The polymerase chain reaction based restriction fragment length polymorphism (FCR-RFLP)method was used to study DLA class Ⅱgene n dogs.Genomic DNA from 11 DLA homozygous reference dogs representing 8 different haplot...The polymerase chain reaction based restriction fragment length polymorphism (FCR-RFLP)method was used to study DLA class Ⅱgene n dogs.Genomic DNA from 11 DLA homozygous reference dogs representing 8 different haplotypes and 2 families with a total of 16 animals were amplified by the oligonuclectide primer pair (HLA-DRB-AMP-A/B) cross-hybriding HLA-DRB specific and fit for the amplification of DLA-DRE1 gene.The corresponding amplified DNA products were 235 base pairs[1].Amplified DNA was digested by 32 different restriction endonucleasts,whith could recognize allelic variations within DLA-DRB.After digesting only with Hae Ⅲ,HhaI,HitfI,RsaIand Sau96 high polymorphism was revealed respectively and 9 distinct RFLP pattern wert shown, which could be correate to the DLA haplotypes studied. The 8 cellular established DLA-D specificities presentin the reference panel were defined unequivocally by PCR-RFLP and correlated with DLADw5 and Dw6 two subtypes.The segregation pattern of four different DLA-DRE types could be demonstrated in two families.Based on these data we conclude that PCRRFLP typing utilizing the above mentioned priiner pair and endonuleases is a valuable tool to define DLA class Ⅱ types in the dog.展开更多
文摘The polymerase chain reaction based restriction fragment length polymorphism (FCR-RFLP)method was used to study DLA class Ⅱgene n dogs.Genomic DNA from 11 DLA homozygous reference dogs representing 8 different haplotypes and 2 families with a total of 16 animals were amplified by the oligonuclectide primer pair (HLA-DRB-AMP-A/B) cross-hybriding HLA-DRB specific and fit for the amplification of DLA-DRE1 gene.The corresponding amplified DNA products were 235 base pairs[1].Amplified DNA was digested by 32 different restriction endonucleasts,whith could recognize allelic variations within DLA-DRB.After digesting only with Hae Ⅲ,HhaI,HitfI,RsaIand Sau96 high polymorphism was revealed respectively and 9 distinct RFLP pattern wert shown, which could be correate to the DLA haplotypes studied. The 8 cellular established DLA-D specificities presentin the reference panel were defined unequivocally by PCR-RFLP and correlated with DLADw5 and Dw6 two subtypes.The segregation pattern of four different DLA-DRE types could be demonstrated in two families.Based on these data we conclude that PCRRFLP typing utilizing the above mentioned priiner pair and endonuleases is a valuable tool to define DLA class Ⅱ types in the dog.
