ROUTINE HLA DRB DQB OLIGONUCLEOTIDE TYPING BY A NONRADIOACTIVE DOT-BLOT MICROMETHOD/

In the last few years the clinical need for HLA genotyping has become evident. However, the routine use of PCR-based DNA typing techniques h as been hampered by economical and/or technical considerations. The cl assical PCR-SSO (product-dot) method has been widely tested and proven to be useful for large-scale HLA DNA typing. However, it is not a sui table method for routine typing of single samples because it takes sev eral days. Using primers and probes for sequences identical to those c ompiled by the Eleventh International Histocompatibility Workshop, we designed a non-radioactive dot-blot technique in which each hybridizat ion reaction is performed in a microtiter plate well containing PCR-am plified DNA that has been previously dotted on a small nylon membrane, so that a large number of oligonucleotide probes tailed with biotin-1 4-dATP can be simultaneously tested against the same sample. We studie d 23 B-lymphoblastoid cell lines of known HLA genotype to test the met hod and, so far, it has been validated on more than 100 patients and h ealthy relatives typed prospectively. This simple, rapid, inexpensive PCR-SSO dot-blot micromethod makes DRB/DQB DNA typing of single sample s possible in a short period of time, and is therefore an attractive a lternative to serological typing in routine medical practice.