Q.52 Reverse transcriptase is used for synthesizing cDNA from RNA.
Which of the following option represents the correct order of the given steps?

A. Annealing of the oligo dT primer to mRNA
B. DNA polymerase I and dNTPs form dsDNA
C. Reverse transcriptase forms the complementary single stranded DNA
D. mRNA is degraded with alkali
E. Downstream analysis of dsDNA

Choose the correct answer from the options given below:

1. A, C, D, B, E
2. A, E, D, B, C
3. D, A, C, B, E
4. B, C, A, E, D

   Reverse transcriptase synthesizes cDNA from mRNA in a specific sequence starting with primer annealing, ensuring accurate first-strand DNA formation before second-strand synthesis and analysis.​​

   Question Breakdown

   The query tests the standard cDNA synthesis protocol using reverse transcriptase, oligo dT primer, and enzymes like DNA polymerase I. Steps involve priming mRNA, reverse transcription to ss cDNA, RNA removal, dsDNA formation, and final use.

   Step-by-Step Explanation

   • A. Annealing of oligo dT primer to mRNA: First step; oligo dT binds the poly-A tail of mRNA at 42-50°C, providing the 3′-OH start for reverse transcriptase.​

   • C. Reverse transcriptase forms complementary single-stranded DNA: Second; enzyme uses dNTPs to extend primer, creating RNA-cDNA hybrid (first-strand cDNA) in 5′-3′ direction.​​

   • D. mRNA is degraded with alkali: Third; NaOH or RNase H removes RNA strand, leaving ss cDNA.​

   • B. DNA polymerase I and dNTPs form dsDNA: Fourth; Pol I’s nick-translation or RNase H nicking enables second-strand synthesis, yielding ds cDNA.​

   • E. Downstream analysis of dsDNA: Final; cloning, PCR, sequencing, etc..​

   Other options fail: A,E,D,B,C skips core synthesis; D,A,C,B,E starts with degradation (impossible sans cDNA); B,C,A,E,D begins with Pol I (needs ss cDNA first).

   Correct Sequence

   A, C, D, B, E matches the protocol.​​

   Reverse transcriptase cDNA synthesis steps order is crucial for molecular biology exams like GATE, converting mRNA to ds cDNA via oligo dT priming and enzymatic steps. This protocol enables gene expression studies without genomic DNA introns.​

   Core Steps Detailed

   1. A. Annealing of oligo dT primer to mRNA: Poly-A tail binding initiates at ~42°C.​

   2. C. Reverse transcriptase forms ss cDNA: MMLV-RT extends primer using dNTPs, 30-60 min at 42-50°C.

   3. D. mRNA degraded with alkali: NaOH/RNase H removes RNA, yielding pure ss cDNA.​

   4. B. DNA pol I + dNTPs form dsDNA: Second strand via nick translation.​

   5. E. Downstream dsDNA analysis: qPCR, cloning, sequencing.​

   Why This Order?

   Primer (A) precedes RT (C); RNA must degrade (D) before second strand (B); analysis (E) last. A-C-D-B-E is standard.​

   Exam Tips

   Visualize: mRNA → hybrid → ss cDNA → ds cDNA. Variants use random primers but oligo dT is classic for poly-A mRNA.​