US 7,432,055 B2 · Dual phase multiplex polymerase chain reaction

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.

Claims22

Dependent claims

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 obtain modified target nucleic acids;
(b) releasing the amplified modified target nucleic acid to a liquid phase;
and
(c) performing a dual phase amplification of the modified target nucleic acid molecules in the liquid phase with universal primers and in the support with complex primers.

1 more element

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 target nucleic acids;
(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 target nucleic acids in solution to the complex primers attached to the solid support resulting in further linear amplification of the modified target nucleic acids bound to the support.

3 more elements

Dependencies are inferred from the claim text.

Dual phase multiplex polymerase chain reaction

Life of this patent

Dual phase multiplex polymerase chain reaction

Life of this patent

Abstract

Claims22