Patent ID: 8182995

Claim:
A method for monitoring the amplification of a set of nucleic acid sequences of interest, said method comprising: (a) contacting a nucleic acid sample in a reaction vessel with a set of pairs of oligonucleotide primers, wherein: each said pair comprises a first oligonucleotide primer that specifically hybridizes with a nucleic acid molecule comprising said nucleic acid sequence of interest, and a second oligonucleotide primer that specifically hybridizes with a sequence comprised by the complement of said nucleic acid sequence of interest, wherein the primer extension product of one oligonucleotide primer, when separated from its complement, can serve as a template for the synthesis of the extension product of the other primer; each said pair of oligonucleotides is specific for one nucleic acid sequence of interest; each oligonucleotide primer pair in said set is selected so that it generates a distinctly sized amplification product in a subsequent amplification regimen; and one oligonucleotide in each said pair of oligonucleotides is detectably labeled; b) subjecting the mixture resulting from step (a) to an amplification regimen comprising at least two iterative cycles of nucleic acid strand separation, oligonucleotide primer annealing and polymerase extension of annealed primers, wherein during the amplification regimen, following at least one of said iterative cycles, an aliquot of said mixture is removed from said reaction vessel and nucleic acid molecules in said aliquot are separated; c) for each aliquot removed and subjected to separation of nucleic acid molecules according to step (b), detecting the incorporation of detectable label in a distinctly sized amplification product present in said aliquot; d) for each distinctly sized amplification product detected in step (c), calculating a cycle number, C t , where the amount of that amplification product crosses a predefined threshold, and correlating the threshold cycle with the amount of a nucleic acid having a sequence of interest in said sample, wherein said method provides an amplification profile and a relative abundance for members of said set of nucleic acid sequences of interest.