DNA fingerprinting

 DNA fingerprinting or DNA profiling is a process used to determine the nucleotide sequence
at a specific part of the DNA that is unique in all human beings.
 The process of DNA fingerprinting was invented by Sir Alec Jeffrey at the University of
Leicester in 1985.

DNA fingerprinting relies on the unique pattern made by a series of DNA fragments after separating
them according to length by gel electrophoresis. DNA samples from different suspects, the victim,
and samples from the crime scene are first purified. The samples are then processed to generate a set
of DNA fragments. When Alec Jeffreys invented DNA fingerprinting in 1985 in England, the DNA
was cut with restriction enzymes to generate fragments because PCR had not yet been invented. In
addition, early DNA fingerprinting used radioactivity for labeling the DNA fragments. Nowadays,
DNA is prepared by PCR, and fluorescent dyes are used for labeling. In addition, modern DNA
fingerprinting uses repeated sequences (short tandem repeats or STRs) for routine identification
purposes.

The principle of DNA Fingerprinting

 The DNA of every human being on the planet is 99.9% the same. However, about 0.1% or 3 x
106base pairs (out of 3 x 109 bp) of DNA is unique in every individual.
 Human genome possesses numerous small non-coding but inheritable sequences of bases
which are repeated many times. They do not code for proteins but make-up 95% of our
genetic DNA and therefore called the ―junk DNA.
 They can be separated as the satellite from the bulk DNA during density gradient
centrifugation and hence called satellite DNA.
 In satellite DNA, repetition of bases is in tandem. Depending upon length, base composition
and numbers of tandemly repetitive units, satellite DNAs have subcategories like
microsatellites and mini-satellites.
 Satellite DNAs show polymorphism. The term polymorphism is used when a variant at a
locus is present with a frequency of more than 0.01 population.
 Variations occur due to mutations. These mutations in the non-coding sequences have piled
up with time and form the basis of DNA polymorphism (variation at genetic level arises due
to mutations).
 The junk DNA regions are thus made-up of length polymorphisms, which show variations in
the physical length of the DNA molecule.
 At specific loci on the chromosome, the number of tandem repeats varies between
individuals. There will be a certain number of repeats for any particular loci on the
chromosome.
 Depending on the size of the repeat, the repeat regions are classified into two groups. Short
tandem repeats (STRs) contain 2-5 base pair repeats, and the variable number of tandem
repeats (VNTRs) have repeats of 9-80 base pairs.
 Since a child receives 50% of the DNA from its father and the other 50% from his mother, so
the number VNTRs at a particular area of the DNA of the child will be different may be due
to insertion, deletion or mutation in the base pairs.
  As a result, every individual has a distinct composition of VNTRs, and this is the main
principle of DNA fingerprinting.
 As the single change in nucleotide may make a few more cleavage site of a given nucleotide
or might abolish some existing cleavage site.
 Thus, if DNA of any individual is digested with a restriction enzyme, fragments pattern
(sizes) will be produced and will be different in cleavage site position. This is the basics of
DNA fingerprinting.
Methods of DNA Fingerprinting
Restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR)
amplification of short tandem repeats (STRs) are two main DNA tests widely used for DNA
fingerprinting.

For the first generation of DNA fingerprints, restriction enzymes were used to generate the variation
in DNA fragment size between individuals. Variations in the DNA base sequence of restriction
enzyme recognition sites result in differences in the size of the fragments. Such sequence differences
are called restriction fragment length polymorphisms (RFLPs). There is approximately one difference
in every 1000 nucleotides between nonrelated individuals. Many different restriction enzymes with
distinct recognition sites exist, and in practice several such enzymes are used on each DNA sample.
Even if mutations have changed a few bases of the target sequence around the cut site, there is usually
still enough similarity for probes to bind. The entire process requires several weeks to finish.
 The steps involved in RFLP-based DNA fingerprinting are as follows:

A pattern of different-sized DNA fragments is generated during DNA fingerprinting. The sequence of
the DNA determines where the restriction enzyme cuts in the first step; thus, different people will
have a different pattern of fragments for the same restriction enzyme. These differences may be used
to identify people
1. The DNA is cut with a restriction enzyme.
2. The DNA fragments are separated by length or molecular weight by gel electrophoresis.
 3. The fragments are visualized by Southern blotting. The separated fragments are
transferred from the gel to nylon paper. Then a radioactively labeled DNA probe is added.
The probe binds to those DNA fragments with complementary sequences.
4. The blot is covered with radiation-sensitive film to give an autoradiograph. This shows
the location of those DNA fragments that reacted with the radioactive probe.

