Li Dong, Zhang Le-ling, Ju Xiu-li, Hou Huai-shui, Shi Qing, Shen Bai-jun
Lab of Cryomedicine, Qilu Hospital, Shandong University, Jinan 250012, China.
Zhonghua Xue Ye Xue Za Zhi. 2007 May;28(5):308-11.
To apply the single cell nested multiplex polymerase chain reaction (PCR) to HLA typing, and analyze the influence factors on the amplification results.
Single cell DNA templates were prepared with different methods. The exon 2, 3 and intron 2 of HLA-A, B, and exon 2 of DRBI were amplified using multiplex PCR. The second round of SSP-PCR HLA typing was carried out according to the large scale routine HLA typing results.
Enzyme lysis method was the most efficient procedure for preparing the single cell DNA template, with a success rate (SR) of 93.3%, while the SRs of alkali lysis and freezing-thaw lysis methods were 83.3% and 73.3%, respectively. The second round amplification using enzyme lysis and SSP-PCR in 20 samples obtained a 95% success rate and a 15% allele drop out rate. The time for performing the whole procedure was less than 6 hours.
The modified nested multiplex PCR technique is efficient for single cell HLA typing and might be applied to clinical preimplantation genetic diagnosis.
将单细胞巢式多重聚合酶链反应（PCR）应用于HLA分型，并分析影响扩增结果的因素。
采用不同方法制备单细胞DNA模板。使用多重PCR扩增HLA-A、B的第2、3外显子和第2内含子以及DRB1的第2外显子。根据大规模常规HLA分型结果进行第二轮序列特异性引物PCR（SSP-PCR）HLA分型。
酶裂解法是制备单细胞DNA模板最有效的方法，成功率（SR）为93.3%，而碱裂解法和冻融裂解法的成功率分别为83.3%和73.3%。在20个样本中使用酶裂解和SSP-PCR进行第二轮扩增，成功率为95%，等位基因脱扣率为15%。整个过程所需时间不到6小时。
改良的巢式多重PCR技术对单细胞HLA分型有效，可能应用于临床植入前遗传学诊断。
