Simplified PCR-SSO procedure for DQA and DQB typing. Simultaneous hybridization of two probes to one membrane.
To simplify DQA and DQB oligotyping, we applied our improved PCR-SSO procedure for DR typing. We used 12 oligonucleotide probes for DQA typing and 18 for DQB typing. Oligonucleotide probes that require the same hybridization and stringent washing conditions were selected as pairs for simultaneous hybridization to a dot-blot membrane containing various DNA samples. One probe of each pair was labeled with digoxigenin and the other with biotin. After hybridization, the dot-blot membranes were incubated with a mixture of conjugates. Specific binding of the corresponding DNA probes was visualized on an X-ray film using a chemiluminescent substrate (CSPD) and by staining using a chromogenic substrate (TMB). This approach, previously employed for DR typing, is also suitable for DQA and DQB oligotyping and significantly reduces the labor inherent in PCR-SSO typing.
Chen
D F
DF
Blutbank-Immunhämatologie-Transfusionsmedizin, Medizinische Hochschule Hannover, Germany.
Endres
W
W
Meyer
S A
SA
Stangel
W
W
D005838:Genotype; D006683:HLA-DQ Antigens / Q000235:genetics*; D059848:HLA-DQ alpha-Chains; D059866:HLA-DQ beta-Chains; D006801:Humans; D008567:Membranes, Artificial; D009693:Nucleic Acid Hybridization / Q000235:genetics*; D015345:Oligonucleotide Probes / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
D006683:HLA-DQ Antigens; D059848:HLA-DQ alpha-Chains; D059866:HLA-DQ beta-Chains; C069050:HLA-DQA1 antigen; C057615:HLA-DQbeta antigen; D008567:Membranes, Artificial; D015345:Oligonucleotide Probes