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Methods, compositions, and kits for amplifying and sequencing polynucleotides

  • US 20060141487A1
  • Filed: 01/26/2005
  • Published: 06/29/2006
  • Est. Priority Date: 01/26/2004
  • Status: Abandoned Application
First Claim
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1. A method of amplifying and sequencing at least a region of a polynucleotide, said method comprising the steps of:

  • a) incubating the polynucleotide with a composition comprising;

    at least one amplification primer, a processive amplification polymerase, a sequencing primer, a sequencing polymerase, deoxynucleoside triphosphates suitable for template-dependent primer extension, and four different terminating nucleotides, each of which terminates template-dependent primer extension at a different template nucleotide, the incubating being carried out at a first temperature suitable for amplifying the polynucleotide with the processive amplification polymerase;

    b) incubating the product of step (a) at a second temperature suitable for forming a plurality of differently-sized extended sequencing primers with the sequencing polymerase;

    c) determining a sequence of said polynucleotide by evaluating said plurality of differently-sized extended sequencing primers.

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