Sarkar F H, Ball D E, Li Y W, Crissman J D
Department of Pathology, Wayne State University School of Medicine, Detroit, MI.
DNA Cell Biol. 1993 Sep;12(7):611-5. doi: 10.1089/dna.1993.12.611.
This report documents the cloning and sequencing of a previously unknown intron of the Her-2/neu gene and compares three different cloning strategies for efficient PCR cloning. Although successful results have previously been documented using blunt-end ligation strategies, the TA cloning system (Invitrogen, CA) offered the best result in our hands using a PCR-amplified DNA fragment of Her-2/neu (ERBB2) gene containing an unreported intron. The TA cloning system is easy to manipulate and the cloned DNA can be easily sequenced without further subcloning into the M13 vector. Additionally, this cloning strategy does not require (i) any prior selection of restriction sites during primer design, (ii) post PCR restriction digestion, and/or (iii) gel purification of PCR-amplified DNA. The newly identified and sequenced DNA of the Her-2/neu neu intron (GenBank Accession No. M95667) may help in designing primers for the analysis of the Her-2/neu gene in biological specimens. Therefore, we recommend the TA cloning system as the preferred choice for cloning any DNA fragments generated by PCR.
本报告记录了Her-2/neu基因一个此前未知内含子的克隆与测序过程，并比较了三种不同的高效PCR克隆策略。尽管此前使用平端连接策略已取得成功，但对于含有一个未报道内含子的Her-2/neu（ERBB2）基因的PCR扩增DNA片段，TA克隆系统（Invitrogen公司，加利福尼亚州）在我们的实验中效果最佳。TA克隆系统易于操作，克隆的DNA无需进一步亚克隆到M13载体中即可轻松测序。此外，这种克隆策略不需要（i）引物设计过程中对限制酶切位点进行任何预先选择，（ii）PCR后进行限制酶消化，和/或（iii）对PCR扩增的DNA进行凝胶纯化。新鉴定并测序的Her-2/neu内含子DNA（GenBank登录号M95667）可能有助于设计用于分析生物标本中Her-2/neu基因的引物。因此，我们推荐TA克隆系统作为克隆任何由PCR产生的DNA片段的首选方法。
