DNA Profiling

I. History –
DNA fingerprinting, also known as DNA profiling or genetic fingerprinting,
emerged as a revolutionary scientific technique in the 1980s. Its roots can be
traced back to the work of British geneticist Alec Jeffreys, who first discovered
the variability in the human DNA sequence that could be exploited for
identification purposes.
The first practical applications of DNA fingerprinting were in criminal
investigations, establishing paternity, and resolving immigration disputes.

II. An Introduction

Also referred to as DNA profiling. The laboratory process known as DNA

fingerprinting uses the nucleotide sequences of particular regions of
human DNA that are unique to each person to determine a person’s likely
identification. DNA fingerprinting is used in criminal investigations, paternity
testing, and other forensic applications. The objective in these situations is to
“match” two DNA fingerprints, such as a DNA sample from a known individual
and one from an unknown individual. It is a forensic method that has been
applied to the zoological, botanical, and agricultural studies of animal and plant
populations. For example,
If you want to identify a criminal at a crime scene, DNA fingerprinting will
work as a means to rectify the identification of the criminal.
1. We collect DNA samples from the crime scene.
2. Then we collect the DNA samples of the suspects.
III. Definition of Basic Terminologies
 DNA (Deoxyribonucleic Acid): The genetic material present in the
cells of living organisms, consisting of a double helix structure formed by
nucleotide base pairs.

Polymorphism: Polymorphism in the context of DNA refers to the coexistence

of different forms or variations within a population. In the realm of genetics,
there are various types of polymorphisms that contribute to the unique genetic
makeup of individuals. Here are some key types:

1. Single Nucleotide Polymorphism (SNP):

 SNPs are the most common type of polymorphism, involving a
single nucleotide change in the DNA sequence. These variations
occur at specific positions in the genome and can influence traits,
susceptibility to diseases, and responses to medications.

2. Short Tandem Repeats (STRs) or Microsatellites:

 STRs consist of short, repeating sequences of DNA, typically 2 to 6
base pairs in length, repeated in tandem. The number of repeats
varies among individuals, making STRs valuable for DNA
fingerprinting and paternity testing.

3. Insertion-Deletion Polymorphisms (Indels):

 Indels involve the insertion or deletion of a small DNA segment in
the genome. These variations can impact gene function and
contribute to genetic diversity.

4. Copy Number Variations (CNVs):

 CNVs refer to the duplication or deletion of relatively large
segments of DNA, ranging from a kilobase to several megabases.
CNVs can influence gene expression levels and are associated with
diseases and phenotypic differences.

5. Variable Number Tandem Repeats (VNTRs):

 Similar to STRs, VNTRs are regions of DNA where sequences of
10 to 100 base pairs are repeated. The number of repeats varies
 among individuals, and these variations are often used in DNA
profiling.

6. Restriction Fragment Length Polymorphisms (RFLPs):

 RFLPs result from variations in the lengths of DNA fragments
generated by restriction enzymes. These variations are detected
through gel electrophoresis and were commonly used in early DNA
fingerprinting techniques.

 PCR (Polymerase Chain Reaction): A laboratory technique used to

amplify a specific DNA segment, facilitating the analysis of small or
degraded samples.

 Gel Electrophoresis: A method for separating DNA fragments based

on their size using an electric field to move them through a gel matrix.

 VNTRs : Variable number of tandem repeat markers (VNTRs) are

located in a genome where a short nucleotide is organized as a tandem
repeat. These can be found on many chromosomes, and they often show
variations in length. Each variant acts as an inherited allele that allows its
use for identification.

 Probes : Probes are a single-stranded sequence of DNA or RNA that is

used to identify specific sequences of DNA or RNA. They are designed
as complementary to the part of the genome of interest, so that when the
probe and the genome are brought together, the probe will hybridise with
the target sequence.
IV. Basic Principle
DNA fingerprinting relies on the identification of variable DNA sequences,
known as polymorphisms, which exhibit differences among individuals.

The most common type of polymorphism used is Short Tandem Repeats

(STRs), where short DNA sequences are repeated at specific locations. These
variations are inherited and unique to each individual, forming the basis for
DNA profiling.