A. Restriction fragment length polymorphism (RFLP)

1. The first step in this process is to isolate the DNA from the sample material to be tested. The
sample size for the RFLP test must be large enough to get the proper result.
2. Once the required size of the sample is available, the DNA is isolated from the sample and
is subjected to restriction digestion using restriction enzymes.
3. The digested DNA sample is then separated by agarose gel electrophoresis, in which the
DNA is divided based on the size.
4. The next step is the transfer of separated DNA from gel slab onto the
nitrocellulose membrane to hybridise with a labelled probe that is specific for one VNTR
region (radioactivity labelled complementary sequence for VNTR region nucleotide
sequence).
5. This technique of transferring and hybridising DNA onto nitrocellulose membrane is known
as Southern blotting, a most widely used DNA detection technique by molecular biologists.
6. After the hybridisation with the radioactive probes, the X-ray film is developed form the
southern blotting and only the areas where the radioactive probe binds will show up on the
film.
7. Now these bands when compared with the other known samples, will give the final result
of the DNA fingerprinting.
Advantages
The RFLP is considered to be more accurate than the PCR, mainly because the size of the sample
used more, use of a fresh DNA sample, and no amplification contamination.
Limitation
The RFLP, however, require more prolonged period to complete the analysis and is costly.
B. Polymerase Chain Reaction (PCR) amplification of short tandem repeats (STRs)
1. Thousands of copies of a particular variable region are amplified by PCR which forms the
basis of this detection.
2. STR with a known repeat sequence is amplified and separated using gel electrophoresis.
o The distance migrated by the STR is examined.
3. For the amplification of STRs using PCR, a short synthetic DNA, called primers are specially
designed to attach to a highly conserved common non-variable region of DNA that flanks the
variable region of the DNA.
4. By comparing the STR sequence size amplified by PCR with the other known samples, will
give the final result of the DNA fingerprinting.
Advantages
 The small amount of specimen is sufficient for the test.
 Takes a shorter time to complete.
 Less costly.
Limitation
 Less accurate than RFLP.
 Possibility of amplification contamination
Applications of DNA Fingerprinting
 DNA Fingerprinting is used by scientists to distinguish between individuals of the same
species using only samples of their DNA. It is a primary method for identifying an individual.
1. Forensic Science:
Biological materials used for DNA profiling are- Blood, Hair, Saliva, Semen, Body tissue cells etc.
DNA isolated from the evidence sample can be compared through VNTR (Variable number of
tandem repeats) prototype. It is useful in solving crimes like murder and rape.
2. Paternity and Maternity Determination:
A Person accedes to his or her VNTRs from his or her parents. Parent-child VNTR prototype analysis
has been used to solve disputed cases. This information can also be used in inheritance cases,
immigration cases.
3. Personal Identification:
It utilises the concept of using DNA fingerprints as a sort of genetic barcode to pinpoint individuals.
4. Diagnosis of Inherited Disorders:
It is also useful in diagnosing inherited disorders in both prenatal and newborn babies. These
disorders may include cystic fibrosis, haemophilia, Huntington’s disease, familial Alzheimer’s, sickle
cell anaemia, thalassemia, and many others.
5. Development of Cures for Inherited Disorders:
By studying the DNA fingerprints of relatives who have a history of some particular disorder, DNA
prototypes associated with the disease can be ascertained.
6. Detection of AIDS:
By comparing the band of HIV “RNA” (converted to DNA using RT-PCR) with the bands formed by
the man’s blood, the person suffering with AIDS can be identified.
7. Breeding Program:
Breeders conventionally use the phenotype to evaluate the genotype of a plant or an animal. As it is
difficult to make out homozygous or heterozygous dominance from appearance, the DNA
fingerprinting allows a fastidious and precise determination of genotype. It is basically useful in
breeding racehorses and hunting dogs.
Forensic Science
DNA profiling as it is now known, was first described in 1985 by Alec Jeffreys and coworkers and it
has had a tremendous impact in forensic genetics. Before that, all the forensic genetic casework
(paternity testing, criminal casework, individual identification) was performed using classical
serological genetic markers. Blood groups, HLA, and polymorphic protein and enzymes were used for
solving forensic genetic casework using immunological and electrophoretic methodologies. These
genetic markers were nevertheless limited when it was necessary to analyze minimal or degraded
material, which is commonly involved in forensic cases. It was, in addition, difficult to analyze
biological material other than blood, and therefore the information obtained from hair, saliva, and
even semen in rape cases was rather limited.
Due to the fact that the polymorphic proteins and enzymes were infrequent, it was necessary to obtain
as much information as possible. For this reason, sophisticated electrophoretic methods such as
isoelectric focusing, immobilines, or hybrid isoelectric focusing were developed and applied. In spite
of this, the information that the forensic geneticists were able to report in many cases was clearly
insufficient.
Since the discovery of polymorphisms in repetitive DNA by Jeffreys and co-workers, highly
informative and robust DNA typing systems have been developed which are quite powerful for the
individualization of biological material of human origin.
DNA typing has advantages over traditional protein assays, first of all because it is more informative
and can be analyzed in minute or degraded material since DNA is physically much more resistant to
degradation than proteins. In addition, the same DNA genotype can be obtained from any tissue (i.e.
blood, saliva, semen, hair, skin, bones) whereas the analysis of protein markers is restricted to cells
where these proteins are expressed.

