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 application in molecular biology, due to the power, speed, and relative simplicity of the PCR methodology. We have set up a novel PCR cloning strategy to isolate homologous genes, which is based on the capture of the cDNA sequence(s) of interest with a biotinylated probe and streptavidin-coupled magnetic beads followed by PCR amplification of the selected 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 screening procedures give rise to high backgrounds. By using this technique, which we propose to call gene-capture PCR (GC-PCR) cloning, we were able to isolate the full-length murine lymphocyte activation gene 3 (LAG-3) cDNA from total RNA of activated thymocytes. The GC-PCR technique represents a powerful tool for easy isolation not only of homologous genes from related species, but also of genes sharing conserved regions of suitable length, gene variants, and gene encoding proteins where only limited knowledge of the amino acid sequence exists.
Mastrangeli
R
R
Istituto di Ricerca Cesare Serono SpA, Ardea, Italy.
Micangeli
E
E
Donini
S
S
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D018189:Immunomagnetic Separation / Q000379:methods; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D000093322:Lymphocyte Activation Gene 3 Protein
D015703:Antigens, CD; D008565:Membrane Proteins; D000093322:Lymphocyte Activation Gene 3 Protein; C000720327:Lag3 protein, mouse