Question

Now. you should be able to answer the following questions: • How the amplification will be done? - How you will determine your target sequence? How the amplification will be specific for certain segment? What are the requirements to carry PCR?

Answer 1

(8.1). How the amplification will be done? Ans - Polymerase Chain Reaction (PCR) is a malecular biology techniques by which a particular segment of DNA is amplified and producer multiple copies of DNA. To amplify a segment of DNA by using PCR, the sample is first heated at 94'e for breaking Of ds DNA ato two single strandool DNA, BADO Afuerunt a couple of minutes temperatune is lowered to 55-65°c. At this temperature, primens binding take place to their complementary single strandeel templates. After annealing Sunday 14! of primem, Sample is heated to 72C for some times. During this time DNA palymerase adors nucleatiden ato the primer and synthesize a new DNA strand using the template. After 30-35 cycles in thermal cycler, millionas copies of DNA are produced (8.2) How you will determine terget sequence of your's Ans: In quantitative real time PCR (RT-PCR), it is possible to measure the amount of DNA produced during each per cycle. Here fluorescent dye on probe in used to label the double stranded DNA molecule These fluorescent dye binds to the new DNA copies, that makes pealtime monitering of DNA of interest

2013 MARCH 10 11 12 13 14 April 2019 15 Monday Agarose gel electrophoresis is done for detection an analysis of per product. so, we can also determine target sequence by using simple agarose gel eleetophoresin. (8.3) How the amplification will be specific for certain Segment ? ARS: PCR in very sensitive, it can amplify specific region target sequence) from a. mixture of many DNA sequences. The primem. used for targeting sequence is specific food in per If annealing temperature in too low, nonspecific Tuesday binding occur. So, annealing temperatua in 5°C lower than tm Value of primer so that especific binding happens. For chemically synthe- sized primem of zont are used. specific primem only hybridize to their exact comple- mentary sequence on the template strand. 8.4) What are the moquirement to camy PCR ?, Ans. Various components required for PCR, which are given below - Total reaction valume - 25 ML (250) O lox buffer - 2.5 ②25 mm ngela-1 ut ② 2.5mm dNTPs-0.5MM

6000 (4) Fondand primes - 0.5 mL 5 Reveme Primera 0.5 ur. 6 Tag DNA Polymegase - 2M Đd H20-25-7 - 18 u Page 2 Stora Denaturation Primer i Temperature (°C) toying ; 5 min 30 sec extension 728 728 1 55-65°C/30 sec min 40 1 © Annealing L -cycle #1 - Time - Thursday Denaturation - Double stranded DNA F break Annealing - Binding of primers takes place Extension - DNA parymerase synthesize a new strand.