Dna fingerprint

Dna fingerprint

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   INTRODUCTION:  TheDNA fingerprinting unlike the usual fingerprinting which is based on the morphological features and primarily restricted to humans is revealing the identity of an organism at the molecular level.  In fact this is the technique of finding the genetic identity.  This is primarily based on the polymorphisms occurring at the molecular level, that is on the base sequences of the genome.
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   METHODOLOGY: The basicmethodology of DNA profiling in plants involve first the extraction of DNA from plant cells, quantification and quality assessment of extract. The further steps are of two types. 1) PCR based.-RAPD, ISSR, SSR 2) Non PCR based. – RFLP.
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   DNA ISOLATION: Highmolecular weight DNA from plant tissue can be isolated in a number of ways. All methods involves basic steps of removal of all cell wall and nuclear membrane around the DNA and the separation of DNA from other cell components such as cell debris, proteins, lipids or RNA without affecting the integrity of the DNA. The most commonly preferred method is CTAB method.
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  PCR: The DNA amplificationby thermal cycling called Polymerase Chain Reaction is in vitro method that can be used to amplify a specific DNA segment from small amounts of DNA template or duplex into millions of copies. It was invented by Kary Mullis. Steps involved in PCR are:  Heat Denaturation.  Annealing.  Primer Extension.
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  NON-PCR:RFLP  To discoverRFLPs, restriction enzymes (RE) are used to cut DNA at specific 4-6 bp recognition sites.  Sample DNA is digested with one or more RE’s and resulting fragments are separated according to molecular size using gel electrophoresis.  After that Southern Blotting is performed where fragments are immobilized on a nylon membrane, followed by hybridization to a labeled probe that identifies the locus under autoradiograph.  Fragment sizes of digested DNA will differ depending on the presence or absence of the proper recognition sequence for the enzyme.
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  Southern blot 1. Restrictionenzyme digestion 2. Agarose gel electrophoresis 3. Transfer DNA onto membrane 4. Hybridized with probe RFLP Restriction Enzyme site 1. PCR amplify using fluorescent primers 2. Capillary electrophoresis RFLP-based DNA fingerprinting PCR-based DNA fingerprinting PCR product of different sizes Fluorescent peaks of different mobilit Radioactive bands of different mobility
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   CONTD….  Differencesin fragment length result from base substitutions, additions, deletions or sequence rearrangements within RE recognition sequences.  Although two individuals of the same species have almost identical genomes, they will always differ at a few nucleotides.  Some of these differences will produce new restriction sites (or remove them), and the banding pattern seen on a genomic Southern will thus be affected.  For any given probe (or gene), it is often possible to test different restriction enzymes until one which gives a pattern difference between two individuals is found, that is, a RFLP.  The less related the individuals, the more divergent their DNA sequences.
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   RAPD:  InRAPD analysis the total DNA from an organism is mixed with (generally) 10 bp single stranded DNA (primer) together with the four different deoxynucleotides and a heat stable DNA polymerase enzyme.  The reaction mixture is placed on a thermocycler (PCR-machine) which can change the temperature of the reaction rapidly according to a predefined PCR programme.  Reannealing at low temperature makes the primer molecules attach to complementary site on the DNA.
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   Elongation isthe period when the polymerase enzyme elongates the attached primers from their 3' end. Denaturation separates the new formed strands.  After a few PCR cycles DNA pieces with well defined length similar to the one between the two original primer sites dominate the mixture. This is because they amplify exponentially.  In most RAPD reactions the result will be amplification of 5 to 10 different DNA segments whose lengths depend on the primer recognition sites available in the genome.  The DNA after amplification is visualized for bands using Agarose Gel Electrophoresis (AGE).
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   SSR:  SSRanalysis is amenable to automation and multiplexing and allows genotyping to be performed on large numbers of lines, and multiple loci to be analyzed simultaneously.  SSRs can be identified by searching among DNA databases (e.g. EMBL and Gene bank), or alternatively small insert (200-600bp) genomic DNA libraries can be produced and enriched for particular repeats.  For separation and visualization of bands Polyacrylamide Gel Electrophoresis (PAGE) is conducted (6% acrylamide solution), stained and the observed the bands.
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   ISSR:  ISSRanalysis is technically simpler than any other marker systems.  The method provides highly reproducible results and generates higher levels of polymorphism compared to other markers in many systems.  The advantage lies in long primer length.  For separation and visualization of bands Agarose gel electrophoresis is conducted.
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   Use ofRFLP in criminal investigation Victim SuspectA SuspectB SpermDNA From crime scene Matching of DNA obtained from Crime scene Suspects
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   ADVANTAGE:  Criminalinvestigation Matching of suspect DNA with that of crime scene  Database of criminals’ DNA fingerprint Matching of suspect DNA with criminal database CODIS (Combined DNA Index system)  Animal pedigree confirmation Check authenticity of pedigrees of dogs, racing horses  Diagnosis of inherited disorders Any association between DNA fingerprints with genetic diseases?  Identification of dead bodies in natural disaster
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   DISADVANTAGES:  Labourintensive  Expense  Large quantity of DNA needed  Highly sensitive to laboratory changes  Cannot be used across populations nor across species  Can be technically challenging
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  DNA fingerprint