DNA FINGERPRINTING

The development and application of DNA fingerprinting has had beneficial and
farreaching effects in forensic science, as well as for paternity and maternity cases and
the identification of disaster victims. In fact it is hard to listen to the news or read a
paper without finding a mention of DNA fingerprinting.

Alec Jeffreys, a British scientist, first coined the phrase DNA fingerprinting in 1984.
DNA fingerprinting is a genetic identification of a person, where as regular inkpad
fingerprinting is identification by a particular phenotype, how fingertips actually
appear.

The genome of two different people is vastly similar and highly conserved however
there are specific genomic regions of highly variable repeats, known as
microsatellites. The number of variable repeats at a defined position on a genome
varies between two different people. These are the areas analyzed during DNA
fingerprinting.

DNA fingerprinting involves the purification of a person’s genome from a multitude

of biological samples, including skin, hair and blood. The genome is then digested
into small fragments, with restriction (cleaving) enzymes, in a process known as
restriction fragment length polymorphism (RFLP). The fragments are separated by
agarose electrophoresis, which separates the fragments based on their size. In the
normal process, the separated fragments are transferred to a DNA binding membrane,
which is probed with a specific label that allows forensic scientist to visualize the
DNA fingerprint.

DNA fingerprinting in forensic laboratories now utilizes a scientific technique known

as the polymerase chain reaction, which allows scientists to amplify small amounts of
DNA and then identify the variable regions. This process is highly automated and
requires tiny amounts of DNA, such as a single hair follicle.

Below are some examples of how DNA fingerprinting has been used. In 1988, a
British baker, Colin Pitchfork, became the first suspect to be convicted using DNA
evidence. In the same case, a local boy was the prime suspect in the case and with the
help of Alec Jeffreys was cleared and of whom Jeffreys said “I have no doubt
whatsoever that he would have been found guilty had it not been for DNA evidence.
That was a remarkable occurrence”.

In 1992, DNA fingerprinting was used to confirm that the Nazi doctor Josef Megele
was buried in Brazil under the name of Wolfgang Gerhard.
 In addition to convicting criminals, freeing the accused and wrongly imprisoned and
identifying human remains DNA fingerprinting has also been used for the following.
Paternity testing, to identify birth parents, food identification, to test for purity ground
beef, evolutionary studies, and to compare similarities of remains to modern day
Homo sapiens, for example the 5000 year old “Iceman”.
This kit allows students to analyze DNA samples taken from a fictitious crime scene
and attempt to identify the criminal or criminals involved. Students will perform a
simplified DNA fingerprinting experiment; they will digest the DNA samples and
separate the fragments with agarose electrophoresis, but will visualize the DNA
fingerprint directly in the gel. This kit does not use the carcinogen ethidium bromide
for visualization, but a non-toxic fluorescent dye that allows students to visualize the
DNA migrating during electrophoresis.

PRE EXPERIMENT SET UP

1. Add 130µl Sterile Water to each vial of DNA; incubate at room temperature for 5
minutes. Rehydrate the tube by gently flicking or vortexing the tube.

2. Label 6 sets of 5 tubes with “Crime Scene 1”, “Crime Scene 2”, “Victim”,
“Suspect 1” and “Suspect 2”. Transfer 20µl of each DNA sample into the
appropriately labeled tube and supply each group with one of each sample tube.

3. Label 6 tubes with “Loading Buffer”. Aliquot 55µl DNA Loading Buffer into
each tube and supply each group with a single tube.

Preparation of agarose gel

Make 1-2 hours before the experiment.

Wear heat protective gloves throughout the agarose melting and pouring
procedure.

Each group requires 5 wells on a 1% agarose gel. The well must accommodate 30 µl
sample.

1. Prepare running buffer: In a clean two liter container, add the entire contents of the
TAE Buffer (50X) and make up to two liters with ultra pure water to make a 1X
TAE Buffer solution. Stir until thoroughly mixed.

