Reverse Transcriptase (EC 2.7.7.49): The Use of Cloned Maloney Murine Leukemia Virus Reverse Transcr

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 suppliers (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-HCl) have been identified. Furthermore, conditions have been established for proper handling and storage of the enzyme.