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J Integr Plant Biol, 2003, 45 (11): 1312-1318, Research Article
Optimization of Multiplex PCR and Multiplex Gel Electrophoresis in Sunflower SSR Analysis Using Infrared Fluorescence and Tailed Primers
ZHANG Lu-Sheng , Vanessa BECQUET , LI Shao-Hua*, David ZHANG**
doi:
Abstract

In an effort to simplify the procedure and to reduce the cost of fluorescence SSR analysis, the conditions of the multiplex PCR and the multiplex gel electrophoresis were optimized in the genetic analysis of sunflower (Helianthus annuus L.) inbred lines. Results indicated that factors for a successful multiplex PCR assay were related to the cycling touchdown annealing temperature, the balance of primer concentration at the various loci, the concentration of PCR buffer and the Taq DNA polymerase. Based on the optimization, a tailed primer strategy was outlined, and the effective ways were proposed to overcome the troubleshootings commonly encountered in the multiplex PCR and the multiplex gel electrophoresis.

应用红外荧光和加尾引物法进行向日葵S S R 遗传分析中的多聚P C R 和多聚凝胶电泳的优化
张潞生  Vanessa BECQUET  李绍华  David ZHANG
(1. GEVES Le Magneraud, Laboratoire BioGEVES, B.P.52, F-17700 SURGERES, France;
2. 中国农业大学农学与生物技术学院, 北京 100094;3. 中国科学院植物研究所, 北京 100093)


摘要: 在向日葵(Helianthus annuus L.)自交系SSR遗传分析中,为了提高SSR荧光分析效率、简化分析程序和降低研究费用,我们进行了多聚PCR和多聚凝胶电泳两项技术的优化研究。结果表明,优化多聚PCR和多聚凝胶电泳的影响因子(如逐步降低的退火温度的循环数、各个多聚位点引物浓度的平衡、PCR缓冲液浓度以及Taq DNA多聚酶的浓度等)可以收到更好的实验效果。基于以上的优化研究,系统地提出了一套优化的加尾引物法的策略。同时,提出了在多聚PCR和多聚凝胶电泳中常常遇到的技术难题的有效防止和解决的方法。
关键词: 简单重复序列(SSR); 加尾引物;多聚PCR;多聚凝胶电泳;向日葵

通讯作者。 E-mail: <david.zhang@geves.fr>。

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