Pm. Giffard et al., Comparison of competitively primed and conventional allele-specific nucleic acid amplification, ANALYT BIOC, 292(2), 2001, pp. 207-215
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.