Separation of Fragments up to 570 Bases in Length by Use of 6% T Non-Cross-Linked Polyacrylamide for DNA Sequencing in Capillary Electrophoresis

Best, Norine; Arriaga, Edgar A.; Chen, David D. Y.; Dovic̀hi, Norman J.

doi:10.1021/ac00094a031

Cited by 64 publications

(34 citation statements)

References 48 publications

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“…5-g In our common knowledge, higher concentration of polyacrylamide is suitable for separating small DNA fragments and lower concentration of polyacrylamide is indispensable for the separation of large DNA fragments.11-13 Low concentration of polyacrylamide gel (3% T and 0.5% C) is applied to the separation of up to 12 kbp DNA fragments1,2,4 and 1.5% linear polyacrylamide gave a fine resolution of DNA fragments up to 23 kbp3, whereas shorter DNA fragments up to 1 kbp should be separated by using 6% linear polyacrylamide.l, 3 Large DNA fragments up to 48.5 kbp could only be separated by using very diluted linear polyacrylamide (0.6%).9 Use of lower concentration of linear polyacrylamide (2%) allowed DNA sequencing of more than 1000 bases per rung, whereas DNA sequencing of less than 500 bases was achieved using 6% linear polyacrylamide as a separation matrix. 5,6 More recently, some cellulose derivatives were proven to be an efficient separation matrix for DNA fragments.l3-19 In this instance, use of ultradilute polymer solution (0.00125%) was necessary for the separation of DNA up to 23 kbp17-19, whereas 0.5% cellulose solution is suitable for the separation of DNA fragments up to 1 kbp.14-16 A pulsed field technique20 coupled with use of ultradilute polymer solution gave an excellent separation of multikilobase and mega base DNA fragment2l-26, which could not be separated without a pulsed filed technique. 23 Both standard textbooks on gel electrophoresis11,12 and comprehensive review on capillary electrophoresisl3 recommend that effective range of separation of DNA fragments is less than 300 -400 by using 8% polyacrylamide in both slab gel electrophoresis and capillary electrophoresis.…”

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confidence: 99%

Is Higher Concentration of Linear Polyacrylamide Not Suitable for the Capillary Electrophoretic Separation of Large DNA?

Ueda

Baba

1997

ANAL. SCI.

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Polyacrylamide is an excellent separation matrix for capillary electrophoresis 110 and conventional gel electrophoresis,11,12 Low crosslinked polyacrylamide gel and linear polyacrylamide gave an excellent resolution of double-stranded DNA fragments using capillary electrophoresis. l-4,9,10 Linear polyacrylamide has been used as a separation matrix for DNA sequencing especially for long reading by capillary electrophoresis.5-gIn our common knowledge, higher concentration of polyacrylamide is suitable for separating small DNA fragments and lower concentration of polyacrylamide is indispensable for the separation of large DNA fragments.11-13 Low concentration of polyacrylamide gel (3% T and 0.5% C) is applied to the separation of up to 12 kbp DNA fragments1,2,4 and 1.5% linear polyacrylamide gave a fine resolution of DNA fragments up to 23 kbp3, whereas shorter DNA fragments up to 1 kbp should be separated by using 6% linear polyacrylamide.l,3 Large DNA fragments up to 48.5 kbp could only be separated by using very diluted linear polyacrylamide (0.6%).9 Use of lower concentration of linear polyacrylamide (2%) allowed DNA sequencing of more than 1000 bases per rung, whereas DNA sequencing of less than 500 bases was achieved using 6% linear polyacrylamide as a separation matrix.5,6More recently, some cellulose derivatives were proven to be an efficient separation matrix for DNA fragments.l3-19 In this instance, use of ultradilute polymer solution (0.00125%) was necessary for the separation of DNA up to 23 kbp17-19, whereas 0.5% cellulose solution is suitable for the separation of DNA fragments up to 1 kbp.14-16 A pulsed field technique20 coupled with use of ultradilute polymer solution gave an excellent separation of multikilobase and mega base DNA fragment2l-26, which could not be separated without a pulsed filed technique.23Both standard textbooks on gel electrophoresis11,12 and comprehensive review on capillary electrophoresisl3 recommend that effective range of separation of DNA fragments is less than 300 -400 by using 8% polyacrylamide in both slab gel electrophoresis and capillary electrophoresis. Additionally, these textbooks and review conclude the use of less than 3% polyacrylamide is essential for the separation of large DNA more than 1 kbp.In this paper, we demonstrate that 7% polyacrylamide is suitable for the rapid separation of large DNA fragments up to 50 kbp by capillary electrophoresis without pulsed electric field. Only 5.4 min is required for the complete resolution of seven DNA fragments in a sample of DNA restriction fragments T4dC/ Bgl I digest containing DNA fragments ranging from 1.26 to 49.31 kbp by constant field capillary electrophoresis and the peak of T4dC DNA (166 kbp) appears at ca. 8.3 min. Such large DNA fragments up to 50 kbp have never been separated without use of lower concentration of separation matrix and pulsed field technique.21-26 Additionally, DNA conformational dynamics in high concentration of polyacrylamide under electric field was observed directly by use of fluorescence...

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confidence: 99%

Is Higher Concentration of Linear Polyacrylamide Not Suitable for the Capillary Electrophoretic Separation of Large DNA?

