Harold Swerdlow

Dolomite Centre Ltd, Royston, England, United Kingdom

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Publications (16)67.41 Total impact

  • Source
    Sang-Ryoul Park, Harold Swerdlow
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    ABSTRACT: A novel miniature electrolytic conductivity probe and its successful operation with modified bipolar pulse conductometry are presented. The probe based on a concentric ring-disk electrode configuration of less than 1 mm in diameter featured extremely small detection volume, and conductivity was measured with a 1 μL solution. Rapid response, easy and quick washing, and virtually no consumption of samples by measurements were additional advantages of the suggested probe design. Poor linearity was observed with conventional AC conductometry due to a large cell constant and a small double layer capacitance of the probe. Measurement circuits for modified bipolar pulse conductometry were designed, and optimization of the various pulse parameters led to a wide linear dynamic range of 0.05–10 mS cm−1, thus achieving high accuracy with a single-point calibration. The suggested conductometric device could be conveniently applied to biological reagents and samples that are usually too little to be measured with conventional conductometers.
    Electroanalysis 11/2007; 19(22):2294 - 2300. DOI:10.1002/elan.200703981 · 2.50 Impact Factor
  • Juan Astorga-Wells, Harold Swerdlow
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    ABSTRACT: A new preconcentration device was developed for analysis of proteins by capillary electrophoresis (CE). The microfluidic device uses an electric field to capture proteins that pass through the system. The capture zone is maintained in the flow stream by the interaction between hydrodynamic and electrical forces. The device consists of a flow channel made of PEEK tubing with two electrical junctions, each of which is covered with a conductive membrane. A syringe pump provides the flow stream and also allows the injection of up to 13.5 microL of a dilute sample. The system can be easily connected to a CE device postcapture for off-line preconcentration of proteins. For the proteins used in this study, preconcentration factors up to 40-fold can be achieved. CE detection limits for bovine carbonic anhydrase, alpha-lactalbumin and beta-lactoglobulins A and B were in the nanomolar range using UV detection at 200 nm. Preconcentration is dependent on both time and initial protein concentration. We show the possibility of using an off-line fluidic preconcentrator device employing counterflow capillary electrophoresis with minimum sample manipulation, achieving detection limits similar to on-line approaches.
    Analytical Chemistry 11/2003; 75(19):5207-12. DOI:10.1021/ac0300892 · 5.83 Impact Factor
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    Sang-Ryoul Park, Harold Swerdlow
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    ABSTRACT: A novel sample pretreatment device is described, and its application to the concentration and purification of crude DNA samples in a flowing stream for subsequent capillary electrophoresis is demonstrated. The device consists of two gap junctions, each covered with a conductive membrane material and built upon a flow channel made of PEEK tubing. Upon the application of an electric field between the junctions, the negatively charged DNA fragments can resist the hydrodynamic flow stream and are trapped between the junctions. DNA fragments dissolved in microliter volumes are captured in a nanoliter-sized band by simply pushing the sample solution through the device. Depending on their electrophoretic mobility, other interfering materials in a crude sample can be removed from the trapped DNA fragments by washing. The selective permeability of the membrane to small ions allows efficient desalting. The concentrated and purified DNA fragments are released by simply turning off or reversing the electric field. Recovery is up to 95%. Performance of the device was evaluated using crude products of fluorescent dye-primer cycle-sequencing reactions. Compared to these crude reaction products, samples purified in the capture device and subsequently collected showed dramatically enhanced signal and resolution when run on a conventional capillary-electrophoresis instrument. Furthermore, the device could be connected in-line to a capillary system for direct injection. The device has great potential for enabling lab-on-a-chip systems to be used with real-world samples.
    Analytical Chemistry 10/2003; 75(17):4467-74. DOI:10.1021/ac034209h · 5.83 Impact Factor
  • Cissi Gardmo, Harold Swerdlow, Agneta Mode
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    ABSTRACT: The sexually dimorphic secretion of growth hormone (GH) that prevails in the rat leads to a sex-differentiated expression of GH target genes, particularly in the liver. We have used subtractive suppressive hybridization (SSH) to search for new target genes induced by the female-characteristic, near continuous, pattern of GH secretion. Microarrays and dot-blot hybridizations were used in an attempt to confirm differential ratios of expression of obtained SSH clones. Out of 173 unique SSH clones, 41 could be verified as differentially expressed. Among these, we identified 17 known genes not previously recognized as differentially regulated by the sex-specific GH pattern. Additional SSH clones may also represent genes subjected to sex-specific GH regulation since only transcripts abundantly expressed could be verified. Optimized analyses, specific for each gene, are required to fully characterize the degree of differential expression.
    Molecular and Cellular Endocrinology 05/2002; 190(1-2):125-33. DOI:10.1016/S0303-7207(02)00004-7 · 4.24 Impact Factor
  • Yan Xiong, S R Park, Harold Swerdlow
