NUCLEIC ACID DETECTION AND QUANTIFICATION

摘要

The present invention relates to methods and uses for the detection or quantification of newly-synthesized double-stranded target nucleic acid molecules in a sample during quantitative real-time polymerase chain reaction (qPCR) amplification. According to the invention, an intercalating dye recognizing double-stranded DNA molecules with higher affinity than single-stranded DNA molecules and a fluorophore-labeled oligonucleotide-probe being sequence specific for a target nucleic acid molecule are simultaneously employed, thus enabling quantification a specific target and total amount of a mixed nucleic acid population, and enabling assessing the cause of suboptimal PCR performance.

主权项

1. Method for the detection or quantification of newly-synthesized double-stranded target nucleic acid molecules in a sample during quantitative real-time polymerase chain reaction (qPCR) amplification, the method comprising the steps of
(i) contacting a sample comprising one or more target nucleic acid molecules with a) an intercalating dye recognizing double-stranded DNA molecules with higher affinity than single-stranded DNA molecules and said intercalating dye has increasing fluorescence upon intercalation with double-stranded nucleic acid molecules; with b) at least one fluorophore-labeled oligonucleotide-probe being sequence specific for the target nucleic acid molecule, and said at least one fluorophore-labeled oligonucleotide-probe undergoes a detectable change in fluorescence upon amplification of said one or more target nucleic acid molecules; and with c) at least one oligonu-cleotide-primer pair; (ii) incubating the mixture of step (i) with a DNA polymerase under conditions sufficient to amplify one or more target nucleic acid molecules; and (iii) detecting the presence or absence or quantifying the amount of the amplified target double-stranded nucleic acid molecules by measuring fluorescence of said intercalating dye and of said fluorophore.