2. Prepare 1% agarose: In a clean, glass 500ml container add 1gm Agarose for every
100ml of the TAE Buffer from step 1. The pack has 5gm Agarose.
 3. Heat the solution in a microwave on full power, using 10-second bursts. Check to
see if all the agarose has dissolved. Repeat until agarose has dissolved.

DO NOT BOIL. The agarose gets very hot, very quickly and can cause severe
burns. Wear heat protective gloves throughout the melting and pouring procedure.
4. Once the agarose has cooled to the point it can be held comfortably in your hand,
add the entire contents of the LabSafe™ Nucleic Acid Stain to the agarose and
swirl to mix.

5. Pour the agarose into the gel casting mould as per the manufacturer’s instructions.
You will need 25 wells that each holds 30µl for each group, use an appropriate
size comb.

6. Once the gels have set, remove the comb, transfer to the running apparatus and
cover with the running buffer until ready to use.

Important points :
Every cell of our body contains DNA and approximately 99.9% of DNA is similar between
two humans. Only 0.1% difference is responsible to make someone unique (except
identical twins) and this 0.1% of DNA plays an important role in DNA Fingerprinting.
As we know, only 3 % of our genome are coded and act for protein synthesis i.e. called
gene and rest of 97% are non-coding, repetitive and junk; this junk DNA are utilized to
perform DNA fingerprinting. The sequence structure and number of repeats varies between
individuals and organisms. On this basis, the DNA print can be prepared.

DNA fingerprinting technique was first developed by British Professor Sir Alec Jeffrey in
1984. He realized that we can detect variations in human DNA, in the form of
minisatellites. Jeffrey created the first DNA profile using the restriction digestion length
polymorphism. His method was actually a combination of RFLP and autoradiography.

Definition -
“It is a molecular method to identify an individual or any living organism from their DNA
sample by looking at unique patterns in their DNA.”
Satellites DNA :-
The satellites DNA are non-coding, repetitive DNA regions, located on telomeres and
centromeres. It helps in proper replication, whereas mutation in those sequences causes
replication errors.
It is of two types present in human genome based on their repeat sequence nature :-
1. Minisatellite
2. Microsatellites
Minisatellite DNA sequences are 10 to 60 base pair long and repeated 5 to 50 times in a
genome. They are highly variable (polymorphic), unique, and GC-rich sequences. It is
generally present in telomeric regions. e.g. VNTRs.

Microsatellites are smaller than minisatellites. It contains repeated DNA sequence of 1-6
base pair and 5 to 10 times in a genome. e.g. STR and SSR. These regions are also
hypervariable, non-coding and telomeric.

Process of DNA Fingerprinting :-

1. Collection of biological samples.

2. DNA extraction.
3. Restriction digestion or PCR amplification.
4. Agarose gel electrophoresis, capillary electrophoresis or DNA sequencing.
5. Interpreting results.

1. Sample collection :-
DNA can be obtained from any biological samples. e.g. any part of plant or animals.

2. DNA extraction :-
DNA can be extracted with different methods like CTAB DNA extraction methods,
Proteinase K DNA extraction methods and Phenol-chloroform DNA extraction method or
may be with any DNA extraction kit.

3. Restriction digestion or PCR amplifications :-

This process can be performed by three different techniques - (i)
RFLP analysis.
(ii) PCR analysis.
(iii)Real Time PCR analysis.
 Sample

DNA extraction

DNA
Purification

RFLP PCR RTPCR

Restriction Primer Preparation Primers for STR is labelled

Digestion based with a specific coloured
fluorescent tag

Agarose gel Polymerase PCR with

Electrophoresis Chain Reaction Labelled Primer

Transfer on nitro- Agarose gel Results obtained on Screen

cellulose paper electrophoresis (Electrophoresis not needed
)

