Immobilization of denatured DNA to macroporous supports: I. Efficiency of different coupling procedures.
Methods commonly used for covalent immobilization of single stranded DNA have been applied to several solid supports (Sephadex G-25 and Cellex 410) as well as to a number of macroporous materials (Sepharose C1-6B, C1-2B; Sephacryl S-500 and S-1000). Coupling efficiencies and stability of covalently bound DNA are compared for both classes of materials. The yields of the immobilization reaction for sonicated DNA are only 10-40% for G-25 and Cellex 410 in contrast to 60-80% for C1-6B and S-500. Under optimal conditions, up to 0.5 mg of DNA can be coupled initially per g of wet macroporous material. The immobilized DNAs are lost from the supports in a biphasic manner, with about 10-20% loss per day during the first 2-3 days at 45 degrees C, followed by only about 1% loss per day at the same temperature thereafter. The influence of the coupling procedure on the generation of mismatch effects has been studied in 2.4 M tetraethylammonium chloride solution for the hybrid formation between immobilized and mobile DNA. The degree of mismatch ranged from 0-3% and depended on the method of immobilization. The unspecific absorption of DNA on macroporous materials is sufficiently low to allow efficient hybrid selection. No size limitations have been observed when plastid mRNAs are selected by cloned fragments of plastid DNA immobilized to macroporous Sephacryl S-500.
D002846:Chromatography, Affinity / Q000379:methods; D004256:DNA Polymerase I / Q000378:metabolism*; D004269:DNA, Bacterial; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003911:Dextrans; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005782:Gels; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012333:RNA, Messenger / Q000302:isolation & purification*; D012685:Sepharose; D013211:Staphylococcus aureus / Q000201:enzymology
D004269:DNA, Bacterial; D003911:Dextrans; D005782:Gels; D012333:RNA, Messenger; D012685:Sepharose; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase