CLONING OF MURINE LAG-3 BY MAGNETIC BEAD BOUND HOMOLOGOUS PROBES AND PCR (GENE-CAPTURE PCR)

In recent years PCR-based gene cloning strategies have found wide appl ication in molecular biology, due to the power, speed, and relative si mplicity of the PCR methodology. We have set up a novel PCR cloning st rategy to isolate homologous genes, which is based on the capture of t he cDNA sequence(s) of interest with a biotinylated probe and streptav idin-coupled magnetic beads followed by PCR amplification of the selec ted molecules. This method does not require sequence information on 5' and 3' regions of the cDNA of interest and permits gene isolation to be sensitive, fast, simple, and specific even when the conventional sc reening procedures give rise to high backgrounds. By using this techni que, which we propose to call gene-capture PCR (GC-PCR) cloning, we we re able to isolate the full-length murine lymphocyte activation gene 3 (LAG-3) cDNA from total RNA of activated thymocytes. The GC-PCR techn ique represents a powerful tool for easy isolation not only of homolog ous genes from related species, but also of genes sharing conserved re gions of suitable length, gene variants, and gene encoding proteins wh ere only limited knowledge of the amino acid sequence exists. (C) 1996 Academic Press, Inc.