Rapid, Non-gel DNA Electrophoresis using Surfactant Systems

Jim Schneider, Chemical Engineering

The Schneider group has developed a system (“micelle-ELFSE”) that rapidly separates DNA in electric fields without using the gels that are standard equipment in nearly all molecular biology labs.  The method first has DNA end-alkylated and then separated using capillary electrophoresis (CE) using running buffers containing less than 3 vol% of nonionic surfactant.  At these concentrations, the surfactant monomers self-assembled into spherical “micelles” that can bind to the alkane group on the DNAs to be separated.  Attachment of micelles to the DNA ends gives rise to a length dependent separation without expensive gels, and that provides 10-100 fold decreases in runtime using CE.  However, most molecular biology labs in education, research, and industry do not have access to CE instrumentation and instead rely on the time-tested slab-gel electrophoresis method to separate and analyze DNA.  This project aims to develop a new platform for micelle-ELFSE that is compatible with commonly available gel readers and power supplies.  In so doing, we hope to make the technology much more available to everyone and speed up slab-gel electrophoresis significantly from several hours to a few minutes.

We will investigate several potential “gel-like” media for this summer research project.  First, we will create agarose gels with a very open pore structure (average pore size 1-10 microns) so that the DNA is not significantly stretched during the run.  Another option that may provide better mechanical strength is to use gels made of poly (dimethyl siloxane) (PDMS).  While water does not penetrate PDMS pores without modification, the requisite nonionic surfactant is expected to coat the PDMS pores so that buffer infiltration can occur.  Other hydrogels will also be explored, time permitting.

Successful gels will give tight, well-resolved bands of end-alkylated DNA in micelle-containing running buffers and have sufficient mechanical strength that they can be handled easily by lab technicians.  The research plan will first fabricate a series of agarose or PDMS gels using standard procedures (varying molecular weight, crosslink density, etc.) and their average pore size measured by measuring their Young’s modulus in an Ingstron tester (available in the CPS lab, Doherty Hall).  Alternatively, pore sizes could be measured by imaging using atomic force microscopy or by mercury porosimetry.  Next, slab-gel electrophoresis will be performed in the Schneider lab to assess the morphology of stained DNA bands and a correlation determined between the pore size, electric field, DNA length, and surfactant concentration using an ANOVA-type statistical analysis to minimize the number of experiments.