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Methods for determining nucleotide sequences of single nucleic acid molecules

  • US 7,223,568 B2
  • Filed: 11/30/2001
  • Issued: 05/29/2007
  • Est. Priority Date: 03/30/2000
  • Status: Expired due to Fees
First Claim
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1. A method for determining a nucleotide sequence of a single nucleic acid molecule, which comprises:

  • (a) immobilizing a nucleic acid molecule onto the surface of a solid;

    (b) annealing a primer to said nucleic acid molecule, wherein said primer has a sequence complementary to a part of a sequence of the nucleic acid molecule;

    (c) providing a solution which contains a DNA polymerase and only one type of dye-labeled dNTP, where N is A, T or U, G or C, or an RNA polymerase and only one type of dye-labeled NTP, where N is A, U, G or C, to said immobilized nucleic acid molecule, and allowing the dye-labeled dNTP or NTP to react with the 3′

    end of said primer, whereby the dye-labeled dNTP or NTP, which forms a base-pair with a base in the nucleic acid molecule at a position where the dye-labeled dNTP or NTP reacts with the 3′

    end of said primer and is bound to the primer by action of the polymerase;

    (d) detecting a bound, dye-labeled dNTP or NTP;

    (e) disrupting the dye molecule of the bound, dye-labeled dNTP or NTP;

    (f) repeating (c) to (e) while changing the type of dye-labeled dNTP or NTP in turn, to sequentially bind dNTPs or NTPs which forms a base-pair with the nucleotides of the nucleic acid molecule; and

    (g) determining a nucleotide sequence of the nucleic acid molecule based on the types of the sequentially bound dNTPs or NTPs.

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