Question

1. A number of different buffers/components are used in the PCR. Explain the function of each:

a. DNA plasmid

b. Primer pair

c. dNTPs mixture

d. Taq polymerase

2. In our PCR reaction the optimum annealing temperature is 56*C. Explain the general factors that help determine the optimum annealing temperature

Answer 1

Ans- DNA plasmid- served as a template for synthesis of a huge number of copies of a specific DNA segment

Primer pair- short single-stranded DNA fragment complementary to our gene of interest. primers provide a free 3^' OH group To which Taq DNA polymerase adds dNTPs

dNTPs mixture- dNTPs mixture contains dATP, dGTP, dCTP and dTTP . these four nucleotides use as a building block for the synthesis of new strands of DNA. dATP, dGTP, dCTP, and dTTP expand the growing strand of DNA with the help of Taq DNA polymerase.

Taq DNA polymerase- Taq DNA polymerase is thermostable polymerase, which means it can even work at a higher temperature.

The primary function of Taq DNA polymerase to synthesize a new strand of DNA from the existing strand of DNA by adding dNTPs to growing DNA.

Ans 2- factors that help determine the optimum annealing temperature

a- length of primers- higher the length of primer higher the annealing temperature

b- GC content - higher the GC content higher the annealing temperature

The formula for calculating Tm or annealing temprature= 4(G+C) + 2(A+T) ⁰C.