运用全长序列鉴定HLA-C罕见基因的实验研究

摘要

Objective To test and analysis 2 rare alleles that donors carry. Methods Genomic DNA was extracted from blood samples by quick DNA purified kit and then tested by HLA-C locus' commercial SBT(sequence-based typing) kit. The purified PCR product was used as the DNA template in the sequencing reaction,forward and reverse sequencing primers in commercial kit were used for the sequencing of exons 2,3 and 4. 4 direct sequencing reactions of PCR product for exons 5 in both directions,exon 6 in forward direction and exon 7 in reverse direction were performed using self—developed kit. Sequencing result was analyzed with Assign-SBT 3. 5. 1. 45(Conexio Genomies) software. The samples were tested by PCR—sequence specific primer(PCR-SSP) again. Results HLA typing results of the samples were HLA-C * 07 : 63 and HLA-C * 01 : 24. Conclusion Full-length sequencing could be used to make sure the ambiguous SBT results. The 2 rare alleles also provide an available basis for the establishment of the HLA high resolution typing database for China Marrow Donor Program.%目的 检测与分析2例骨髓志愿捐献者携带的罕见等位基因.方法 用快速DNA提取试剂盒从全血样本中提取基因组DNA,经HLA-C基因商品化测序分型试剂盒扩增,纯化后的扩增产物作为模板用试剂盒配套的第2、3、4外显子正反向测序引物及自行研制的第5外显子正反向、第6外显子正向和第7外显子反向测序引物进行测序,结果导入Assign-SBT 3.5.1.45软件分析,并用PCR序列特异性引物技术(SSP)对标本进行确认.结果 分型结果显示其中一例为HLA-C* 07:63,另一例为HLA-C* 01:24.结论 临床移植配型遇罕见基因时应测定全长序列以提高分型结果的准确性,此两例罕见等位基因为中华骨髓库建立HLA高分辨数据库提供了有效依据.