About VNTR

VNTR or the Variable Number of Tandem Repeats are the repeated DNA
sequences at a defined locus. The repeats are clustered together and oriented in
the same direction. Individual repeats can be added or removed through
replication and recombination errors. This forms alleles with different numbers
of repeats.
The DNA segments vary in different individuals and are hence beneficial in
identifying individuals in case of a crime scene or a paternity dispute. This is
known as DNA fingerprinting.
The tandem repeat sequences of DNA are also termed “satellite DNA”. These
are of three main types:
 Satellite
 Minisatellite
 Microsatellite
Types of VNTR

1. Satellite DNA
These are highly repetitive DNA sequences and each DNA sequence consists
of several thousand base pairs. A satellite can measure up to 100 million base
pairs. These are found occurring in the regions of heterochromatin. The Y
chromosome has abundant satellites. This makes it convenient for researchers
to study paternal genetic transmission in mammals.
The density of the DNA is the function of its base and sequence. The satellite
DNA with its highly repetitive DNA has a reduced density compared to the rest
of the genome. Therefore, the name “satellite DNA” is coined.
The satellite DNA has several biological functions:
 Satellite DNA is present in the centromeric and pericentromeric regions.
It regulates the functions of the centromere.
 They help in the formation of heterochromatin.
 Satellite RNA transcripts are found in plants, vertebrates, and
invertebrates

2. Minisatellite

In a minisatellite, each repeat ranges from 9 to 100 base pairs. It is an array of

tandem repeats 500 to 300,000 base pairs long.
Minisatellites have been found in association with important features of the
human genome such as gene regulation, imprinting, and chromosomal fragile
sites. They provided the first highly polymorphic, multiallelic markers for
linkage studies.
Most of the minisatellites are GC rich. They possess a strong strand
symmetry.
3. Microsatellite
The repeats are very short, with 2-6 base pairs each. The whole array ranges
from 10,000 to 100,000 base pairs long. They are therefore called short tandem
repeats or simple sequence repeats.
They are usually found in insect and plant chromosomes, and euchromatin
regions of vertebrates.
Microsatellites are important to population geneticists because of the variable
number of repeats among the individuals of a population.
Microsatellite markers are inherited from both parents. Therefore, they are
useful for paternity tests. Highly polymorphic loci increase our ability for
parental analysis.
Microsatellites are not affected by natural selection. But they are influenced by
gene flow, genetic drift, and mutation.

Importance of VNTR
 VNTRs are found on many chromosomes and vary in length among
different individuals. Each variant helps in personal or parental
identification.
 VNTRs are an important source of genetic marker RFLP which is used in
the linkage analysis of genomes. A banding pattern unique to each
individual is produced by the VNTRs.
 VNTR has its applications in forensic science, DNA fingerprinting, and
other genetics and biology research.
V. Steps Involved

1. 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.

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 separated based on the size.

4. 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).

5. 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.

6. 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.

7. Now these bands when compared with the other known samples,
will give the final result of the DNA fingerprinting.
VI. The Process

Step 1- DNA Isolation

DNA is taken from the cell and purified via chemical processing and
centrifugation.

Step 2- Amplification

Using the polymerase chain reaction, many copies of the extracted DNA are
produced (PCR).
Step 3- DNA digestion by Restriction of Endonuclease Enzyme
The restriction endonuclease enzyme breaks down the DNA into smaller
pieces. These enzymes cut the DNA at particular locations, chopping it up into
different lengths.

Step 4- DNA Fragment Separation

Depending on their size, the DNA fragments are next separated using a
process called electrophoresis.
In the presence of an electric field, a technique called electrophoresis is used
to separate charged molecules. DNA fragments are positioned on a transparent
gel bed on a plate for electrophoresis, which involves applying an electric
current to the gel. DNA pieces gravitate toward the positive electrode because
each one of their negative charges is unique. Eventually, the pieces and gel are
distributed at various locations by their sizes.
Step 5

The agents that separate the DNA fragments into single strands are then
introduced.

Step 6- Transferring (blotting) the isolated DNA fragments from the gel
to synthetic membranes such as nylon or nitrocellulose

The isolated DNA fragments are then transferred from the gel to a nylon
membrane or nitrocellulose, and this method is known as Southern blotting.
In this method, the gel is coated with a nylon membrane that attracts the DNA
fragments, much as how blotting paper dries wet ink.

Step 7- Radiolabeled Probe Hybridizations

In this stage, the radioactive isotope is added to the DNA fragments via
hybridization so that their positions may be seen on an X-ray image. To
accomplish this, a nylon membrane is added to a bath that contains probes
(probes are short pieces of single standard complementary DNA, tagged with
radioactivity that bind to a specific chain of DNA VNTR sequences according
to the base-pairing rule).
Step 8- Hybridized DNA fragment detection

The membrane is exposed to the X-ray film to create an autoradiograph, which

when developed reveals a distinctive pattern of dark and bright bands that
reflect the makeup of DNA. The DNA fingerprints are represented by the dark
bands on the X-ray film.
VII. Application
1. Forensic Science:

Biological samples such as blood, hair, saliva, sperm, and body tissue
cells are used for DNA profiling. It is possible to compare the DNA
recovered from the evidence sample using the VNTR (Variable
number of tandem repeats) prototype. It aids in the investigation of
crimes like rape and murder.

