Patent ID: 7267945

Claim:
A method of determining the presence and sequence of at least one target polynucleotide in a sample comprising: combining nucleic acid from the sample with at least one set of reaction compositions comprising a first reaction composition and second reaction composition, both specific for the at least one target polynucleotide, wherein the first reaction composition comprises amplification primers specific to the at least one target polynucleotide and lacks a fluorescent indicator, and the second reaction composition comprises a fluorescent indicator and amplification primers specific to the at least one target polynucleotide, and wherein the first reaction composition and the second reaction composition are separate reaction compositions; amplifying the at least one target polynucleotide present in the reaction compositions using the amplification primers to obtain at least one amplification product; irradiating the at least one amplification product of the second reaction composition such that the fluorescent indicator produces a fluorescent signal, wherein the intensity of the fluorescent signal is related to the quantity of the at least one amplification product; monitoring the amplifying of the second reaction composition by detecting the fluorescent signal from the fluorescent indicator; determining whether the at least one amplification product is present in both the first reaction composition and the second reaction composition from the intensity of signal from the fluorescent indicator in the second reaction composition; and determining the sequence of the at least one amplification product of the first reaction composition if the at least one amplification product is present in the first reaction composition.