Expression sequence tag-specific full-length cDNA cloning: actin cDNAs.
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.
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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D000199:Actins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics*; D020224:Expressed Sequence Tags; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012107:Research Design
D000199:Actins; C505032:CFTR protein, human; D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator