Design of High-Annealing-Temperature PCR Primers and Their use in the Development of a Versatile Low-Copy-Number Amplification Protocol

High-annealing-temperature (HAT) primers for polymerase chain reaction (PCR) were designed and tested. These HAT primers were used to develop an amplification procedure that employs hypervariable extension times in combination with two temperature amplification cycles, master mixes and hot start to achieve highly reproducible, low-copy-number PCR amplification. The employment of HAT primers drastically reduced cycling times and essentially eliminated nonspecific amplification products even in the presence of vast excesses of nonspecific DNA sequences. Hypervariable extension times provided a simple, noninvasive approach for dynamically modifying the reaction environment during amplification. In addition, this procedure makes extremely efficient use of enzyme, reducing the amount of Taq or Replitherm polymerase to 0.4 and 0.12 U per 50 μl reaction, respectively. The use of PCR master mixes increased the reproducibility and portability of the assay. And finally, a modified form of hot start was employed to reduce primer oligomer formation. HAT primers were designed for the amplification of human cytomegalovirus (HCMV). These primers were used to develop a highly specific, low-copynumber PCR assay that employed a combined annealing/extension temperature of 70 °C or 72 °C. Nonspecific bands, other than primer oligomer bands, were not detected even in the presence of a vast excess of human genomic DNA. These primers can be employed to calibrate the temperature on different thermocylcers as well as to take into account environmental factors that influence amplification. This would be especially advantageous when porting protocols to different machines and simplifying the use of PCR in field studies as well as for identifying factors that influence the local stability at the 3′ end of primers.