Question

Which of the following enzyme is used in second strand cDNA synthesis

• A. RNase A
• B. S1 nuclease
• C. RNase H
• D. AMV-RT

Hint:

RNase H is a highly specific ribonuclease that digests only the RNA strand in RNA-DNA hybrids, leaving double-stranded DNA and single-stranded RNA unaffected.

Answer 1

The Correct Option is C

Step 1: Understanding the Concept:
Complementary DNA (cDNA) synthesis involves converting a single-stranded mRNA template into double-stranded DNA. After synthesizing the first DNA strand using reverse transcriptase, the original RNA template must be processed to allow synthesis of the second DNA strand.

Step 2: Detailed Explanation:
- In the Gubler-Hoffman method for second-strand synthesis:
1. The enzyme RNase H is added to the RNA-DNA hybrid.
2. RNase H specifically cleaves the phosphodiester bonds of the RNA strand within the hybrid, partially digesting it.
3. This leaves behind short RNA fragments that remain annealed to the first-strand DNA, acting as primers.
4. DNA Polymerase I then uses these RNA primers to synthesize the second DNA strand, replacing the RNA primers with DNA.
5. DNA Ligase seals the nicks to complete the double-stranded cDNA.
- S1 nuclease is used to cleave hairpin loops, but it is not the enzyme that initiates second-strand synthesis.

Step 3: Final Answer:
The enzyme used in second strand cDNA synthesis is RNase H.