Expression sequence tag-specific full-length cDNA cloning: actin cDNAs

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 β-actin-based capture primers on cytoplasmic RNA were the isolation of both β- and γ-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.