HLA-C high resolution typing: analysis of exons 2 and 3 by sequence based typing and detection of polymorphisms in exons 1-5 by sequence specific primers
HLA-C high resolution typing: analysis of exons 2 and 3 by sequence based typing and detection of polymorphisms in exons 1-5 by sequence specific primers
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DOI：
10.1111/j.1399-0039.1998.tb03040.x
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发表时间：
1998-09-01
期刊：
影响因子：
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通讯作者：
Ferrara, GB
中科院分区：
文献类型：
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作者：
Delfino, L;Morabito, A;Ferrara, GB
本研究中开发的基于 HLA-C 高分辨率序列的分型涉及包含外显子 1 到内含子 3 的独特 DNA 扩增以及涵盖外显子 2 和 3 的四个荧光测序反应。使用了染料引物和染料终止子测序技术并比较了结果。这种方法可以鉴定迄今为止描述的所有 50 个 HLA-C 等位基因变体，除了通过非编码核苷酸变化区分的两个等位基因对 (Cw*12021=12022、Cw*15051 = 15052) 和三个等位基因对 (Cw*0701 = 0706、Cw*1701 = 1702 和 Cw*1801 = 1802）在外显子 2 和 3 中共享相同的核苷酸序列。为了对这些等位基因变体进行完整的亚型分析，使用了基于序列特异性引物 (PCR-SSP) 的扩增。到目前为止，我们的小组中没有检测到模糊的等位基因杂合组合。将通过该方法获得的 HLA-C 分型数据与之前对测序样本进行的血清学和低分辨率 PCR-SSP 分型数据进行比较。
HLA-C high resolution sequence based typing developed in this study involves a unique DNA amplification encompassing exon 1 to intron 3 and four fluorescent sequencing reactions covering exon 2 and 3. Both dye primer and dye terminator sequencing techniques were performed and results compared. This approach allowed the identification of all of the 50 HLA-C allelic variants so far described, except for two allele pairs that are distinguished by non-coding nucleotide changes (Cw*12021=12022, Cw*15051 = 15052) and three allele pairs (Cw*0701 = 0706, Cw*1701 = 1702 and Cw*1801 = 1802) that share the same nucleotide sequence in exon 2 and 3. For complete subtyping of these allelic variants, an amplification based on sequence specific primers (PCR-SSP) was used. No ambiguous heterozygous combinations of alleles were detected in our panel so far. HLA-C typing data obtained by this method were compared with data from serological and low resolution PCR-SSP typing, which had been performed previously on the samples sequenced.