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.