![]() ![]() Both primers have to be matched in their G+C content and should have similar annealing temperature. The principle features o ideal primers are that these not only have to be complementary to the flanking sequence of the target DNA but must not be self-complementary otherwise they will form primer dimmers. Then thermo stable DNA polymerase called Taq polymerase from thermophilic bacterium Thermus aquaticus with optimum temperature of 72☌ and can also survive after prolonged exposure to 96☌ provided the means to automate the reaction. One problem with the early PCR reaction was the temperature needed to denature the DNA also denatures the DNA polymerase. Thus in another round not only parental target strand but also newly synthesized strands also act as template DNA. It is important to note that since the new strand extends beyond the target DNA, they will contain a region next to their 3′-end that is complimentary to the other primer. The third step, DNA synthesis or extension is carried out by a thermo stable DNA polymerase, most commonly Taq DNA polymerase.ĭNA synthesis proceeds from both primers until new strands have been extended along and beyond the target DNA to be amplified. The annealed oligonucleotide acts as a primer since they provide free 3′-end hydroxyl group to DNA polymerase. The second annealing step allows the hybridization of the two oligonucleotide primers, present in excess to bind to their complimentary sites which flank the DNA. Reactions that are not optimized may give rise to other products along with amplified target sequence or even may not produce amplified product. The precise temperature is critical and each PCR system has to be defined and optimized. The temperature is then cooled to between 40-60☌. In first cycle the double stranded template DNA strand is first denatured by heating the reaction to above 90☌ so that the region to be specifically amplified can be made accessible. Each of the three steps are repeated 30-40 times or cycles. ![]()
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