Question

What are the components of a PCR reaction? How would you calculate the final concentrations of the components in the PCR reaction and in the restriction digest? What happens during each step of a PCR cycle? What parameters vary amount restriction enzymes?

Answer 1

PCR stands for Polymerase Chain Reaction. it is a molecular technique that is used to amplify the target gene sequence.

Components of PCR: the chemical components invovled in the reaction tube are

1. DNA template
2. taq DNA polymerase enzyme
3. primers
4. dNTPs ( deoxy nucleoetides like dATPs, dCTPs, dTTPs, dGTPs.)
5. reaction buffer and Mgcl2.

DNA template is the target DNA molecule. taq DNA ploymerase is the enzyme that is used to amplify the DNA invitro and it works at higher temperatures.

primers are the single strand oligo nucleotides that are used to bind to the target DNA (single strand) and provide dNTPs for the synthesis of new daughter DNA molecules.

reaction buffer is used to optimize the reaction mixture. Mgcl2 is the co-factor that is used to enhance the amplification reaction.

the calculation of the final concentrations of the components of the PCR reaction is done using the type of enzyme used and buffer used. the concentration and final volume is taken as the parametre and reaction mixture is prepared.

PCR cycle consists of three steps mainly.

1. denaturation
2. annealing
3. extension.

at denaturation step, the entire DNA get denaturated at 94-98°C. the hydrogen bonds present between the complimentary bases get broken due to high temperatures and leads to denaturation. thus 2 single strand DNA molecules are formed.

in the annealing step; the 2 different primers like forward and reverse primers are annealed at single strand DNA templates. these primers cover the target DNA region. the annealing temperature will be from 55-65°C

in extension step; the temperature is raised to 72°C for the optimal activity of the taq DNA polymerase enzyme. at this step, the new daughter DNA molecules are synthesized. in some there will be final elongation step will be performed in order to make sure that remaining single strand DNA molecule get extended.

thus, the target DNA molecule get doubled. in the second cycle; the 2 daughter DNA molecules get denaturated and thus the target DNA is amplified in several copies.

like wise; the PCR is set for 30 to 35 cycles in order to obtain millions of target DNA molecules.

the paramters like size of the traget DNA molecule ( in terms of base pairs), the amount of the DNA template, type of target DNA molecule having either blunt DNA or staggered DNA ends, temperature, co factors and buffers are used differently for different kind of restriction enzymes. these will differ with different restriction enzymes.