20µl PCR Reactions 5μg RNA Quantitative PCR Molecular Genetic Analysis

Introduction

In qPCR, DNA amplification is monitored at each cycle of PCR. When the DNA is in the log linear phase of amplification, the amount of fluorescence increases above the background. The point at which the fluorescence becomes measurable is called the threshold cycle (CT) or crossing point. By using multiple dilutions of a known amount of standard DNA, a standard curve can be generated of log concentration against CT. The amount of DNA or cDNA in an unknown sample can then be calculated from its CT value.

Relative fluorescent quantitation (or quantitative fluorescence PCR (QF-PCR) is a technique used in a variety of fragment analysis applications that requires accurate peak height comparisons across multiple samples. Applications that utilize this technique include screening for loss of heterozygosity (LOH) using microsatellites or SNPs, aneuploidy assays, and detecting large chromosomal deletions.

Real-time PCR also lends itself to assays to determine relative quantities of DNA. A reaction may be performed using primers unique to each target region to be amplified and tagged with different fluorescent dyes. Several commercially available quantitative thermal cyclers include multiple detection channels. In this multiplex system, the amount of target DNA/cDNA produced during amplification can be compared to the amount of a housekeeping sequence like GAPDH or ß-actin.

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We have developed a novel "real time" quantitative PCR method. The method measures PCR product accumulation through a dual-labeled fluorogenic probe (i.e., TaqMan Probe). This method provides very accurate and reproducible quantitation of gene copies. Unlike other quantitative PCR methods, real-time PCR does not require post-PCR sample handling, preventing potential PCR product carry-over contamination and resulting in much faster and higher throughput assays. The real-time PCR method has a very large dynamic range of starting target molecule determination (at least five orders of magnitude). Real-time quantitative PCR is extremely accurate and less labor-intensive than current quantitative PCR methods.