High‐resolution single‐stranded DNA analysis on 4.5 cm plastic electrophoretic microchannels

Shi, Yining; Anderson, Rolfe C.

doi:10.1002/elps.200305553

Cited by 50 publications

(49 citation statements)

References 23 publications

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“…Thus, the 4% pDMA matrix gives the best resolution of both smaller and larger fragments and is the best of the three sequencing matrices. Table 2 also reports sequencing times in these matrices, which are up to 66% lower than in any other chip sequencing report published (8,(18)(19)(20)(21)29). Because most microchip-based DNA sequencing studies have used longer channels as well as different electric field strengths and temperatures in efforts to optimize each individual system, it is difficult to compare migration times in our system directly with their published sequencing times.…”

Section: Dna Sequencing Resultsmentioning

confidence: 99%

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

Fredlake

Hert

Kan

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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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

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Section: Dna Sequencing Resultsmentioning

confidence: 99%

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

Fredlake

Hert

Kan

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…Woolley and Mathies (1995) demonstrated the first application of DNA sequencing using a microfabricated glass device in 1995, reporting single-base resolution using their four-color scanner technology. Data quality and readlengths have improved significantly since then, because of an increase in the effective separation lengths with run times of 30 minutes or less (Table 2) (Woolley and Mathies 1995;Liu et al 1999;Schmalzing et al 1999;Backhouse et al 2000;Koutny et al 2000;Liu et al 2000;Salas-Solano et al 2000;Simpson et al 2000;Boone et al 2002;Paegel et al 2002;Shi and Anderson 2003). For example, Liu et al (1999) reported 99.4% accuracy over 500 bases in 20 minutes, with an increase in separation length from 3.5 cm to 6.5 cm.…”

Section: Microfluidic Separation Platformsmentioning

confidence: 99%

“…For example, Liu et al (1999) reported 99.4% accuracy over 500 bases in 20 minutes, with an increase in separation length from 3.5 cm to 6.5 cm. More recent developments by Boone et al (2002) and Shi and Anderson (2003) have shown the first DNA sequencing applications on plastic chips (Table 2). These chips can be fabricated with high geometric aspect ratios (i.e., deep and narrow channels) at significantly lower cost.…”

Section: Microfluidic Separation Platformsmentioning

confidence: 99%

Emerging technologies in DNA sequencing

Metzker¹

2005

Genome Res.

410

262

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Demand for DNA sequence information has never been greater, yet current Sanger technology is too costly, time consuming, and labor intensive to meet this ongoing demand. Applications span numerous research interests, including sequence variation studies, comparative genomics and evolution, forensics, and diagnostic and applied therapeutics. Several emerging technologies show promise of delivering next-generation solutions for fast and affordable genome sequencing. In this review article, the DNA polymerase-dependent strategies of Sanger sequencing, single nucleotide addition, and cyclic reversible termination are discussed to highlight recent advances and potential challenges these technologies face in their development for ultrafast DNA sequencing.

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“…Recently, Shi and Anderson [35] have reported the use of a polymeric microdevice for the separation of ssDNA fragments and successfully achieved single basepair resolution using a polyolefin device coated with poly-DMA/ diethylacrylamide. The authors attributed their ability to achieve single base resolution primarily to their matrix selection, a 4% w/v of 5.5 MDa linear polyarcylamides (LPAs); however, they did not investigate the significance of their coating material and whether the same resolution was achievable using the native polyolefin.…”

Section: Introductionmentioning

confidence: 99%

Surface modification of poly(methyl methacrylate) microfluidic devices for high‐resolution separations of single‐stranded DNA

2007

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While polymer-based microfluidic devices offer some unique opportunities in developing low-cost systems for a variety of application areas, the ability to sort electrophoretically with high efficiency a number of different targets has remained somewhat elusive with an example consisting of achieving single base resolution as required for DNA sequencing. While the reasons for this are many-fold, it is clear that some type of coating is required on the polymer substrate to suppress the EOF and/or minimize potential solute/wall interactions. To this end, we report on a simple grafting procedure to allow the formation of polymer coats, which in this example used linear polyarcylamides (LPAs), onto a poly(methyl methacrylate) (PMMA) microfluidic device. The procedure involved creating an amine-terminated PMMA surface by appropriately functionalizing the PMMA through either a chemical or photochemical process. The aminated surface could then be used to covalently anchor methacrylic acid, which was used as a scaffold to produce LPAs on the surface through radical polymerization of acrylamide. The resulting surfaces demonstrated EOFs that were nearly an order of magnitude smaller than native PMMA. In addition, these LPA-coated devices could produce highly reproducible migration times of over approximately 20 runs with plate numbers exceeding 10(5) m(-1). Using gel electrophoretic analysis of a single base track generated from an M13mp18 template using Sanger cycle sequencing and dye-primer chemistry, the resolution value obtained for bases 199 and 200 was 0.18 while for bases 208 and 209 it was 0.21. For the native PMMA, these bands were found to comigrate.

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High‐resolution single‐stranded DNA analysis on 4.5 cm plastic electrophoretic microchannels

Cited by 50 publications

References 23 publications

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

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

Emerging technologies in DNA sequencing

Surface modification of poly(methyl methacrylate) microfluidic devices for high‐resolution separations of single‐stranded DNA

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