DNA fingerprinting - by Mr. Debajit Rabha Asst. Prof. of Botany PDUAM, Amjonga PDF

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Mr. Debajit Rabha
Asst. Prof. of Botany
PDUAM, Amjonga...

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DNA-fingerprinting (also called DNA profiling or DNA typing)  Concept of DNA fingerprinting  Principle of DNA fingerprinting  Technique of DNA fingerprinting  Applications of DNA fingerprinting  Concept of DNA fingerprinting: DNA fingerprinting is a technique used especially for identification (as for forensic purposes) by isolating and identifying the base-pairs sequence in certain areas of DNA of an individual. So nowadays it is an ideal and most reliable way to distinguish an individual from other. Alec Jeffrey (1984) invented DNA fingerprinting technique.  Principle of DNA fingerprinting: DNA polymorphism is the basis of DNA fingerprinting. DNA polymorphism is any variation in the (DNA) nucleotide sequence between individuals. This difference can be single or more base-pair changes which have to present with a frequency of more than 0.01 % of a population in certain locus. In human genome 90% of the DNA is same (about 99.9% nucleotide bases). DNA fingerprinting is based upon the rest 10% difference in the human DNA (about 0.1% or 3 x 106 base pairs out of 3 x 109 bp). These 0.1% DNA base-pairs are highly variable and unique for each individual known as DNA polymorphism. This uniqueness in the base sequence doesn't only occur in genes but also in repetitive DNA also known as satellite DNA. DNA polymorphism occurred in Satellite DNA are used in DNA fingerprinting. Satellite DNA is a unit of short non-coding but inheritable tandemly repeated sequences of DNA bases (repetitive DNA). Depending on the size of the repeat, the repeat regions Satellite DNA are classified into two groups. 1. Short tandem repeats (STRs) contain 2-5 base pair repeats referred to as Microsatellites and 2. Variable number of tandem repeats (VNTRs) have repeats of 9-80 base pairs often referred as Minisatellites.  Since a child receive 50% of the DNA from its father and the other 50% from his mother, so the number of VNTRs at a particular area of the DNA of the child will be different may be due to insertion, deletion or mutation in the base pairs. As a result, every individual has a distinct composition of VNTRs and this is the main principle of DNA fingerprinting. When DNA of any individual is digested with a restriction enzyme, resultant DNA fragments pattern (sizes) will be different in cleavage site position. This is the basics of DNA fingerprinting.

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Technique of DNA fingerprinting: Following are the summary of DNA fingerprinting:

Isolating the DNA ↓ Digesting the DNA with the help of restriction endonuclease enzymes ↓ Separating the digested fragments as per the fragment size by the process of electrophoresis ↓ separated fragments onto synthetic membranes like nylon by the process of Southern blotting ↓ Hybridising the fragments using labelled VNTR probes ↓ Analysing the hybrid fragments using autoradiography ↓ X-ray film represent the DNA fingerprints

Procedure involved in DNA fingerprinting: 1. Step1: DNA is isolated or obtained from any of the sources (blood, semen traces/vaginal swab from cloths, saliva, hair, and skin/tissue.) by using specific techniques such as a high-speed refrigerated centrifuge. 2. If the DNA is in poor condition or available only in very small quantity, then many copies of those unique repeating sequence are produced by using the technique of PCR (polymerase chain reaction). 3. Then DNA molecules are cleaved into many fragments, at specific sites using the siterecognizing restriction endonuclease enzyme. 4. These fragments are then sorted out according to their length on an agarose gel slab by the technique called electrophoresis. The fragments get arranged along the gel according to their length and electric charges. 5. Then the DNA, which is double stranded, is then split into single strands by using alkaline chemicals. 6. The isolated DNA fragments in the gel are copied onto a nylon nitrocellulose sheet placed on the gel. This technique is called Southern blotting. 7. Special DNA probes (also called hybridization probes) are prepared. These contain repeated sequences of nucleotide complementary to possible VNTRs. These probes are made radioactive. The radioactive DNA probes bind to the single stranded VNTRs on the nylon sheet. This is called hybridization. An X-ray film is exposed to the nylon sheet to mark the places where the radioactive DNA probes have bound to the DNA fragment. These places are marked as dark bands when X-ray film is developed. This process is called autoradiography. 8. The dark bands on the X-ray film represent the DNA fingerprints, or DNA profiles using which the suspect is identified.

 Applications DNA fingerprinting has got lots of applications. 1. This procedure is mostly used in forensic to identify the criminals. 2. It is also used for the paternity test. 3. It is used in the study of breeding patterns of animals facing the danger of extinction. 4. It is also used in determining lineages of humans and other animals to ascertain the process of evolution by checking the "genetic markers" which are passed from the ancestors. 5. It is used to diagnose the genetic disorders and hereditary disorders like haemophilia, sickle cell anaemia, cystic fibrosis etc. 6. It is also used to determine about the antibiotics to which bacteria are resistant. Advantages of DNA fingerprinting: The biggest advantage of this process is that criminals can be easily be identified through this process. Earlier normal fingerprints were used for this process. Fingerprints can be changed through surgery. But there's no way to change one's DNA sequence. Therefore, it's a promising method....