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Polynucleotide sequencing

  • US 6,833,246 B2
  • Filed: 03/29/2002
  • Issued: 12/21/2004
  • Est. Priority Date: 09/29/1999
  • Status: Expired due to Term
First Claim
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1. A method for determining the sequence of a target polynucleotide on an array, comprising the steps of:

  • (i) contacting the array with a first DNA polymerase and one or more detectably-labelled bases A, T, G and C, under conditions that permit a polymerisation reaction to occur, to thereby incorporate a labelled base into a strand complementary to the target polynucleotide;

    (ii) removing non-incorporated bases and the first DNA polymerase and detecting an incorporation event;

    (iii) contacting the array of step (ii) with a second DNA polymerase having 3′

    to 5′

    exonuclease activity and non-labelled blocked bases A, T, G and C, under conditions whereby the second DNA polymerase is capable of cleaving one labelled base from the strand complementary to a target polynucleotide and incorporating a corresponding non-labelled blocked base into the strand complementary to the target polynucleotide, thereby replacing the labelled base with a non-labelled blocked base, wherein the non-labelled blocked base comprises a removable blocking group which prevents further base incorporation from occurring;

    (iv) removing the removable blocking group from the non-labelled blocked base; and

    (v) repeating steps (i)-(iv) sequentially, to determine the sequence.

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