中国药房2018，Vol.29Issue(10)：1309-1312,4.DOI:10.6039/j.issn.1001-0408.2018.10.04
广西产拳卷地钱DNA的SCoT-PCR引物筛选及反应体系优化
SCoT-PCR Primer Selection and Reaction System Optimization of DNA of Marchantia convoluta in Guangxi
摘要
Abstract
OBJECTIVE:To establish PCR reaction system of DNA of Marchantia convoluta in Guangxi marked with SCoT polymorphism marker technique by screening primer and optimizing reaction condition. METHODS:Modified CTAB method was used to extract DNA of M. convolute from Guangxi;gel electrophoresis and UV spectrophotometry were used to investigate purity and concentration of DNA. Using sample DNA as template,PCR amplification of 36 SCoT primers was conducted,and suitable primers were screened after electrophoresis,staining and imaging of products. The orthogonal experiment of 5 factors and 4 levels was conducted by 5 main factors as DNA concentration,Mg2 + concentration,dNTP concentration,primer concentration,Taq DNA polymerase concentration. The condition of SCoT-PCR reaction system was optimized. RESULTS:Extracted sample DNA bands were neat without RNA contamination,degradation or dispersion of fluorescence;sample well was clear. UV absorbance ratio ranged 1.7-2.0 at 260 nm and 280 nm;purity and concentration of DNA were both suitable for follow-up test. PCR results of 36 primers showed that product band of No. 4 primer was neat without diffuse fluorescence but with best luminance,so No. 4 primer was used for PCR reaction. The optimal SCoT-PCR reaction system contained 30.00 μg/mL DNA,2.00 mmol/L Mg2+,0.20 mmol/L dNTP, 0.40 μmol/L primer,0.50 U/mL Taq DNA polymerase(total reaction volume of 20 μL). CONCLUSIONS:Suitable SCoT-PCR primer of DNA is screened,and reaction system is optimized. It provides technologic basis for variety identification and genetic relationship analysis of M. convoluta in Guangxi.关键词
拳卷地钱/SCoT/PCR/引物筛选/体系优化Key words
Marchantia convolute/SCoT/PCR/Primer selection/System optimization分类
医药卫生引用本文复制引用
谢凤凤,李鹏,黎理,朱华,尚小红,李平凤..广西产拳卷地钱DNA的SCoT-PCR引物筛选及反应体系优化[J].中国药房,2018,29(10):1309-1312,4.基金项目
国家自然科学基金资助项目(No.81260620) （No.81260620）
广西壮瑶药重点实验室项目(No.桂科基字〔2014〕32号) （No.桂科基字〔2014〕32号）
壮瑶药协同创新中心项目(No.桂教科研〔2013〕20号) （No.桂教科研〔2013〕20号）
广西重点学科项目(No.桂教科研〔2013〕16号) （No.桂教科研〔2013〕16号）
广西"八桂学者"项目(No.桂科政字〔2013〕25号) （No.桂科政字〔2013〕25号）
