High resolution HLA-DQB1 typing by combination of PCR-RFLP and PCR-SSCP.
A reliable method for high-resolution HLA-DQB1 typing using a combination of PCR- restriction fragment length polymorphism (RFLP) and PCR-single strand conformation polymorphism (SSCP) analysis is described. The second exon of the DQB1 gene was subjected to PCR using generic primers and digested with two restriction enzymes, MspA1I and HaeIII, and the DQB1 alleles were divided into seven groups. According to the RFLP patterns, appropriate group specific primers for DQ5, 6 and DQ2, 3, 4 groups were used to selectively amplify the alleles and the SSCP technique was used to distinguish the individual alleles. A total of 88 quality control samples of various ethnic groups distributed in the International Cell Exchange and HLA DNA Exchange programs and the ASHI/CAP Proficiency Tests were investigated by the PCR-RFLP/SSCP method. The concordance between our typing results and the consensus results of the surveys were 100%, and a total of 14 DQB1 alleles in 49 homozygous and heterozygous combinations were all correctly identified by the method described. This method is accurate, economical and relatively easy to interpret and well suited for routine clinical and research uses.
Park
M H
MH
Department of Clinical Pathology, Seoul National University College of Medicine, Korea. parkmhee@snu.ac.kr
Whang
D H
DH
Kang
S J
SJ
D006683:HLA-DQ Antigens / Q000145:classification* / Q000235:genetics; D059866:HLA-DQ beta-Chains; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D018807:Polymorphism, Single-Stranded Conformational
D006683:HLA-DQ Antigens; D059866:HLA-DQ beta-Chains; C069051:HLA-DQB1 antigen