Cloning full-length, cap-trapper-selected cDNAs by using the single-strand linker ligation method
Cloning full-length, cap-trapper-selected cDNAs by using the single-strand linker ligation method
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DOI：
10.2144/01306st01
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发表时间：
2001-06-01
期刊：
影响因子：
2.7
通讯作者：
Hayashizaki, Y
中科院分区：
文献类型：
--
作者：
Shibata, Y;Carninci, P;Hayashizaki, Y
我们开发了单链连接子连接方法（SSLLM），该方法使用DNA连接酶在单链全长cDNA上添加dsDNA连接子。这些连接子具有随机的6-bp (dN(6)或dGN(5)) 3'悬垂，可以连接到任何cDNA序列，从而促进产生滴度超过1 × 10(6)个独立克隆的cDNA文库。我们证实了利用SSLLM克隆的cDNA插入片段的5‘端是全长的，并且包含了5’未翻译区。该方法最大的优点是消除了GC尾部，简化了全长克隆的测序和蛋白质翻译。此外，我们的方法比传统的RNA连接酶反应更有效地标记dna。
We have developed the single-strand linker ligation method (SSLLM), which uses DNA ligase to add a dsDNA linker to single-stranded (ss) full-length cDNA. The linkers have random 6-bp (dN(6) or dGN(5)) 3' overhangs that can ligate to any cDNA sequence, thereby facilitating the production of cDNA libraries with titers exceeding 1 x 10(6) independent clones. We confirmed that the 5' ends of cDNA inserts cloned by using SSLLM are full-length and include the 5' untranslated regions. The great advantage of our method is that the elimination of the GC tail simplifies the sequencing and protein translation of the full-length clones. Further our method tags ss cDNAs more efficiently than does the traditional RNA ligase reaction.