Affinity chromatography of DNA labeled with chemically cleavable biotinylated nucleotide analogs.
DNA labeled with the chemically cleavable biotinylated nucleotide Bio-12-SS-dUTP was chromatographed on biotin cellulose affinity columns using either avidin or streptavidin as the affinity reagent. Although both proteins were equally effective in binding the Bio-12-SS-DNA to the affinity resin, two important differences were found. First, nonbiotinylated DNA bound to avidin, but not to streptavidin, in buffers containing 50 mM NaCl. Second, Bio-12-SS-DNA was released much more slowly from the streptavidin affinity column than from the avidin column upon washing with buffer containing dithiothreitol. This difficulty in reducing the disulfide bond of Bio-12-SS-DNA bound to streptavidin is most likely due to steric protection of the disulfide bond by the protein. This conclusion is supported by our finding that DNA labeled with another biotinylated nucleotide analog, Bio-19-SS-dUTP, is rapidly and efficiently recovered from a streptavidin column. In Bio-19-SS-DNA, the distance between the disulfide bond and the biotin group is approximately 10 A greater than that in Bio-12-SS-DNA. Therefore, Bio-19-SS-dUTP and streptavidin form the basis of an efficient affinity system for the isolation and subsequent recovery of biotinylated DNA in the presence of low ionic strength buffers.
D001426:Bacterial Proteins; D001710:Biotin / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D002482:Cellulose; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity / Q000379:methods; D004247:DNA / Q000302:isolation & purification*; D003856:Deoxyuracil Nucleotides / Q000138:chemical synthesis; D004220:Disulfides; D019809:Streptavidin
D001426:Bacterial Proteins; D003856:Deoxyuracil Nucleotides; D004220:Disulfides; C049787:biotin-19-SS-dUTP; D001710:Biotin; D002482:Cellulose; D004247:DNA; D019809:Streptavidin; C049786:biotin-12-SS-dUTP