Mutagenesis of bovine basic fibroblast growth factor through hybrid clonemid construction: a new approach to DNA manipulation.
Basic fibroblast growth factor (bFGF) is a heparin-binding protein which is mitogenic for a wide variety of cells derived from the mesoderm and neuroectoderm. It is also known as an angiogenic and neurotrophic molecule. The mutagenesis of bovine bFGF gene into the human bFGF gene and the expression of human bFGF have been carried out in this laboratory. In this study, efficient linkage (ligation) of single strands of DNA at their terminal complementary areas was found to be obtained easily, at low temperature. Based upon this discovery, we have advanced a general method of DNA manipulation. The major principle is to make use of complementary sequences in a DNA linker to circularise a cut vector or use the single-strand DNA fragments to rebuild the plasmid through heteroduplex plasmid (hybrid clonemid) construction. The use of single DNA strands in DNA manipulation produces high flexibility in the course of hybrid clonemid construction. It enables DNA cloning and mutagenesis to be carried out concurrently. The primer-induced and polycation-reliant unidirectional deletion (PPUD) phenomenon is also observed in this study. The factors that affect the efficiency of mutagenesis are discussed. This work offers a new understanding of DNA manipulation.
Zhao
Q
Q
Department of Molecular Biology, Basic Medicine Institute, China Rehabilitation Research Centre (CRRC), Beijing, PR China.
D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods*; D004277:DNA, Single-Stranded / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D016222:Fibroblast Growth Factor 2 / Q000235:genetics*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics
D004277:DNA, Single-Stranded; D016222:Fibroblast Growth Factor 2