Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate.

TLDR

Dextran sulfate at 10 % concentration can accelerate hybridization of randomly cleaved double‑stranded DNA probes to immobilized nucleic acids by up to 100‑fold without increasing background. The authors present a method for transferring electrophoretically separated double‑stranded DNA bands from agarose gels onto diazobenzyloxymethyl paper. The transfer relies on in‑situ controlled cleavage by sequential dilute acid and alkali treatments, which depurinate and cleave the DNA, allowing rapid, size‑independent release from the gel. Covalently attaching the DNA to the paper prevents loss during hybridization and washing, enabling repeated use of a single paper, while dextran sulfate enhances probe hybridization up to 100‑fold.

Abstract

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.