CapSelect: a highly sensitive method for 5' CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs.
CapSelect: a highly sensitive method for 5' CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs.
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DOI：
10.1093/nar/27.21.e31
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发表时间：
1999-11-01
影响因子：
14.9
通讯作者：
Mueller, M W
中科院分区：
文献类型：
--
作者：
Schmidt, W M;Mueller, M W
在这里，我们提出CapSelect作为一种新的实验方法，用于在pcr介导的mRNA序列分析中选择性富集全长cdna。该方法结合了在锰存在的情况下，通过逆转录酶在全长cDNA的3‘端特异性地添加3到4个非模板化的dCMP残基的5’- cap依赖性，以及通过末端脱氧核苷酸转移酶使用rATP控制cDNA末端的核糖核苷酸尾部。利用生成的末端序列基序(5’-dC(3-4)rA(3-4))，全长cdna通过T4 DNA连接酶选择性地锚定在双链DNA适配器上(带有dT（3-4)dG(3)3’-悬垂）。所描述的技术是高效的，区分过早终止产物和富集全长cdna。
Here we present CapSelect as a novel experimental approach for the selective enrichment of full-length cDNAs in PCR-mediated analysis of mRNA sequences. The method combines the 5'-CAP-dependent addition of specifically three to four non-templated dCMP residues to the 3'-end of full-length cDNAs by reverse transcriptases in the presence of manganese and the controlled ribonucleotide tailing of cDNA ends by terminal deoxynucleotidyl transferase using rATP. By virtue of the generated terminal sequence motif (5'-dC(3-4)rA(3-4)), full-length cDNAs are selectively anchored to a double-stranded DNA adapter (with a dT(3-4)dG(3)3'-overhang) by T4 DNA ligase. The technique described is highly efficient, discriminates premature termination products and enriches full-length cDNAs.