46 - Reverse Transcription of mRNA by Thermus aquaticus DNA Polymerase followed by Polymerase Chain Reaction Amplification

Abstract

Publisher Summary The structural analysis of messenger RNA (mRNA) plays an important role in the examination of gene structure and expression. The key step is the reverse transcription of mRNA into complementary DNA (cDNA), which subsequently can be cloned and analyzed. One difficulty that can prevent the efficient copying of RNA sequences into cDNA is the ability of single-stranded RNA to adopt stable intramolecular stem-loop structures. This can cause pausing of the reverse transcriptase and thus result in premature termination of the reverse transcript. One solution to this problem would be to perform the reverse transcription reaction at an elevated temperature at which such structures would be disrupted, allowing the enzyme to copy through to the 5′ end of the RNA molecule. This chapter describes an experiment to reverse transcribe mRNA into cDNA with primers specific for the human glucose-6-phosphate dehydrogenase gene. Polymerase possess reverse transcriptase activity and this enzyme is useful for the analysis of mRNA structure.