During investigation of area where crime is committed, lots of evidences found at crime scene which are collected by crime scene investigating team and then analyzed in forensic laboratories. Trace evidences which found at crime scene need separation of molecules for analysis as they are in complex form. Electrophoresis is one of the techniques which is used for separation of molecules even sample size is very small.
Electrophoresis – Theory of electrophoresis is given by Ferdinand Frederic Reuss in 1807 and then electrophoretic apparatus was Developed by the Arne Tiseleus in 1937 also known as Tiseleus apparatus. It is an electrokinetic process which separates charged particle in a fluid using a field of electrical charge and electric field exerts electrostatic coulomb force to separate charged particles.
The rate of migration of a particle is depends on :-
- Strength of field
- Size & shape of a molecule
- Net charge
- Ionic strength
- Temperature of medium in which molecules are moving.
Common mediums used in Electrophoresis:-
◊ Cellular Acetate:- This medium helps in good separation of proteins from biological fluids & is also allows migration of even large serum proteins.
◊ Agarose gel:- This gel obtained from agar-bearing marine algae- Rhodophyta. This gel is a polysaccharide taken out from seaweed and it has large pore structure.
This medium is mainly used for electrophoresis of DNA & this media also allows large molecules to move easily through it. But this media is not good for small molecules.
◊ Polyacrylamide gel or PAGE :- This media is used for separation of mixture of proteins & also helps to identify how proteins bind to DNA.
This media is most suitable for quantitative analysis. And it is used in understanding how bacteria is becoming resistant to antibiotics through plasmid analysis.
It has small range of separation but high resolving power.
◊ 2D – Electrophoresis:- It separates molecules along an X-axis & Y-axis. It separates molecules by charge & by size.
Types of Electrophoresis:-
1.Affinity Capillary Electrophoresis or Electrophoretic Mobility Shift Assay
It is a versatile technique to study the non-covalent interaction of receptors to neutral and charged ligands.
This method is based on changes in the electrophoretic pattern of molecules through complex formation. The binding of a molecule charged or uncharged will change the electrophoretic properties of a molecule.
2. Capillary Electrophoresis
Used to separate ionic specific by their charge and frictional forces and hydrodynamic radius. It has high resolution separation.
If two ions are of the same size, one with greater charge will move the fastest. If ions have same charge , smaller particles have less friction & overall faster migration rate.
It is coined by Grabar & Williams.
It is used for isolation and characterization of proteins on basis of electrophoresis and their reaction with antibodies.
All types of immuno-electrophoresis need immunoglobulins, the antibody which react with proteins to be isolated and characterized.
Types of Immuno-Electrophoresis :-
It is a 1-D quantitative immune electrophoresis. Antigen is electrophoresed into gel containing antibodies. And the reaction result in a rocket shaped precipitin formation.
The distance from the starting well to the front of the rocket shaped arc is related to antigen concentration.
This method is used for quantitative estimation of antigen in the serum.
It helps in determining the concentration of specific protein in a protein mixture.
►Fused Rocket Immuno-Electrophoresis
It is modified version of Rocket immune-electrophoresis.
Changes in the electrophoretic pattern of proteins via specific interaction or complex formation with other macromolecules or ligands.
4. Pulsed field gel electrophoresis (PFGE) :- This type is used for isolation of large DNA molecules by application of an electric field to a gel matrix that intermittently changes its direction. Due to periodic changing of field direction, the different lengths of DNA react to the change at different rates. Large piece of DNA will be slower to rearrange their charge when field direction is changed , while smaller pieces faster. Each band will begin to separate even at very large lengths.
5. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis(SDS-PAGE):-
- SDS is a detergent which coats the linear protein sequence with a layer of SDS.
- SDS denature sequence by binding to the hydrophobic region of protein.
- Due to negative charge of SDS it becomes the dominant charge of the complex.
- Binding of number of SDS molecule is directly proportional to the size of protein.
- All different sized proteins covered with SDS would run at about same mobility.
- Proteins instead of water run through an inert polymer known as polyacrylamide.
- Charged molecules migrates to the electrode with the opposite charge when placed in an electric field.
- SDS-PAGE allows to eliminate the influence of structure and charge for separation.
Proteins are separate on basis of difference in their molecular weight.
6. Native Polyacrylamide Gel electrophoresis or Non-denaturing gel :-
The native charge on the protein divided by its mass, governs the movement of protein in terms of speed and direction.
Native gel is used for studying the composition and structure of native protein.
7. Electro focusing gel or Isoelectric focusing :-
A gel which has a pH gradient from one end to another. As a protein travels through this pH gradient, its different ionizable group either pick up or loose protons. Finally, they will find a pH where its charge is zero and it will get focused at that point.
Technique used isoelectric focusing is – 2D- PAGE.
8. DNA agarose gel :-
This method uses to separate large fragments of DNA from one another by size. These gels contain additional denaturing compound such as urea. Two pieces of DNA that differ in size by 1 base can be distinguished from each other by this method.
Application of Electrophoresis in Forensic Science :-
- For DNA fingerprinting by separating DNA fragments to investigate crime scene.
- For taxonomic studies by DNA profiling to distinguish different species.
- To study structure and functions of proteins, RNA & DNA.
- For analysis of drugs of abuse.
- For analyzing explosive compounds and residues.
MSc (Forensic Science)
NTA UGC-NET Qualified