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| Primer design and PCR condition optimization for plant site-specific methylation |
| Received:February 02, 2016 |
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| KeyWord:plant methylation;primer design;Taq polymerase;PCR condition |
| Author Name | Affiliation | E-mail | | WANG He-tong | 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 | | | SONG Jie | Liaoning University, Shenyang 110036, China | | | CUI Wei-na | Shanghai Institute of Technology, Shanghai 201418, China | | | CAO Xia | Shenyang Agricultural University, Shenyang 110866, China
Institute of Vegetable Science, Tong Liao Academy of Agricultural Sciences, Tongliao 028000, China | | | HE Lei | Liaoning University, Shenyang 110036, China | | | JIA Chun-yun | Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China | | | HUI Xiu-juan | Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China | | | TAI Pei-dong | Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China | | | CHENG Zhi-bo | Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China | | | LIU Wan | Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China | liuwan63@hotmail.com |
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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 polymerases. 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® 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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