Patent ID: 7655443
Filing Date: 2010-02-02
Classification: C12Q

Abstract:
1. A method for sequencing and quantitation of a target nucleic acid analyte in a sample, comprising the steps of: (a) generating a set of nucleic acid sequencing fragments by processing one or more aliquots of the sample using a set of reagents containing a set of forward and reverse sequencing primers that flank a region of interest of the target nucleic acid between the primers, wherein the primers are sufficiently complementary to the respective 5′ ends of the sense and anti-sense strands of the target nucleic acid to produce through a plurality of thermocycles a mixture of primer extension nucleic acid sequencing fragments terminating at each of a particular base type within the target nucleic acid and incorporating a detectable label indicative of the terminating base type that is capable of detection to determine the sequence of the nucleic acid and to determine the amount of target nucleic acid present in the sample, said processing being carried out within a quantitative regime wherein the plurality of thermocycles is performed at cycle numbers where a plot of log of the amount of product as a function of cycle number is linear; (b) determining the sequence of the target nucleic acid analyte by separating the labeled nucleic acid sequencing fragments in the modified sample in order of size and detecting the order of separation of the labels associated with the separated sequencing fragments generated in (a); and (c) determining the quantity of the target nucleic acid analyte present in the sample by correlating the intensity of a signal obtained from the label of one or more of the sequencing fragments generated in (a) with the amount of the target nucleic acid analyte present in the sample, using a reference standard, wherein the sequencing fragments used to determine the sequence of the target nucleic acid and the sequencing fragments used to determine the quantity of target polynucleotide are produced using primers having the same nucleic acid sequence.