Comparison of competitively primed and conventional allele-specific nucleic acid amplification

A simulation of competitively primed allele-specific DNA amplification has been constructed and its behavior examined, This has shown that when the ra tio of the amount of homoduplex misprime product to the total amount of amp limer is low, it increases by approximately one-fourth of the mispriming fr equency with each doubling of the total amount of amplimer, When the ratio is high acid reverse mispriming becomes significant, it asymptotes toward a value <0.5. An analogous simulation was carried out on conventional allele -specific DNA amplification. As expected, the ratio of the amount of amplim er in the positive and negative reactions closely approximates the misprimi ng frequency provided that amplification is exponential in both cases. This suggests that conventional allele-specific amplification has somewhat high er inherent specificity than competitively primed amplification, However, c onventional allele-specific reactions are subject to a "catch-up" phase in which the positive reaction slows or stops, thus reducing the specificity. It was hypothesized that competitively primed reactions may be easier to op timize than conventional allele-specific reactions. This conjecture was sup ported experimentally. In addition, it was shown that the specificity of co mpetitively primed reactions is a function of the degree of amplification. (C) 2001 Academic Press.