RNA‐Dependent DNA Polymerases

George Tzertzinis1, Stanley Tabor2, Nicole M. Nichols1

1 New England Biolabs, Ipswich, Massachusetts, 2 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 3.7
DOI:  10.1002/0471142727.mb0307s84
Online Posting Date:  October, 2008
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Abstract

Reverse transcriptases (RTs) are multifunctional enzymes, but are mainly used as RNA‐directed DNA polymerases in first‐strand cDNA synthesis. Specifically, oligodeoxynucleotides are used as primers for extension on RNA templates. The DNA synthesized from an RNA template is referred to as complementary DNA (cDNA) and is often used as a template for PCR or converted to dsDNA for cloning. This unit describes appropriate reaction conditions for RTs from Moloney murine leukemia virus (MMLV) and avian myeloblastosis virus (AMV), along with applications such as synthesizing cDNA, 3′ fill‐in reactions, and labeling the 3′ terminus of DNA fragments with 5′ protruding ends, and DNA sequencing. Curr. Protoc. Mol. Biol. 84:3.7.1‐3.7.4. © 2008 by John Wiley & Sons, Inc.

Keywords: first‐strand cDNA; RT‐PCR; MMLV RT; AMV RT; fill‐in reaction

     
 
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Table of Contents

  • Enzyme: Reverse Transcriptase
  • Literature Cited
  • Figures
     
 
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Materials

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Figures

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Literature Cited

Literature Cited
   Blain, S.W. and Goff, S.P. 1993. Nuclease activities of Moloney murine leukemia virus reverse transcriptase. J. Biol. Chem. 268:23586‐23592.
   Krug, M.S. and Berger, S.L. 1989. Ribonuclease H activities associated with viral reverse transcriptases are endonucleases. Proc. Natl. Acad. Sci. U.S.A. 86:3539‐3543.
   Roberts, J.D., Preston, B.D., Johnston, L.A., Soni, A., Loeb, L.A., and Kunkel, T.A. 1989. Fidelity of two retroviral reverse transcriptases during DNA‐dependent DNA synthesis in vitro. Mol. Cell. Biol. 9:469‐476.
   Roth, M.J., Tanese, N., and Goff, S.P. 1985. Purification and characterization of murine retroviral reverse transcriptase expressed in Escherichia coli. J. Biol. Chem. 260:9326‐9335.
   Schatz, O., Mous, J., and Le Grice, F.J. 1990. HIV‐1 RT‐associated ribonuclease H displays both endonuclease and 3′‐5′ exonuclease activity. EMBO J. 9:1171‐1176.
   Tabor, S. and Richardson, C.C. 1987. DNA sequencing with a modified T7 DNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 84:4767‐4771.
   Taylor, J.M., Illmensee, R., and Summers, J. 1976. Efficient transcription of RNA into DNA by avian sarcoma virus polymerase. Biochem. Biophys. Acta 442:324‐330.
   Verma, I.M. 1977. Reverse transcriptase. In The Enzymes, Vol. 14A (P.D. Boyer, ed.) pp. 87‐104. Academic Press, New York.
   Whiting, S.H. and Champoux, J.J. 1994. Strand displacement synthesis capability of Moloney murine leukemia virus reverse transcriptase. J. Virol. 68:4747‐4758.
   Zagursky, R.J., Baumeister, K., Lomax, N., and Berman, M.L. 1985. Rapid and easy sequencing of large linear double‐stranded DNA and supercoiled plasmid DNA. Gene Anal. Tech. 2:89‐94.
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