Six Mamu-A locus alleles defined by a polymerase chain reaction sequence specific primer method

Rhesus monkeys are relevant models for human diseases and transplantation. In each case, a complete understanding of these models requires knowledge of macaque MHC. Due to high polymorphism and multiple genes per haplotype, it has been difficult to develop a rapid typing method for rhesus monkey MHC class I. We developed a simple and rapid PCR-SSP strategy for rhesus monkey Mamu-A locus typing. Fifty-two rhesus monkeys were included in the study. Six rhesus monkey allel-specific primer pairs were designed based on published Mamu-A locus gene sequences. Allele-specific PCR products ranged in size from 346 to 788 bp; 5′ and 3′ Mamu-A locus allele specific primers were located in the second and third exons, respectively. Specific PCR product gel purification was followed by direct sequencing, without subcloning, in both directions. Our data showed variability in the number of Mamu-A alleles ranging from 1 to 4 per genotype. The highest frequencies were observed for Mamu-A∗02, -A∗04, and -A∗03 alleles. Thus, we report here the first PCR-SSP typing method for Mamu-A∗02, -03, -04, -05, -06, and -07 array of class I alleles. This technique appears to be a highly reproducible and discriminatory method for detecting this subset of class I A locus genes in rhesus monkeys.