Expression sequence tag-specific full-length cDNA cloning: actin cDNAs |
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Authors: | Xu Z Jablons D M Gruenert D C |
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Institution: | Department of Surgery and Mount Zion Cancer Center, University of California, 2340 Sutter Street, P.O. Box 1674, San Francisco, CA 94115, USA. zxu@cc.ucsf.edu |
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Abstract: | Current strategies for cDNA cloning are based on construction of cDNA libraries and colony screening. The process of obtaining a full-length cDNA clone can be highly time and labor intensive. Using the human actin gene as a model target cDNA, we have developed an RNA-capture method for rapid cloning of full-length cDNAs. The approach involves the capture of mRNA with expressed sequence tag (EST)-derived, biotin labeled antisense "capture" primers and streptavidin-coated magnetic beads. Full-length cDNA is then synthesized from purified EST-specific mRNA and cloned directly into plasmid vectors. The results of using beta-actin-based capture primers on cytoplasmic RNA were the isolation of both beta- and gamma-actin cDNA clones. Of the 16 actin-specific cDNA clones analyzed, 15 (93%) were full-length. This approach for cloning full-length cDNAs from available ESTs or partial cDNA sequences will facilitate a more rapid and efficient characterization of gene structure and function. |
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