CAPTURE PRIMERS AND CAPTURE SEQUENCE LINKED SOLID SUPPORTS FOR MOLECULAR DIAGNOSTIC TESTS
First Claim
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.
1 Assignment
0 Petitions
Accused Products
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).
110 Citations
35 Claims
-
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; 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. - 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, 28, 29, 30)
-
-
31. A system comprising:
-
a) at least one sequencing device; and b) a primer pair comprising a capture primer and a reverse primer, wherein said capture primer comprises;
i) a 3′
region configured to hybridize to a target nucleic acid such that it can be extended by a polymerase to form a first amplification product, and ii) a 5′
region comprising a capture sequence, wherein said 5′
region is configured to not hybridize to said target nucleic when said 3′
region of said capture primer is hybridized to said target nucleic acid, and wherein said reverse primer is configured to hybridize to said first extension product and be extended to form a second extension product.
-
-
32. A system, comprising:
-
a) a primer pair comprising a capture primer and a reverse primer, wherein said capture primer comprises;
i) a 3′
region configured to hybridize to a target nucleic acid such that it can be extended by a polymerase to form a first amplification product, and ii) a 5′
region comprising a capture sequence, wherein said 5′
region is configured to not hybridize to said target nucleic when said 3′
region of said capture primer is hybridized to said target nucleic acid, and wherein said reverse primer is configured to hybridize to said first extension product and be extended to form a second extension product;b) a reaction vessel or substrate; and c) a detector configured to detect detectable signals produced in or on the reaction vessel or substrate, which detectable signals correspond to at least some nucleobases incorporated into a nucleic acid to generate nucleobase incorporation data. - View Dependent Claims (33, 34)
-
-
35. A system, comprising:
-
(a) a sequencing device configured to generate nucleic acid sequence data corresponding to the nucleic acid sequence of one or more amplicons produced using at least one purified oligonucleotide primer pair that comprises a capture primer and a reverse primer, wherein said capture primer comprises;
i) a 3′
region configured to hybridize to a target nucleic acid such that it can be extended by a polymerase to form a first amplification product, and ii) a 5′
region comprising a capture sequence, wherein said 5′
region is configured to not hybridize to said target nucleic when said 3′
region of said capture primer is hybridized to said target nucleic acid, and wherein said reverse primer is configured to hybridize to said first extension product and be extended to form a second extension product; and(b) a controller operably connected to said sequencing device, said controller configured to query a database with said nucleic acid sequence data to identify said target nucleic acid.
-
Specification