Patent ID: 6830887
Filing Date: 2004-12-14
Classification: C12Q

Abstract:
A method for quantitative and qualitative analysis of a nucleic acid analyte in a sample suspected to contain the nucleic acid analyte, comprising the steps of:(a) preparing a reaction mixture containing the sample and a known amount of an internal quantitative standard; (b) combining a first aliquot of the reaction mixture with a set of amplification reagents, said reagents including a first primer pair which is effective to amplify a first region of the nucleic acid analyte if present in the sample to produce a first amplified sample fragment and to amplify at least a portion of the internal quantitation standard to produce a control fragment; (c) amplifying the nucleic acid analyte and the internal quantitation standard in the reaction mixture using the first primer pair to produce an amplification product mixture containing first amplified sample fragments and control fragments when the nucleic acid analyte is present in the sample, and only control fragments when the nucleic acid analyte is not present in the sample; (d) analyzing the relative amounts of first amplified sample fragments and control fragments in the amplification product mixture to quantify the amount of nucleic acid analyte in the sample; and (e) determining the sequence of the first amplified sample fragments in the amplification mixture to determine the qualitative characteristics of any nucleic acid analyte in the sample using at least a first sequencing primer, wherein the internal quantification fragment is derived from the analyte nucleic acid by the incorporation of a plurality of sequence variations, including at least a first sequence variation effective to render the internal quantitation standard distinguishable from the first amplified sample fragment, and a second sequence variation effective to substantially eliminate the production of sequencing products from interaction of the internal quantitation standard and the first sequencing primer.