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Reverse transcription with thermostable DNA polymerase-high temperature reverse transcription

  • US 5,310,652 A
  • Filed: 06/24/1993
  • Issued: 05/10/1994
  • Est. Priority Date: 08/22/1986
  • Status: Expired due to Term
First Claim
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1. A homogeneous single addition method for copying a target RNA molecule in a sample to produce a cDNA, and then amplifying the resulting cDNA, the method comprising the steps of:

  • (a) treating said sample in a reaction mixture comprising a first and second primer, wherein said first primer is sufficiently complementary to said target RNA to hybridize therewith and initiate synthesis of a cDNA molecule complementary to said target RNA, and said second primer is sufficiently homologous to said target RNA to hybridize to said cDNA and initiate synthesis of an extension product, and a thermostable DNA polymerase having reverse transcriptase activity in the presence of all four deoxyribonucleoside triphosphates, in an appropriate buffer, wherein said buffer comprises Mn+2, at a temperature sufficient for said thermostable DNA polymerase to initiate synthesis of an extension product of said first primer to provide a cDNA molecule complementary to said target RNA;

    (b) treating said reaction mixture at an appropriate temperature to provide single-stranded cDNA;

    (c) treating said reaction mixture at an appropriate temperature for said thermostable DNA polymerase to initiate synthesis of an extension product of said second primer to provide a double-stranded cDNA molecule; and

    (d) amplifying the double-stranded cDNA molecule of step (c) by a polymerase chain reaction.

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