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      DNA cloning using in vitro site-specific recombination.

      1 , ,
      Genome research
      Cold Spring Harbor Laboratory

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          Abstract

          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.

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          Author and article information

          Journal
          Genome Res
          Genome research
          Cold Spring Harbor Laboratory
          1088-9051
          1088-9051
          Nov 2000
          : 10
          : 11
          Affiliations
          [1 ] Life Technologies, Inc., Rockville, Maryland 20850, USA. jhartley@lifetech.com
          Article
          10.1101/gr.143000
          310948
          11076863
          48ec4f20-f34b-4eed-9967-aa103be831fe

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