title: DNA adsorption to the reservoir walls causing irreproducibility in studies of protein-DNA interactions by methods of kinetic capillary electrophoresis.
abstract: Methods of kinetic capillary electrophoresis (KCE) facilitate kinetic studies of protein-DNA interactions and highly efficient selection of DNA aptamers for protein targets. Here, we report a previously unnoticed source of error that affects the precision and accuracy of KCE-based measurements. The error manifests itself in cases that require the use of low concentrations of DNA. In such measurements, the reproducibility of the signal generated by the same fluorescently labeled DNA sample can have a relative standard deviation (RSD) as high as 40%. We have investigated the cause of the irreproducibility and found that it is attributed to DNA adsorption to the surface of the sample vials, in which protein-DNA mixtures are prepared prior to a KCE experiment. The use of commercially available "high DNA recovery" sample vials does not resolve the problem. We have found that the problem can be significantly alleviated by the passivation of the vial surface with blocking agents, such as masking DNA or bovine serum albumin (BSA). The described adsorption of DNA to the surface of sample vials may also be important in other procedures that deal with low DNA concentrations, such as aptamer selection and quantitative PCR.
authors.affiliation: Department of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Ontario M3J 1P3, Canada.
authors.forename: Mirzo
authors.initials: M
authors.lastname: Kanoatov
authors.forename: Sergey N
authors.initials: SN
authors.lastname: Krylov
mesh_terms: D000327:Adsorption; D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry; D002417:Cattle; D004247:DNA / Q000378:metabolism*; D019075:Electrophoresis, Capillary; D007700:Kinetics; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012710:Serum Albumin, Bovine / Q000378:metabolism*
chemical_list: D052157:Aptamers, Nucleotide; D012710:Serum Albumin, Bovine; D004247:DNA