cDNA Library Construction—Order of Steps from mRNA Choose the correct sequential order for constructing a double-stranded cDNA from a eukaryotic mRNA template using oligo-dT priming and tailing methods.
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ABind oligo-dT, treat with reverse transcriptase, digest with RNase, add G residues to the 3' end, bind oligo-dC, treat with DNA polymerase
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BTreat with reverse transcriptase, digest with RNase, add G residues to the 3' end, bind oligo-dC, treat with DNA polymerase, then bind oligo-dT
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CDigest with RNase, add G residues to the 3' end, treat with reverse transcriptase, add G residues again, then treat with DNA polymerase
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DBind oligo-dC, treat with reverse transcriptase, digest with RNase, add G residues to the 3' end, bind oligo-dT, then treat with DNA polymerase
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EBind random hexamers, add A residues to the 5' end, treat with DNA ligase, then add oligo-dG
Answer
Correct Answer: Bind oligo-dT, treat with reverse transcriptase, digest with RNase, add G residues to the 3' end, bind oligo-dC, treat with DNA polymerase
Explanation
Introduction / Context:Complementary DNA (cDNA) synthesis converts processed eukaryotic mRNA into DNA for cloning and expression. A classical method uses an oligo-dT primer to anneal to the mRNA poly(A) tail, followed by first-strand synthesis, RNA removal or nicking, and tailing to prime second-strand synthesis. Correct step order ensures successful, full-length cDNA generation.
Given Data / Assumptions:
- mRNA has a poly(A) tail at its 3' end.
- Reverse transcriptase synthesizes first-strand cDNA using oligo-dT primer.
- RNase (often RNase H) partially degrades RNA in RNA–DNA hybrids.
- Terminal transferase can add homopolymeric dG tails; oligo-dC can then prime the second strand for DNA polymerase.
Concept / Approach:Map the workflow: prime first strand with oligo-dT → synthesize cDNA with reverse transcriptase → remove RNA template → tail the cDNA with dG → anneal oligo-dC → extend second strand with DNA polymerase. This produces double-stranded cDNA ready for cloning adapters or linkers.
Step-by-Step Solution:
Step 1: Bind oligo-dT to the poly(A) tail.Step 2: Use reverse transcriptase to synthesize first-strand cDNA.Step 3: Digest the RNA in the hybrid with RNase H.Step 4: Add dG tails to the 3' end of cDNA with terminal transferase.Step 5: Anneal oligo-dC to the dG tail.Step 6: Extend with DNA polymerase to make the second strand.Verification / Alternative check:Classical cDNA library protocols and kit manuals mirror this sequence, with variations (e.g., second-strand synthesis by RNase H/DNA Pol I method) but the listed tailing order remains valid for homopolymer tailing approaches.
Why Other Options Are Wrong:
- Starting without oligo-dT or misordering tailing and reverse transcription disrupts priming.
- Adding 5' tails or ligase at early steps is not part of this method.
- Binding oligo-dC before first-strand synthesis is illogical since no dG tail exists yet.
Common Pitfalls:Confusing random-primed cDNA approaches with oligo-dT tailing workflows; mixing up dA/dG tailing directions.
Final Answer:Bind oligo-dT, treat with reverse transcriptase, digest with RNase, add G residues to the 3' end, bind oligo-dC, treat with DNA polymerase