C3=CC2=C1C1=CC=C(C(O)=O)C=C1C(O)=O PWZJEXGKUHVUFP-UHFFFAOYSA-N 0.000 abstract description 4
- 239000000975 dye Substances 0.000 description 37
- 230000005284 excitation Effects 0.000 description 15
- 238000002360 preparation method Methods 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 5
- 230000002068 genetic effect Effects 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 4
- 239000007850 fluorescent dye Substances 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 2
- 238000005251 capillar electrophoresis Methods 0.000 description 2
- 238000001215 fluorescent labelling Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108091092878 Microsatellite Proteins 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
the invention discloses a dye labeling method, which is characterized in that a long-wavelength dye, Atto633, Atto590 or ROX is labeled at the 5' end of a primer sequence, and another short-wavelength dye, such as 6-FAM, is labeled in the middle of the primer sequence. The internal standard detection effect prepared by the dye marking method is close to the internal standard detection effect of the energy transfer dye marking, the detection signal is improved by 5-10 times compared with the long-wavelength dye signal marked by a primer alone, the detection process is simple and rapid, the cost is low, the application and popularization are facilitated, and the practicability is higher.
Description
Technical Field
the invention belongs to the field of biotechnology, and particularly relates to a dye marking method.
Background
Fluorescence labeling short tandem repeat locus multiplex amplification, detection of amplification products through capillary electrophoresis, and wide application in forensic DNA detection (patent numbers 201410003973.0 and 201410645266.1), even clinical detection (patent number 200710028600.9).
a common amplification system is a four-color amplification using 6-FAM, HEX or JOE, TAMRA and ROX as fluorescent labels, and a fifth color fluorescence liz (ABI, USA) as an internal standard label. The capillary electrophoresis apparatus used was a series 3100, 3130 and 3500 by ABI, USA. The detection instrument 3500 adopts 505nm laser excitation, and other systems adopt 488/515nm laser excitation.
Each dye has a maximum excitation wavelength and a maximum emission wavelength, and the wavelength difference between the two is within 30 nm. The maximum excitation and emission wavelengths of the Dyes 6-FAM, HEX or JOE, TAMRA, ROX, Atto590 and Atto633 are shown in the following table (data from IDTDNA website http:// www.idtdna.com/site/Catalog/Modifications/Dyes):
When the machine is used for detection, the fluorescence with the corresponding maximum emission wavelength is received, and the excitation efficiency of the excitation light source to the fluorescence is greatly different: the closer the wavelength of the excitation light, the higher the efficiency of the emitted fluorescence, the same amount of the ROX-labeled product fluorescence signal is 10 times lower than that of the 6-FAM-labeled product. Liz is an energy transfer dye with a maximum excitation wavelength of about 500nm and a detection wavelength of 650 nm. Although the Atto633 dye detection wavelength was the same as liz, the internal standard was made to have a very low detection signal. And energy transfer dyes are expensive to purchase.
The forensic detection kit requires detection signals with a signal ratio of over 30% for various fluorescence labeling products. In order to improve the balance, it is considered to select a dye with stronger fluorescence signal or an energy transfer dye in the development of the kit, or to add more primers labeled by Atto590, ROX, TAMRA than 6-FAM, HEX in the configuration of the primers. For the former solution, the fluorescent dye with good efficiency is a patent product of a large foreign company, such as LIZ, U.S. ABI patent. For the latter scheme, a further increase is difficult because the ROX marker signal is low, barely reaching 30%. Therefore, a dye labeling method with good detection effect, low price and easy popularization and application is needed.
Disclosure of Invention
Aiming at the technical problems, the invention provides a dye marking method which effectively reduces the cost and has good detection effect.
The technical scheme of the invention is as follows:
A dye labeling method comprising the steps of:
step a, selecting a short-wavelength dye with shorter excitation wavelength, such as 6-FAM;
b, selecting a long-wavelength dye with longer excitation wavelength, such as one of Atto633, Atto590 and ROX;
And c, marking the dye B at the 5' end position of the primer sequence, and marking the dye A at the middle position of the primer sequence.
Preferably, the middle position of the primer sequence includes any primer sequence position except for the 5 'end and the 3' end in the primer sequence.
preferably, the middle position of the primer sequence is the position of the primer sequence 2-10 bases away from the 5' end in the primer sequence.
