| 植物位点特异性甲基化研究的引物设计及PCR体系优化 |
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| 引用本文： | 王鹤潼,宋婕,崔伟娜,曹霞,何蕾,贾春云,惠秀娟,台培东,成智博,刘宛.植物位点特异性甲基化研究的引物设计及PCR体系优化[J].农业环境科学学报,2016,35(9):1686-1693. |
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| 作者姓名： | 王鹤潼 宋婕 崔伟娜 曹霞 何蕾 贾春云 惠秀娟 台培东 成智博 刘宛 |
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| 作者单位： | 1. 中国科学院沈阳应用生态研究所污染生态与环境工程重点实验室，沈阳 110016; 辽宁何氏医学院，沈阳 110163;2. 辽宁大学,沈阳,110036;3. 上海应用技术学院,上海,201418;4. 沈阳农业大学，沈阳 110866; 通辽市农业科学研究院蔬菜所，内蒙古通辽 028000;5. 中国科学院沈阳应用生态研究所污染生态与环境工程重点实验室,沈阳,110016 |
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| 基金项目： | 国家自然科学基金项目（21347007，41272255，41472237）；国家科技重大专项（2012ZX7505-001） |
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| 摘 要： | 通过设计大量引物,探索在植物甲基化引物设计中长、短引物的设计方法及其相应的PCR条件,同时比较不同Taq酶对甲基化率影响。结果表明:17~30 bp短引物适合使用Touch-down程序PCR,但特异性差、成功率低;31~50 bp长引物使用55℃退火60℃延伸的PCR条件成功率最高,其中反向引物的长度及Tm小于正向引物较佳;高保真酶因其3′→5′外切酶活性不宜用于甲基化研究,而Epi TaqTMHS在PCR结果中表现最佳。
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| 关 键 词： | 植物甲基化 引物设计 Taq 酶 PCR 条件 |
| 收稿时间： | 2/2/2016 12:00:00 AM |
Primer design and PCR condition optimization for plant site-specific methylation |
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| WANG He-tong,SONG Jie,CUI Wei-n,CAO Xi,HE Lei,JIA Chun-yun,HUI Xiu-juan,TAI Pei-dong,CHENG Zhi-bo and LIU Wan.Primer design and PCR condition optimization for plant site-specific methylation[J].Journal of Agro-Environment Science( J. Agro-Environ. Sci.),2016,35(9):1686-1693. |
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| Authors: | WANG He-tong SONG Jie CUI Wei-n CAO Xi HE Lei JIA Chun-yun HUI Xiu-juan TAI Pei-dong CHENG Zhi-bo and LIU Wan |
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| Institution: | Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China;Liaoning He University, Shenyang 110163, China,Liaoning University, Shenyang 110036, China,Shanghai Institute of Technology, Shanghai 201418, China,Shenyang Agricultural University, Shenyang 110866, China;Institute of Vegetable Science, Tong Liao Academy of Agricultural Sciences, Tongliao 028000, China,Liaoning University, Shenyang 110036, China,Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China,Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China,Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China,Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China and Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China |
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| Abstract: | The present study was to explore design methods for short and long primers in plant methylation research and their corresponding PCR conditions employing large number of designed primers and to analyze the effects on methylation rates using three different Taq poly-merases. Results showed:a Touch-down PCR was suitable when using short primers of 17 ~ 30 bp, however, low specificity and success rates were observed; PCR performed the best when using long primers of 31~50 bp, provided that length and Tm of forward primers﹥those of reverse primers and 55 ℃ and 60 ℃ served as annealing and prolonging temperature, respectively; Super-Fidelity Taq polymerase like Prime STAR R HS isn't suitable for methylation research because of its 3'→5'exonuclease activities. However, PCR performed the best when using EpiTaqTM HS. |
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| Keywords: | plant methylation primer design Taq polymerase PCR condition |
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