Question

The discovery of reverse transcriptase was worthy of the 1975 Nobel Prize as both destroyed the central dogma and was a key advancement in the field of molecular biology.

A. When making cDNA libraries from mRNA, we often run into the 5’ end problem, where the 5’ end of an mRNA is potentially undersampled/underrepressented. This prevents us from identifying information at the 5’ end reliably, including the transcriptional start site for the gene encoding the mRNA. Describe how we might solve the 5’ end problem for a polyadenylated (polyA) mRNA

B. We may have to use random hexamer primers to initiate the reverse transcriptase reaction. How might the use of random hexamers cause a 3’ end problem?

Answer 1

A. To solve the 5' end problem a random hexamer primer should be used for the 1st strand cDNA synthesis. This hexamer primer will hybridize to various positions of the mRNA and will synthesize various fragments of 1st strand cDNA having different lengths. This synthesis is done by Reverse transcriptase. After 1st strand synthesis, Reverse transcriptase is inactivated and then poly-A polymerase is added. Poly-A polymerase now will add a poly-A tail to the 3' end of the 1st strand of the cDNA. This is followed by RNase H treatment, which degrades mRNA strands. Now all of the 1st strands of cDNA have poly-A tails. Now PCR has to be done in the presence of oligo dT primer and random hexamer primer. This PCR will amplify the cDNA fragments and all cDNA should posses the complementary sequence of the 5' end of the mRNA. This method is known as 5' RACE.

B. Random hexamer primer will find its complementary sequence to any region of the mRNA. It may hybridize to far away from the 3' poly-A tail. So, the method which is described in A, have all the cDNAs lacking the 3' sequence information of the mRNA. This creates a 3' end problem.