Capture primers and capture sequence linked solid supports for molecular diagnostic tests
First Claim
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1. A method comprising:
- a) contacting a sample suspected of containing a target nucleic acid with a capture primer and a reverse primer, wherein said capture primer comprises;
i) a 3′
region configured to hybridize to said target nucleic acid such that it can be extended by a polymerase, and ii) a 5′
region comprising a capture sequence; and
wherein said contacting is under conditions such that;
i) said 3′
region of said capture primer hybridizes to said target nucleic acid and is extended to generate a first amplification product, andii) said reverse primer hybridizes to said first amplification product and is extended to generate a second amplification product, wherein said second amplification product comprises a 3′
capture sequence complement capable of hybridizing to said capture sequence; and
b) treating said sample under conditions such that said second amplification product is separated from said first amplification product;
c) contacting said second amplification product with a solid support comprising a plurality of bound capture sequences under conditions such that said 3′
capture sequence complement of said second amplification product hybridizes to one of said bound capture sequences to generate a hybridized solid support; and
d) treating said hybridized solid support under conditions such that one of said bound capture sequences is extended along said second amplification product to generate a target sequence that is linked to said solid support; and
e) treating said target sequence linked to said solid support under conditions such that at least part of the nucleic acid sequence of said target sequence is determined by a method comprising contacting said target sequence with at least one nucleotide incorporating biocatalyst, labeled nucleotides, and at least one primer nucleic acid that is at least partially complementary to at least a subsequence of said target sequence, under conditions whereby said nucleotide incorporating biocatalyst extends said primer nucleic acid to produce an extended primer nucleic acid by incorporating said labeled nucleotides at a terminal end of said extended primer nucleic acid.
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Abstract
The present invention provides systems, methods, and compositions for performing molecular tests. In particular, the present invention provides methods, compositions and systems for generating target sequence-linked solid supports (e.g., beads) using a solid support linked to a plurality of capture sequences and capture primers composed of a 3′ target-specific portion and a 5′ capture sequence portion. In certain embodiments, the target sequence linked solid support is used in sequencing methods (e.g., pyrosequencing, zero-mode waveguide type sequencing, nanopore sequencing, etc.) to determine the sequence of the target sequence (e.g., in order to detect the identity of a target nucleic acid in sample).
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Citations
27 Claims
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1. A method comprising:
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a) contacting a sample suspected of containing a target nucleic acid with a capture primer and a reverse primer, wherein said capture primer comprises;
i) a 3′
region configured to hybridize to said target nucleic acid such that it can be extended by a polymerase, and ii) a 5′
region comprising a capture sequence; and
wherein said contacting is under conditions such that;i) said 3′
region of said capture primer hybridizes to said target nucleic acid and is extended to generate a first amplification product, andii) said reverse primer hybridizes to said first amplification product and is extended to generate a second amplification product, wherein said second amplification product comprises a 3′
capture sequence complement capable of hybridizing to said capture sequence; andb) treating said sample under conditions such that said second amplification product is separated from said first amplification product; c) contacting said second amplification product with a solid support comprising a plurality of bound capture sequences under conditions such that said 3′
capture sequence complement of said second amplification product hybridizes to one of said bound capture sequences to generate a hybridized solid support; andd) treating said hybridized solid support under conditions such that one of said bound capture sequences is extended along said second amplification product to generate a target sequence that is linked to said solid support; and e) treating said target sequence linked to said solid support under conditions such that at least part of the nucleic acid sequence of said target sequence is determined by a method comprising contacting said target sequence with at least one nucleotide incorporating biocatalyst, labeled nucleotides, and at least one primer nucleic acid that is at least partially complementary to at least a subsequence of said target sequence, under conditions whereby said nucleotide incorporating biocatalyst extends said primer nucleic acid to produce an extended primer nucleic acid by incorporating said labeled nucleotides at a terminal end of said extended primer nucleic acid. - 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, 26, 27)
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Specification