6 Reverse Transcriptase

This chapter deals with the purification, enzymatic activities, mechanism, and research applications of reverse transcriptase to molecular biology. Reverse transcriptase transcribes RNA into complementary DNA (cDNA). It exhibits three enzymatic activities—namely, (1) conversion of RNA into DNA (RNA-dependent DNA polymerase), (2) conversion of single-stranded DNA to double-stranded DNA (DNA-dependent DNA polymerase), and (3) selective removal of RNA moiety from an RNA–DNA hybrid (RNase H). In addition, reverse transcriptase also exhibits DNA endonuclease and swivelase activities. Reverse transcriptase resides in the core of the virion and can be easily solubilized by nonionic detergents like Nonidet P40 or Triton X-100. The detergent-lysed virions essentially manifest all three major activities associated with purified reverse transcriptase and yields homogeneous preparations of reverse transcriptase relatively fast and in fairly high yields. Some of the applications of reverse transcriptase in molecular biology include (1) synthesis of cDNA, (2) synthesis of double stranded DNA, (3) and end labeling.