H Swerdlow

University of Utah, Salt Lake City, UT, United States

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Publications (9)33.84 Total impact

  • S C Huang, H Swerdlow, K D Caldwell
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    ABSTRACT: The binding of 5'-end biotinylated DNA fragments was compared between streptavidin (SA)-coated commercial M-280 magnetic latex particles with a diameter of 2.8 microns and adsorption-coated polystyrene (PS) latex standard particles whose diameter is 0.272 microns. Amino acid analysis showed the protein content of the commercial particles to correspond to 4x monolayer coverage, while the adsorption-coated PS particles displayed monolayer coverage, or 8 pmol/cm2. A fluorescence-based method was developed to quantify the adsorption of FITC-labeled SA, biotin, and biotinylated DNA. The validity of the method was substantiated for the labeled protein by both amino acid analysis and a colorimetric protein assay. While the specific binding of biotin was 0.38 mol per mole of SA on the adsorption-coated 0.272-microns particles and slightly higher (0.6 mol per mole SA) on the 2.8-microns particles, the specific binding of the bulky biotinylated 300-bp DNA was more favorable on the smaller particle (0.12 mol per mole SA for 0.272 microns versus 0.04 mol per mole SA for 2.8 microns).
    Analytical Biochemistry 12/1994; 222(2):441-9. · 2.58 Impact Factor
  • H Swerdlow, K Dew-Jager, R F Gesteland
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    ABSTRACT: Current automated fluorescent instruments are all based on slab gels that are used once and then discarded. Practitioners of capillary gel electrophoresis often reuse their gels for multiple samples. As slab gels are made thinner to increase speed, the ability to reload new samples after each run will become more desirable. Techniques have previously been developed for reloading and stabilizing capillary gels. The application of these methods to slab gel electrophoresis is reported. Gels are shown to be reusable for at least four consecutive automated runs. The stability of various slab gel formulations and their ability to survive multiple loadings with sequencing samples are compared. Formamide-containing gels are shown to be superior to their urea counterparts. The potential that running buffer additives have for improving automated DNA sequencing is discussed. Residual template DNA in sequencing samples can produce gel instability, reduce resolution and decrease signal. These effects are examined.
    BioTechniques 05/1994; 16(4):684-5, 688-93. · 2.40 Impact Factor
  • H Swerdlow, K Dew-Jager, R F Gesteland
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    ABSTRACT: Cycle sequencing using Taq DNA polymerase has gained popularity recently due to reduced template requirements, improved signal and its ability to directly sequence PCR fragments. A major drawback to the technique is the time required for performing reactions in a block-based thermal cycler. To help cycle sequencing compete with other methods, we have modified the protocol to be performed in capillaries using an air-based thermal cycling instrument. This instrument has been developed and optimized for rapid, specific amplification of DNA by PCR. The resulting cycle sequencing methodology is faster than block-based approaches; a reaction can be completed in 25 min, compared with about 2 h in a conventional instrument. Thus, the speed of the technique is competitive with standard uncycled T7 or Taq reactions. Accuracy of the sequencing data is improved; two problem areas in the sequence obtained with a block cycler are ameliorated by the capillary methodology. This technique represents a novel approach to cycle sequencing that will further the development of capillary-based analytical methods.
    BioTechniques 10/1993; 15(3):512-9. · 2.40 Impact Factor
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    ABSTRACT: Recent interest in capillary gel electrophoresis has been fueled by the Human Genome Project and other large-scale sequencing projects. Advances in gel polymerization techniques and detector design have enabled sequencing of DNA directly in capillaries. Efforts to exploit this technology have been hampered by problems with the reproducibility and stability of gels. Gel instability manifests itself during electrophoresis as a decrease in the current passing through the capillary under a constant voltage. Upon subsequent microscopic examination, bubbles are often visible at or near the injection (cathodic) end of the capillary gel. Gels have been prepared with the polyacrylamide matrix covalently attached to the silica walls of the capillary. These gels, although more stable, still suffer from problems with bubbles. The use of actual DNA sequencing samples also adversely affects gel stability. We examined the mechanisms underlying these disruptive processes by employing polyacrylamide gel-filled capillaries in which the gel was not attached to the capillary wall. Three sources of gel instability were identified. Bubbles occurring in the absence of sample introduction were attributed to electroosmotic force; replacing the denaturant urea with formamide was shown to reduce the frequency of these bubbles. The slow, steady decline in current through capillary sequencing gels interferes with the ability to detect other gel problems. This phenomenon was shown to be a result of ionic depletion at the gel-liquid interface. The decline was ameliorated by adding denaturant and acrylamide monomers to the buffer reservoirs. Sample-induced problems were shown to be due to the presence of template DNA; elimination of the template allowed sample loading to occur without complications.(ABSTRACT TRUNCATED AT 250 WORDS)
