LOCALIZED SEQUENCE REGIONS POSSESSING HIGH-MELTING TEMPERATURES PREVENT THE AMPLIFICATION OF A DNA MIMIC IN COMPETITIVE PCR

The polymerase chain reaction is an immensely powerful technique for i dentification and detection purposes. Increasingly, competitive PCR is being used as the basis for quantification. However, sequence length, melting temperature and primary sequence have all been shown to influ ence the efficiency of amplification in PCR systems and may therefore compromise the required equivalent co-amplification of target and mimi c in competitive PCR. The work discussed here not only illustrates the need to balance length and melting temperature when designing a compe titive PCR assay, but also emphasises the importance of careful examin ation of sequences for GC-rich domains and other sequences giving rise to stable secondary structures which could reduce the efficiency of a mplification by serving as pause or termination sites. We present data confirming that under particular circumstances such localised sequenc e, high melting temperature regions can act as permanent termination s ites, and offer an explanation for the severity of this effect which r esults in prevention of amplification of a DNA mimic in competitive PC R. It is also demonstrated that when Tao DNA polymerase is used in the presence of betaine or a proof reading enzyme, the effect may be redu ced or eliminated.