Cloning of the 5' flanking region of the murine laminin B1 gene by genomic PCR.
Induction of the murine laminin B1 gene in F9 cells occurs 24-48 hours after the retinoic acid (RA) addition. In order to reveal the mechanism of the late induction of the laminin B1 gene, it is necessary to understand fully the promoter structure of it. We report here that the promoter region of the laminin B1 was rapidly isolated utilizing the genomic PCR technique. MgCl2, formamide concentration, and annealing temperature were optimized for PCR. The result showed that MgCl2 concentration profoundly affects the efficiency in amplifying the specific DNA. The annealing temperature (51 degrees C-63 degrees C) did not significantly affect the yield. Under the optimal conditions, about 50 ng of the specific DNA was obtained from 1 microgram of total genomic DNA after 20 cycles of amplification, indicating that approximately 2 x 10(5) fold specific amplification had occurred. Southern blot analysis and sequence data proved that the amplified DNA fragment contained the promoter region of the laminin B1 gene.
Okano
R
R
Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan.
Mita
T
T
Matsui
T
T
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D005796:Genes; D007797:Laminin / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic
D007797:Laminin