Beyond the Identification of Transcribed Sequences: Functional and Expression Analysis

9th Annual Workshop, October 28-31, 1999

Co-sponsored by the U.S. Department of Energy


TEMPLE

GATEWAY cloning: A high-throughput gene transfer technology for rapid functional analysis and protein expression

James Hartley, Gary Temple, Michael Brasch, et al.

Life Technologies, Inc., Rockville, Maryland, USA

Each step of characterization of new ORFs requires subcloning into specialized vectors that impart functional properties to the cloned segment. We describe a new method, called Recombinational Cloning (RC), that uses in vitro site-specific recombination to speed the accurate transfer of DNA segments between vector backbones. DNA segments flanked by recombination sites in an Entry Clone can be "automatically" transferred into new vector backgrounds simply by adding the desired "Destination" vector and recombinase, incubating for 1 hour, and transforming any standard E. coli strain. Strong selections ensure that the desired subclones are recovered at high efficiency (typically >90%), reducing or eliminating downstream analysis of candidate clones. The recombination is conservative (no net addition or loss of nucleotides) and transfer occurs without affecting the cloned DNA segment (in contrast to PCR-based approaches). Thus once Entry Clones are created, these clones can be validated and then transferred, unchanged, into any number of vectors. This permits the generation of large collections of validated ORFs (e.g., from model organisms) that can serve as a common source of clones for research. Collaborations to build such collections are in progress.

By incorporating 25 bp attB recombination sites into the 5' end of PCR primers, RC also permits efficient, directional cloning of PCR products (as Entry Clones). The resulting Entry Clones then can be rapidly transferred into any number of Destination Vectors for further analysis.

The RC method is fast, convenient, and can be automated, allowing numerous DNA segments to be cloned and then transferred in parallel into many different vector backgrounds. The resulting subclones maintain reading frame, allowing amino- and carboxy-fusions. Essentially any vector can be readily converted to a Destination vector. Approaches for optimization of protein expression, rapid functional analysis, and the integration of technology platforms will be discussed.


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