2. Personal Identification:

It makes use of the idea that DNA fingerprints can be used to identify
people, acting as a kind of genetic bar code.

3. Determining Paternity and Maternity:

A person accepts their VNTRs from their parents. Cases that were in
disagreement have been resolved via parent-child VNTR prototype
analysis. Additionally, immigration and inheritance proceedings may
make use of this information.

4. Breeding Program:

Breeders typically assess a plant or animal’s genotype using its

phenotype. Since homozygous or heterozygous dominance is difficult
to distinguish from appearance, the genotype can be determined with
great care and accuracy using DNA fingerprinting. Hunting dogs and
racehorses can both benefit from it.

5. Diagnosis of Hereditary Disorders:

It can be used to identify inherited diseases in both newborn and

prenatal children. Cystic fibrosis, hemophilia, Huntington’s disease,
familial Alzheimer’s, sickle cell anemia, thalassemia, and a host of
other conditions may fall under this category.
6. The creation of treatments for inherited diseases:

DNA prototypes linked to the disease can be identified by examining

the DNA fingerprints of family members who have a history of a
particular disorder.

7. Detection of AIDS:

A person with AIDS can be diagnosed by comparing the HIV “RNA”

band (converted to DNA via RTPCR) with the bands formed by the
man’s blood.
VIII. Issues that can happen in Forensic DNA Sample

Degraded samples and DNA mixes are the two most frequent problems that
forensic experts run against when examining DNA evidence.

Degraded DNA

Analyzing degraded DNA samples was nearly difficult until the development
of contemporary PCR techniques. Techniques like RFLP, or restriction
fragment length polymorphism High molecular weight DNA was necessary
for the sample for restriction fragment length polymorphism, the first
approach for DNA analysis in forensic science, to produce accurate results.
However, high molecular weight DNA is not present in degraded samples
because the DNA is too fragmented to perform RFLP with sufficient accuracy.
Polymerase chain reaction analysis of degraded DNA samples was not
possible before the development of contemporary PCR techniques. The little
DNA snippets still present in damaged samples may be isolated and amplified,
thanks in part to multiplex PCR. Multiplex PCR techniques differ significantly
from more traditional techniques like RFLP. While RFLP required at least 100
ng of DNA to do an analysis, multiplex PCR has the potential to amplify less
than 1 ng of DNA.

DNA Mixtures

When evaluating unknown or suspicious DNA samples, forensic scientists

frequently run into the problem of mixtures. A DNA sample with two or more
individual contributions is referred to as a mixture. This frequently happens
when a DNA sample is taken from a substance that has been handled by
multiple people or when a sample combines the DNA of the victim and the
attackers. It can be difficult to identify individual profiles in DNA samples
that contain multiple people, so only experts with extensive training should
analyze mixtures will be challenging to analyze mixtures with two or three
different individuals

Restriction Enzymes
It is an enzyme that breaks DNA into small pieces at or close to particular
recognition regions in molecules called restriction sites. Within the larger
endonuclease group of enzymes, restriction enzymes are one subgroup.
Restriction enzymes are often divided into five categories, which differ from
one another in terms of their structure and whether or not they cleave DNA
substrates at their recognition sites. Each sugar-phosphate backbone, or each
strand of the DNA double helix, is cut by a restriction enzyme twice to cut
DNA. While it is true that each person’s DNA is truly unique, there are some
parts of your DNA that you have in common with everyone else on the earth.
Since the sequence or order of certain bases is known, scientists have created
molecules called restriction enzymes that hunt for those locations. As a result,
when a scientist is examining someone’s DNA, they add restriction enzymes
that locate those sites that everyone has and split the DNA strand in two. For
all kinds of species, there are hundreds of restriction enzymes, and each one
is designed to look for and cut a particular DNA sequence.

Short Tandem Repeats

Humans and other species have several different forms of identical DNA. The
term “Short Tandem Repeat” or “STR” refers to the kind of DNA
fingerprinting that is most frequently utilized. A portion of the human genome
has been shown to have repeats of the same sequence, but with varying
numbers of repetitions. Therefore, while the sequence “CAGT” may exist in
all humans, its repetition rates may vary. It can be 10 times for you and 15
times for your neighbor. Researchers can recognize these minute variations
and use them to uniquely identify you.