METHOD, INSTRUMENT AND COMPUTER PROGRAM PRODUCT FOR QUANTIFICATION OF PCR PRODUCTS

摘要

This document relates to melting curve analysis of nucleic acids. The method according to first aspect of the invention comprises analyzing a nucleic acid melting curve measured from a sample, the melting curve comprising a sum signal of at least two nucleic acid melt signals and a background signal as a function of temperature. The method further comprises optimizing at least one constant in a temperature-dependent exponential correction function so as to minimize the variation of the nucleic acid melting curve at a temperature region where the target nucleic acids in the sample remain essentially double stranded, and generating a corrected nucleic acid melting curve representative of the nucleic acid melt signal by applying said exponential correction function over the region of the measured melting curve where the strands of the nucleic acids dissociate. According to further aspects, the invention relates to a curve fitting algorithm for precise estimation of melting peak areas and a mathematical transformation for linearization of calibration curve data to enhance the linear measuring range of a competitive PCR assay. The invention provides a powerful tool for analyzing PCR-amplified sample containing two or more different nucleic acids having similar but distinguishable melting temperature.