The correct sequence of steps involved in polymerase chain reaction (PCR) are:
A. DNA polymerase is used to extend the primers using oligonucleotides.
B. Desired DNA fragments are denatured.
C. Amplified fragments are ligated with the vector for cloning.
D. Oligonucleotides are added
Choose the correct answer from the options given below:
Answer & explanation
Correct answer: option 4
The correct answer is Option (4) → B, D, A, C
B. Desired DNA fragments are denatured. — first. Heating separates the two strands of the DNA.
D. Oligonucleotides are added — second. The short primers bind (anneal) to their matching sequences on the single strands.
A. DNA polymerase is used to extend the primers using oligonucleotides. — third. The thermostable polymerase extends the primers; repeating these steps amplifies the DNA about a billion times.
C. Amplified fragments are ligated with the vector for cloning. — last. The amplified DNA can then be joined to a vector for further cloning.
NCERT Extract:
Amplification of Gene of Interest Using PCR – “PCR stands for Polymerase Chain Reaction. In this reaction, multiple copies of the gene (or DNA) of interest is synthesised in vitro using two sets of primers (small chemically synthesised oligonucleotides that are complementary to the regions of DNA) and the enzyme DNA polymerase. The enzyme extends the primers using the nucleotides provided in the reaction and the genomic DNA as template. If the process of replication of DNA is repeated many times, the segment of DNA can be amplified to approximately billion times, i.e., 1 billion copies are made. Such repeated amplification is achieved by the use of a thermostable DNA polymerase (isolated from a bacterium, Thermus aquaticus), which remain active during the high temperature induced denaturation of double stranded DNA. The amplified fragment if desired can now be used to ligate with a vector for further cloning (Figure 9.6).”