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Length determination of nucleic acid repeat sequences by discontinuous primer extension

  • US 6,309,829 B1
  • Filed: 12/03/1998
  • Issued: 10/30/2001
  • Est. Priority Date: 05/27/1997
  • Status: Expired due to Term
First Claim
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1. A method for determining the number of repeat units in a repeat region of a target nucleic acid comprising the steps of:

  • (A) contacting a plurality of different-sequence primers with a polynucleotide sample under conditions effective for said primers to anneal to primer-complementary regions in one or more target polynucleotides, to form one or more target-primer hybrid(s), wherein either (1) each different-sequence primer contains (i) a target binding segment and (ii) a tag segment having a nucleotide sequence that uniquely identifies the target binding segment, or (2) one or more polynucleotides in the sample are tagged polynucleotides that contain a tag segment having a nucleotide sequence that uniquely identifies the attached polynucleotide, (B) performing a first primer extension reaction on said hybrid(s) using a first primer extension reagent;

    (C) separating the target-primer hybrid(s) and unreacted first primer extension reagent;

    (D) performing a second primer extension reaction on said hybrid(s) using a second primer extension reagent, wherein at least one of the first or second primer extension reagents includes an extendible nucleotide having a label attached thereto;

    (E) separating the target-primer hybrid(s) from unreacted second primer extension reagent;

    (F) measuring a signal produced by the label;

    (G) treating the label so as to render the label undetectable; and

    (H) repeating a cycle of steps (A) through (G) until the signal detected in the target-primer hybrid(s) is substantially less than a signal detected in a previous cycle, wherein prior to step (F), at least an aliquot of either (1) said different-sequence primers or (2) said tagged sample polynucleotides are contacted with an addressable array of immobilized, different tag complements, and each different tag complement contains a sequence that is complementary to one of said tag segments, under conditions effective to hybridize the tag segments to corresponding tag complements on the support.

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