A rapid and efficient cloning method with a solid-phase DNA probe: application for cloning the 5'-flanking region of the gene encoding human fibronectin.
A rapid and efficient gene-cloning method was developed using oligodeoxyribonucleotide probes (approx. 30-mer) immobilized by their 5' terminal to the gel (LiChrosphare Si1000; used for high-performance liquid chromatography). The method allowed us to concentrate DNA fragments having a particular base sequence in an eppendorf tube with very high efficiency within 2 h. For example, genomic DNA encoding the human fibronectin gene (fn) was concentrated by this method and cloned into lambda Charon4A vector. Screening of the concentrated genomic DNA library for the 5' region of the human fn gene showed that approx. 30% of 3000 recombinant phages tested were positive clones. Various factors affecting cloning efficiency were investigated.
Tsurui
H
H
Department of Physics, Faculty of Science, University of Tokyo, Japan.
Hara
E
E
Oda
K
K
Suyama
A
A
Nakada
S
S
Wada
A
A
D001483:Base Sequence; D002021:Buffers; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular / Q000379:methods*; D015342:DNA Probes; D004274:DNA, Recombinant; D005353:Fibronectins / Q000235:genetics*; D015698:Genomic Library; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds
D002021:Buffers; D015342:DNA Probes; D004274:DNA, Recombinant; D005353:Fibronectins; D013757:Tetraethylammonium Compounds; D019789:Tetraethylammonium