COMPLETE SUBTYPING OF THE HLA-A LOCUS BY SEQUENCE-SPECIFIC AMPLIFICATION FOLLOWED BY DIRECT SEQUENCING OR SINGLE-STRAND CONFORMATION POLYMORPHISM ANALYSIS
COMPLETE SUBTYPING OF THE HLA-A LOCUS BY SEQUENCE-SPECIFIC AMPLIFICATION FOLLOWED BY DIRECT SEQUENCING OR SINGLE-STRAND CONFORMATION POLYMORPHISM ANALYSIS
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DOI：
10.1111/j.1399-0039.1995.tb02483.x
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发表时间：
1995-08-01
期刊：
影响因子：
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通讯作者：
SALAMA, A
中科院分区：
文献类型：
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作者：
BLASCZYK, R;HAHN, U;SALAMA, A
与HLA I类系统相关的各种原因使得分子方法在HLA I类分型中的应用变得复杂。在这里，我们提出了一种基于pcr的HLA-A分型策略，考虑了两个最多态性外显子的序列变化，从而可以对HLA-A位点进行完整的分型。该方法是基于序列特异性扩增鉴定血清学定义的HLA-A特异性。这些组特异性引物产生的PCR产物具有扩增后特异性步骤所需的序列信息。引物对位于一个外显子内，要么是外显子2，要么是外显子3，这避免了多态内含子序列的扩增，从而允许随后的单链构象多态性分析，并便于直接测序。我们用这种方法研究了48个细胞系和153个临床样本。每个个体进行23次PCR反应以确定Al-A80的血清学特异性。在所有细胞系和85个临床样本中，在两个不同的场合进行分型，可重复性为100%。除了231种可能的血清学组合中的13种外，所有Al-A80的纯合和杂合组合都可以通过特定的扩增模式来区分。PCR分型结果与血清学分型结果有12%的差异。群体特异性PCR片段的固相测序或SSCP分析允许HLA-A位点的完整亚型。该策略可以根据第2和第3外显子的序列变化识别所有48个HLA-A等位基因。HLA-A位点可能存在1128个纯合和杂合等位基因组合。在这1128个等位基因组合中，只有4个未解决。
A variety of reasons related to the HLA class I system has complicated the application of molecular approaches to HLA class I typing. Here we present a PCR-based HLA-A typing strategy considering the sequence variations of the two most polymorphic exons which allows complete subtyping of the HLA-A locus. The method is based on a sequence-specific amplification identifying the serologically defined HLA-A specificities. The PCR products generated by these group-specific primers bear the sequence information necessary for a postamplification specificity step. The primer pairs are located within one exon, either exon 2 or exon 3, which avoids amplification of polymorphic intron sequences allowing subsequent single-strand conformation polymorphism analysis and facilitating direct sequencing. Using this method we investigated 48 cell lines and 153 clinical samples. 23 PCR reactions are performed per individual for the assignment of the serological specificities Al-A80. The reproducibility was 100% in all cell lines and 85 clinical samples typed on two separate occasions. With the exception of 13 out of 231 possible serological combinations all homozygous and heterozygous combinations of Al-A80 can be distinguished by specific amplification patterns. Comparing the PCR based typing results with those of serology in 12% a discrepancy was found. Solid-phase sequencing or SSCP analysis of the group-specific PCR fragments allowed complete subtyping of the HLA-A locus. This strategy can identify all 48 HLA-A alleles based on the sequence variations of the 2nd and 3rd exon. 1128 homozygous and heterozygous allele combinations are possible for the HLA-A locus. Only 4 out of these 1128 allele combinations remained unresolved.