中国烟草学报Issue(4)：75-78,4.DOI:10.3969/j.issn.1004-5708.2014.04.015
基于毛细管电泳的TILLING实验技术的建立
Establishment of TILLING experimental technique based on capillary electrophoresis
摘要
Abstract
cellI enzyme that specifically cleaves mismatch in DNA double strands is one of the most important components of TILLING experiment platform. Crude extract of cellI enzyme was obtained from celery growing in Guiyang of Guizhou province. A pair plasmids with a known single base difference were used as substrate to verify cellI restriction enzyme activity of crude extracts, and cleavage products were detected by agarose gel electrophoresis and capillary electrophoresis, which confirmed cellI activity in crude extracts. An effective cellI digestion system was thus established: cellI enzyme effectively cut mismatch DNA in 15 µL digestion reaction solution including 8 µL PCR product, 4.5 µL ddH2O, 1.5 µL 10×cleavage buffer (pH 7.5, 500 mmol/ L KCL, 100 mmol / L Tris-Cl, 15 mmol / L MgCl2 ) and 1µL the 20 times dilution of cellI crude extraction after 60-min incubation under 42℃.关键词
TILLING/芹菜CEL I核酸酶/ABI遗传分析仪/毛细管电泳Key words
TILLING/celery cellI nuclease/ABI genetic analyzer/capillary electrophoresis分类
生物科学引用本文复制引用
付强,邹颉,张吉顺,王轶,任学良..基于毛细管电泳的TILLING实验技术的建立[J].中国烟草学报,2014,(4):75-78,4.基金项目
贵州省科技厅农业攻关项目（黔科合NY字[2011]3047号）；中国烟草总公司重点项目（中烟办[2010]221号）；中国烟草总公司重大专项（中烟办[2012]146号）和贵州省优秀青年科技人才培养对象专项资金 （）
