Question

What is the sequence of the PCR process?

Answer 1

1. Denaturation of the template into single strands.
2. Annealing of primers to each original strand for new strand synthesis.
3. Extension of the new DNA strands from the primers.

Answer 2

PCR, short for polymerase chain reaction, is a technique employed to study specific segments of DNA. The process of PCR unfolds in three distinct steps:

Step 1: Denaturation

• In this initial stage, a double-stranded DNA sample containing the gene of interest is utilized.
• The sample is subjected to high temperatures ranging from 94 to 98 degrees Celsius for approximately 20 to 30 seconds.
• The elevated temperature disrupts the hydrogen bonds holding the DNA base pairs together, leading to the separation of the two strands.

Step 2: Annealing

• Following denaturation, the temperature is reduced slightly.
• Primers, short DNA sequences with complementary base pairs to the DNA template, are introduced.
• Additionally, DNA polymerase enzyme is incorporated into the mix, facilitating the hybridization of primers to the DNA template and initiating DNA synthesis.

Step 3: Extension

• During this phase, a thermostable Taq DNA polymerase enzyme is added to the sample at temperatures ranging from 75 to 80 degrees Celsius.
• The Taq polymerase extends the primers by adding nucleotides in a 5’ to 3’ direction, synthesizing complementary DNA strands.
• This process is repeated in cycles, typically 20 to 30 times, allowing for the amplification of the DNA target region by up to a billion-fold.