基于T4 DNA连接酶的RNA标记改进全长cDNA的制备
Improved full-length cDNA production based on RNA tagging by T4 DNA ligase.
作者信息
Clepet Christian, Le Clainche Isabelle, Caboche Michel
机构信息
Unité de Recherches en Génomique Végétale, INRA/CNRS, 2 Rue Gaston-Crémieux, F-91057 Evry Cedex, France.
出版信息
Nucleic Acids Res. 2004 Jan 2;32(1):e6. doi: 10.1093/nar/gng158.
Abstract
Second-strand cDNA priming is a central problem for full-length characterization of transcripts. A new strategy using bacteriophage T4 DNA ligase and partially degenerate adapters is proposed for grafting a sequence tag to the end of polyribonucleotides. Based on this RNA tagging system and previously described protocols, a new method for full-length cDNA production has been implemented. Validation of the method is shown in Arabidopsis thaliana by the construction of a full-length cDNA library and the analysis of 154 clones and by 5'-RACE-PCR run on a documented experimental system.
摘要
第二链cDNA引发是转录本全长表征的核心问题。本文提出了一种使用噬菌体T4 DNA连接酶和部分简并衔接子的新策略，用于在多聚核糖核苷酸末端嫁接序列标签。基于此RNA标记系统和先前描述的方案，实现了一种全长cDNA制备的新方法。通过构建全长cDNA文库、分析154个克隆以及在一个有记录的实验系统上进行5'-RACE-PCR，在拟南芥中验证了该方法。
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