Hartley J L, Temple G F, Brasch M A
Life Technologies, Inc., Rockville, Maryland 20850, USA.
Genome Res. 2000 Nov;10(11):1788-95. doi: 10.1101/gr.143000.
As a result of numerous genome sequencing projects, large numbers of candidate open reading frames are being identified, many of which have no known function. Analysis of these genes typically involves the transfer of DNA segments into a variety of vector backgrounds for protein expression and functional analysis. We describe a method called recombinational cloning that uses in vitro site-specific recombination to accomplish the directional cloning of PCR products and the subsequent automatic subcloning of the DNA segment into new vector backbones at high efficiency. Numerous DNA segments can be transferred in parallel into many different vector backgrounds, providing an approach to high-throughput, in-depth functional analysis of genes and rapid optimization of protein expression. The resulting subclones maintain orientation and reading frame register, allowing amino- and carboxy-terminal translation fusions to be generated. In this paper, we outline the concepts of this approach and provide several examples that highlight some of its potential.
由于众多基因组测序项目，大量候选开放阅读框被识别出来，其中许多尚无已知功能。对这些基因的分析通常涉及将DNA片段转移到各种载体背景中进行蛋白质表达和功能分析。我们描述了一种称为重组克隆的方法，该方法利用体外位点特异性重组高效地完成PCR产物的定向克隆以及随后将DNA片段自动亚克隆到新的载体骨架中。众多DNA片段可并行转移到许多不同的载体背景中，为基因的高通量、深入功能分析和蛋白质表达的快速优化提供了一种方法。所得的亚克隆保持方向和阅读框对齐，允许产生氨基和羧基末端翻译融合体。在本文中，我们概述了这种方法的概念，并提供了几个例子以突出其一些潜力。
