ABSTRACT
Allelic variability has traditionally been determined phenotypically, but often may be more accurately investigated via molecular biology techniques, either indirectly by restriction fragment length polymorphism (RFLP) analysis or directly by polymerase chain reaction-based (PCR) techniques. In the determination of allelic polymorphism by PCR amplification with sequence-specific primers (PCR-SSP), oligonucleotide primers are designed to obtain amplification of specific alleles or groups of alleles. The typing method is based on the principle that a completely matched primer will be more efficiently used in the PCR reaction than a primer with one or several mismatches, especially in the first critical cycles. Twenty-one primer mixes are used for DR "low-resolution" PCR-SSP typing: 17 for assigning DR1-DRw18 and 3 for identifying the DRB3, DRB4, and DRB5 groups of alleles. The annealing temperature of PCR cycle is also of critical importance in the PCR-SSP typing system.
