Routine HLA DRB/DQB oligonucleotide typing by a non-radioactive 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 has been hampered by economical and/or technical considerations. The classical 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 suitable method for routine typing of single samples because it takes several days. Using primers and probes for sequences identical to those compiled by the Eleventh International Histocompatibility Workshop, we designed a non-radioactive dot-blot technique in which each hybridization reaction is performed in a microtiter plate well containing PCR-amplified DNA that has been previously dotted on a small nylon membrane, so that a large number of oligonucleotide probes tailed with biotin-14-dATP can be simultaneously tested against the same sample. We studied 23 B-lymphoblastoid cell lines of known HLA genotype to test the method and, so far, it has been validated on more than 100 patients and healthy relatives typed prospectively. This simple, rapid, inexpensive PCR-SSO dot-blot micromethod makes DRB/DQB DNA typing of single samples possible in a short period of time, and is therefore an attractive alternative to serological typing in routine medical practice.
García-Pacheco
J M
JM
Unidad de Inmunología, Hospital do Meixoeiro, Vigo, Spain.
Mantilla
P
P
García-Olivares
E
E
Manzano-Fernández
M N
MN
D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000032:analysis; D017931:DNA Primers; D015342:DNA Probes; D005260:Female; D005838:Genotype; D006683:HLA-DQ Antigens / Q000737:chemistry*; D006684:HLA-DR Antigens / Q000737:chemistry*; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D008214:Lymphocytes / Q000737:chemistry; D008297:Male; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000032:analysis*; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D015342:DNA Probes; D006683:HLA-DQ Antigens; D006684:HLA-DR Antigens; D009841:Oligonucleotides; D004247:DNA