qPCR Technical Guide

Abstract

The routine study of DNA became practical with the invention of the polymerase chain reaction (PCR) by Kary Mullis in 1983. With the advent of PCR, it was possible to multiply a given DNA segment from com- plex genetic material millions of times in a few hours using simple equipment. PCR provided researchers with the ability to generate enough genetic material to study gene function and the effects of mutations, offering new possibilities in basic research and diagnostics. Despite these advances, quantitation of DNA or RNA in cells remained a difficult task until 1993 when Russell Higuchi, et al.,1 introduced real-time, or kinetic, moni- toring of DNA amplification. Higuchis experiments revealed that the relationship between the amount of target DNA and the amount of PCR product generated after a specific number of amplification cycles is linear. This observation formed the basis for real-time quanti- tative PCR (qPCR). Numerous qPCR detection chemistries and instruments are now available to answer a wide range of questions. For instance, qPCR can be used to measure viral load or bacterial pathogens in a clinical sample, to verify microarray data, for allelic discrimination or to deter- mine RNA (via cDNA) copy numbers to analyze the level of gene product in a tissue sample. The Quantitative PCR Technical Guide from Sigma-Aldrich is intended to provide new users with an introduction to qPCR, an understanding of available chemistries, and the ability to apply qPCR to answer research questions. The guide also contains numerous tips and tools for the experienced qPCR user.