Question

in pcr practical, Briefly describe the reaction mechanism leading to the synthesis of a new DNA strand by Taq DNA polymerase. In your answer include:

i. The mechanism of the synthesis reaction,

ii. The direction of the synthesis reaction

iii. The direction in which sequence information is read from the template strand.

Answer 1

The polymerase chain reaction (PCR) is a technique in molecular biology which is used to amplify specific, target DNA fragments from low quantities of initial source DNA or RNA (after the formation of complementary DNA (cDNA) with the help of reverse transcription in case of RNA). Even in the contamination, the target sequence is amplified.

Steps involve in PCR

Initial Denaturation:

The reaction temperature is increased to 95 °C and the reaction is incubated for 2–5 min (based on complexity) to ensure that all complex present as double-stranded DNA (dsDNA) molecules are converted into single strands.

Cycling:

1. Denaturation: The reaction temperature is increased to 95 °C, which melts all dsDNA into single-stranded DNA (ssDNA). It breaks down the hydrogen bonds between the strands.
2. Annealing: The temperature is lowered to approximately 5 °C below the melting temperature (T_m) of the primers that is 45–60 °C so that the primers binds to the template. There are two primers: forward and reverse primer.
3. Extension: The temperature is increased to 72 °C, which is optimum for Taq polymerase because DNA polymerase does not work at this high-temperature that includes the addition of the nucleotide to the primer for extension complementary to the template. The Taq polymerase bind the nucleotide in 5' to 3' direction.

Repeat: The bound strands are again denatured and then new priers are bind to the single strands and then extended wit the help of Taq polymerase. The cycles are repeated approximately 35 cycles.