Bias in quantitative capillary zone electrophoresis caused by electrokinetic sample injection

Huang, Xiaoxia; Gordon, Manuel J.; Zare, Richard N.

doi:10.1021/ac00155a023

Cited by 241 publications

(117 citation statements)

References 16 publications

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“…To find out tbe optimal quantitative repeatability for a specific instrument it isnecessary lo check a number of sample introduction and separation parameters. A number of reports are dealing witb tbe quantitative repeatability in electroehromalography, which may vary over a wide range [1][2][3][4][5][6][7][8]11,12]. The most significant parameters influencing the sample introduction are tbe applied voltage and tbe injection time at an electrokinetic sample introduction or tbe height difference of tbe sample and buffer solution levels and tbe injection time when hydrostatic sample introduction is applied.…”

mentioning

confidence: 99%

The Repeatability of the Quantitative Analysis in Electrochromatography

Coufal¹,

Claessens²,

Cramers³

1993

Journal of Liquid Chromatography

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mentioning

confidence: 99%

The Repeatability of the Quantitative Analysis in Electrochromatography

Coufal¹,

Claessens²,

Cramers³

1993

Journal of Liquid Chromatography

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“…This may be a consequence of variability in the PCR amplifications or the inefficiency of the simple ethanol cleanup procedure to fully remove salt . Residual salt in the sample might influence the electrical resis- Cold Spring Harbor Laboratory Press on May 12, 2018 -Published by genome.cshlp.org Downloaded from tance in the loading channel resulting in salt-dependent DNA velocities during the 2-min-long loading process and thereby introduce some variability of sample concentration during injection (Huang et al 1988). The DNA velocities in the separation channel would not be affected, as only a miniscule amount of sample salt enters the separation channel during injection.…”

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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“…3. The injection volume, 30 pL, was estimated using standard methods [19]. The calibration curve was linear (r = 0.9914, n = 16) with a near zero intercept.…”

Section: Detection Limit Of Labeled G M1mentioning

confidence: 99%

Yoctomole analysis of ganglioside metabolism in PC12 cellular homogenates

et al. 2007

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We report an ultrasensitive method for the analysis of glycosphingolipid catabolism. The substrate G(M1) and the set of seven metabolites into which it can be degraded (G(A1), G(M2), G(A2), G(M3), LacCer, GlcCer, and Cer) were labeled with the highly fluorescent dye tetramethylrhodamine. CE with LIF detection was used to assay these compounds with 150 +/- 80 yoctomole mass (1 ymol = 10(-24) mol = 0.6 copies) detection limits and 5 +/- 3 pM concentration detection limits. An alignment algorithm based on migration of two components was employed to correct for drift in the separation. The within-day and between-day precision in peak height was 20%, in peak width 15%, and in adjusted migration time 0.03%. After normalization to total sample injected, the RSD in peak height reduced to 2-6%, which approaches the limit set by molecular shot noise in the number of molecules taken for analysis. PC12 cells were incubated with the labeled G(M1). Fluorescent microscopy demonstrated uptake by the cells. CE was used to separate a cellular homogenate prepared from these cells. A set of peaks was observed, which were tentatively identified based on comigration with the standards. Roughly 120 pL of homogenate was injected, which contained a total of 150 zmol of labeled substrate and products. Metabolite that preserves the fluorescent label can be detected at the yoctomole level, which should allow characterization of this metabolic pathway in single cells.

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Bias in quantitative capillary zone electrophoresis caused by electrokinetic sample injection

Cited by 241 publications

References 16 publications

The Repeatability of the Quantitative Analysis in Electrochromatography

The Repeatability of the Quantitative Analysis in Electrochromatography

Toward Real-World Sequencing by Microdevice Electrophoresis

Yoctomole analysis of ganglioside metabolism in PC12 cellular homogenates

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