DNA Fingerprinting
DNA fingerprinting (also called DNA profiling) is the process of determining an individual's DNA characteristics. DNA profiling is a forensic technique in criminal investigations, comparing criminal suspects' profiles to DNA evidence so as to assess the likelihood of their involvement in the crime.
DNA fingerprinting is performed in 4 basic steps. They are as follows –
1. DNA isolation/extraction
2. DNA quantification
3. DNA amplification
4. DNA fragment analysis
DNA Isolation
The simplest explanation of DNA extraction is, “extracting DNA from cells”.
“Isolation of DNA by breaking the cell membrane and nuclear membrane with the help of chemicals, enzyme or physical disruptions is defined as a DNA extraction”.
The image represents the general outline of how DNA can be extracted from the cell.
It can be done by 2 methods – chemical and physical
• The DNA Isolation DNA from cells are extracted by using lysis buffer and DTT.
• The instruments used are Thermomixer refrigerated centrifuge , automate, Maxwell
A) MAGNETIC BASED METHOD
DNA QUANTIFICATION :-
DNA quantification is done by RTPCR method i.e. Real Time PCR.
This process tells us about the amount of DNA, tells the amount of Y chromosome i.e. whether it is male’s DNA or not and whether it is human or animal i.e. if no reading are obtained then it is a disputed sample.
QUANTIFICATION PROTOCOL :-
Three constituents:
Reaction Mixture - 5 microliter
Primer Mixture - 4 microliter Trio kit DNA Sample - 1 microliter
Reaction mixture is a master mix containing dNTP, DdNTP, Taq polymerase , Mg+2.
DNA AMPLIFICATION/ POLYMERASE CHAIN REACTION
The sections or genes which cause physiological changes like skin tone, eye color, height, etc are long sequences which are difficult to analyze and are time consuming. So, Alec Jeffry found out that out of all our DNA, only 5% of it is useful to our body and the rest of it is junk DNA which has small sequences which are called STR (Short Tandem Repeats) and they have 10- 100 base pairs only and so easy and quick analysis can be done.
PCR-
Standard ingredients in the mixture are:
• the DNA segment of interest
• specific primers
• heat-resistant DNA polymerase enzyme
• the four different types of DNA nucleotides
• the salts needed to create a suitable environment for the enzyme to act.
STEPS OF PCR :-
Step 1: Denaturation
As in DNA replication, the two strands in the DNA double helix need to be separated.
The separation happens by raising the temperature of the mixture, causing the hydrogen bonds between the complementary DNA strands to break. This process is called denaturation.
Step 2: Annealing
Primers bind to the target DNA sequences and initiate polymerisation. This can only occur once the temperature of the solution has been lowered. One primer binds to each strand.
Step 3: Extension
New strands of DNA are made using the original strands as templates. A DNA polymerase enzyme joins free DNA nucleotides together. This enzyme is often Taq polymerase, an enzyme originally isolated from a thermophilic bacteria called Thermus aquaticus. The order in which the free nucleotides are added is determined by the sequence of nucleotides in the original (template) DNA strand.
DNA FRAGMENT ANALYSIS
It is sometimes also called DNA sequencing but these are two different things although highly similar. DNA sequencing denotes the sequencing of the entire DNA which is very time consuming and not possible for all samples whereas, DNA fragment analysis is the analysis of those 25 markers only.
DNA fragmentation is a technique of artificial breaking of DNA in order to use it in various downstream genetics and well as genomics applications.”
The instrument used is 3500XL GENETIC ANALYSER. Its sensitivity is 0.5ng to 1-2ng. So, due to its sensitivity range, if the sample is concentrated then we need to dilute it using any of the
following- distilled water/ nuclear water or milique water.
FRAGMENT ANALYSIS PROTOCOL-
We add the following chemicals (according to Global Filer kit) in the PCR tubes with sample in it-
- Formamide/hidi-8.5 microliters
- Allelic Ladder- 1 microliter
- Size standard/Liz- 0.5 microliter
References:
https://bpchalihacollege.org.in/online/attendence/classnotes/files/1643777309.pdf
https://ebookee.com/Detailed-Polymerase-Chain-Reaction-Concepts_5364127.html
Written by:
Gauri Vijay Maske
Volunteer of AFRS.
