Question

1.In terms of primer design, what can be done (at least in the case of most genes) to ensure that any amplification of contaminating genomic DNA is not mistaken for amplification of cDNA in RT-PCR reactions? Explain why this works.

b.What properties make actin a suitable housekeeping gene for expression analysis? What is another example of a housekeeping gene?

c.Eukaryotic cells contain several classes of RNA, of which messenger RNA (mRNA) is just one. What are these classes of RNA and what proportion of the total RNA does each of them typically comprise?

2.. From what source were reverse transcriptases originally obtained from ? (now they are made recombinantly)? Briefly describe the process of reverse transcription.  

Answer 1

2.Ans-
Reverse transcriptases were obtained from two RNA tumor viruses such as R-MLV and again RSV in 1970.

Reverse transcription is a process to produce complementary DNA (cDNA) from an RNA template by using reverse transcriptase (RT) enzyme.
1.   Lysyl tRNA primer involves to hybridize the complementary part of the virus RNA genome and called the primer binding site or PBS.
2.   Reverse transcriptase enzyme then adds DNA nucleotides onto the 3' end of the primer to synthesize DNA complementary to the U5 is a non-coding region and R region of the viral RNA.
3.   The U5 and R regions are degraded by RNAseH on the 5’ end of the RNA.
4.   The PBS moves to the 3’ end of the viral genome and synthesized new DNA strands that hybridize to the complementary R region on the RNA.
5.   The complementary DNA which is added in 2nd step is further extended, and then the majority of viral RNA is degraded by RNase H, leaving only the PP sequence.
6.   After degradation of a majority of viral RNA by RNAseH, the remaining part of viral RNA use as a primer is involved to synthesize the second DNA strand
7.   Then PBS jump from the second strand hybridizes with the complementary PBS on the first strand.
8.   Both strands are extended to produce a double-stranded DNA copy of the original viral RNA genome that incorporated into the host's genome with the help of integrase enzyme.