ISOELECTRIC-FOCUSING SUBTYPES OF HLA-A CAN BE DEFINED BY OLIGONUCLEOTIDE TYPING
ISOELECTRIC-FOCUSING SUBTYPES OF HLA-A CAN BE DEFINED BY OLIGONUCLEOTIDE TYPING
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DOI：
10.1111/j.1399-0039.1993.tb01992.x
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发表时间：
1993-03-01
期刊：
影响因子：
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通讯作者：
YANG, SY
中科院分区：
文献类型：
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作者：
OH, SH;FLEISCHHAUER, K;YANG, SY
本研究描述了一种简单直接的序列特异性寡核苷酸探针（SSOP）分型HLA I类基因a位点的方法。采用血清学和等电聚焦（IEF）方法鉴定HLA- I类抗原的200多个细胞的基因组DNA，用于HLA- a序列的位点特异性PCR扩增。扩增产物与来自外显子2和3高变区的28个sops进行点杂交。HLA-H假基因的三个等位基因与HLA-A2和A10存在明显的连锁不平衡共扩增，但不影响HLA-A等位基因的分型。在低温四甲基氯化铵（TMAC）杂交和洗涤步骤中使用短ssop（每个15个核苷酸），可以通过其杂交模式明确地定义30个ief可定义的HLA-A抗原同型。此外，将分型结果与现有HLA-A等位基因核苷酸序列进行比较表明，所使用的条件可以忠实地检测探针和模板之间的单个密码子不匹配。因此，这些等位基因可以通过ssop产生的独特杂交模式来识别。任何新的HLA-A等位基因的核苷酸序列分析将进一步允许其快速和明确的SSOP分型鉴定。
This study describes a simple and direct method for sequence-specific oligonucleotide probe (SSOP) typing of the A locus of HLA class I genes. Genomic DNA from a panel of over 200 cells which have been characterized by the methods of serology and isoelectric focusing (IEF) for the HLA class I antigens was used for locus-specific PCR amplification of HLA-A sequences. Dot blot hybridization of the amplified products was performed with 28 SSOPs derived from hypervariable regions in exon 2 and 3. Co-amplification of three alleles of HLA-H pseudogene in apparent linkage disequilibrium with HLA-A2 and A10 was observed but did not interfere with the typing of HLA-A alleles. Using short SSOPs (15 nucleotides each) in single temperature tetramethylammonium chloride (TMAC) hybridization and washing steps, 30 IEF-definable isotypes of HLA-A antigens could be unambiguously defined by their hybridization patterns. Moreover, comparison of the typing results with available nucleotide sequences of HLA-A alleles showed that the conditions used allowed faithful detection of single codon mismatches between probe and template. Thus, these alleles can be identified by their unique hybridization patterns generated by the SSOPs. Nucleotide sequence analysis of any new HLA-A allele will further permit its rapid and unambiguous characterization by SSOP typing.