定向cDNA文库构建的高效方法。

High-yield method for directional cDNA library construction.

作者信息

Bellemare G, Potvin C, Bergeron D

机构信息

Département de Biochimie, Faculté des Sciences et de Génie, Université Laval, Québec, Canada.

出版信息

Gene. 1991 Feb 15;98(2):231-5. doi: 10.1016/0378-1119(91)90178-e.

DOI:10.1016/0378-1119(91)90178-e

PMID:1849863

Abstract

Improvement of a cDNA synthesis procedure using a single stranded (ss) vector primer [Bellemare et al., Gene 52 (1987) 11-19] is reported. This vector (pPBS27), upon linearization with XbaI using an appropriate restriction site-directed fragment, releases a thymidilic tail used to prime cDNA synthesis. DNA polymerase I and RNase H replace the RNA strand and replicate the vector before double-stranded (ds) blunt-end ligation with T4 DNA ligase. More than 10(7) cfu/microgram of vector can be obtained with an efficient transformation protocol using either globin-encoding or 7.5-kb poly(A)-tailed RNA. This improved cloning method is easier, faster and a few hundred times more efficient than the original procedure as it involves ds rather than ss DNA for transformation.

摘要

报道了一种使用单链（ss）载体引物改进cDNA合成程序的方法[贝勒马尔等人，《基因》52（1987）11 - 19]。该载体（pPBS27），使用合适的限制性位点定向片段经XbaI线性化后，释放出一个胸腺嘧啶尾用于引发cDNA合成。DNA聚合酶I和RNase H取代RNA链并在与T4 DNA连接酶进行双链（ds）平端连接之前复制载体。使用编码珠蛋白的RNA或7.5 kb聚（A）尾RNA，通过高效转化方案，每微克载体可获得超过10⁷ cfu。这种改进的克隆方法比原始方法更简便、更快，效率高出数百倍，因为它涉及双链而非单链DNA用于转化。