Incubation with Results visualized Analysis of results on the

labelled probe in UV light basis of peak formation on
RT PCR Screen

X -Ray Film

Auto
radiography

Fig :- Showing Different DNA Fingerprinting Techniques

RFLP analysis :-
RFLP is the first method used for DNA fingerprinting. It has been performed with
restriction enzymes, which is used to cut the DNA. This resulted in thousands of pieces of
DNA with a variety of different lengths. With the help of agarose gel electrophoresis these
DNA has been separated on the basis of their size. Further the piece of DNA has been
transferred to nylon membrane. The nylon membrane was incubated with radioactive
probes (Probes are small fragments of minisatellite DNA tagged with radioactive
phosphorous). The probes only attach to the complementary pieces of DNA and here, they
attached to the minisatellites in the genome. The minisatellites attached with probes were
then visualized by exposing the nylon membrane to X-ray film. When exposed to
radioactivity a pattern of dark bands appeared on the film at the sight of the labelled DNA.
This pattern is called the DNA fingerprint. To compare two or more different DNA
fingerprints the different DNA samples were run side-by-side on the same electrophoresis
gel.

Fig 2 :- Showing RFLP analysis with the help of X-ray film

PCR based analysis :-
PCR based DNA fingerprinting technique is most popular among all. It is simpler than
RFLP-autoradiography and gives reliable results than traditional techniques. It is faster
and more accurate.
PCR based DNA fingerprinting relies on microsatellites like STRs (Short tandem repeats)
and VNTRs. Unlike the RFLP DNA fingerprinting method, it does not use restriction
enzymes to cut the DNA. Polymerase chain reaction (PCR) has been used to produce many
copies of specific STR and VNTR sequences. Many STRs and VNTRs sequences of our
interest are available on NCBI database and primers can be designed on basis of these
sequenced data. PCR has been performed with the set of prepared primes and
further agarose gel electrophoresis set up to obtain results. On the basis of size of
DNA fragments, different DNA bands appear in a gel.
However, PCR based gel electrophoresis is mostly used for VNTR analysis. STRs are small in
length so, it difficult to distinguish in agarose gel. For this purposes, RT PCR based analysis
performed.

Fig :- Showing PCR technique for DNA Fingerprinting with Agrose gel
electrophoresis
RT-PCR based analysis :-
As we know, STRs are small in length so, it is very difficult to visualize on agarose
gel. Real-time PCR is able to amplify DNA as well as counts the number of amplicons.
Therefore the number of repeats present in a sample can be calculated. It is accurate,
quick, reliable, and cheaper than other methods. Moreover, it can even quantify a smaller
amount of DNA from any sample. Fluoro-labeled probes are used in RT-PCR. Once the
fluoro-labeled probe is paired with complementary sequence, it signals the machine
to monitors the amplification process of that particular fragment. Based on the
fluorescent signals received, it creates different peaks for different repeats on the monitor
(therefore
Gel electrophoresis doesn’t require in this technique). We can calculate the number of
repeats present in different samples.
 a)

Fig :- (a) RT-PCR with fluorescent labelled primer.

DNA fingerprinting in Plants :-
DNA fingerprints have great value in plant breeding due to its somatic stability
and high variability, which can be used to identify individuals. Its databases act as
important tools for plant molecular research because it provide important technical and
information support for variety quality control, crop breeding and molecular marker-
assisted breeding. DNA fingerprinting plays an essential role in determining the genetic
character of particular species in a broader way.

Other applications of DNA fingerprinting :-

DNA fingerprinting has applications in paleontology, archaeology, various
fields of biology, medical diagnostics and forensics science. In criminal investigations, the
DNA fingerprint of a suspect's blood or other body material is compared to that of the
evidence from the crime scene to see how closely they match.

(i) The technique can also be used to establish paternity.

(ii) Identify a dead body that’s too old or damaged to be recognizable.
(iii) Match tissues of organ donors with those of people who need transplants.

(iv) Identify diseases that are passed down through your family.

(v) Help find cures for those diseases, called hereditary conditions.
(vi) DNA fingerprinting is extremely accurate. Most countries now keep DNA
records on file in much the same way police keep copies of actual fingerprints.

Scientists and researchers are using DNA fingerprinting for various

applications. It has been updated time to time, to make the technique more accurate. PCR is
a powerful tool and gives sensitivity, accuracy, precision, and low-cost assay to DNA.