Molecular and Cellular Probes

For the amplification of HIV-1 gag gene, and other challenging targets, a simple new hot start PCR protocol is presented which consists simply and entirely of the buffer system. This novel buffer composition and reaction assembly protocol for PCR includes magnesium and phosphate combined at high concentration in addition to standard buffer reagents. The resulting magnesium-containing precipitate provides a hot start for PCR, since the magnesium in the precipitate is unavailable to DNA polymerase until thermal cycling. No extra manipulations at the thermal cycler or changes to standard thermal cycling profiles are necessary. Upon normal cycling, the magnesium becomes fully available within the first 3 cycles. The method effectively prevents premature primer extension by several DNA polymerases or mixtures of DNA polymerases tested, including Klentaq1, KlentaqLA, Pfu, and full-length wild-type DNA polymerase. Once the precipitate is formed, the hot start buffer is stable and functional for at least a week at −20°, 4°, or 25°. We demonstrate that the method is as effective as a manual hot start (addition of magnesium at or above primers» annealing temperatures) for several target gene amplifications which require a hot start.