Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate.
We describe a technique for transferring electrophoretically separated bands of double-stranded DNA from agarose gels to diazobenzyloxymethyl-paper. Controlled cleavage of the DNA in situ by sequential treatment with dilute acid, which causes partial depurination, and dilute alkali, which causes cleavage and separation of the strands, allows the DNA to leave the gel rapidly and completely, with an efficiency independent of its size. Covalent attachment of DNA to paper prevents losses during subsequent hybridization and washing steps and allows a single paper to be reused many times. Ten percent dextran sulfate, originally found to accelerate DNA hybridization in solution by about 10-fold [J.G. Wetmur (1975) Biopolymers 14, 2517-2524], accelerates the rate of hybridization of randomly cleaved double-stranded DNA probes to immobilized nucleic acids by as much as 100-fold, without increasing the background significantly.
D001080:Apurinic Acid / Q000378:metabolism; D004247:DNA / Q000302:isolation & purification*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D003911:Dextrans; D004586:Electrophoresis / Q000379:methods*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D008722:Methods; D008747:Methylcellulose / Q000031:analogs & derivatives; D009693:Nucleic Acid Hybridization
D003911:Dextrans; D001080:Apurinic Acid; D008747:Methylcellulose; D004247:DNA; D004262:DNA Restriction Enzymes