Methods in Molecular Biology • 16 Enzymes of Molecular Biology

CHAPTER 6

Reverse Transcriptase (EC 2.7.7.49)

The Use of Cloned Moloney Murine Leukemia Virus

Reverse Transcriptase to Synthesize DNA from RNA

Gary F. Gerard and James M. D'Alessio

1. Introduction
   The conversion of mRNA into cDNA is the essential first step in the
   study of eukaryotic cell products expressed from cloned genes. The
   key enzyme used first in this process, retroviral RNA-directed DNA
   polymerase (reverse transcriptase), catalyzes the synthesis of a DNA
   copy of an RNA template in the presence of a suitable primer. Reverse
   transcriptase (RT) was discovered in 1970 (1,2) and was first used to
   copy a eukaryotic cell mRNA in 1971 (3-5); the first cDNA clones
   prepared using RT were reported in 1976 (6). Until recently, the only
   enzyme available was purified from avian myeloblastosis virus (AMV).
   The overall quality and consistency of commercially available AMV
   RT preparations have improved dramatically in the last six years,
   although considerable differences in performance characteristics still
   exist among enzyme preparations from different commercial suppli-
   ers (7). In addition, parameters for carrying out first-strand cDNA
   synthesis from poly(A) ÷ mRNA populations with AMV RT have been
   optimized thoroughly (8); standard optima exist for pH, nucleotide
   concentration, and monovalent and divalent cation concentration; also
   essential stimulatory additives (such as sodium pyrophosphate and
   spermidine-HC1) have been identified. Furthermore, conditions have

From: Methods in Molecular Biology, VoL 16: Enzymes of Molecular Biology

Edited by: M. M. Burrell Copyright ©1993 Humana Press Inc., Totowa, NJ

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