Question

What are the different conditions that were used in Polymerase Chain Reaction

Answer 1

Different conditions that were used in Polymerase Chain Reaction:

• PCR stands for Polymerase Chain Reaction.
• It is a method used for cloning DNA by copying a specific DNA sequence via in vitro reaction and amplifying the target DNA sequence present in small quantities.
• The conditions used in PCR are :

1. Presence of  Thermostable DNA polymerase enzyme: It extends the primers using nucleotides provided in the reaction, and using the genomic DNA as a template. The thermostable enzyme remains active in the high temperature required for DNA denaturation. It is isolated from a bacterium, Thermus aquaticus.
2. Mg^2+: It is an important cofactor for DNA polymerase.
3. Two sets of primers: Two short single-stranded DNA oligonucleotide primers (typically 20 nucleotides long), one complementary to  the 5′ end of one strand of target DNA to be amplified and  another primer complementary to the opposing strand of  target DNA at its 3′ end. They are used for binding and extension of target DNA sequence.
4. Different range of temperatures for different steps:

• Denaturation: 92°C-95°C for about 1 minute.
• Hybridisation/Annealing: 45°C- 65°C
• Extension: 65°C- 75°C