LDR结合毛细管电泳技术用于检测母体血浆中胎儿父系DNA点突变的可行性研究  

The feasibility of PCR/LDR/capillary electrophoresis for non-invasive prenatal diagnosis of β-thalassaemia IVS-Ⅱ-654(C→T) mutation.

作　　者：易萍[1] 李力[1] 郑英如[1] 颜耀华[1] 赵艳[2] 周元国[2] 陈竹钦[1] 

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400042

出　　处：《现代妇产科进展》2009年第6期401-406,共6页Progress in Obstetrics and Gynecology

基　　金：国家自然科学基金资助项目(No:30672249)

摘　　要：目的：研究DNA连接酶检测反应（LDR）技术结合毛细管电泳用于检测母体血浆中胎儿父系DNA点突变的可行性。方法：将Cy5荧光标记的LDR引物稀释成0.1-500fmol,用毛细管电泳技术（CEQ8000型遗传分析仪）检测稀释引物;然后以β地中海贫血IVS-Ⅱ-654（C→T）突变为例,将含该突变不同浓度的扩增产物为LDR模板,用LDR技术特异地识别突变,其中LDR一侧引物用Cy5荧光标记,最后用毛细管电泳技术检测连接产物。结果：遗传分析仪检测Cy5荧光标记的LDR引物灵敏度至少达到0.1fmol;该分析仪能检测含0.1fmol IVS-Ⅱ-654（C→T）突变的扩增产物为模板的LDR产物,产物峰面积随模板浓度增加而增加,而且未检测到以1000fmol无IVS-Ⅱ-654（C→T）突变的扩增产物为模板的LDR产物。结论：PCR/LDR结合毛细管电泳技术用于检测低丰度基因点突变有很高的灵敏度,有望用于检测母体血浆中胎儿DNA点突变,从而可能大大拓展母体血浆中胎儿DNA在无创性产前诊断中的应用范围。Objective：To study the feasibility of PCR/LDR/capillary electrophoresis for the detection of point mutations of paternally inherited fetal DNA in maternal plasma. Methods ： The LDR primers labelled by Cy5 were diluted into 0.1 - 500fmol, and the diluted primers were directly analyzed on an CEQ8000 automated capillary DNA sequencer （Beckman Coulter）. Different concentrations of the purified PCR products contained IVS-Ⅱ-654（C→T） were used as LDR templates and the LDR products were analyzed by automated capillary DNA sequencer. Results：The capillary electrophoresis could detect 0.1fmol of the diluted primers at least. All PCR products which contain the mutation IVS-Ⅱ-654（C→T） produced LDR product peaks in eleetropherogram, while there was no LDR product peak in 1000fmol purified PCR products which did not contain IVS-Ⅱ-654（C→T）. Conclusion：It indicates LDR/eapillary electrophoresis may be capable of identifying a single mutation unambiguously at a sensitivity ratio of 1：10000. PCR/LDR/capillary electrophoresis is able to discriminate point mutations of fetal paternal DNA in maternal plasma.

关 键 词：连接酶检测反应 血浆DNA 产前诊断 点突变 

分 类 号：R714.15[医药卫生—妇产科学]