【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。更多还原

【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 Tmof forward primers>those of reverse primers and 55 ℃ and 60 ℃ served as annealing and prolonging temperature, respectively; Super-Fidelity Taq polymerase like Prime STARRHS isn′ t suitable for methylation research because of its 3′ →5′ exonuclease activities. However, PCR performed the best when using Epi TaqTMHS.更多还原