Patent ID: 7674582
Filing Date: 2010-03-09
Classification: C12Q,G01N

Abstract:
1. A method for monitoring the amplification of a set of at least four different nucleic acid sequences of interest, said method comprising: (a) contacting a nucleic acid sample in a reaction vessel with a set of at least four 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; and each oligonucleotide primer pair in said set of at least four oligonucleotide pairs is selected so that it generates a distinctly sized amplification product in a subsequent amplification regimen; (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 a plurality of said iterative cycles, an aliquot of said mixture is removed from said reaction vessel and nucleic acid molecules in said aliquot are separated by capillary electrophoresis; (c) detecting distinctly sized primer extension products in each said aliquot , said products corresponding to each of said set of at least four different nucleic acids of interest present in said sample; and (d) for each distinctly sized primer extension product, calculating a cycle number where the amount of that primer extension product crosses a predefined threshold, and correlating the threshold cycle with the amount of target nucleic acid in said sample, wherein said method provides an amplification profile and a relative abundance for members of said set of at least four different nucleic acids of interest in said sample.