Igor V. Kourkine

Critical factors for high-performance physically adsorbed (dynamic) polymeric wall coatings for capillary electrophoresis of DNA

Physically adsorbed (dynamic) polymeric wall coatings for microchannel electrophoresis have distinct advantages over covalently linked coatings. In order to determine the critical factors that control the formation of dynamic wall coatings, we have created a set of model polymers and copolymers based on N,N‐dimethylacrylamide (DMA) and N,N‐diethylacrylamide (DEA), and studied their adsorption behavior from aqueous solution as well as their performance for microchannel electrophoresis of DNA. This study is revealing in terms of the polymer properties that help create an “ideal” wall coating. Our measurements indicate that the chemical nature of the coating polymer strongly impacts its electroosmotic flow (EOF) suppression capabilities. Additionally, we find that a critical polymer chain length is required for polymers of this type to perform effectively as microchannel wall coatings. The effective mobilities of double‐stranded (dsDNA) fragments within dynamically coated capillaries were determined in order to correlate polymer hydrophobicity with separation performance. Even for dsDNA, which is not expected to be a strongly adsorbing analyte, wall coating hydrophobicity has a deleterious influence on separation performance.

Technical challenges in applying capillary electrophoresis-single strand conformation polymorphism for routine genetic analysis.

Recent and future advances in population genetics will have a significant impact on health care practices and the economics of health care provision only if a spectrum of patient‐tailored, effective methods of DNA screening for sequence alterations has been developed. Genetic screening by capillary electrophoresis‐single strand conformation polymorphism (CE‐SSCP), which is based upon the differences in electrophoretic mobilities of wild‐type and mutant DNA species, offers an important complement to other presently available techniques such as Sanger sequencing and DNA hybridization arrays due to its simplicity, versatility, and low cost of analysis. A two‐part review of CE‐SSCP that discusses its advantages and limitations is presented. Emphasis is placed on technological aspects of CE‐SSCP (including such rarely addressed issues as sample preparation protocols and the nature of the polymeric DNA separation matrix) as well as on the potential of CE‐SSCP for routine genetic analysis. An attempt is made to organize and present the information in sufficient detail to allow the use of SSCP for routine genetic screening even by those inexperienced in CE. Some discussion of CE‐based heteroduplex analysis (HA) is also presented.

Measurement of protein-ligand binding constants from reaction-diffusion concentration profiles.

Protein-ligand dissociation constants, K(d), are determined precisely and down to the picomolar range from reaction-diffusion (RD) concentration profiles created by proteins diffusing through hydrogels functionalized with protein ligands. The RD process effectively amplifies the molecular-scale binding events into macroscopic patterns visible to the naked eye. The method is applicable to various protein-ligand pairs and does not require any prior knowledge about the protein structure.