摘要
建立了HSP70-2基因多态性的毛细管电泳-激光诱导荧光（CE-LIF）检测方法。采用试剂盒法提取人血清标本中全基因组DNA作为模板,选择特异引物进行PCR扩增反应,产物用Pst I限制性内切酶酶切;酶切产物用高灵敏度的SYBR Gold荧光染料标记后,用毛细管电泳-激光诱导荧光法检测。在优化的毛细管电泳-激光诱导荧光条件下,酶切产物在25 min内即可完成检测。GeneRular 100bp DNA ladder在同一天内连续测定6次,迁移时间和峰面积的RSD分别为1.8%~2.9%和2.8%~7.9%;连续6日测定迁移时间与峰面积的RSD分别为2.1%~4.3%和3.5%~9.3%。本研究共检测200份样品,其中G/G分型3份,A/G分型25份,A/A分型172份,检测结果与凝胶电泳结果一致。CE-LIF检测方法具有电泳时间短、试样消耗少、绿色环保等优点,能用于HSP70-2基因多态性的检测。
A new method for the detection of HSP70 -2 gene polymorphism bycapillary electrophoresis-laser induced fluorescence has been proposed. Whole genome DNA extracted from serum was used as templates, and thespecific primers werechosen for PCR amplification. The PCR products were digested by Pst I restriction endonuclease. Then the enzyme-digested products were labeled with highly sensitive nucleic acid dye SYBR Gold and detected by CE-LIF. The enzyme-digested products could be detected within 25 min under the optimal conditions. The intraday relative standard deviations （RSD） of migration time and peak area for DNA ladder werein the ranges of 1.8% -2.9% and 2.8% ~ 7.9% ; and the interday RSD of migration time and peak area werein the ranges of 2.1% - 4.3 % and 3.5 % ~ 9.3 %, respectively. A total of 200 specimens were detected by the proposed method in this research. Among them, A/A, A/G and G/G genotypes were 172, 25 and 3, respectively, which had good agreement with the results by agarose gel electrophoresis. This proposed method requires small amount of reagents and less analytical time, and is environmental friendly. The results demonstrated the possibility for rapid, accurate and specific detection of HSP70-2 gene polymorphism by CE-LIF.
出处
《分析试验室》
CAS
CSCD
北大核心
2014年第10期1135-1138,共4页
Chinese Journal of Analysis Laboratory
基金
国家自然科学基金(青年基金)项目(81102162)
四川大学青年教师启动基金项目(2010SCU11013)资助