Preferably, the preparation of the internal standard marked by Atto-633 comprises the steps of using PUC19 plasmid DNA as a template, using an amplification primer S1-S15, using a common marking primer Atto-633FAM, marking the 5' end of the primer by Atto-633, marking the middle sequence of the primer by 6-FAM dye, and respectively amplifying 14 fragments of 75bp, 100bp, 139bp, 150bp, 160bp, 200bp, 250bp, 300bp, 340bp, 350bp, 400bp, 450bp, 490bp and 500 bp; the preparation of the ROX-labeled internal standard comprises the steps of taking PUC19 plasmid DNA as a template, taking amplification primers from S1 to S15, taking a common primer D: 5' ROX-AGCTG (6-FAM) CATTAATGAATCGGCCAA, and amplifying 14 fragments of 75bp, 100bp, 139bp, 150bp, 160bp, 200bp, 250bp, 300bp, 340bp, 350bp, 400bp, 450bp, 490bp and 500bp respectively.
the dye marking method has the advantages that the detection effect of the dye marking method is close to that of the marked energy transfer dye, the detection process is simple and rapid, the cost is low, the application and the popularization are facilitated, and the practicability is high.
drawings
FIG. 1 is an electrophoretogram of a ROX-labeled internal standard of the dye labeling method of the present invention;
FIG. 2 is an electrophoretogram of an Atto-633 labeled internal standard of the dye labeling method of the present invention;
FIG. 3 is an electrophoretogram of an internal standard of primer amplification carried out by labeling 5' end Atto-633 and labeling 6-FAM in a middle sequence in the dye labeling method.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
as shown in the figure, the invention discloses a dye marking method, which comprises the following steps:
Step a, selecting a short-wavelength dye 6-FAM with shorter excitation wavelength;
B, selecting a long-wavelength dye with longer excitation wavelength, wherein the long-wavelength dye comprises one of an Atto-633 labeled internal standard and a ROX labeled internal standard;
And c, marking the dye B at the 5' end position of the primer sequence, and marking the dye A at the middle position of the primer sequence.
the middle position of the primer sequence comprises any primer sequence position except for the 5 'end and the 3' end in the primer sequence.
The middle position of the primer sequence is the position of the primer sequence which is 2-10 bases away from the 5' end in the primer sequence.
The preparation of the internal label marked by the Atto-633 comprises the steps of using PUC19 plasmid DNA as a template, using an amplification primer S1-S15, using a common marking primer Atto-633FAM, using the Atto-633 marker at the 5' end of the primer, using a 6-FAM dye to mark a primer intermediate sequence, and respectively amplifying 14 fragments of 75bp, 100bp, 139bp, 150bp, 160bp, 200bp, 250bp, 300bp, 340bp, 350bp, 400bp, 450bp, 490bp and 500 bp; the preparation of the ROX-labeled internal standard comprises the steps of taking PUC19 plasmid DNA as a template, taking amplification primers from S1 to S15, taking a common primer D: 5' ROX-AGCTG (6-FAM) CATTAATGAATCGGCCAA, and amplifying 14 fragments of 75bp, 100bp, 139bp, 150bp, 160bp, 200bp, 250bp, 300bp, 340bp, 350bp, 400bp, 450bp, 490bp and 500bp respectively.
Selection of marker position
Using PUC19(GENEBANK L09137.2) as template and primer S1
AGCTGCATTAATGAATCGGCCAA
and 1R-GAGCGAGGAAGCGGAAGAGC. The 5' end of the P19F primer is labeled with a fluorescent dye ROX, and the middle italic bold base labels 6-FAM. The specific markers are shown in the following table:
the labeled primers A-I and the primers 1R-13R are respectively amplified, and PUC19 plasmid and enzyme are purchased from Dalibao organisms (the cargo numbers are 3219 and RR070A respectively).
