Beyond the Identification of Transcribed Sequences:
Functional, Evolutionary and Expression Analysis
12th International Workshop
October 25-28, 2002
Washington, DC

List of Abstracts * Speakers * Organizers * Authors * Original Announcement

Development of a Novel High Throughput Directional Cloning Platform

Ron Hernan, Leanne Snyder, Ken Heuermann, and Brian Ward
Sigma-Aldrich Biotechnology St. Louis, MO
Telephone: 314-289-8496, x8610
Fax: 314-286-7645

The cloning and expression of a large number of genes has been an enormous challenge for gene function studies in the post-genomics era. One of the major limitations has been the lack of a pair of universal restriction enzymes for generating the cohesive ends required for directional cloning. Traditionally, different enzymes are used for directional cloning of each gene, which restricts throughput and increases workload.  To overcome this problem, thionucleotides dATPaS and dGTPaS are incorporated during PCR amplification with restriction enzyme sites sequences engineered into the primers. Digestion of the amplified product with Exonuclease III produces 5’ ends that are restriction enzyme site compatible and capable of ligation into vectors digested with corresponding restriction enzymes. With this approach, cloning efficiencies greater then 90% (transformants with positive inserts in the correct orientation). Because this method of cloning is universal, it is highly adaptable to robotic applications. Using this technology we have developed a system to both systematically and directionally clone open reading frames in a universal fashion without consideration of internal restriction enzyme sites.  We have cloned 384 E. coli ORFs using this technology with a 74 % success rate.  In addition, we applied this technology to successfully clone selected ORF’s from mammalian systems into expression vectors with comparable success.

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