Patent ID: 6309829
Filing Date: 2001-10-30
Classification: C12Q

Abstract:
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.