Question

The correct order of steps in Polymerase Chain Reaction (PCR) is
A. Denaturation, Extension, Annealing
B. Extension, Denaturation, Annealing
C. Denaturation, Annealing, Extension
D. Annealing, Extension, Denaturation

Answer 1

Hint: PCR is also called "molecular photocopying," it is a fast as well as inexpensive process for the "amplification" or copying small segments of DNA. The process requires significant amounts of DNA samples. This is used for molecular or genetic analyses, studying isolated pieces of DNA.

Complete Answer:
The sample is first heated to amplify a fragment of DNA using PCR so that the DNA denatures, or separates into two single-stranded DNA pieces. First an enzyme called Taq polymerase synthesizes two new DNA strands, using templates as the original strands. This method results in the replication of the original DNA, with one old and one new strand of DNA comprising one of the new molecules. Then each one of these strands can be used to make two new copies of DNA, which keeps on repeating. The process of denaturing and synthesizing new DNA is repeated up to 30 or 40 times, resulting in more than one billion precise copies of the original segment of DNA.

The following three steps consist of polymerase chain reaction or PCR:
1. Denaturation - When heated to 92°C, the two DNA strands of template DNA separate from each other because H-bonds between nucleotide base pairs are broken to separate the double helical structure.
2. Annealing - The two primers anneal each of the single strands of DNA at the end of 3’OH.
3. Extension - By adding nucleotides to form full strands of DNA, the primers are extended by DNA polymerase. The sequence of steps therefore consists of denaturation, annealing, extension.

So, option C is the correct answer.

Note: For the activity of DNA polymerase $Mg^{2+}$ is required. The whole PCR cycling process is automated and can be finished in just a few hours. The process of PCR is operated by a thermocycler where the reaction temperature can be changed every few minutes.