Ueda

Baba

1997

ANAL. SCI.

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“…Resolution of 0.5 is defined as the point at which the migration time difference between two Gaussian peaks equals their average full widths at half maximum (Luckey et al 1993). This resolution is commonly set as a minimum requirement for accurate robust sequencing (Best et al 1994), although specialized base-calling software has been reported to operate to a resolution as small as 0.25 (B.L. Karger, unpubl.).…”

Section: Resultsmentioning

confidence: 99%

Toward Real-World Sequencing by Microdevice Electrophoresis

Schmalzing¹,

Tsao²,

Koutny³

et al. 1999

Genome Res.

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We report results using a microdevice for DNA sequencing using samples from chromosome 17, obtained from the Whitehead Institute Center for Genome Research (WICGR) production line. The device had an effective separation distance of 11.5 cm and a lithographically defined injection width of 150 µm. The four-color raw data were processed, base-called by the sequencing software Trout, and compared to the corresponding ABI 377 sequence from WICGR. With a criteria of 99% accuracy, we achieved average continuous reads of 505 bases in 27 min with 3% linear polyacrylamide (LPA) at 150 V/cm, and 460 bases in 22 min with 4% LPA at 200 V/cm at a temperature of 45°C. In the best case, up to 565 bases could be base-called with the same accuracy in <25 min. In some instances, Trout allowed for accurate base-calling down to a resolution R as low as R = 0.35. This may be due in part to the high signal-to-noise ratio of the microdevice. Unlike many results reported on capillary machines, no additional sample cleanup other than ethanol precipitation was required. In addition, DNA fragment biasing (i.e., discrimination against larger fragments) was reduced significantly through the unique sample injection mechanism of the microfabricated device. This led to increased signal strength for long fragments, which is of great importance for the high performance of the microdevice.Significant advancement in the technology of DNA analysis is expected from the use of microfabricated electrophoretic devices for sequencing and genotyping. In this approach photolithography, combined with wet-etching and thermal wafer bonding, is used to construct enclosed intricate microchannel structures in glass and fused-silica substrates; these structures are then utilized for electrophoresis (Harrison et al. 1993). It has been speculated that these devices will allow DNA separations approaching the theoretical limits of electrophoresis and in a format that will reduce analysis time and extend parallelism and automation (Freemantle 1999), which might hence increase throughput well beyond current capillary array machines. For example, in recent experiments we have demonstrated genotyping at 10-to 100-fold reduced analysis times on microdevices when compared to capillaries and slab gels, respectively (Schmalzing et al. 1997. DNA sequencing of single-color pGEM and four-color M13 DNA standard sequencing samples has been demonstrated on 3.5-, 11.5-, and 7-cm-long microdevices (Woolley et al. 1995;Schmalzing et al. 1998;Liu et al. 1999). The feasibility of ultra-high sample throughput has been proven through still modest multiplexing up to 96 microchannels (Simpson et al. 1998;Koutny et al. 1999). However, to the best of our knowledge, all published studies on DNA sequencing by microdevices have been performed using DNA standard samples such as M13 or pGEM. Practical sequencing must deal with additional factors such as variable salt and template concentrations Salas-Solano et al. 1998), highly samplespecific compression regions, and the interplay between electr...

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“…Although both manufacturing procedures are similar, LPA is less prone to formation of bubbles. LPA solution provides the longest ssDNA read length unsurpassed by other linear or branched polymer solutions [74][75][76], and has been the workhorse matrix in DNA sequencing. Unfortunately, LPA has no self-coating ability, so it has to be used in precoated separation channels.…”

Section: Linear Polyacrylamide (Lpa)mentioning

confidence: 99%

Polymer solutions and entropic‐based systems for double‐stranded DNA capillary electrophoresis and microchip electrophoresis

Baba

2004

Electrophoresis

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We give an overview of recent development of low-viscosity polymer solutions and entropic trapping networks for double-stranded DNA (dsDNA) separations by conventional capillary electrophoresis and microchip electrophoresis. Theoretical models for describing separation mechanisms, commonly used noncross-linked polymer solutions, thermoresponsive (viscosity-adjustable) polymer solutions, and novel entropic trapping networks are included. The thermoresponsive polymer solutions can be loaded at one temperature into microchannels at lower viscosities, and used in separation at another temperature at entanglement threshold concentrations and higher viscosities. The entropic-based separations use only arrays of regular obstacles acting as size-separations and do not need viscous polymer solutions. These progresses have potential in integration to automated capillary and microfluidic chip systems, enabling better reusability of separation microchannels, much shorter DNA separation times, and higher reproducibility due to less matrix degradation.

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Separation of Fragments up to 570 Bases in Length by Use of 6% T Non-Cross-Linked Polyacrylamide for DNA Sequencing in Capillary Electrophoresis

Cited by 64 publications

References 48 publications

Is Higher Concentration of Linear Polyacrylamide Not Suitable for the Capillary Electrophoretic Separation of Large DNA?

Is Higher Concentration of Linear Polyacrylamide Not Suitable for the Capillary Electrophoretic Separation of Large DNA?

Toward Real-World Sequencing by Microdevice Electrophoresis

Polymer solutions and entropic‐based systems for double‐stranded DNA capillary electrophoresis and microchip electrophoresis

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