Identification of new Mamu-DRB alleles using DGGE and direct sequencing

Abstract

Rhesus macaques represent important animal models for biomedical research. The ability to identify macaque major histocompatibility complex (Mhc) alleles is crucial for fully understanding these models of autoimmune and infectious disease. Here we describe a rapid and unambiguous way to distinguish DRB alleles in the rhesus macaque using the polymerase chain reaction, denaturing gradient gel electrophoresis (DGGE), and direct sequencing. The highly variable second exon of Mamu-DRB alleles was amplified using generic DRB primers and alleles were separated by DGGE. DNA was then reamplified from plugs removed from the gel and alleles were determined using fluorescent-based sequencing. Validity of this typing procedure was confirmed by identification of all DRB alleles for three macaques previously characterized by cloning and sequencing techniques. Importantly, our analysis revealed DRB alleles not previously identified in the three reference animals. Using this technique, we identified 40 alleles in fifteen unrelated macaques. On the basis of phylogenetic tree analyses, 14 new DRB alleles were assigned to 10 different Mhc-DRB lineages. Interestingly, two of the new DRB6 lineages had previously been identified in prosimians and pigtailed macaques. Whereas traditional DRB typing methods provide limited information, our new technique provides a simple and relatively rapid way of identifying DRB alleles for tissue typing, determining individual identification and studies of disease association and susceptibility. This new technique should also contribute to ongoing studies of Mhc function and evolution in many different species of nonhuman primates.

Mhc class I haplotypes associated with survival time in simian immunodeficiency virus (SIV)-infected rhesus macaques

“…25,29,55 For the novel class I alleles, a total of 23 primer pairs were created (Table 1). Allele-specific PCR products ranged in size between 408 and B1300 bp.…”

“…DRB-specific primers (MDRB-5 0 and 3 0 -MDR) or actin gene primers served as internal controls. 55,56 HPLCpurified primers were obtained from VBC-GENOMICS Bioscience Research (Vienna, Austria). Reaction mixtures of 25 ml contained 100-150 ng DNA, PCR buffer, Qsolution, 200 mM dNTP, 750-800 mM allele-specific forward and reverse primer, 80-120 nM of the internal control primers and 1.25 unit Taq polymerase (Qiagen).…”

Unprecedented Polymorphism of Mhc-DRB Region Configurations in Rhesus Macaques

“…PCR of exon 2 of Mamu-DRB was conducted with generic primers as previously described (27) (N. Otting, N. G. de Groot, M. C, Noort, G. G. M. Doxiadis, and R. E. Bontrop, manuscript in preparation). For amplification with sequence-specific primers (SSP) of Mamu-DRB6 genes with the 62-bp deletion (25), the following primers were used with restriction sites for SalI and XbaI, respectively: (5Ј-MDRB6) 5Ј-TTG GTC GAC GCT AAG TGY GAS TGT CMT A-3Ј and (3Ј-MDRB6) 5Ј-CTC TCT AGA CCS RYA ATT GTA AYT CTG T-3Ј.…”

“…Separation of Mamu-DRB alleles with DGGE was conducted as described, with minor modifications (27). Briefly, GC-clamped PCR products of DRB exon 2 were electrophoresed in a 9% acrylamide (acrylamide-bisacrylamide, 37.5:1) gel with a 40 -65% parallel denaturing gradient of urea and formamide at a constant temperature of 57°C for ϳ3.5 h. When DNA fragments are electrophoresed through an increasing gradient of denaturants, the alleles dissociate at different rates because of sequence-specific differences in denaturation.…”

“…The colony has been pedigreed based on segregation of serologically defined Mamu-A, -B, and -DR Ags, and 10 Mamu-DR specificities have been defined (26). Subsequently, a large segment of the BPRC breeding colony has been analyzed by molecular techniques as denaturing gradient gel electrophoresis (DGGE) to screen for Mamu-DRB exon 2 variation (27). Animals that show unknown DGGE profiles were subjected to nucleotide sequence analysis.…”