RESUMO
A simple and efficient oligonucleotide array was developed to identify single nucleotide polymorphisms (SNPs) encoded within the highly polymorphic human major histocompatibility complex (MHC) using HLA-B as a model system. A total of 137 probes were designed to represent all known polymorphisms encoded in exons 2 and 3. PCR products were amplified from human genomic DNA and allowed to hybridize with the oligonucleotide array. Hybridization was detected by fluorescence scanning, and HLA-B alleles were assigned by quantitative analysis of the hybridization results. Variables known to influence the specificity of hybridization, such as oligonucleotide probe size, spacer length, surface density, hybridization conditions, and array uniformity and stability were studied. The efficiency and specificity of identifying HLA-B SNPs using the oligonucleotide arrays was evaluated by blinded analysis of 100 samples from unrelated individuals representing all HLA-B phenotypes. The oligonucleotide array method described in this paper provides unambiguous detection of complex heterozygous SNP combinations. This methodological approach may be applied to other highly polymorphic gene systems.
