Question

The correct sequence of enzymes used for cDNA library preparation is

• A. Reverse transcriptase, RNAs H, DNA polymerase, Terminal transferase
• B. RNAs H, DNA polymerase, Reverse transcriptase, Terminal transferase
• C. DNA polymerase, Terminal transferase, Reverse transcriptase, RNAs H
• D. Reverse transcriptase, Terminal transferase, RNAs H, DNA polymerase

Hint:

• cDNA synthesis from mRNA: 1. Primer annealing (e.g., oligo(dT)). 2. First strand synthesis by Reverse Transcriptase}. 3. mRNA degradation by RNase H}. 4. Second strand synthesis by DNA Polymerase}.
• Further steps for library construction often involve modifying cDNA ends (e.g., adding linkers, tailing with Terminal Transferase}) and ligating into vectors.

Answer 1

The Correct Option is A

To prepare a cDNA library, a specific sequence of enzymatic reactions is used to transcribe RNA into complementary DNA. The correct sequence involves the following enzymes:

1. Reverse Transcriptase: This enzyme is responsible for synthesizing a complementary DNA (cDNA) strand from the RNA template.
2. RNase H: This enzyme degrades the RNA strand of the RNA-DNA hybrid, leaving behind the single-stranded cDNA.
3. DNA Polymerase: This enzyme synthesizes the second DNA strand, creating a double-stranded DNA molecule from the single-stranded cDNA.
4. Terminal Transferase: This enzyme can be used to add nucleotides to the 3' ends of the DNA strands, providing a homopolymer tail which is useful for cloning procedures.

The correct sequence of enzymes used for cDNA library preparation is: Reverse transcriptase, RNAs H, DNA polymerase, Terminal transferase.