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High temperature reverse transcription using mutant DNA polymerases

  • US 20020012970A1
  • Filed: 03/30/2001
  • Published: 01/31/2002
  • Est. Priority Date: 04/18/2000
  • Status: Active Grant
First Claim
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1. A method for reverse transcribing an RNA, that comprises:

  • (a) providing a reverse transcription reaction mixture comprising said RNA, a primer, a divalent cation, and a mutant thermoactive DNA polymerase, wherein said mutant DNA polymerase is characterized in that i) in its native form said DNA polymerase comprises an amino acid sequence that is SEQ ID NO;

    1;

    ii) the amino acid at position 4 of said amino acid sequence is mutated in comparison to said native sequence to an amino acid other than E, A, G, or P; and

    (b) treating said reaction mixture at a temperature sufficient for said mutant DNA polymerase to initiate synthesis of an extension product of said primer to provide a cDNA molecule complementary to said RNA.

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