Ultrafast DNA sequencing on a microchip by a hybrid separation mechanism that gives 600 bases in 6.5 minutes

Abstract

To realize the immense potential of large-scale genomic sequencing after the completion of the second human genome (Venter's), the costs for the complete sequencing of additional genomes must be dramatically reduced. Among the technologies being developed to reduce sequencing costs, microchip electrophoresis is the only new technology ready to produce the long reads most suitable for the de novo sequencing and assembly of large and complex genomes. Compared with the current paradigm of capillary electrophoresis, microchip systems promise to reduce sequencing costs dramatically by increasing throughput, reducing reagent consumption, and integrating the many steps of the sequencing pipeline onto a single platform. Although capillary-based systems require Ϸ70 min to deliver Ϸ650 bases of contiguous sequence, we report sequencing up to 600 bases in just 6.5 min by microchip electrophoresis with a unique polymer matrix/adsorbed polymer wall coating combination. This represents a two-thirds reduction in sequencing time over any previously published chip sequencing result, with comparable read length and sequence quality. We hypothesize that these ultrafast long reads on chips can be achieved because the combined polymer system engenders a recently discovered ''hybrid'' mechanism of DNA electromigration, in which DNA molecules alternate rapidly between reptating through the intact polymer network and disrupting network entanglements to drag polymers through the solution, similar to dsDNA dynamics we observe in single-molecule DNA imaging studies. Most importantly, these results reveal the surprisingly powerful ability of microchip electrophoresis to provide ultrafast Sanger sequencing, which will translate to increased system throughput and reduced costs.DNA separation mechanism ͉ microchip electrophoresis ͉ entangled polymer solution ͉ DNA imaging

Modeling the separation of macromolecules: A review of current computer simulation methods

“…However, the absence of crosslinking may be important, especially in strong fields typically used for capillary electrophoresis. Recent videomicroscopy experiments [266,267] seem to suggest that the DNA is able to drag matrix polymers even well above the entanglement concentration. Simulations are needed to help understand this process.…”

Ultrasensitive, amplification-free assays for detecting pathogens.

“…Initial attem pts focused on using a pol ydimethylacrylamide (PDMA) sieving m atrix at concentrations of 3-4%, which perform ed very well in initial exp eriments with se paration of DNA ladders described in [6], an d has also been successful as a sieving m atrix for DNA sequencing separations [33]. With the hybridization assay, results with this m atrix were not reproducible: sometimes successful separations were obtained, as in Figure 7 above, although the mobility shift of the hybrid peak was not always consisten t, and som etimes the hybrid peak failed to appear.…”

Recent advances in microfluidic technologies for biochemistry and molecular biology

“…The rapid development of sequencing technology was instrumental to the early completion of the Human Genome Project, and will undoubtedly continue to play an important role in molecular biology and biochemistry for many years to come. Various microfluidic tools for DNA sequencing have been successfully transferred to planar-chip formats (6)(7)(8)(9). For example, Quake and co-workers sequenced DNA with a 4 base pair read length using a sequencing-by-synthesis paradigm (6).…”