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

The Fluorogenic 5 ' -nuclease polymerase chain reaction (PCR) assay has bee n shown to be useful for quantifying a given DNA target in a sample. Here w e show how an existing PCR protocol can be amended for quantification by in corporating distinctive dual-labeled, sequence-specific oligonucleotide pro bes and resulting in a two- to threefold broader and more reliable dynamic range than that of conventional end-point analysis of PCR products. Moreove r, we show a multiplex situation in which two targets, one normal and one m utated, can be amplified and quantified simultaneously and in the same reac tion tube. Use of this novel approach For quantitative PCR applications eli minates the need for post-PCR processing and has clinical- and research-bas ed diagnostic applications, particularly for measuring levels of mutations in a mixture. Published 2001 Wiley-Liss, Inc.(dagger).