The beginnings of real-time PCR.

Featured Article: Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996;6:986 –94. During the mid-1990s the PCR was becoming a mature technology, impacting such areas such as cloning and automated sequencing. Many scientists were making efforts to tame the quantitative power of PCR. The power of exponential amplification was tremendous, but it had proven hard to control in quantitative applications. Many attempts were made to use socalled end-point quantification. This approach attempted to determine a starting target concentration based on the quantification of the final amplified product. Several aspects of PCR complicated the success of these efforts. A major source of difficulty was that PCR often reaches a plateau stage at which exponential amplification ceases and there is very little to no further product accumulation. Therefore, the product concentration at plateau is similar for all starting target amounts. In essence, it is impossible to accurately quantify starting target concentrations if PCR reaches plateau. Another complication is that PCR, like any enzyme-driven reaction, is sensitive to reaction inhibition. Many of the most interesting biological samples and methods for nucleic acid purification possessed known inhibitors of PCR (e.g., heme from blood). Southern and Northern blots with radioactive probes were the method of choice for many aspiring scientists wishing to have semiquantitative nucleic acid data, although these methods came with their own inherent pitfalls. It was during this time period that new thoughts began to surface. David Gelfand and Pam Holland and colleagues at Cetus had described the 5 -to-3 nuclease activity of DNA polymerase and the method they used to harness this activity to degrade a hybridization probe placed in a PCR (1 ). Their discovery was eventually and cleverly named “TaqMan,” honoring one of the first electronic video games, Pacman. Additionally, Russ Higuchi and colleagues, formerly from Cetus but now at Roche, described “simultaneous amplification and detection” of PCR products achieved by use of the accumulation of ethidium bromide fluorescence generated during a PCR (2, 3 ). Ken Livak and colleagues at ABI described the use of fluorescent energy transfer for generating a signal system for probe degradation in the 5 -to-3 nucleolytic PCR assay (4 ). Major advances in PCR equipment were also taking place. A group at Roche led by Bob Watson developed a kinetic PCR thermal cycler that captured real-time EtBr fluorescence. Another group, led by Carl Witwer and Kirk Ririe, developed a capillary-based rapid thermal cycler that dramatically decreased times required for PCR reactions. It was during this period that Roche’s efforts to bring PCR technology into the diagnostics arena began to bear fruit. A recently described disease, AIDS, was the major focus of the efforts of many researchers and pharmaceutical companies. A clear and powerful application of PCR was in the detection and quantification of HIV, the causative agent of AIDS. PCR afforded a sensitive detection method. Shirley Kwok, John Sninsky, and colleagues at Roche developed a quantitative reverse-transcription PCR test for measuring HIV viral RNA copy numbers. The use of a coamplified control sequence of known copy number and detection before plateau phase helped overcome some of the hurdles to successful PCR quantification. This test eventually became the Cobas Amplicor HIV-1 Monitor test. The development of this test was a pivotal moment in bringing the power of PCR technology to diagnostic applications. The test has been used as part of many clinical studies and has clearly demonstrated that viral burden is an important end-point of therapy response. During this period many additional quantitative PCR advances were made; an example is the work of Mike Piatak and his colleague Jeffrey Lifson. They too were focused on HIV viral burden and they described another means of employing PCR for the quantitative detection of HIV. They published a report of the development of a quantitative competitive PCR as a tool for monitoring HIV viral burden (5 ). My laboratory at Genentech was fortunate enough to have connections with both Applied BioSystems and Roche. We were selected as the test site for a prototype real-time quantitative PCR instrument being developed by Applied BioSystems. Both Chris Heid and I realized the potential of this application if it truly provided sensitive, reliable, and accurate quantification of 1 Roche Molecular Systems, Pleasanton, CA. * Address correspondence to the author at: Roche Molecular Systems, 4300 Hacienda Drive, Pleasanton, CA 94588. E-mail [email protected]. 2 This paper has been cited 2895 times since its publication. Received January 29, 2009; accepted February 4, 2009. Previously published online at DOI: 10.1373/clinchem.2008.122226 Clinical Chemistry 55:4 833–834 (2009) Citation Classic