Question

Exercise 20: What are the steps of the PCR-RFLP experiment? When is the DNA amplified? How is the DNA amplified? Understand the process of PCR (steps). When is the DNA cut with the restriction enzyme? What is the restriction enzyme in tas this exercise?

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es Steps in PCR-RELP expenment- Isolation of DNA Invitro amplify isolated ONA by Performing per.. © Digestion with restriction enzyme CONA fragmentation) 4 Perform Gel Electrophoresis for separation of DNA fragement's. ® Analysis Q. When is the DNA ampliry? Ans per has 3 steps in each eyele - - Denaturing ② Annealing ④ Extending (DNA Synthesis). After Successfully completion of these 3 steps in each cycle you DNA will be amplity. Q. How per is work process of per Ans. Per is a invitro method of DNA amplification . All you need for amplification - Isolated DNA as template for DNA Synthesis Primers (Short nucleotide sequence about 15-25 nucleotides) Primers are specific sequence that bind to template DNA for initiating amplification .. * Heat Stable DNA polymerase ( Taq polymerase) DATP C Deoxyribonucleotides) Ô Buffer, * Steps and Process of per. ④ Denaturing: typically at about 93²980 (varies for different DNA). At this temperature DNA is heated to separate into two single- Single Strand. This single strand DNA Used as template. @ Annealing : at about so-7o e depending upon muiting . temperature (Tm) Annealing temperature must be low enough to enable Cination between primer and template.

☺ Extending (DNA Synthesis) : typically at about 70-75°C . The Taq polymerase cheal stable) extend the DNA spand by adding duitp. Present in the mineture. After these three steps Constitute one cycle of the PER Amplication. Q. When is the ONA cut with restriction enzyme? Ars In for- RALP. require large amount of Ona, isolation that much DNA for RFLP insis time consuming. To overcome this problem, PeR could be use to amplify the sufficient DNA for RFLP. Hence, we call PCR-RFLp. - After Performing per, the DNA cut with restriction enzyme. Ans: Restriction enzyme, cuts the double stranded DNA into fragements after a recognizing specific nucleotide Sequences. The short sequences recognized by restriction enzyme - known as restriction site. (usually about 4-8 nucleotide) » In this exercise, the amplity DNA digest with restriction enzyme at specific restriction site. - After this put this fraganeufs in gel efeeprophoren is . for further analysis,