A simple, efficient method for coupling DNA to cellulose. Development of the method and application to mRNA purification.
A simple, efficient method to couple covalently DNA to cellulose is described. It utilizes the bifunctional oxirane 1,4-butanediol diglycidyl ether to activate cellulose and subsequently to link DNA to the cellulose. The optimal conditions for the latter reaction included use of a dehydration technique whereby DNA and activated cellulose were allowed to react on a glass slide in 0.1 N NaOH. Initial volume of the reaction was important; less than or equal to 250 microliters/50 mg cellulose was necessary for maximum efficiency. At DNA concentrations of less than or equal to 4 micrograms/mg cellulose, efficiency of binding was 90%. Binding studies using nucleotide homopolymers indicated that the order of the relative efficiencies of binding was poly(dT) greater than poly(dC) = poly(dA) greater than poly(dG). DNAs subjected to the binding conditions had an average of 0-1 breaks/molecule (for a 915-base DNA). A cloned double-stranded cDNA was coupled to cellulose by this technique. The cDNA was coupled to cellulose by this technique. The DNA-cellulose matrix was successfully used to purify the complementary mRNA from total poly(A)-enriched RNA by affinity chromatography. This method is very simple and highly efficient and can be conveniently adapted for the covalent coupling of various DNA species to cellulose for affinity chromatography.
D000818:Animals; D001053:Apolipoproteins / Q000235:genetics; D001717:Birds; D002482:Cellulose; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D004247:DNA; D007700:Kinetics; D008079:Lipoproteins, VLDL / Q000235:genetics; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*
D001053:Apolipoproteins; D008079:Lipoproteins, VLDL; D012333:RNA, Messenger; C027610:apolipoprotein VLDL II; D002482:Cellulose; D004247:DNA