Characteristics of selective polymerase chain reaction (PCR) using two-base anchored primers and improvement of its specificity

We have developed a reliable method for eliminating base-mispair amplification in selective polymerase chain reaction (PCR), which is utilized for amplifying unknown sequence fragments produced by restriction enzyme reaction. The proposed procedure applies amplified fragment length polymorphism (AFLP) with high fidelity. Selective PCR utilizes the known polymerase reaction characteristic that the complementary strand extension is strongly affected by matching a template with the 3′-terminus of the primers. However, false positive amplification is frequently observed because the specificity of terminal bases for discrimination of fragments (usually, 1-3 anchor sequences) is not enough to separate each fragment. A protocol for the selective PCR separation of every fragment was therefore investigated. A single-base mismatch was artificially introduced on the 4th base position from the 3′ end of the primers to improve the hybridization specificity of anchored 2-bases at the 3′ termini of primers. PCR reaction was carried out at 66°C to prevent false positive amplification. The concentration of the primers having anchored-base sequences of AA, AT, TA, and TT must be three times larger than that of other primers because the T_m values for these sequences are lower than the others. As all the fragments can be separated into groups with high fidelity, the improved selective PCR will be applied to gene finding and analyzing differences on genome sequences based on AFLP.