Below are the steps involved in cloning gene sequences using mRNA. Arrange the steps in the appropriate order. 1. The double-stranded cDNA is treated with a restriction endonuclease to generate compatible ends for annealing and ligation.
2) mRNA is isolated from the cell and converted back into double-stranded sequences using reverse transcriptase to generate complementary DNA.
3) The RNA-DNA hybrid is treated with a nuclease to cleave the RNA strand, producing RNA fragments.
4) Reverse transcriptase completes the single-stranded cDNA when it reaches the 5 ^' end of the mRNA transcript.
5) RNA fragments serve as primers for DNA synthesis of the second strand of cDNA using DNA polymerase.

Question

Quizplus · Accepted Answer

The first step involves isolating mRNA and converting it back into double-stranded sequences using reverse transcriptase to generate complementary DNA (Step 2). The next step involves the completion of the single-stranded cDNA using reverse transcriptase (Step 4). The third step involves treating the RNA-DNA hybrid with a nuclease to cleave the RNA strand, producing RNA fragments (Step 3). These RNA fragments then serve as primers for DNA synthesis of the second strand of cDNA using DNA polymerase (Step 5). Finally, the double-stranded cDNA is treated with a restriction endonuclease to generate compatible ends for annealing and ligation (Step 1). Therefore, the correct order is 2, 4, 3, 5, 1, which corresponds to option B.

Below Are the Steps Involved in Cloning Gene Sequences Using