Patent ID: 8067206

Claim:
A method for coamplification of two or more target nucleic acids having different sequence compositions present at comparable copy numbers wherein the maximum difference between the lowest and highest copy number is 10-fold said method comprising at least 15 primary amplification cycles, each amplification cycle comprising the sequential steps of: (A) heating a reaction mixture of two or more target nucleic acids, or their primer extension products, at a first temperature, T 1 , for denaturation of the strands of the target nucleic acids or their primer extension products, and (B) priming the denatured strands with a set of primers specific to and hybridizable with opposing strands of each target nucleic acid to be amplified, by cooling to a second temperature, T 2 , and (C) either as a continuation of step (B) or in a separate step, forming primer extension products in a reaction mixture of PCR reagents, by incubation at a third temperature, T 3 , provided that when priming and primer extension product formation are carried out in the same step, T 2 and T 3 are the same, and wherein the reaction mixture in at least one of the primary amplification cycles comprises from 1 to 20 weight % of a nonionic, polymeric volume exclusion agent, a thermostable hot start DNA polymerase, and optionally a sequence specific labeled probe which binds with the primer binding regions and which is detectable after hybridization, and (D) in the course of the reaction in each amplification cycle or in an amplification cycle after the last primary amplification cycle detecting one or more of the primer extension products as an indication of one or more of the target nucleic acids.