The PCR reaction solution was prepared as follows:
Pre-denaturation was programmed with an ABI 9700 PCR instrument for 2min at 95 ℃ followed by 30 cycles of amplification at 94 ℃ for 5s, 60 ℃ for 20s, 72 ℃ for 20s, and final extension for 5 min at 68 ℃. The amplification product was diluted 50-fold and then analyzed by detection using a genetic analyzer 3100 (applied biosystems, USA). The sample was injected at a voltage of 3kv for 10 seconds and electrophoresed at a voltage of 15kv for 1500 s.
in FIG. 1, the left vertical line is the peak height, and the size of the fragment is marked on each peak. Peak height 5000-; as a result of the electrophoresis, the signals detected by the primers in the groups A-I are all shown as the wavelength of ROX dye. The detection signal for the B-I product was higher than for A. After the intermediate marker 6-FAM, there was a 4-10 fold increase in signal intensity. The signal at the 6-FAM label position 2-10 bases away from the 5' end is stronger than that at 12-16. The B-I labeled primer, 6-FAM excitation wavelength, was closer to the detection laser wavelength of the genetic analyzer 3100. After laser excitation of 6-FAM, the resulting excitation light excites the ROX dye. Stronger than the detection of the laser-excited ROX signal with the genetic analyzer 3100 alone. The distance between the 6-FAM and the ROX has an effect on the enhancement procedure of the detection signal. In this experiment, the distance of 6 bases is the best. The efficiency of energy transfer decreases too far away and the 12-16 signals are relatively low.
Preparation of ROX-labeled internal standards
Using PUC19(GENEBANK L09137.2) as a template, primers were designed as shown in the following table, sharing primer D: 5' ROX-AGCTG (6-FAM) CATTAATGAATCGGCCAA, and amplifying 14 fragments of 75bp, 100bp, 139bp, 150bp, 160bp, 200bp, 250bp, 300bp, 340bp, 350bp, 400bp, 450bp, 490bp and 500bp respectively.
The PCR reaction solution was prepared as follows:
taking 1ul of each amplification product, mixing and adding water to 50ul to obtain the ROX-labeled 75-500bp internal standard. The analysis was performed using a genetic analyzer 3100 (applied biosystems, USA). The sample was injected at a voltage of 3kv for 10 seconds and electrophoresed at a voltage of 15kv for 1500 s. The detection diagram is shown in figure 1. 14 fragments labeled with ROX, peak height at 5000-.
Preparation of Atto633 internal Standard
Using PUC19(GENEBANK L09137.2) as a template, primers were designed as shown in the following table, and common labeled primers were used
atto633FAM, wherein 5 'of a primer is marked by Atto633, the sixth 6 basic groups from the 5' end are marked by 6-FAM dye, and 14 fragments of 75bp, 100bp, 139bp, 150bp, 160bp, 200bp, 250bp, 300bp, 340bp, 350bp, 400bp, 450bp, 490bp and 500bp are respectively amplified. The primer Atto633 is commonly marked, the 5' of the primer is marked by the Atto633, and 14 fragments of 75bp, 100bp, 139bp, 150bp, 160bp, 200bp, 250bp, 300bp, 340bp, 350bp, 400bp, 450bp, 490bp and 500bp are respectively amplified.
The PCR reaction solution was prepared as follows:
taking 1ul of each amplification product, mixing and adding water to 50ul to obtain the ROX-labeled 75-500bp internal standard. The analysis was performed using a genetic analyzer 3100 (applied biosystems, USA). The sample was injected at a voltage of 3kv for 10 seconds and electrophoresed at a voltage of 15kv for 1500 s. In FIG. 2, the left vertical line indicates the peak height, and the size of the fragment (250bp unlabeled) is marked on each peak. Peak height 70-80RFU, from 75-500bp, total 14 fragments; the left vertical line in FIG. 3 is the peak height, and the size of the fragment (250bp unlabeled) is marked on each peak, and the peak height 700 and 800RFU, from 75-500bp, total 14 fragments. The signal intensity of the two is different by ten times. In conclusion, on the basis of the conventional 5' fluorescence labeled primer, the dye label with a short wavelength is added in the middle, so that the signal intensity of detection can be improved. The dye marking method has the advantages of detection effect close to that of the marked energy transfer dye, simple and quick detection process, low cost, more contribution to application and popularization and stronger practicability. The preparation methods of the ROX mark internal standard and the Atto-633 mark internal standard introduced in the invention can be used for forensic STR detection.
while embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (4)
1. a dye labeling method is characterized in that one long-wavelength dye is labeled at the 5' end of a primer sequence, and the other short-wavelength dye is labeled in the middle of the primer sequence; the long-wavelength dye is Atto-633 or ROX, and the short-wavelength dye is 6-FAM.
