ELECTROPHORESIS AND ITS FORENSIC APPLICATIONS.pptx

This presentation is about electrophoresis and its forensic application

1. DEPARTMENT OF FORENSIC SCIENCE “ ELECTROPHORESIS AND ITS FORENSIC APPLICATIONS” Presented By- Presented To- PALLAVI KUMARI DEPARTMENT OF 19MSFS001 FORENSIC SCIENCE M.Sc. Forensic science Semester 1 st (2019-2020) Sam Higginbottom University of Agriculture, Technology and Science

2. INTRODUCTION Electrophoresis is a physical method of analysis which involves separation of the compounds that are capable of acquiring electric charge in conducting electrodes. History Electrophoresis is an important analytical tool used in all branches of biology. Electrophoresis was observed for the first time in 1807 by Russian professors Peter Ivanovich Strakhov and Ferdinand Frederic Reuss at Moscow State University, who noticed that the application of a constant electric field caused clay particles dispersed in water to migrate

3. DEFINITION Electrophoresis may be defined as the migration of the charged particles through a solution under the influence of an external electrical field. Ions that are suspended between two electrodes tends to travel towards the electrodes that bears opposite charge. THE AIM OF CARRYING OUT ELECTROPHORESIS INCLUDE : To determine the number, amount and mobility of components in a given sample or to separate them. To obtain information about the electrical double layers surrounding the particles. Determination of molecular weight of proteins and DNA sequencing.

4. PRINCIPLE OF ELECTROPHORESIs Comprehensive term that refers to the migration of charged particles of any size in liquid medium under the influence of an electric field. Depending on kind of charge the molecule carry, they move towards either to cathode or to anode. The rate of migration of an ion in electrical field depending on factors: Net charge of molecule. Size and shape of particle. Strength of electrical field.

5. Properties of supporting medium Temperature of operation This can be represented by the following equation V= E.q /f For molecules with similar confirmation , f varies with size but not with the shape. The electrophoretic mobility (µ) of a molecule is directly proportional to the charge density (charge/mass ratio). Molecules with different charge/mass ratio migrate under the electric field at different rates and hence get separated .

6. TYPES OF ELECTROPHORESIS 1) Zone Electrophoresis Paper Electrophoresis Gel Electrophoresis a- AGE b- PAGE c- SDS PAGE d- 2D Electrophoresis Thin layer Electrophoresis Cellulose acetate Electrophoresis 2) Moving boundary Electrophoresis Capillary Electrophoresis Isotachophoresis Immuno Electrophoresis Isoelectric Focusing

7. 1. PAPER ELECTROPHORESIS In this the sample is applied as a circular support on a strip of whatman filter paper moistened with the buffer solution. The ends of the paper are immersed in separate reservoirs containing buffer and in which the electrodes are fitted . Upon passing electric current, the ions in the sample migrate towards oppositely charged electrodes . This method is suitable for separation of low molecular weight compounds such as amino acids and nucleotides .

8. Forensic APPLICATIONS OF PAPER ELECTROPHORESIS Paper Electrophoresis is used to identify and separate drugs in the field of FORENSIC CHEMISTRY. This technique is also used to isolate and compare hemoglobin in the field of FORENSIC HEMATOLOGY .

9. 2. GEL ELECTROPHORESIS Separation is brought about through molecular sieving technique, based on the molecular size of the substances. Gel material act as a “molecular sieve”. Gel is a colloid in a solid form (99% is water). Different types of gels which can be used are; Agar and Agarose gel, starch, polyacrylamide gels. A porous gel acts as a sieve by retarding or, in some cases by completely obstructing the movement of macromolecule of while allowing smaller molecules to migrate freely.

10. a. AGAROSE GEL ELECTROPHORESIS Agarose gels are more porous and have a large pore size and therefore used to fractionate large macromolecules such as nucleic acids. Agarose gels are cast by boiling agarose in presence of a buffer then poured into a mold and allowed to harden to form a matrix. Higher the agarose larger is the pore size. When the electric feild is applied across the gel, negatively charged molecules move towards oppositely charged electrodes on the basis of their molecular size. Smaller DNA molecules travel faster than larger ones . Commonly used buffers- T ris A cetate , T ris B orate or T ris P hosphate at a concentration of 50mM over a pH range of

11. b . POLYACRYLAMIDE GEL ELECTROPHORESIS The components used in the formation of this gel are known to be neurotoxins. The most commonly used components to synthesize the matrix are acrylamide monomer, ammonium persulphate and tetramethylenediamine (TEMED). These free radicals can activate acryl amide monomer including them to react with other acrylamide monomer forming long chains. This chains become cross-linked if the reaction is carried out in the presence of N-N’- methylenebisacrylamide .

12. Forensic APPLICATIONS OF GEL ELECTROPHORESIS Gel electrophoresis used in FORENSIC science is a way to analyze DNA. Because the DNA of each person is unique, the patterns of separation created using Gel Electrophoresis are unique. In the case of a crime, the forensic scientist can perform an Electrophoresis of DNA from the crime scene and from the suspect's DNA, then determine if the two are an exact match .

13. 3. CAPPILARY ELECTROPHORESIS The technique that has very high resolving power and speed . CE involve Electrophoresis of sample in small narrow capillary tubes and high voltage from kV is applied . This technique is used to separate biological material including amino acids, proteins, nucleic acids as well as drugs or even metals. Very small amount of sample are required for analysis.

14. FORENSIC APPLICATION of CAPILLARY electrophoresis Used for FORENSIC drugs, toxicological samples, fibres analysis. Used for the analysis of explosive and gunshot residues.

15. Case Study In FORENSIC Science many biological evidences play an important role, as they are the source of DNA. The DNA is isolated from different sources which include Blood, Blood stains, Semen, Tissues, Bones and Hair in different cases like Rape, Missing identity, Murder and Paternity. In this sexual assault case study blood and semen are the source of DNA for DNA Finger Printing. A girl aged 23 had been brutally raped by a person of age 26 at Kurnool district in Andhra Pradesh, India. After filing a complaint by the girl, the semen stained under garment wore by the girl at the time of rape, was collected, DNA was isolated from the semen in the inner ware and the STR profiles were taken as Electropherograms by using Capillary Electrophoresis technique. On the other hand Blood sample was collected from the suspect in this rape case, DNA was isolated from it and STR profiles were taken as Electropherograms . Both the Electropherograms are compared and the suspect is proved to be the culprit in this case.

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