Nowadays, DNA analysis has become the standard method in forensic genetics as it is currently used
by most of the labs for the majority of forensic genetic expertise and especially in criminal forensic
casework (stain analysis and hairs) and identification.

The final product of a DNA fingerprint is an autoradiograph that contains at least five essential lanes.
The markers are standardized DNA fragments of known size, which have been radioactively labeled.
They help determine the size of the various fragments. The “control” is DNA from a source known to
react positively and reliably to the DNA probes and shows whether the test has worked as expected.
The experimental lanes have samples from the victim, the defendant, and the crime scene. In this
example, blood from the defendant’s clothing was compared with his own blood and the victim’s
blood. The DNA from the clothing actually matches that of the victim.
 j

Actual DNA fingerprint showing that the pattern of DNA fragments of the victim (V) were found on
the defendant’s clothing (jeans/shirt). The first two and last two lanes are the standard size markers
(labeled λ and 1 kb). The lane marked TS is a positive control showing that the fingerprint technique
was successful. The lane marked D is the defendant’s DNA pattern. Reprinted with permission from
Quick Publishing, LC.

Two variants of DNA fingerprinting have been used: single-locus probing (SLP) and multiple-locus
probing (MLP). In SLP, each probe is specific for a single site, that is, a single locus, in the genome.
Because humans are diploid, each SLP probe normally gives rise to two bands from each person,
provided that the chosen locus shows substantial allelic variation. Occasional persons will be
homozygous and show only a single band. For full identification using SLPs, it is necessary to run
several reactions, each using a different SLP probe.
Multilocus probes bind to multiple sites in the genome and consequently generate multiple bands
from each individual. Because it is not known which particular band comes from which particular
locus, interpretation is difficult. Furthermore, statistical analysis is impractical, and data cannot be
reliably standardized for reference. In practice, fingerprints generated by MLP must be directly
compared with others run on the same gel. Historically, MLP was used before SLP. However, SLP
analyses use smaller amounts of material and are easier to interpret and compare. Statistical analysis
and population frequencies are possible using SLP data. Consequently, MLP methods have largely
been displaced by SLP analysis.

In addition to preparing DNA for fingerprinting, PCR may be used to generate DNA segments for
direct comparison by DNA sequencing or hybridization. PCR is especially useful for amplifying small
regions of DNA with high person-to-person variability. If the sequences of two samples match in
several highly variable regions, they are probably from the same person.

In this approach, DNA from a forensic sample is amplified by PCR and compared with DNA from the
suspect. For hybridization, spots of DNA samples are bound to a membrane and tested for binding a
DNA probe that is tagged with a fluorescent dye (radioactive probes were used in early work). In such
dot blots, the probe either binds or doesn’t bind, so any spot is either positive or negative.
Alternatively, segments of DNA that have been amplified by PCR can be fully sequenced.

PCR plus Dot Blots for Identification

This example compares whether or not evidence from a crime scene matches the victim or suspect.
DNA from the victim, the suspect, and the evidence was isolated. PCR was used to amplify a short
segment of the DNA using primers flanking a highly variable region. The PCR products from the
evidence, victim, suspect, and a control were each applied as four separate spots to a nylon
membrane. The membrane was treated with four different probes, such that each probe was in contact
with each of the different PCR products. Probes bound specifically to those spots that had sequences
matching the probe. Notice that the pattern of the suspect and the evidence are identical. Therefore,
the suspect’s DNA was present at the scene of the crime.