title: High-throughput cloning and expression library creation for functional proteomics.
abstract: The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particularly important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single-gene experiments, creating the need for fast, flexible, and reliable cloning systems. These collections of ORF clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial, we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator(TM) DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This tutorial is part of the International Proteomics Tutorial Programme (IPTP12).
authors: Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, AZ 85287-6401, USA.
authors: Fernanda
authors: F
authors: Festa
authors: Jason
authors: J
authors: Steel
authors: Xiaofang
authors: X
authors: Bian
authors: Joshua
authors: J
authors: Labaer
mesh_terms: D010582:Bacteriophage lambda; D002474:Cell-Free System; D003001:Cloning, Molecular / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004798:Enzymes / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D040901:Proteomics / Q000379:methods*; D011995:Recombination, Genetic; D014734:Vibrio cholerae / Q000235:genetics
chemical_list: D004798:Enzymes; D004262:DNA Restriction Enzymes