Real-time quantitative PCR.

Those who have worked in the field of quantitative polymerase chain reaction (PCR) since the early 1990s have accepted many of the tedious aspects of the early assays as routine. Difficulties associated with early quantitative PCR techniques included: (i) ensuring that the PCR was within the linear range of amplification (that portion where the PCR signal is directly proportional to the input copy number) and (ii) finding a suitable method to detect the product once linear amplification was achieved. To ensure linearity, researchers would amplify serial dilutions of cDNA (1) or alter the amplification cycle for each gene (2). Others would add a competitor template (3), perform limiting dilution assays (4), or use a PCR mimic with a similar primer sequence that would be coamplified along with the product (5). Detection of the amplicon generally resorted to running gels and DNA detection using a stain, radioactivity, or probe. For those who have converted to using real-time quantitative PCR, the old days are but a memory. In real-time PCR, worrying about linear amplification, running gels, working with radioactivity, and gathering numbers are a thing of the past. The ability to generate data within a 2to 3-h period is now a reality. Other advantages of real-time PCR include enhanced sensitivity, high throughput, use of a closed-tube system, reduced variation, the ability to simultaneously multiplex reactions, and lack of post-PCR manipulations. The technology to detect PCR products in real time, i.e., during the reaction, has been available for more than 5 years, but has seen a dramatic increase in use over the past 2 years. A Medline search using the key words TaqMan or real-time and PCR yielded 19 citations in 1996, 28 citations in 1997, and 52, 157, and 409 citations in 1998, 1999, and 2000, respectively. At the time of this writing, there were 551 citations in 2001 (Fig. 1). Various manufacturers are touting realtime PCR instrumentation and affiliated technology