DNA typing of primate major histocompatibility complex (Mhc)-DQA1 locus by PCR and dot blot hybridization.
Non-human primates (NHPs) are increasingly utilized as models to investigate different aspects of immune responses against self (autoimmunity) and foreign antigens. These animals provide valuable models for testing the efficacy of candidate vaccines against pathogens such as human immunodeficiency virus (HIV) and also fertility regulating agents (immunocontraceptives). In order to fully understand the effects of vaccination, it may be necessary to elucidate the immunogenetic background of these animals. The major histocompatibility complex (Mhc) molecules play an important role in the generation of effective immune responses. Serological techniques have been used in the identification of human leukocyte antigens (HLA) necessary for cross-matching organs and tissues for transplantation. However, the application of this technique for typing monkey Mhc alleles has been hampered by unavailability of well characterized immunological reagents. Polymerase chain reaction (PCR)-based techniques such as restriction fragment length polymorphism (RFLP) and sequence-specific oligonucleotide probe hybridization (SSOP) have been extensively used for typing HLA-DP, DQ and DR alleles. A commercially available Kit (AmpliTypeR) designed for amplification and typing of HLA DQalpha alleles is routinely used in typing DNA samples for forensic casework. In the present study, we have evaluated this kit for possible application in routine typing of primate DQA1 alleles. Genomic DNA from ten African primate species (23 individuals) was isolated from peripheral blood lymphocytes and polymorphic second exon of DQA1 locus amplified using GH26 and GH27 PCR primers. The PCR products were hybridized on a nylon membrane containing immobilized sequence-specific oligonucleotide probes. Our results show seven of the nine probes hybridizing with primate DQA1 alleles, indicating that typing of equivalent primate alleles can be accomplished at lower stringency conditions. However, it may be necessary to design additional oligonucleotides probes (based on available primate DQA1 sequences) to improve the discriminating power of this kit for use in routine typing of Old World monkey DQA1 alleles.
Mwenda
J M
JM
Institute of Primate Research, National Museums of Kenya, Nairobi. jmmwenda@arcc.or.ke
Shotake
T
T
Yamamoto
T
T
Uchihi
R
R
Bambra
C S
CS
Katsumata
Y
Y
D000818:Animals; D005838:Genotype; D006683:HLA-DQ Antigens / Q000235:genetics*; D059848:HLA-DQ alpha-Chains; D006801:Humans; D008285:Major Histocompatibility Complex / Q000235:genetics*; D015336:Molecular Probe Techniques; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D011323:Primates / Q000235:genetics*; D011933:Reagent Kits, Diagnostic
D006683:HLA-DQ Antigens; D059848:HLA-DQ alpha-Chains; C069050:HLA-DQA1 antigen; D015345:Oligonucleotide Probes; D011933:Reagent Kits, Diagnostic