Real-time PCR:Layout 1

(PCR) was born in 1983 when Kary Mullis was taking a ride in the mountain range in California1,2. It is as simple as brilliant. Based on the natural ability of the polymerase enzyme to copy nucleic acids, Kary Mullis reasoned that using a heat stable polymerase it should be possible to automate the reaction to perform multiple copying events by cycling the temperature. The double stranded DNA molecule is strand separated by heating to 95°C, then the temperature is lowered to allow short synthetic DNA oligonucleotide primers to anneal to complementary sequence in the DNA template, and finally the temperature is set to 72°C, at which the heat stable Thermus aquaticus (Taq) polymerase extends the primers into full length copies. Since both strands are copied, the number of DNA molecules is doubled in each cycle. Using PCR, virtually any DNA can be amplified starting from a single copy to a large number of molecules that can readily be analysed or used for engineering. Development of PCR was a major breakthrough as a qualitative analytical tool, but it was not quantitative. The amount of PCR product produced depended on the amount of reagents added rather than on the amount of starting material. In the beginning of the 90s Russell Higuchi discovered that PCR can be performed in the presence of nucleic acid stains that become fluorescent upon binding the DNA. The fluorescence from the dyes could be measured throughout the reaction, making it possible to monitor the accumulation of the PCR product in real time. By registering the number of PCR amplification cycles required to obtain a particular amount of product characterised by certain dye fluorescence, it was possible to calculate the number of target molecules the sample contained initially. The approach was named quantitative real-time PCR or qPCR for short. The analytical sensitivity of qPCR was only limited by sampling effects, since a single molecule was sufficient to generate product, and its dynamic range was virtually unlimited. Reproducibility was also impressive, considering the technique gives exponential response. A year ago I summarised in DDW the emerging qPCR applications and in this news story I follow up on those and other important happenings in the qPCR field during the past year.