Rapid, Free-Solution Electrophoretic Separations of Kilobase DNA by Transiently Attached Wormlike Micelles

Carnegie Mellon University, Pittsburgh PA

Investigators

Abstract

Proposal Number: 1605351, Scheider Rapid, Free-Solution Electrophoretic Separations of Kilobase DNA by Transiently Attached Micelles This project will develop an ultrafast method to analyze long-strand DNA without the use of gels but with shorter analysis times. Many important DNA sequencing methods require a length-based sorting of DNA longer than 1000-10,000 bases. The most common method to accomplish this is pulsed-field gel electrophoresis, which requires 12-36 hours to complete and presents a significant bottleneck. The proposed method instead uses low viscosity, water-based solutions of surfactants instead of gels, and is expected to perform the same function as mentioned in gel electrophoresis but with runtimes of less than five minutes. Additionally, the low-viscosity solutions used are easy to inject into capillaries and microchips and do not suffer from clogging and degradation with repeated use. Incorporation of this rapid, gel-free method into critical DNA sequencing procedures could greatly decrease the cost of sequencing methods used in forensic identification and biology research such as genome mapping and pathogen identification. One Ph.D student, three M.S. students, and five undergraduates will be trained in colloid and interface science, organic chemistry, and bioseparations as they contribute to the project. Single-molecule DNA visualization studies will be built into demonstrations for K-12 student outreach, and lab modules for undergraduate courses will be developed based on microchip electrophoresis. This project will research a rapid, non-gel means to separate double strand DNA greater than 10-100 kilobases (kB) in length as required for genome mapping and other applications using inexpensive, widely available capillary electrophoresis equipment. The method uses transiently attached wormlike micelles as drag-tags, which, when attached to DNA by a terminal n-alkane group, provide a length-dependent electrophoretic mobility without the use of a sieving matrix. Specific objectives of the project aims are: (1) to design and implement substituted peptide nucleic acid staples to attach alkyl groups to the ends of 48.5 kB lambda-DNA and its digests; (2) to screen a series of nonionic micelle buffers to obtain the largest drag-tag size possible; (3) to develop a theory predicting the mobility of the DNA by consideration of DNA-drag-tag segregation effects in networks of varying pore size and micelle lifetimes; and (4) to perform single-molecule imaging of DNA to assess the extent of DNA-drag-tag segregation. The proposed work will identify optimal buffers and run conditions for a given set of DNA lengths to be separated and identify limits of DNA length and runtime that can be achieved. Insights into the perturbation of wormlike micelle entanglements by vicinal hydrophobes may also help better understand the rheological behavior of these solutions in the presence of contaminants.