MA Haixin,PANG Shengqun,YAN Lijuan,et al.Establishment and Optimization of PCR-SSCP Amplification Reaction System in Processing Tomato[J].Northern Horticulture,2015,39(15):88-91.[doi:10.11937/bfyy.201515024]
加工番茄PCR-SSCP分子标记体系的建立与优化
- Title:
- Establishment and Optimization of PCR-SSCP Amplification Reaction System in Processing Tomato
- 文献标志码:
- A
- 摘要:
- 以加工番茄为试验材料，采用PCR-SSCP分子标记技术，对DNA提取方法、PCR反应程序、聚合酶链式反应-单链构象多态性分子标记体系的电泳条件、变性剂等诸多因素进行了研究，筛选和建立了多态性检出率高、带型清晰、重复性好的PCR-SSCP反应体系和反应程序。结果表明：采用CTAB法提取番茄叶片DNA时，在CTAB缓冲液中加入5 mol/L NaCl，第一次抽提离心速度和离心时间分别为8 000 r/min和15 min，可以获得高质量的DNA，PCR扩增程序为94℃ 5 min，94℃ 1 min，52℃ 30 s，72℃ 1 min，72℃ 10 min，29个循环，退火温度52℃，引物大小100~300 bp、98℃变性10 min，非变性聚丙烯酰胺凝胶浓度8%，电泳1.5~2.5 h，聚丙烯酰胺凝胶中不添加甘油为加工番茄PCR-SSCP 分子标记体系最佳的条件组合。
- Abstract:
- Taking processing tomato as material，single strand conformation ploymorphism (SSCP)technique was used,the effect of many factors such as extraction method of DNA,reaction process of PCR,electrophoretic factors(denaturalization temperature and time,electrophoresis temperature,electrophoresis time,electrophoresis buffer and electrophoresis power),denaturant on SSCP technique was studied.The PCR-SSCP reaction system and reaction process which were high polymorphism detection rate,good repeatability,clear stripe were screened and established.The results showed that adding 5 mol/L NaCl and the first centrifugation speed and centrifugal extraction time was 8 000 r/min,15 minutes could obtain high quality DNA in CTAB buffer when extracting tomato leaves’ DNA using the CTAB method,the amplification program was 94℃ predenature 5 minutes,94℃ denature 1 minutes,52℃ annealing 30 s,72℃ extension of 1 minutes,a total of 29 cycles，final extension at 72℃ 10 minutes.Primer size was 100-300 bp.Denaturalization temperature was 98℃,denaturalization time was 10 minutes,non-denaturing polyacrylamide gel concentration of 8%.Electrophoresis time was 1.5-2.5 hours.No added glycerin,the bands of the single strand DNA were clear and easy to read in processing tomato.
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备注/Memo
第一作者简介：马海新（1990-），男，新疆昌吉人，硕士研究生，研究方向为蔬菜遗传育种。E-mail:294524958@qq.com. 责任作者：庞胜群（1970-），女，安徽萧县人，硕士，副教授，现主要从事蔬菜遗传育种等研究工作。E-mail：pangshqok@shzu.edu.cn. 基金项目：石河子大学资助项目（gxjs2012-yz08）；新疆生产建设兵团科技局资助项目(2014AB004)；科技支疆计划资助项目（2014AB004）。