Primer Extension Reactions

                         For allelic discrimination, the amplicons containing the target polymorphic site serve as templates for PE reactions. Prior to the PE reactions, the amplicons are purified by treatment at 37°C with exonuclease I and shrimp alkaline phosphatase in order to remove the unincorporated single strand oligo primers and deoxynuclotides respectively.

                         The enzymes are then inactivated at 80°C. After purification, the PCR products are mixed with Thermo Sequenase (GE Healthcare) and appropriate ddNTPs. The PE reactions are carried out in a thermal cycler with a universal thermal cycling condition that includes denaturation at 96°C, annealing at 43°C followed by extension at 60°C.

                         The single strand extended products are analyzed by DHPLC under completely denaturing condition.