DNA FINGERPRINTING

Introduction
In 1984, Alec Jeffreys developed the technique of DNA
fingerprinting in his laboratory at the University
of Leicester. These techniques have revolutionised the way that
the police solve crimes.
Alec and his team were studying inherited variation and had
shifted their focus from the products
of genes?
, specifically blood groups, to DNA?
itself. As humans we share around 99.9 per cent of the
same genetic material. But what Alec was interested in was the
remaining percentage, the part that
contains our unique genetic code. Alec wanted to find this code
and understand it.
Alec started by examining the DNA sequence found in the
myoglobin gene of seals.
The myoglobin enables them to hold oxygen in their bodies for
long periods of time. The
myoglobin gene has developed and adapted over millions of
years, mice have a similar gene, and so do
we. Within the DNA sequence of the seal myoglobin gene Alec
identified a repeating sequence or
‘stutter’ in the sequence. He realised that these stutters were
unique to an individual and therefore
could be used to distinguish one seal from another. Perhaps
similar sequences in humans could be used
to distinguish one person from another.
The scientific name for these stutters is a minisatellite. These are
short, repetitive sequences of DNA,
(usually 10 to 60 base pairs?
long), that occur at more than 1,000 locations throughout the
human genome?
. Minisatellites are highly variable so tend to differ from person to
person, which means
that by comparing minisatellites it is possible to identify a
particular individual.
he tried to cut the DNA up into smaller and smaller pieces using
specific enzymes?
. But time after time
he came up against a wall – the enzymes weren’t cutting the DNA
as much as he hoped. This meant he
wasn’t able to get a clear picture of the stutter.
In September 1984 he decided to try something different based
on his knowledge of the myoglobin
gene. He took a DNA sample from one of his lab technicians and
placed it alongside DNA from their
mother and father as well as the DNA from a tobacco plant, a cow
and a seal.
What Alec found was that the probe had bound to all the
minisatellite sequences that had a similar
sequence to his probe. This resulted in a pattern of dark bands
that was completely unique to the
individual. The technician shared parts of their pattern with their
mother and father showing that they
were related. In contrast, the tobacco, cow and seal all had
completely different patterns, showing that
they were not related at all. Finally, the penny dropped and Alec
realised he had produced the first DNA
fingerprints.
In 1983 , first criminal case was solved by dna finger printing
method.

PRINCIPLE OF DNA FINGERPRINTING

The DNA of every human being on the planet is 99.9% 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 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. A microsatellite is a
repeating sequence of 2-6 base pairs in the genome. Since it
is a type of tandem repeats with short sequences of nucleotides,
microsatellites
are also known as short tandem repeats (STRs). The repeats of
single
nucleotides are called single nucleotide polymorphism (SNP).
Furthermore,
microsatellites occur throughout the genome. In the human
genome, the
dinucleotide repeats occur in every 30, 000 base pairs. Moreover,
microsatellites are a highly mutative region in the genome.
Unique
microsatellite sequences occur within families. Therefore, we use
the analysis
of microsatellites for paternity testing. Furthermore, the extension
of
trinucleotide microsatellite repeats causes severe human
disorders like Fragile
X syndrome and Huntington’s disease. Minisatellite is a repeating
sequence of 10-100 base pairs in the
genome. Here, the repeating unit is somewhat large and it is
called
a DNA motif. Another name for minisatellite is variable number
tandem repeats (VNTRs). The number of VNTRs is highly
variable among individuals. The repetitive unit of a minisatellite is
GC rich. Due to the highly variable nature of minisatellites among
individuals, scientists use them for DNA fingerprinting. They also
use minisatellites as genetic markers during the linkage analysis.
Some minisatellite sequences are involved in the formation of ras
oncogene-associated cancer. 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 specific
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 variable number of tandem repeats (VNTRs) have repeats of
9-80 base
pairs.

Methods of DNA Fingerprinting

A. Restriction fragment length polymorphism (RFLP)
The first step in this process is to isolate
the DNA from the sample material to be
tested. The sample size for RFLP test must
be large enough to get the proper result.
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.
The digested DNA sample is
then separated by agarose gel
electrophoresis, in which the DNA is
separated based on the size. The next step is transfer of
separated DNA from
gel slab onto the nitrocellulose membrane to
hybridize with a labeled probe that is specific for
one VNTR region (radio activity labeled
complimentary sequence for VNTR region
nucleotide sequence).
This technique of transferring and hybridizing
DNA onto nitrocellulose membrane is known as
southern blotting, a most widely used DNA
detection technique by molecular biologists.
After the hybridization 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.
Now these bands when compared with the other
known samples, will give the final result of the
DNA fingerprinting.

B. Polymerase Chain Reaction (PCR) amplification of short

tandem repeats (STRs)
Thousands of copies of a particular variable region are amplified
by PCR which forms the basis of this detection.
STR with a known repeat sequence is amplified and separated
using gel-electrophoresis. The distance migrated by the STR is
examined. For the amplification of STRs using PCR, a short
synthetic DNA, called
primers are specially designed to attach to a highly conserved
common
nonvariable region of DNA that flanks the variable region of the
DNA.
By comparing the STR sequence size amplified by PCR with the
other
known samples, will give the final result of the DNA fingerprinting.
Application 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.

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.

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.

Diagnosis of Inherited Disorders:

It is also useful in diagnosing inherited disorders in both
prenatal and newborn babies. These disorders may include
cystic fibrosis, hemophilia, Huntington’s disease, familial
Alzheimer’s, sickle cell anemia, thalassemia, and many others

Reference
https://nptel.ac.in/courses/102103017/pdf/lecture%2038.pdf
http://www.indiastudychannel.com/resources/155090-
Theprinciples-techniques-application-DNA-fingerprinting.aspx