Covalent Attachment of DNA to Agarose

Abstract

DNA has been covalently linked to insoluble matrices of agarose (Sepharose) in high yield using cyanogen bromide activation. Both double-stranded and single-stranded DNA have been coupled with yields up to 225 nmol/mg dry weight Sepharose or 3-8 pmol nucleotide phosphate/ml bed volume. The DNA-Sepharose has been used for (a) the affinity chromatography of various enzymes (Escherichiu coli DNA polymerase I and RNA polymerase) from crude extracts or after initial purification steps, resulting in high yields and degrees of purification, and for (b) nucleic acid hybridization. The DNA-Sepharose is stable to high temperature, prolonged storage, and in the case of singlestranded DNA, can be washed with NaOH to destroy nuclease activity and to release any digested oligonucleotides or mononucleotides.The affinity chromatography of DNA-binding proteins using DNA entrapped physically in a matrix of cellulose was introduced by Alberts et al. [l] and Litman [2]. The latter author described the additional feature of cross-linking of the cellulose-bound DNA by ultraviolet irradiation. The DNA-cellulose technique has been reviewed by Alberts and Herrick [3].Other methods have been described for immobilizing DNA and RNA using a variety of solid supports such as agarose [4], polyacrylamide [5], and cellulose acetate in combination with agar [6,7]. While the cellulose and agarose derivatives have a high capacity (0.5-5 mg DNA/ml bed volume) they are normally useful only at reduced temperatures and neutral pH, that is, conditions under which desorption is minimized. A major disadvantage of polyacrylamide is a low capacity since only 0.05 mg of DNA/ml bed volume can be entrapped.In the case of DNA and RNA hybridization the linkage of one of the nucleic acid partners to an insoluble support offers many advantages. The use in such Abbreviations. CNBr, cyanogen bromide ; abbreviations for polynucleotides follow CBN recommendations, see Eur. J. Biochem. 15,203-208 (1970).Enzymes. E. coli DNA polymerase I (EC 2.7.7.7); E. coli RNA polymerase (EC 2.7.7.6); E. coli polynucleotide phosphorylase (EC 2.7.7.8) ; calf thymus terminal deoxyribonucleotidyl transferase (EC 2.7.7.31).In this paper all polynucleotide amounts are expressed in moles of nucleotide phosphate. hybridization experiments of high temperatures and organic solvents dictates that the nucleic acids be covalently attached to the support. Gilham introduced the chemical coupling of nucleic acids to cellulose using a water-soluble carbodiimide [8]. Such derivatives have been used to select DNA's from mixtures [9] and recently, the procedure has been extended to cellulose powders, thus simplifying many technical aspects of the hybridization [ 101. Oligonucleotides polymerized and coupled to cellulose with dicyclohexylcarbodiimide have also been used as solid-state primers and templates for polymerases allowing the covalent attachment of specific single-stranded and double-stranded nucleic acids to a matrix at one end without interference with the secondary and tertiary structure ...

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“…Mcm2-containing fractions from the MonoQ step were pooled (approximately 3 ml, 2.7 mg/ml) and dialyzed overnight against buffer H. The dialysate was applied to a 10-ml ssDNA-Sepharose column, 43 washed with 100 ml of buffer H, and eluted with 100 ml of 0-500 mM NaCl gradient.…”

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