Nucleic acid sequencing with simultaneous quantitation

Abstract

Simultaneous sequencing and quantitation of a nucleic acid analyte in a sample using the same reagents for both assays is achieved by processing a sample containing, or suspected of containing the nucleic acid analyte of interest using a single set of reagents through a plurality of thermocycles to obtain a mixture of labeled polynucleotides which are used for the determination of both sequence information about the target nucleic acid and the amount of target nucleic acid present in the sample. The fragments are separated on the basis of size, for example by electrophoresis, and the label associated with the separated fragments is detected. The positions of the separated nucleic acid fragments are evaluated to obtain information about the sequence of the target nucleic acid analyte, and the intensity of a signal derived from the label associated with one or more of the separated fragments is evaluated to determine the quantity of the target nucleic acid analyte in the sample. Only one label is needed for both sequencing and quantitation, although two or more labels may be used if bidirectional sequencing is concurrently performed.