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. 1992 Sep 25;20(18):4873–4880. doi: 10.1093/nar/20.18.4873

Two-label peak-height encoded DNA sequencing by capillary gel electrophoresis: three examples.

D Chen 1, H R Harke 1, N J Dovichi 1
PMCID: PMC334245  PMID: 1408803

Abstract

We report a modification to the peak-height encoded DNA sequencing technique of Tabor and Richardson. As in the original protocol, the sequencing reaction uses modified T7 polymerase with manganese rather than magnesium to produce very uniform incorporation of each dideoxynucleoside. To improve sequencing accuracy, two fluorescently labeled primers are employed in separate sequencing reactions. As an example, one sequencing reaction uses a FAM-labeled primer with dideoxyadenosine triphosphate and dideoxycytosine triphosphate; the concentrations of ddATP and ddCTP are adjusted to produce a 2:1 variation in the relative intensity of fragments. The second sequencing reaction uses a TAMRA labeled primer with dideoxythymidine triphosphate and dideoxyguanidine triphosphate; the concentrations of ddTTP and ddGTP are adjusted to produce a 2:1 variation in relative intensity of fragments. The pooled reaction products are separated by capillary gel electrophoresis and identified by one of three different detector systems. Use of a 2:1 peak height ratio typically produces a sequencing accuracy of 97.5% for the first 350 bases; a 3:1 peak height ratio improves accuracy to 99.5% for the first 400 bases. For these experiments, capillary electrophoresis is performed at an electric field of 200 V/cm; two to three hours are required to separate sequencing fragments up to 400 nucleotides in length.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ansorge W., Zimmermann J., Schwager C., Stegemann J., Erfle H., Voss H. One label, one tube, Sanger DNA sequencing in one and two lanes on a gel. Nucleic Acids Res. 1990 Jun 11;18(11):3419–3420. doi: 10.1093/nar/18.11.3419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chen D. Y., Swerdlow H. P., Harke H. R., Zhang J. Z., Dovichi N. J. Low-cost, high-sensitivity laser-induced fluorescence detection for DNA sequencing by capillary gel electrophoresis. J Chromatogr. 1991 Oct 18;559(1-2):237–246. doi: 10.1016/0021-9673(91)80074-q. [DOI] [PubMed] [Google Scholar]
  3. Cohen A. S., Najarian D. R., Karger B. L. Separation and analysis of DNA sequence reaction products by capillary gel electrophoresis. J Chromatogr. 1990 Sep 7;516(1):49–60. doi: 10.1016/s0021-9673(01)90203-1. [DOI] [PubMed] [Google Scholar]
  4. Drossman H., Luckey J. A., Kostichka A. J., D'Cunha J., Smith L. M. High-speed separations of DNA sequencing reactions by capillary electrophoresis. Anal Chem. 1990 May 1;62(9):900–903. doi: 10.1021/ac00208a003. [DOI] [PubMed] [Google Scholar]
  5. Karger A. E., Harris J. M., Gesteland R. F. Multiwavelength fluorescence detection for DNA sequencing using capillary electrophoresis. Nucleic Acids Res. 1991 Sep 25;19(18):4955–4962. doi: 10.1093/nar/19.18.4955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Luckey J. A., Drossman H., Kostichka A. J., Mead D. A., D'Cunha J., Norris T. B., Smith L. M. High speed DNA sequencing by capillary electrophoresis. Nucleic Acids Res. 1990 Aug 11;18(15):4417–4421. doi: 10.1093/nar/18.15.4417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Swerdlow H., Gesteland R. Capillary gel electrophoresis for rapid, high resolution DNA sequencing. Nucleic Acids Res. 1990 Mar 25;18(6):1415–1419. doi: 10.1093/nar/18.6.1415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Swerdlow H., Wu S. L., Harke H., Dovichi N. J. Capillary gel electrophoresis for DNA sequencing. Laser-induced fluorescence detection with the sheath flow cuvette. J Chromatogr. 1990 Sep 7;516(1):61–67. doi: 10.1016/s0021-9673(01)90204-3. [DOI] [PubMed] [Google Scholar]
  9. Swerdlow H., Zhang J. Z., Chen D. Y., Harke H. R., Grey R., Wu S. L., Dovichi N. J., Fuller C. Three DNA sequencing methods using capillary gel electrophoresis and laser-induced fluorescence. Anal Chem. 1991 Dec 15;63(24):2835–2841. doi: 10.1021/ac00024a006. [DOI] [PubMed] [Google Scholar]
  10. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Effect of pyrophosphorolysis and metal ions. J Biol Chem. 1990 May 15;265(14):8322–8328. [PubMed] [Google Scholar]
  11. Tabor S., Richardson C. C. Effect of manganese ions on the incorporation of dideoxynucleotides by bacteriophage T7 DNA polymerase and Escherichia coli DNA polymerase I. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4076–4080. doi: 10.1073/pnas.86.11.4076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Wu S., Dovichi N. J. High-sensitivity fluorescence detector fluorescein isothiocyanate derivatives of amino acids separated by capillary zone electrophoresis. J Chromatogr. 1989 Oct 20;480:141–155. doi: 10.1016/s0021-9673(01)84284-9. [DOI] [PubMed] [Google Scholar]

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