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The Gradient Feature: Use in Optimization of Allelic Discrimination Assays, Rev B

Luis A Ugozzoli, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA 94547 USA


Introduction
Real-Time PCR
PCR has been greatly refined since its advent 16 years ago. Several improvements in the technology and associated equipment have significantly changed the way PCR is performed today relative to earlier protocols. One of the most remarkable PCR advancements has been the development of real-time PCR (Higuchi et al. 1993), a system that permits the detection of nucleic acids while they accumulate during amplification. Various applications of real-time PCR have been published recently that show this technique to be a powerful tool for the quantitation of RNA and DNA targets as well as for the detection of single nucleotide polymorphisms (SNPs) (Foy and Parkes 2001, Bustin 2003). The advantages of using real-time PCR over the basic PCR technology are numerous. For example, the chances for generating carryover contamination are reduced because no post-PCR handling is required. In addition, the real-time detection of PCR products eliminates post-PCR detection as a potential error source. For quantitative experiments, the dynamic range of real-time PCR assays is several orders of magnitude greater than the range generated by end-point measurements.

PCR Optimization
Under optimal reaction conditions, a high PCR efficiency allows the synthesis of micrograms of DNA from a few starting molecules of template DNA or RNA. However, reaction optimization requires careful design of primers as well as the adjustment of the cycling conditions and buffer composition. For the development of PCR cycling conditions, one of the most important
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