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

A cDNA (complementary DNA) library is a collection of DNA copies made from the mRNA molecules isolated from a cell or tissue type. It represents the genes that are being expressed (transcribed into mRNA) at a particular time. The general steps and enzymes involved in cDNA synthesis and library preparation are: 1. mRNA Isolation: Isolate total RNA and then purify mRNA (e.g., using its poly(A) tail). 2. First-Strand cDNA Synthesis:

• An oligo(dT) primer (complementary to the poly(A) tail) or random primers are annealed to the mRNA.
• Reverse Transcriptase (an RNA-dependent DNA polymerase) synthesizes a single-stranded DNA copy (the first cDNA strand) complementary to the mRNA template. This creates an mRNA-cDNA hybrid.

3. Second-Strand cDNA Synthesis:

• The mRNA strand in the mRNA-cDNA hybrid is typically degraded or removed. This can be done using:
  • RNase H (RNAs H): An enzyme that specifically degrades the RNA strand of an RNA-DNA hybrid.
  • Alkaline hydrolysis (less common now).
• The remaining single-stranded cDNA is then used as a template to synthesize the second complementary DNA strand. This is done using:
  • DNA Polymerase I (often its Klenow fragment, which has polymerase and 3'\(\rightarrow\)5' exonuclease activity but lacks 5'\(\rightarrow\)3' exonuclease activity). Self-priming (hairpin loop formation at the 3' end of the first cDNA strand) or specific priming methods can be used.

This results in double-stranded cDNA (dsDNA). 4. Modification of cDNA ends (for cloning):

• The ends of the dsDNA may need to be made blunt (e.g., using T4 DNA polymerase).
• Linkers or adaptors (short, synthetic DNA sequences containing restriction enzyme sites) may be ligated to the ends of the dsDNA to facilitate cloning into a vector.
• Alternatively, homopolymer tailing can be done using Terminal Deoxynucleotidyl Transferase (Terminal Transferase). This enzyme adds a string of identical nucleotides (e.g., poly(dC)) to the 3' ends of the dsDNA, which can then be annealed to a vector tailed with a complementary homopolymer (e.g., poly(dG)).

5. Ligation into Vector and Library Construction: The modified dsDNA fragments are ligated into a suitable cloning vector (e.g., plasmid, phage), and these recombinant molecules are introduced into host cells (e.g., E. coli) to create the cDNA library. Let's evaluate the sequence in option (a): "Reverse transcriptase, RNAs H, DNA polymerase, Terminal transferase"

• Reverse transcriptase: First-strand synthesis. Correct.
• RNAs H: Degradation of mRNA template after first strand. Correct.
• DNA polymerase: Second-strand synthesis. Correct.
• Terminal transferase: End modification (tailing) for cloning. A possible subsequent step. Correct sequence.

This sequence of enzyme usage is logical and standard for many cDNA library preparation protocols. Other options reorder these in ways that are not standard. For example, DNA polymerase before Reverse Transcriptase makes no sense for starting from mRNA. \[ \boxed{\text{Reverse transcriptase, RNAs H, DNA polymerase, Terminal transferase}} \]