Polymerase signaling assay
First Claim
1. A method for analyzing the sequence of a template comprising:
- a) capturing the template with a sequencing reagent to form a captured template, said sequencing reagent being immobilized to a solid surface and comprising;
(i) a capture moiety capable of forming a stable duplex with a region of the template nucleic acid molecule;
(ii) a primer region comprising from 3 to 7 bases; and
between said capture moiety and said primer region (iii) a spacer region that minimizes template independent noise; and
b) scanning the captured template using a primer-polymerase complex for regions of complementarity to the primer region and forming a duplex;
c) extending the primer region by at least one nucleotide moiety by means of a template-homology dependent extension reaction to form an extended primer; and
d) detecting the extended primer, wherein said detecting of the extended primer indicates the presence of one or more regions of complementarity to the primer region in the captured template;
wherein the steps of the method are repeated for sequencing reagents that are bound in an array pattern onto to said solid surface so that a pattern of signals is generated for the template.
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Abstract
The present invention relates to the field of nucleic acid sequence analysis. The invention provides methods for performing a polymerase signal assay (PSA) to analyze nucleotide sequences using solid phase sequence arrays comprising a plurality of sequence reagents with primer sequences 4-6 bases in length. The methods of the invention generate a binary signal pattern which can be used to identify nucleic acid sequences and/or mutations and polymorphisms of a nucleic acid sequence. Mutations and polymorphisms which can be identified by the methods of the invention include single nucleotide polymorphisms (SNP'"'"'s), base deletions, base insertions, and heterozygous as well homozygous polymorphisms.
30 Citations
25 Claims
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1. A method for analyzing the sequence of a template comprising:
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a) capturing the template with a sequencing reagent to form a captured template, said sequencing reagent being immobilized to a solid surface and comprising;
(i) a capture moiety capable of forming a stable duplex with a region of the template nucleic acid molecule;
(ii) a primer region comprising from 3 to 7 bases; and
between said capture moiety and said primer region(iii) a spacer region that minimizes template independent noise; and
b) scanning the captured template using a primer-polymerase complex for regions of complementarity to the primer region and forming a duplex;
c) extending the primer region by at least one nucleotide moiety by means of a template-homology dependent extension reaction to form an extended primer; and
d) detecting the extended primer, wherein said detecting of the extended primer indicates the presence of one or more regions of complementarity to the primer region in the captured template;
wherein the steps of the method are repeated for sequencing reagents that are bound in an array pattern onto to said solid surface so that a pattern of signals is generated for the template. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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Specification