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    ABSTRACT: An on-column sample concentration method for capillary-based DNA sequencing was studied. This base-stacking method allows direct injection of unpurified products of dye-primer sequencing reactions onto capillaries without any pretreatment. On-column concentration of DNA fragments is achieved simply by electrokinetic injection of hydroxide ions. A neutralization reaction between these OH- ions and the cationic buffer component Tris+ results in a zone of lower conductivity, within which field focusing occurs. DNA fragments are concentrated at the front of this low-conductivity zone. With sample injection times as long as 360 s at 50 V/cm, resolution could still be restored by the stacking process. Using a 36/47-cm-long uncoated capillary, with poly(dimethylacrylamide) as the separation matrix, and electric field of 160 V/cm, a resolution of at least 0.5 could be generated for fragments up to 650 nucleotides long. Both resolution and signal strength are excellent relative to conventional injection of highly purified samples. No significant degradation of the capillary performance was observed over at least 20 sequencing runs using this new sample injection methods.
    Analytical Chemistry 10/1998; 70(17):3605-11. DOI:10.1021/ac980376j · 5.83 Impact Factor
  • Harold Swerdlow, Barbara J. Jones, Carl T. Wittwer
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    ABSTRACT: A simple, reliable, automated genetic analysis instrument has been designed and prototyped. The system uses novel fluidic technology, coupling thermal cycling, reaction purification, in-line loading, and capillary electrophoresis in a single instrument. Samples in the loop of an injection valve are amplified inside a rapid air thermal cycler. A liquid chromatographic separation eliminates contaminants and excess salt. The sample is loaded in an efficient, continuous, flow-through manner onto a polymer-filled separation capillary. Detection by laser-induced fluorescence produces signal-to-noise ratios of 1000:1 or greater. Refilling of the polymer-filled capillary is automatic; during the run, the system is reconditioned for injection of another sample. Since all components and connections are fluidic, automation is natural and simple. The instrument is reliable and fast, performing PCR reaction cycling, purification and analysis, all in 20 min. Reproducibility (CV) of retention times is 2% (n = 129) and of peak areas 9% (n = 34). Bubbles and particulates are eliminated by the chromatography column. Adaptation of the instrument prototype for separation of DNA-sequencing reactions is described; cycle sequencing and electrophoresis of a single lane are complete in 90 min. Implications and challenges for development of fully automated fluidic instruments for genomic sequencing are discussed.
    Analytical Chemistry 04/1997; 69(5):848-55. DOI:10.1021/ac961104o · 5.83 Impact Factor
  • 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. DOI:10.1006/abio.1994.1514 · 2.31 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. · 2.75 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. · 2.75 Impact Factor
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    ABSTRACT: Porcine renodoxon is a kidney mitochondrial iron-sulfur protein (ISP) that functions to transfer electron to cytochromes P450 of the vitamin D pathway. A full-length cDNA clone to porcine renodoxin was isolated in the current investigation and used to study the protein's primary structure and immunological properties. The cysteine ligands for the iron-sulfur center, and the surface protein-binding and phosphorylation sites occupied identical positions in both porcine renodoxin and bovine adrenodoxin. Furthermore, porcine renodoxin was functionally indistinguishable from bovine adrenodoxin and the mature forms of both proteins had the same encoded length and shared approximately 91% sequence similarity. A synthetic peptide to the surface protein-binding region was used to demonstrate the antigenicity of the domain in both the porcine and the bovine ISPs. However, porcine renodoxin displayed only limited immunological identity to other regions of bovine adrenodoxin as measured by competitive enzyme-linked immunosorbent assay. Part of this immunological distinction was attributed to the COOH-terminal processing of porcine renodoxin, an action which negated expression of a COOH-terminal antigenic site that is present in bovine adrenodoxin. Other antigenic differences were linked to charged-residue substitutions that were located in predicted surface domains. The highest frequency of surface-residue substitutions in ferredoxin proteins was predicted for porcine renodoxin, which could provide a basis for understanding why the pig protein appears more antigenically divergent than other ferredoxins.
    Archives of Biochemistry and Biophysics 04/1992; 293(2):213-8. DOI:10.1016/0003-9861(92)90387-C · 3.04 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. DOI:10.1021/ac00024a006 · 5.83 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 01/1992; 13(8):475-83. DOI:10.1002/elps.11501301101 · 3.16 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 A 11/1991; 559(1-2):237-46. DOI:10.1016/0021-9673(91)80074-Q · 4.26 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.
    Proceedings of SPIE - The International Society for Optical Engineering 07/1991; DOI:10.1117/12.44240 · 0.20 Impact Factor
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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 A 10/1990; 516(1):61-7. DOI:10.1016/S0021-9673(01)90204-3 · 4.26 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. DOI:10.1093/nar/18.6.1415 · 8.81 Impact Factor

Publication Stats

875 Citations
67.41 Total Impact Points

Institutions

  • 2007
    • Dolomite Centre Ltd
      Royston, England, United Kingdom
  • 2002–2003
    • Karolinska Institutet
      • Department of Biosciences and Nutrition
      Solna, Stockholm, Sweden
  • 1992–2003
    • University of Utah
      • • Department of Human Genetics
      • • Department of BioEngineering
      Salt Lake City, Utah, United States
  • 1991–1992
    • University of Alberta
      • Department of Chemistry
      Edmonton, Alberta, Canada
  • 1990
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States