Answer to Question #340222 in Genetics for lilhomosapien

Question

3. Why during the PCR is there a temperature change for certain times and what is the function of these cycles of different temperatures? explain.

• During the PCR, the temperature change for certain times is designed in order to carry out the exponential amplification of the small DNA fragment by chain polymerization.

• Cycles are rendered in a basic three step process which is repeated 25-40 times:

• 1st step: separates the DNA double strand for a period of 15-40 seconds at a temperature of 95º C -97º C.

• 2nd step: designed primers react with the single stranded DNA thus sticking complementary to other bases in a period of 0.5-2min. at a temperature of 40º C- 65º C.

• 3rd step: dNTPs are placed between the primers synthesizing complementary sequence to template DNA strands for a period of 1-2 min. at a temperature of 72º C.

Answer 1

• At the first step at the temperature of 95º C-97º C, DNA denaturation occurs more efficiently, that is, the destruction of hydrogen bonds between nucleotides, followed by separation of two DNA strands. At this temperature, Taq polymerase can work;
• At the second step the temperature range of 40ºС-65ºС is optimal for hybridization of primers with DNA, since, firstly, it does not exceed the optimal temperature for Taq polymerase operation. Secondly, this range includes temperatures that are 5 degrees lower than the so-called melting temperature, which characterizes the temperature at which only half of the primers hybridize;
• At the third stage, at the temperature of 72ºС, DNA synthesis occurs with the greatest efficiency, since this temperature is optimal for the operation of Taq polymerase.