细胞裂解法对单细胞PCR扩增效率的影响  

The assessment of the amplified efficacy of single cell PCR by diffent lysis buffers.

作　　者：易萍[1] 李力[1] 王华[2] 邓兵[3] 

机构地区：[1]第三军医大学大坪医院野战外科研究所妇产科,重庆400042 [2]第三军医大学大坪医院野战外科研究所分子生物学中心,重庆40004 [3]重庆医科大学附属儿童医院,重庆400014

出　　处：《中国优生与遗传杂志》2005年第2期27-28,32,共3页Chinese Journal of Birth Health & Heredity

基　　金：国家科技部 973项目 (2 0 0 1CB5 10 3 0 5 )

摘　　要：目的　研究细胞裂解法对单细胞PCR扩增效率的影响 ,寻求较好的细胞裂解方法。方法　应用显微分离技术分离单个淋巴细胞 ,分别使用冻融变性和蛋白酶K裂解法处理单细胞 ,应用多重PCR同时扩增X染色体特异重复序列(DXZ1)和Y染色体特异重复序列 (DYZ1)对单细胞进行性别诊断 ,比较其扩增效率。结果　使用显微操作法获得 80个男性淋巴细胞。采用冻融变性处理单细胞 ,4 0个淋巴细胞经多重PCR扩增后有 32个产生DXZ1和DYZ1两条电泳带 ,正确率为80 %。采用蛋白酶K裂解法 ,4 0个淋巴细胞经多重PCR扩增后均产生DXZ1和DYZ1两条电泳带 ,正确率为 10 0 %。两者的扩增效率有显著的差异性 (χ2 =6 .81,P<0 .0 1)。结论　Rechitsky等的蛋白酶K裂解法比较简便 ,费时短 ,扩增效率高 ,适合细胞裂解及PCR扩增模板的制备。Objective: To assess the amplified efficacy of singlecell PCR by diffent lysis buffers and choose the better lysis method. Methods: Single lymphocyte was isolated under the inverted microscope equipped with micromanipulations. Two sorts of cell lysis methods (freezing-thawing and proteinase K lysis) were compared by their efficacy of multiplex amplification of DXZ1 and DYZ1 from a single cell. Results: Under the inverted microscope equipped with micromanipulations, 80 lymphocytes were isolated. The amplification efficacy was 100% with cell lysis of proteinase K, and 80% with cell lysis of freezing-thawing. The amplification efficacy of two cell lysis methods showed significant difference (χ2=6.81,P<0.01). Conclusions:Proteinase K cell lysis method is simple and rapid. It can result in high amplification efficacy and meet to cell lysis and preparation of DNA template for amplification.

关 键 词：PCR扩增 淋巴细胞 单细胞 增效 多重PCR 进行性 性别诊断 细胞裂解 电泳 重复序列 

分 类 号：Q25[生物学—细胞生物学]