High and intermediate resolution DNA typing systems for class I HLA-A, B, C genes by hybridization with...

DNA typing systems for alleles of the HLA class I loci A, B, C at intermediate (IR) and high resolution (HR) levels were developed. The approaches combine locus-specific amplification of genomic DNA by the polymerase chain reaction (PCR) and hybridization with sequence-specific oligonucleotide probes (SSOP). The SSOP were designed to match nucleotide sequences at all polymorphic sites of exons 2 and 3 at these loci. Alleles and genotypes for these loci are assigned by their unique hybridization patterns. Some genotypes with particular allele combinations resulted in the same hybridization pattern. These genotype ambiguities were resolved by performing additional group-specific amplifications with appropriate GSA primers and hybridization with informative SSOP. At intermediate resolution level, many groups of alleles of HLA-A and B with the same serologic equivalence resulted in the same hybridization pattern. Both HR and IR typing approaches required the design, validation and testing of locus- and group-specific primers and sequence-specific oligonucleotide probes (SSOP). Single locus-specific amplification and hybridization with sets of 67 SSOP for HLA-A, 99 for HLA-B and 57 for HLA-C allowed us to identify unequivocally the majority of A, B, C alleles at HR level. To resolve ambiguous genotypes at HLA-B, we performed 4 GSA with 5' primers at codon 45-46 and hybridization with selected sets of SSOP. About 22,415 high resolution typing results were obtained (4,953A, 6,621B, 10,841C). In these samples, 63 HLA-A, 170 HLA-B and 40 HLA-C alleles were observed. In the course of these studies, more than 30 new alleles have been identified. In IR testing, sets of 39 SSOP for HLA-A typing and 59 SSOP for HLA-B typing allowed us to obtain maximal resolution of the majority of common genotypes. To achieve IR level, the majority of SSOP selected were those that span codons encoding amino acid residues located in the alpha helical segments of the class I molecules. A total of 50,522 samples were typed for HLA-A and B at IR level. Approximately 2.0% of them carried ambiguous genotypes associated with alternative switches of Bw4/w6 related sequences. All these ambiguities were resolved by Bw4/Bw6 GSA by 2 primer pairs (77N-IALR-83/3B.1; 77S-DLRG-83/3B.1) and hybridization with 9 selected SSOP Testing was performed by individual hybridization of replicate dot blot membranes with the SSOP of the corresponding set. The approach was robust and cost-effective in large-scale HLA class I molecular typing. The resolution provided by HLA-A, B IR reached serologic split-level or higher. The description of primers and probes for HLA class I typing may be utilized as starting elements for development of second generation methods with a more rapid turn around time.