DNA Testing :Procedure of DNA Fingerprinting

DNA Testing :Procedure of DNA Fingerprinting

When you read about DNA testing , mitocondrial DNA etc you come across many definitions like this one:

DNA Fingerprinting is a technique employed to assist in the identification of individuals on the basis of their respective DNA profiles.

What does this difficult sentence really mean? A simple Explanation is needed .

A Simple Explanation of the Procedure DNA Fingerprinting

Before going into the detail of the process of DNA fingerprinting  and DNA testing ,you need to know the meaning of the terms:

•  Variable number of tandem repeats 

• Restriction Endonuclease Enzyme.

Variable number of tandem repeats

Only about 1 percent of DNA in humans encodes for mRNA. The remaining 99 percent is called “junk” DNA.

This junk has areas that contain repeated patterns.  For instance, the four building blocks of DNA (Guanine, Cytosine, Adenosine, and Thymine) may repeated with this motif: TAA

 The number of times that this pattern repeats can vary in different individuals.  For instance, in the homologous chromosome we get one from mother and one from father ,if in the mother’s chromosome it repeats 3 times and in the father’s chromosome it repeats  2 times .The individual will have( 3 ,2) repeats in the Homologous chromosome pair.

The number of repeats are inherited from a persons parents.  You can test several people to find out if they are the parents.

                        Restriction Endonuclease Enzyme

An enzyme  that can recognize specific base sequences in DNA and cut the DNA at that site. A restriction enzyme acts as a biochemical scissors.

To get DNA fragments containing the VNTRs( TAA )we can use a restriction enzyme just like scissors to extract (TAA) from the rest of the DNA.

Polymerase chain reaction, or PCR: is a laboratory technique used to make multiple copies of a segment of DNA. PCR is very precise and can be used to amplify, or copy, a specific DNA target from a mixture of DNA molecules

Step by Step Procedure For DNA Fingerprinting

DNA molecules are isolated from a sample of blood, semen or other body fluids or tissues by using specific techniques such as a high speed refrigerated centrifuge.

If the DNA is in poor condition or available only in minute quantities,the particular DNA fragment can be amplified by making several copies of it with the help Polymerase chain reaction.(PCR)

DNA molecules are cleaved (Cut into fragments)at specific sites (VNTR’s site)with the help of a site recognizing restriction endonuclease enzyme .The DNA fragment contain the VNTRs.

DNA fragments are now sorted out on a agarose gel slab by  a technique called electrphoresis.Electrophoresis is a technique used in laboratories in order to separate macromolecules based on size.

The technique applies a negative charge so proteins move towards a positive charge.DNA is negatively charged, therefore, when an electric current is applied to the gel,DNA will migrate towards the positively charged electrode. Shorter strands of DNA move more quickly through the gel than longer strands resulting in the fragments being arranged in order of size.

Double stranded DNA is now split into single strands by using alkaline chemicals.

 The isolated DNA fragments in the gel are copied onto a nylon nitrocellulose sheet by placing  the nylon nitrocellulose sheet on top of the Agarose gel  .This is called Southern Blotting.

Special DNA probes are prepared in the lab ,these contain repeated sequence of nucleotide complementary to those on the VNTRs(ATT in our example).These probes are made radioactive .The radioactive DNA probes bind to the repeated sequencenes on the nylon sheet.This is called Hybridisation.

An X -RAY flim is exposed to the nylon sheet to mark the places where the radioactive DNA probes have bound to the DNA fragment .These places look like  dark bands when X -RAY flims are developed.This is known as Autoradiography.

The Dark bands on the X-RAY film represent the DNA fingerprint or the DNA profiles.