Quantitative plasmid mixture analysis using the fluorogenic 5'-nuclease polymerase chain reaction assay

The fluorogenic 5'-nuclease polymerase chain reaction (PCR) assay has been shown to be useful for quantifying a given DNA target in a sample. Here we show how an existing PCR protocol can be amended for quantification by incorporating distinctive dual-labeled, sequence-specific oligonucleotide probes and resulting in a two- to threefold broader and more reliable dynamic range than that of conventional end-point analysis of PCR products. Moreover, we show a multiplex situation in which two targets, one normal and one mutated, can be amplified and quantified simultaneously and in the same reaction tube. Use of this novel approach for quantitative PCR applications eliminates the need for post-PCR processing and has clinical- and research-based diagnostic applications, particularly for measuring levels of mutations in a mixture.