克隆(编程)
生物
功能基因组学
计算生物学
克隆载体
遗传学
多克隆站点
基因
转化(遗传学)
分子克隆
限制性酶
基因组DNA
载体(分子生物学)
基因组学
基因组
基因表达
计算机科学
重组DNA
程序设计语言
作者
Songbiao Chen,Pattavipha Songkumarn,Jianli Liu,Guo‐Liang Wang
出处
期刊:Plant Physiology
[Oxford University Press]
日期:2009-04-29
卷期号:150 (3): 1111-1121
被引量:409
标识
DOI:10.1104/pp.109.137125
摘要
Abstract With the recent availability of complete genomic sequences of many organisms, high-throughput and cost-efficient systems for gene cloning and functional analysis are in great demand. Although site-specific recombination-based cloning systems, such as Gateway cloning technology, are extremely useful for efficient transfer of DNA fragments into multiple destination vectors, the two-step cloning process is time consuming and expensive. Here, we report a zero background TA cloning system that provides simple and high-efficiency direct cloning of PCR-amplified DNA fragments with almost no self-ligation. The improved T-vector system takes advantage of the restriction enzyme XcmI to generate a T-overhang after digestion and the negative selection marker gene ccdB to eliminate the self-ligation background after transformation. We demonstrate the feasibility and flexibility of the technology by developing a set of transient and stable transformation vectors for constitutive gene expression, gene silencing, protein tagging, protein subcellular localization detection, and promoter fragment activity analysis in plants. Because the system can be easily adapted for developing specialized expression vectors for other organisms, zero background TA provides a general, cost-efficient, and high-throughput platform that complements the Gateway cloning system for gene cloning and functional genomics.
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