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 0.45-4.5×10g (equivalent to 10-10genomes) of a bacterial genomic DNA are disclosed.