[HLA-DR typing using PCR-SSO with simultaneous hybridization of each dot blot membrane with a digoxigenin- and a...

Here we present a PCR-SSO procedure designed to simplify HLA-DR typing. We labelled 8 of a total of 16 oligonucleotide probes with digoxigenin, the others with biotin, and formed 8 pairs, each containing a digoxigenin- as well as a biotin-labelled probe. Each pair was hybridized simultaneously to one of eight dot blot membranes containing the DNA to be typed. Specific binding was detected by incubation with a mixture of the appropriate conjugates followed by sequential addition first of a chemiluminescent substrate to detect the digoxigenin-labelled probe and then a chromogenic substrate for detection of the biotin-labelled probe. With this procedure, the specific binding of two different probes to the same dot blot membrane could be evaluated, considerably reducing the labor inherent in PCR-SSO typing.