CHARACTERISTICS OF SELECTIVE POLYMERASE CHAIN REACTION (PCR) USING TWO-BASE ANCHORED PRIMERS AND IMPROVEMENT OF ITS SPECIFICITY

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