470451 Long-Read DNA Separations Using Micelle-Elfse in Microchip Electrophoresis

Tuesday, November 15, 2016: 1:45 PM
Embarcadero (Parc 55 San Francisco)
Randall Gamble1, Lingxiao Yan1, Ruohui Zheng2 and James W. Schneider1, (1)Chemical Engineering, Carnegie Mellon University, Pittsburgh, PA, (2)Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA

Our group has developed a gel-free method (“micelle-ELFSE”) that provides rapid separation of end-alkylated DNA fragments in running buffers containing 1-3 vol% of nonionic wormlike surfactant micelles. These micelles transiently attach to the alkyl group and serve as a drag-tag so that the free-solution mobility of the fragments in length dependent. Micelles undergo rapid fluctuations in size so that the drag imparted to each fragment in the sample is highly uniform. We have recently showed that the method can resolve Sanger sequencing products over 700 bases in length, with single-base resolution, in less than 15 mins using standard bench-top capillary electrophoresis equipment. Micelle-ELFSE is simple to implement in bench-top capillary electrophoresis instrumentation, uses low-cost materials, and the buffers used do not clog in the presence of contaminants. Micelle size can be tuned to fit a given separation by temperature or formulation.

In this presentation, we will discuss our efforts to apply micelle-ELFSE to much longer DNA fragments, well over 1000 bases in length. Kilobase DNA separations are required for many applications in biology and medicine, including gene mapping, pathogen identification, and processing of chromosomal DNA digests in next-generation sequencing technologies. In DC mode, gel-based DNA electrophoresis fails for fragments above 15,000 bases in length due to biased reptation effects. Pulsed-field electrophoresis methods are capable of separating fragments above 15,000 bases, but with runtimes of several days. An alternative is the use of microfabricated post arrays and entropic traps, but these are expensive to implement and have low throughput. Micelle-ELFSE methods are not expected to suffer from biased reptation limits as the wormlike micelles used have large effective pore sizes and short micelle lifetimes. To test this concept, we created a ladder with fragments 1000, 2000, 3000, 4000, 6000, and 10,000 bases in length and were able to separate it, with baseline resolution, in 3 minutes using bench-top capillary electrophoresis instrumentation. The success of the method relies on an apparent DNA-tag segregation that causes the peak spacing to be larger than predicted by theories that assume the tag and DNA to be hydrodynamically indistinct. Additionally, we have found that use of very long wormlike micelles, which form an entangled network capable of sieving unalkylated DNA, does not impact the effectiveness of micelle-ELFSE. Access to very large micelle drag-tags greatly extends the lengths of DNA that can be resolved.

We have since created ladders exceeding 30,000 bases in length and will optimize large-micelle buffers for their separation for presentation in this talk. The ultimate goal is to determine the fastest runtimes and DNA lengths that can be resolved using micelle-ELFSE. We will also report an observed buffer dependence of the DNA-tag segregation that suggests new mechanisms for segregation. Finally, we will discuss efforts to perform these separations in microfluidic devices, where snap-shot imaging of the full microchannel can further reduce runtime.

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