2. The dye labeling method of claim 1, wherein: the middle position of the primer sequence comprises any primer sequence position except for the 5 'end and the 3' end in the primer sequence.
3. The dye labeling method of claim 1 or 2, characterized in that: the middle position of the primer sequence is the position of the primer sequence which is 2-10 bases away from the 5' end in the primer sequence.
4. The dye labeling method of claim 1, wherein: preparing an internal standard marked by Atto-633, wherein PUC19 plasmid DNA is used as a template, an amplification primer is S2-S16, a common marking primer is S16, the 5' end of the primer is marked by Atto-633, and a primer intermediate sequence is marked by 6-FAM dye to respectively amplify 14 fragments of 75bp, 100bp, 139bp, 150bp, 160bp, 200bp, 250bp, 300bp, 340bp, 350bp, 400bp, 450bp, 490bp and 500 bp; preparing an internal standard marked by the ROX, wherein the internal standard marked by the ROX comprises the steps of taking PUC19 plasmid DNA as a template, taking amplification primers S1-S15 and sharing a primer S1 to respectively amplify 14 fragments of 75bp, 100bp, 139bp, 150bp, 160bp, 200bp, 250bp, 300bp, 340bp, 350bp, 400bp, 450bp, 490bp and 500 bp;
The sequence of the primer S1 is 5' ROX-AGCTG (6-FAM) CATTAATGAATCGGCCAA;
The sequence of the primer S2 is GAGCGAGGAAGCGGAAGAGC;
the sequence of the primer S3 is GAACGACCGAGCGCAGCGAG;
The sequence of the primer S4 is TTACCGCCTTTGAGTGAGCT;
the sequence of the primer S5 is GGATAACCGTATTACCGCCT;
The sequence of the primer S6 is CTGATTCTGTGGATAACCGTATTAC;
The sequence of the primer S7 is TGCTGGCCTTTTGCTCACAT;
the sequence of the primer S8 is GCCTATGGAAAAACGCCAGC;
The sequence of the primer S9 is CCACCTCTGACTTGAGCGTC;
the sequence of the primer S10 is GGGGAAACGCCTGGTATCTT;
The sequence of the primer S11 is GAGCTTCCAGGGGGAAACGC;
The sequence of the primer S12 is CGGACAGGTATCCGGTAAGC;
The sequence of the primer S13 is CCTACAGCGTGAGCATTGAGAA;
the sequence of the primer S14 is AGCCCAGCTTGGAGCGAA;
the sequence of the primer S15 is TTCGTGCACAGCCCAG;
the sequence of the primer S16 is 5' Atto633-AGCTG (6-FAM) CATTAATGAATCGGCC AA.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1644709A (en) * | 2004-06-15 | 2005-07-27 | 上海申友健海生物技术有限责任公司 | Individual DNA identification by short serial repeated sequential point isogenic gradient and determination reagent box |
| CN101050474A (en) * | 2007-03-23 | 2007-10-10 | 鼎生科技（北京）有限公司 | Method for preparing internal standard of molecular weight, and internal standard of molecular weight prepared by using the method |
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2016
- 2016-11-07 CN CN201610988458.1A patent/CN106566827B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1644709A (en) * | 2004-06-15 | 2005-07-27 | 上海申友健海生物技术有限责任公司 | Individual DNA identification by short serial repeated sequential point isogenic gradient and determination reagent box |
| CN101050474A (en) * | 2007-03-23 | 2007-10-10 | 鼎生科技（北京）有限公司 | Method for preparing internal standard of molecular weight, and internal standard of molecular weight prepared by using the method |
Non-Patent Citations (2)
| Title |
|---|
| John M等.Forensic DNA typing by capillary.《Electrophoresis》.2004,第25卷 * |
| 杨翩等.荧光标记DNA分子量内标的设计与制备.《生命科学仪器》.2014,第12卷 * |
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