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.

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:

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