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Methods of using improved polymerases

  • US 8,476,045 B2
  • Filed: 07/27/2012
  • Issued: 07/02/2013
  • Est. Priority Date: 11/28/2001
  • Status: Expired due to Term
First Claim
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1. A method of performing a quantitative real-time polymerase chain reaction on a target nucleic acid present in a solution that comprises a DNA-binding fluorescent dye, the method comprising:

  • (a) contacting the target nucleic acid with a thermally stable DNA polymerase, wherein the DNA polymerase is joined to a sequence non-specific double-stranded nucleic acid binding domain that specifically binds to polyclonal antibodies generated against either Sso7d (SEQ ID NO;

    2) or Sac7d (amino acids 7-71 of SEQ ID NO;

    10) and enhances the processivity of the DNA polymerase compared to an identical DNA polymerase not having the sequence non-specific double-stranded nucleic-acid binding domain fused to it,wherein the solution comprises the DNA-binding fluorescent dye, which exhibits altered fluorescence emissions when the dye is bound to double-stranded DNA, and is of a composition that permits the sequence non-specific double-stranded nucleic acid binding domain to bind to the target nucleic acid and the polymerase domain to extend a primer that is hybridized to the target nucleic acid sequence;

    (b) incubating the solution under conditions in which the primer is extended by the DNA polymerase,(c) exposing the solution to a suitable excitation light and measuring fluorescence emission from the DNA-binding fluorescent dye and;

    (d) performing at least one additional cycle of amplification comprising steps (a) to (c).

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