Use of gradient PCR to determine optimum annealing temperature

摘要

Problems with the amplification of a specific DNA fragment using the polymerase chain reaction (PCR, Hoffmann-LaRoche, Inc., Nutley, NJ) are an everyday occurrence in the laboratory. Nonspecific secondary bands may form after the PCR reaction, which hinder, or even prevent, further analysis (cycle sequencing, mutation detection, etc.) or an unequivocal assessment of the PCR result. In such cases, PCR conditions must be optimized. This is normally achieved by titrating the concentration of magnesium, template, primer, dNTP, or Taq polymerase; the use of hot start PCR or touch-down PCR; addition of detergents; reduction in PCR cycles; or gradual increase of the annealing temperature. The selection of the annealing temperature is possibly the most critical component for optimizing the specificity of a PCR reaction. In most cases, this temperature must be tested empirically. The PCR is.normally started at 5 deg C below the calculated temperature of the primer melting point (T_m). However, the possible formarion of unspecific secondary bands shows that the optimum temperature is often much higher than the calculated temperature (>12 deg C). This paper describes the use of the gradient function of the Eppendorf~R Mastercycler~R Gradient (Eppendorf-Netheler-Hinz, Hamburg, Germany) to optimize a PCR reaction.