Patent DE-60325679-D1 (DE)
Inventor: ENGEL HOLGER
IPC: C12Q1/68
Filed: 2003-09-23
Granted: 2009-02-26
Abstract: Coamplification of target nucleic acids (NA) having different sequence compositions, present at comparable copy numbers, is new. Coamplification (M1) of two or more target nucleic acids having different sequence compositions which are present at comparable copy numbers, where the maximum difference between the lowest and the highest copy number is 10 fold, involves performing at least 15 primary amplification cycles 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 (T1), for denaturation of the strands of the target nucleic acids or their primer extension product; (b) priming the denatured strands with a set of unmodified or modified (e.g. carrying a fluorophore moiety) primers specific to and hybridizable with opposing strands of each target nucleic acid to be amplified, by cooling to a second temperature (T2); and (c) either as a continuation of above step or in a separate step forming primer extension products in a reaction mixture of PCR reagents, by incubation at a third temperature (T3), when priming and primer extension product formation are carried out in the same step, where T2 and T3 are the same and the reaction mixture in at least one of the primary amplification cycles comprises: (i) from 1-20 wt.% (wt.%) of a nonionic polymeric volume exclusion agent; or (ii) from 1-20 wt.% of a nonionic polymeric volume exclusion agent, a hot start DNA polymerase and a sequence specific probe which binds within the primer binding regions and which generates a fluorescent signal after hybridization. Optionally, (M1) involves 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, 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), either as a continuation of above step or in a separate step, forming primer extension products in a reaction mixture of PCR reagents, by incubation at a third temperature (T 3), when priming and primer extension product formation are carried out in the same step, where T 2and T 3are the same, and the reaction mixture in at least one of the primary amplification cycles comprises from 1-20 wt.% of a nonionic polymeric volume exclusion agent, a hot start DNA polymerase and optionally a sequence specific probe which binds within the primer binding regions and which generates a fluorescent signal after hybridization, and 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. Independent claims are also included for: (1) an amplification reaction composition (I) which is buffered to a pH of 7.5-9, comprising one or more sets of primers, a thermostable hot-start-DNA polymerase, several dNTP's and 1-20 wt.% of a nonionic polymeric volume exclusion agent and optionally a probe; (2) a kit (II) for performing (M1), comprising (I) and a capture reagent having an oligonucleotide immobilized on a water-insoluble substrate; (3) a self-contained test device (III) consisting of (I) and a capture reagent having an oligonucleotide immobilized on a water-insoluble substrate, in separate compartments which is connected in (III), such that (I) can be brought into contact with the capture reagent after amplification without opening (III); and (4) a kit for preparing (I), comprising at least one hot-start DNA polymerase and at least one polymeric exclusion reagent.