Question

The order for the construction of cDNA fragment from mRNA is to:

• A. bind oligo-dT, treatment with reverse transcriptase, digest with RNase, add G residues to the 3' end, bind oligo-dC, treat with DNA polymerase
• B. treat with reverse transcriptase, digest with RNase, add G residues to the 3' end, bind oligo-dC, treat with DNA polymerase and bind oligo-dT
• C. digest with RNase, add G residues to the 3' end, treat with reverse transcriptase, add G residues to the 3' end and treat with DNA polymerase
• D. bind oligo-dC, treat with reverse transcriptase, digest with RNase, add G residues to the 3' end, bind oligo-dT and treat with DNA polymerase

Hint:

During cDNA synthesis, reverse transcriptase is used to convert mRNA into cDNA, while RNase degrades the RNA template to allow the synthesis of the second DNA strand.

Answer 1

The Correct Option is A

The construction of cDNA from mRNA involves the following steps:
(1) Bind oligo-dT: This binds to the poly-A tail of the mRNA.
(2) Treatment with reverse transcriptase: Reverse transcriptase synthesizes the complementary DNA (cDNA) strand using the mRNA as a template.
(3) Digest with RNase: This enzyme degrades the RNA template after the first cDNA strand is synthesized.
(4) Add G residues to the 3' end: Guanine residues are added to the 3' end to allow the binding of oligo-dC. 5. Bind oligo-dC: This binds to the G residues added earlier. 6. Treat with DNA polymerase: DNA polymerase synthesizes the second strand of the cDNA. Thus, the correct order is: Option 1: bind oligo-dT, treatment with reverse transcriptase, digest with RNase, add G residues to the 3' end, bind oligo-dC, treat with DNA polymerase.
Conclusion: The correct answer is Option 1 as it follows the proper sequence of steps in constructing cDNA from mRNA.