DNA cloning using in vitro site-specific recombination.

By J. Hartley, G. Temple, M. Brasch. Genome Research, 2000

1,150 citations recorded by the source corpus.

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.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/96ebb56ca00cbb23391d16fafb6ba979665adc32

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Citations in the corpus

Using deep sequencing to characterize the biophysical mechanism of a transcriptional regulatory sequence (2010, background): “Using deep sequencing to characterize the biophysical mechanism of a transcriptional regulatory sequence”
A Versatile Viral System for Expression and Depletion of Proteins in Mammalian Cells (2009, methodology): “These vectors are based on the Gateway System [17] (Invitrogen) whereby a cDNA, shRNA or miRNA is cloned into an Entry vector which is recombined in vitro with the viral Destination vector of choice.”
Gateway Vectors for Plant Genetic Engineering: Overview of Plant Vectors, Application for Bimolecular Fluorescence Complementation (BiFC) and Multigene Construction (2012, background): “They are attB1 and attB2, attP1 and attP2, attL1 and attL2, and attR1 and attR2; a recombination reaction can occur only in the combinations of attB1 and attP1, attB2 and attP2, attL1 and attR1, or attL2 and attR2, since recombination strictly depends on att sequences (Hartley et al., 2000; Walhout ”
Gateway-compatible vectors for plant functional genomics and proteomics. (2006, background): “Fortunately, such barriers have been 616 ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd lowered considerably by the advent of Gateway cloning technology (Hartley et al., 2000).”
Engineering BioBrick vectors from BioBrick parts (2008, background): “The Gateway® recombinational cloning system and associated vectors are arguably the closest example of a vector standard in biological research [30,31].”
C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression (2003, methodology): “Results Genome-wide ORFeome cloning To simultaneously address these three problems, we designed an alternative strategy referred to as Gateway-based ORFeome clonin”
The Tol2kit: A multisite gateway‐based construction kit for Tol2 transposon transgenesis constructs (2007, methodology): “First, recombination-based cloning using the multisite Gateway system (Hartley et al., 2000; Cheo et al., 2004) greatly simplifies the generation of expression constructs.”
Gateway vectors for plant transformation (2009, methodology): “The Gateway cloning method uses these att sites and clonases for construction of plasmid in vitro (Hartley et al. 2000, Walhout et al. 2000).”
Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library. (2004, methodology): “Recently developed recombinational cloning technologies (Liu et al. 1998; Hartley et al. 2000; Walhout et al. 2000a; Zhang et al. 2002) allow such versatile cloning approaches at HT (Reboul et al. 2003; Dricot et al. 2004).”
Cell-Free Protein Synthesis: Applications Come of Age (2011, methodology): “The Gateway vector system, available from Invitrogen (Hartley et al., 2000), utilizes integrase enzymes for one-step insertion of a desired gene into a vector while avoiding excess restriction digest and ligation reactions.”