广西产拳卷地钱DNA的SCoT-PCR引物筛选及反应体系优化
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篇名: 广西产拳卷地钱DNA的SCoT-PCR引物筛选及反应体系优化
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摘要: 目的:通过筛选引物并优化反应条件,建立以目标起始密码子多态性(SCoT)技术进行标记的广西产拳卷地钱DNA的聚合酶链式反应(PCR)体系。方法:采用改进的十六烷基三甲基溴化铵(CTAB)法提取广西产拳卷地钱药材样品DNA,以凝胶电泳法与紫外分光光度法考察样品DNA的纯度和浓度;以样品DNA为模板,对36条SCoT引物进行PCR扩增反应,并对产物进行电泳、染色、成像以筛选合适引物;以DNA浓度、Mg2+浓度、dNTP浓度、引物浓度、Taq DNA聚合酶浓度等5 个主要因素进行5因素4水平的正交试验,对SCoT-PCR反应体系条件进行优化。结果:所提取的样品DNA条带整齐,无RNA污染、无降解、无弥散荧光,加样孔清晰;260、280 nm紫外波长处吸光度比值在1.8~2.0范围内,样品DNA纯度和浓度均适合后续试验;36条引物PCR结果显示,4号引物所得产物条带整齐、无弥散荧光且亮度最高,故以其为引物进行PCR反应;最佳SCoT-PCR反应体系为30.00 μg/mL DNA、2.00 mmol/L Mg2+、0.20 mmol/L dNTP、0.40 μmol/L引物、0.50 U/mL Taq DNA聚合酶(总反应体积20 μL)。结论:筛选得到合适的样品DNA的SCoT-PCR引物并优化了反应体系,可为广西产拳卷地钱的品种鉴定、遗传多样性评价及亲缘关系分析提供技术基础。
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.
期刊: 2018年第29卷第10期
作者: 谢凤凤,李鹏,黎理,朱华,尚小红,李平凤
AUTHORS: XIE Fengfeng,LI Peng,LI Li,ZHU Hua,SHANG Xiaohong,LI Pingfeng
关键字: 拳卷地钱;SCoT;PCR;引物筛选;体系优化
KEYWORDS: Marchantia convolute; SCoT; PCR; Primer selection; System optimization
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