    Electrophoresis 09/1992; 13(8):475-83. · 3.26 Impact Factor
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    ABSTRACT: Capillary gel electrophoresis is demonstrated for the four-spectral-channel sequencing technique of Smith, the two-spectral-channel sequencing technique of Prober, and the one-spectral-channel sequencing technique of Richardson and Tabor. Sequencing rates up to 1000 bases/h are obtained at electric field strengths of 465 V/cm. At lower electric field strengths, capillary electrophoresis produces useful data for fragments greater than 550 nucleotides in length with 2 times better resolution than slab gel electrophoresis. An on-column detector produces detection limits of 200 zmol (1 zmol = 10(-21) mol = 600 molecules) for the four-spectral-channel technique. A postcolumn detector, based on the sheath flow cuvette, produces detection limits of 20 and 2 zmol for the two- and one-spectral-channel techniques, respectively.
    Analytical Chemistry 01/1992; 63(24):2835-41. · 5.70 Impact Factor
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    ABSTRACT: A low cost, 0.75-mW helium neon laser, operating in the green region at 534.5 nm, is used to excite fluorescence from tetramethylrhodamine isothiocyanate-labelled DNA fragments that have been separated by capillary gel electrophoresis. The detection limit (3 sigma) for the dye is 500 ymol [1 yoctomole (1 ymol) = 10(-24) mol] or 300 analyte molecules in capillary zone electrophoresis; the detection limit for labeled primer separated by capillary gel electrophoresis is 2 zmol [1 zeptomole (1 zmol) = 10(-21) mol]. The Richardson-Tabor peak-height encoded sequencing technique is used to prepare DNA sequencing samples. In 6% T, 5% C acrylamide, 7 M urea gels, sequencing rates of 300 bases/hour are produced at an electric field strength of 200 V/cm; unfortunately, the data are plagued by compressions. These compressions are eliminated with addition of 20% formamide to the sequencing gel; the gel runs slowly and sequencing data are generated at a rate of about 70 bases/hour.
    Journal of Chromatography 11/1991; 559(1-2):237-46. · 4.61 Impact Factor
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    ABSTRACT: Low zeptomole (1 zmol equals 10-21 mol = 600 molecules) detection limits are produced for DNA sequencing by capillary gel electrophoresis. A 750 (mu) w green helium-neon laser ((lambda) equals 543.5 nm) is used to excite tetramethylrhodamine-labeled DNA fragments in a sheath-flow cuvette. A cooled photomultiplier tube is used to detect fluorescence in a single spectral channel. Sequencing data is generated at a rate of about 70 bases/hour.
    Proc SPIE 07/1991;
  • H Swerdlow, S L Wu, H Harke, N J Dovichi
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    ABSTRACT: Capillary polyacrylamide gel electrophoresis separation of dideoxycytidine chain-terminated DNA fragments is reported. A post-column laser-induced fluorescence detector based on the sheath flow cuvette was used to minimize background signals due to light scatter from the gel and capillary. A preliminary mass detection limit of 10(-20) mol of fluorescein-labeled DNA fragments was obtained. The system was used to analyze an actual DNA sequencing sample. Theoretical plate counts of 2 x 10(6) were produced. Gel stability limits the performance of the current system.
    Journal of Chromatography 10/1990; 516(1):61-7. · 4.61 Impact Factor
  • Source
    H Swerdlow, R Gesteland
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    ABSTRACT: Capillary gel electrophoresis has been demonstrated for the separation and detection of DNA sequencing samples. Enzymatic dideoxy nucleotide chain termination was employed, using fluorescently tagged oligonucleotide primers and laser based on-column detection (limit of detection is 6,000 molecules per peak). Capillary gel separations were shown to be three times faster, with better resolution (2.4 x), and higher separation efficiency (5.4 x) than a conventional automated slab gel DNA sequencing instrument. Agreement of measured values for velocity, resolution and separation efficiency with theory, predicts further improvements will result from increased electric field strengths (higher voltages and shorter capillaries). Advantages of capillary gel electrophoresis for automatic DNA sequencing instruments and for genomic sequencing are discussed.
    Nucleic Acids Research 04/1990; 18(6):1415-9. · 8.28 Impact Factor

Publication Stats

273 Citations
2 Downloads
378 Views
33.84 Total Impact Points

Institutions

  • 1992–1994
    • University of Utah
      • Department of Human Genetics
      Salt Lake City, UT, United States
    • University of Alberta
      • Department of Chemistry
      Edmonton, Alberta, Canada
  • 1990
    • Howard Hughes Medical Institute
      Maryland, United States