Sequence analysis of a novel HLA-B * 9534 allele and establishment of group specific primers polymerase chain reaction method / 中华微生物学和免疫学杂志

ABSTRACT

Objective To analyze the molecular genetic basis of novel allele HLA-B * 9534 and establish the allele group specific primer PCR method. Methods Genomic DNA was extracted from whole blood by commercial DNA extraction kit. The HLA-B exons 1 to 8 coding sequences of the proband were am-plified by PCR and the amplification product was purified with double enzymes digestion and both strands of exons 2, 3 and 4 were sequenced. The exon 2-4 amplification of the HLA-B * 9534 was performed with al-lele group specific primers PCR and the PCR product was directly sequenced for exon 2 to 4. Results The proband has two HLA-B alleles. The result was assigned for HLA-B * 1518 and B * 4601 combination with a mismatch in 593A/G heterozygote by DNA sequencing of exon 2 to 4 with loci primers. After separating the two alleles of the proband with allele group specific primers polymerase chain reaction method, HLA-B * 4601 and HLA-B * 9534 alleles were identified after sequencing. The HLA-B * 9534 is identical to HLA-B * 1518 except for one nucleotide substitutions in exon 3 at position 593 A→G, this results in amino acid substitution at cedon 174 from Asn to Ser. The sequences of the novel allele have been submitted to GenBank (EU046491) and the allele has been officially nominated by the WHO Nomenclature Committee. Conclusion Identification of a novel HLA-B * 9534 allele and allele group specific primer PCR for HLA-B * 9534 was re-liable.