A polymerase chain reaction-sequence-specific oligonucleotide procedure for HLA class II typing using biotin- and digoxigenin-labeled probes simultaneously in hybridization.
To simplify PCR-SSO HLA-DRB generic typing, we labeled eight of 15 oligonucleotide probes with DIG and the others with biotin, and hybridized each dot blot with both a biotin-labeled probe and a DIG-labeled probe simultaneously. We chose oligonucleotide pairs which require the same hybridization and stringent washing conditions and do not compete with each other during hybridization. After incubation with a mixture of anti-DIG Fab fragment-alkaline phosphatase and streptavidin-peroxidase conjugates, specific binding of the DIG-labeled probe was revealed by a chemiluminescent substrate (CSPD) and specific binding of the biotin-labeled probe was subsequently visualized by a chromogenic substrate (TMB). The sensitivity of both probes was similar and gave comparable hybridization signals. Using this simplified procedure, the number of hybridizations or dot blots can be reduced to half the usual amount and the labor involved in PCR-SSO typing significantly reduced.
Chen
D F
DF
Bloodbank-Immunohematology-Transfusion Medicine, Medical University of Hannover, Germany.
Endres
W
W
Meyer
S A
SA
Stangel
W
W
D001483:Base Sequence; D001710:Biotin; D002460:Cell Line; D004247:DNA / Q000032:analysis; D004076:Digoxigenin; D005838:Genotype; D006684:HLA-DR Antigens / Q000032:analysis*; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D006684:HLA-DR Antigens; D015345:Oligonucleotide Probes; D001710:Biotin; D004247:DNA; D004076:Digoxigenin