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Technical Note: DNA Typing for Ovine MHC DRB1 Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP)

S. Konnai^*,,1, Y. Nagaoka^*,1,2, S. Takesima^*, M. Onuma and Y. Aida^*

^* Retrovirus Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 Japan
Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido 060-0818 Japan

Corresponding author: Y. Aida; e-mail: aida{at}postman.riken.go.jp.

The ovine major histocompatibilty complex (Ovar) class II DRB1 second exon was amplified by polymerase chain reaction (PCR) from DNA samples of 52 Suffolk sheep. Polymerase chain reaction products were characterized by the restriction fragment length polymorphism (RFLP) technique using nine restriction enzymes, RsaI, HaeIII, SacI, SacII, DdeI, NciI, Hin1I, EcoRI, and BstNI, yielding 13 types. Sequencing of cloned PCR products identified 16 Ovar-DRB1 alleles. Collectively, all PCR-RFLP patterns exactly matched those predicted from DNA sequences. These findings strongly indicate that the PCR-RFLP method using a combination of nine restriction endonucleases is a very powerful tool in Ovar typing.

Key Words: Ovar-DRB1 exon 2 • PCR-RFLP • genetic polymorphism • restriction enzyme

Abbreviation key: MHC = major histocompatibility complex, Ovar = ovine MHC, SSCP = single-strand conformational polymorphism.