Amplitaq Gold™: Biochemical characterization and PCR optimization

AmpliTaq Gold™ is a thermally activated form of recornbinant Thermus aquaticus DNA polymerase. The enzvme is provided in an inactive state, and is activated during the high temperature denaturation phase of the PCR. amplification. This ensures that active enzyme is never present at temperatures below the desired stringent annealing temperature, allowing for simplified, high throughput room temperature PCR set-up, and eliminating non specific prod uct formation at lower temperatures. The incorporation of this automated "hot start" feature directly into the amplification reaction results in higher specificity, and thus, higher yield of desired product. This increased specificity expands the utility of the PCR process: for sensitive, low ropy detection, accu rate quantitation, for increased throughput by performing multiplex PCR, and for the preparation of PCR product for DNA sequencing. Biochemical characterization and activation kinetics of the modified en/ynie indicate that three variables are important for activation: time, temperature, and pH. These variables can be manipulated for optimal application specific amplification. Additionally, hot start PCR results in a considerably higher amplification success rate, with minimized optimization of primer design and MgCI 2 concentration.