Non-radioactive oligotyping for HLA-DR1-DRw10 using polymerase chain reaction, digoxigenin-labelled oligonucleotides and chemiluminescence detection.
We describe a rapid non-radioactive DNA typing of the serological types DR1-DRw10 using polymerase chain reaction (PCR)-amplified DNA and 15 sequence-specific oligonucleotides (SSO) which are labelled enzymatically at their 3' end with one digoxigenin (DIG). The hybridized SSOs were detected using anti-DIG alkaline phosphatase and Fab fragments and visualization was obtained with the chemiluminescent substate 3-(2'-spiroadamantan)-4-(3''-phosphoryloxy)-phenyl-1,2-di o xetan (AMPPD). The results were identical with those of the previously used 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)/4-nitrobluetetrazolium chloride (NBT) system. The use of AMPPD is more rapid and allows the repeated rehybridization of the membrane-bound DNA.
Nevinny-Stickel
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Labor für Immungenetik, Kinderpoliklinik, München, Germany.
Hinzpeter
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M
Andreas
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Albert
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ED
D001483:Base Sequence; D004076:Digoxigenin; D006684:HLA-DR Antigens / Q000235:genetics*; D006239:Haplotypes; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry; D016133:Polymerase Chain Reaction
D006684:HLA-DR Antigens; D015345:Oligonucleotide Probes; D004076:Digoxigenin