31] 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. Central to this process are the reverse transcriptases. This chapter focuses on the reverse transcription of mRNA by Thermus aquaticus DNA polymerase followed by polymerase chain reaction (PCR) amplification . T.aquaticus DNA polymerase has been shown to possess reverse transcriptase activity and this enzyme (or engineered variants and other thermostable polymerases) should prove extremely useful for the analysis of mRNA structure. Escherichia coli DNA polymerase has the potential to use RNA as a template for the synthesis of DNA in vitro and have alluded to the possibility that Thermus aquaticus DNA polymerase may possess reverse transcriptase activity. The advent of the PCR and the subsequent commercial availability of thermostable DNA polymerases permitted an investigation into the utilization of T. aquaticus DNA polymerase to copy RNA into complementary (cDNA).