Dual phase multiplex polymerase chain reaction
First Claim
1. A method to amplify a target nucleic acid molecule in a sample, the method comprising:
- (a) performing linear multiplex amplification of the target nucleic acid on a support with complex primers and universal primers to synthesize modified targets;
(b) releasing the amplified modified targets to a liquid phase;
(c) performing amplification of the target nucleic acid molecules in the liquid phase with universal primers; and
(d) annealing the amplified modified targets to the support wherein further linear amplification with complex primers occurs.
3 Assignments
0 Petitions
Accused Products
Abstract
Highly specific and sensitive methods were developed for multiplex amplification of nucleic acids on supports such as microarrays. Based on a specific primer design, methods include five types of amplification that proceed in a reaction chamber simultaneously. These relate to four types of multiplex amplification of a target DNA on a solid support, directed by forward and reverse complex primers immobilized to the support and a fifth type—pseudo-monoplex polymerase chain reaction (PCR) of multiple targets in solution, directed by a single pair of unbound universal primers. The addition of the universal primers in the reaction mixture increases the yield over the traditional “bridge” amplification on a solid support by approximately ten times. Methods that provide multitarget amplification and detection of as little as 0.45-4.5×10−12 g (equivalent to 102-103 genomes) of a bacterial genomic DNA are disclosed.
66 Citations
32 Claims
-
1. A method to amplify a target nucleic acid molecule in a sample, the method comprising:
-
(a) performing linear multiplex amplification of the target nucleic acid on a support with complex primers and universal primers to synthesize modified targets;
(b) releasing the amplified modified targets to a liquid phase;
(c) performing amplification of the target nucleic acid molecules in the liquid phase with universal primers; and
(d) annealing the amplified modified targets to the support wherein further linear amplification with complex primers occurs.
-
-
2. A method to amplify a target nucleic acid molecule in a sample, the method comprising:
-
(a) immobilizing a plurality of first and second complex primers to a support;
(b) annealing a single stranded target nucleic acid molecule to a specific segment of a first complex primer on the support;
(c) extending the first complex primer to synthesize a first complementary nucleic acid molecule on the support;
(d) forming a bridge from the first complementary nucleic acid molecule to a specific segment of a second complex primer on the support;
(e) extending the second complex primer to synthesize a second complementary nucleic acid molecule on the support;
(f) amplifying the first and second complementary nucleic acid molecules immobilized to the support using unbound universal primers to synthesize a plurality of modified targets;
(g) releasing the modified targets to a liquid phase of a reaction chamber;
(h) amplifying the modified targets in the liquid phase of the reaction chamber using unbound universal primers; and
(i) annealing the unattached modified targets to the first and second complex primers in the support wherein further amplification occurs. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
-
-
18. A module comprising a support to which a plurality of complex primers are attached, wherein the complex primers comprise a 5′
- -universal segment and a 3′
-segment specific to a target nucleic acid molecule, and a reaction chamber comprising a plurality of universal primers. - View Dependent Claims (19, 20)
- -universal segment and a 3′
-
21. A microarray comprising a support with a plurality of attachment sites;
- wherein a first attachment site comprising a plurality of tethered forward and reverse complex primers, wherein the forward complex primer comprises a 5′
-universal segment identical to a 5′
-universal segment of the forward complex primer in a second attachment site and a 3′
-segment specific for a first target nucleic acid molecule, and the reverse complex primer comprises a 5′
-universal segment identical to a 5′
-universal segment of the reverse complex primer in a second attachment site and a 3′
-segment specific for the first target nucleic acid molecule. - View Dependent Claims (22, 23, 24, 25)
- wherein a first attachment site comprising a plurality of tethered forward and reverse complex primers, wherein the forward complex primer comprises a 5′
-
26. A diagnostic kit comprising in discrete compartments:
-
(a) a support comprising a plurality of attachment sites bearing tethered complex primers;
(b) a reaction chamber; and
(c) a plurality of universal primers. - View Dependent Claims (27)
-
-
28. A method to amplify a target nucleic acid molecule in a sample using a combination of a plurality of universal primers present in a solution and a plurality of complex primers immobilized to a solid support, wherein the complex primers comprise a 5′
- -universal segment and a 3′
-segment specific to a target nucleic acid in the sample, the method comprising;
(a) annealing the target nucleic acid molecule reversibly to the solid support via hybridization of the 3′
-specific segment of a first immobilized complex primer to the target nucleic acid, where an extension of the first complex primer results in a linear amplification of the target nucleic acid molecule and an irreversible binding of the amplified target nucleic acids to the solid support through the first complex primer;
(b) forming a bridge between extended first complex primer bound in the solid support and a second complex primer via hybridization of the complementary sequences of the extended first complex primer and a 3′
-segment of the second complex primer followed by extension of the second complex primer, resulting in further amplification of the target nucleic acid;
(c) amplifying the extended first and second complex primers bound to the support using universal primers to synthesize a plurality of modified targets;
(d) releasing the amplified modified targets to the solution, wherein further pseudo-monoplex amplification with universal primers occurs; and
(e) annealing 3′
-end regions of the amplified modified targets in solution to the complex primers attached to the solid support resulting in further linear amplification of the modified targets bound to the support. - View Dependent Claims (29, 30, 31, 32)
- -universal segment and a 3′
Specification