Skip to main content
SpringerLink
Log in

16 DNA Analysis Under Highly Denaturing Conditions in Bare Fused Silica Capillaries

  • Protocol
Capillary Electrophoresis of Nucleic Acids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 162))

  • 636 Accesses

Abstract

Denaturing electrophoresis has widely been used for DNA sequencing and mutation detection. Specific electromigration behavior of completely or partially dissociated DNA molecules is the prerequisite of high-selective separations, based on their size and/or conformation differences. Various denaturing techniques have been developed for both slab gel (SGE) and capillary electrophoresis (CE) formats. The highest possible denaturing ability of the background electrolytes (BGE) of both the sample solution and electrophoresis buffer is used for sequencing, and for analyses of restriction fragment length polymorphism (RFLP), or for length polymorphism of fragments amplified by polymerase chain reaction (PCR-FLP) (1–5). In the technique of single-strand conformation polymorphism (SSCP) analysis, completely denatured samples are loaded into a native sieving medium where ssDNA fragments adopt a conformation that is determined by their nucleotide sequence (6,7). Thus, not only complementary strands, but also strands carrying mutations are separated under optimum conditions. The SSCP technique is very sensitive, and even the point mutations, i.e., the substitutions of a single nucleotide in a sequence, can be detected. In the constant denaturant (CDCE) (8,9) or denaturing gradient capillary electrophoresis (DGCE) techniques (10,11), a native sample is loaded into a sieving medium with a moderate denaturing ability. Consequently, dsDNA molecules dissociate according to their melting temperature, which sensitively reflects the mutations in the DNA sequence. The optimum denaturing power of a BGE is controlled by the temperature (constant or programmed) of the run, and by the concentration of denaturing agent(s). Based on the extent of dissociation, mutant DNA sequences are separated reproducibly from wild-type sequences, with high resolution.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Wenz H., Robertson J. M., Menchen S., Oaks F., Demorest D. M., Scheibler D., Rosenblum B. B, Wike C., Gilbert D. A., and Efcavitch J. W. (1998) High-precision genotyping by denaturing capillary electrophoresis. Genome Res. 8, 69–80.

    PubMed  CAS  Google Scholar 

  2. Rosenblum B. B., Oaks F., Menchen S., and Johnson B. (1997) Improved single-strand DNA sizing accuracy in capillary electrophoresis. Nucleic Acids Res. 25, 3925–3929.

    Article  PubMed  CAS  Google Scholar 

  3. Schmalzing D., Koutny L., Adourian A., Chisholm D., Matsudaira P., and Ehrlich D. (2001) Genotyping by microdevice electrophoresis, in Capillary Electrophoresis of Nucleic Acids, Vol. 2 (Mitchelson K. R. and Cheng J., eds.), Humana Press, Totowa, NJ, pp. 163–173.

    Chapter  Google Scholar 

  4. Wang Y., Wallin J. M., Ju J., Sensabaugh G. F., and Mathies R. A. (1996) High-resolution capillary array electrophoretic sizing of multiplexed short tandem repeat loci using energy-transfer fluorescent primers. Electrophoresis 17, 1485–1490.

    Article  PubMed  CAS  Google Scholar 

  5. Mansfield E. S., Vainer M., Enad S., Barker D. L., Harris D., Rappaport E., and Fortina P. (1996) Sensitivity, reproducibility, and accuracy in short tandem repeat genotyping using capillary array electrophoresis. Genome Res. 6, 893–903.

    Article  PubMed  CAS  Google Scholar 

  6. Kuypers A. W., Willems P. M., Vanderschans M. J., Linssen P. C. M., Wessels H. M. C., Debruijn C. H., Everaerts F. M., and Mensink E. J. (1993) Detection of point mutations in DNA using capillary electrophoresis in a polymer network. J. Chromatogr. B 621, 149–156.

    Article  CAS  Google Scholar 

  7. Hebenbrock K., Williams P. M.,and Karger B. L. (1995) Single strand conformational polymorphism using capillary electrophoresis with two-dye laser-induced fluorescence detection. Electrophoresis 16, 1429–1436.

    Article  PubMed  CAS  Google Scholar 

  8. Muniappan B. P. and Thilly W. G. (1999) Application of constant denaturant capillary electrophoresis (CDCE) to mutation detection in humans. Genet. Anal. 14, 221–227.

    PubMed  CAS  Google Scholar 

  9. Coller H. A., Khrapko K., Torres A., Frampton M. W., Utell M. J., and Thilly W. G. (1998) Mutational spectra of a 100-base pair mitochondrial DNA target sequence in bronchial epithelial cells: a comparison of smoking and nonsmoking twins. Cancer. Res. 58, 1268–1277.

    PubMed  CAS  Google Scholar 

  10. Righetti P. G. and Gelfi C. (1998) Analysis of clinically relevant, diagnostic DNA by capillary zone and double-gradient gel slab electrophoresis. J. Chromatogr. A 806, 97–112.

    Article  PubMed  CAS  Google Scholar 

  11. Gelfi C., Cremoresi L., Ferrari M., and Righetti P. G. (2001) Point mutation detection by temperature-programmed capillary electrophoresis, in Capillary Electrophoresis of Nucleic Acids, Vol. 2 (Mitchelson K. R. and Cheng J., eds.), Humana Press, Totowa, NJ, pp. 73–88.

    Chapter  Google Scholar 

  12. Liu S., Shi Y., Ja W. W., and Mathies R. A. (1999) Optimization of high-speed DNA sequencing on microfabricated capillary electrophoresis channels. Anal. Chem. 71, 566–573.

    Article  PubMed  CAS  Google Scholar 

  13. Kheterpal I. and Mathies R. A. (1999) Capillary array electrophoresis DNA sequencing. Anal. Chem. 71, 31A–37A.

    PubMed  CAS  Google Scholar 

  14. Tan H. and Yeung E. S. (1998) Automation and integration of multiplexed on-line sample preparation with capillary electrophoresis for high-throughput DNA sequencing. Anal. Chem. 70, 4044–4053.

    Article  PubMed  CAS  Google Scholar 

  15. Salas-Solano O., Carrilho E., Kotler L., Miller A. W., Goetzinger W., Sosic Z., and Karger B. L. (1998) Routine DNA sequencing of 1000 bases in less than one hour by capillary electrophoresis with replaceable linear polyacrylamide solutions. Anal. Chem. 70, 3996–4003.

    Article  PubMed  CAS  Google Scholar 

  16. Klepárník K., Berka J., Foret F., Doskar J., Kailerova J., Rosypal S., and Bocek P. (1998) DNA cycle sequencing of a common restriction fragment of Staphylococcus aureus bacteriophages by capillary electrophoresis using replaceable linear polyacrylamide. Electrophoresis 19, 695–700.

    Article  PubMed  Google Scholar 

  17. Schmalzing D., Adourian A., Koutny L., Ziaugra L., Matsudaira P., and Ehrlich D. (1998) DNA sequencing on microfabricated electrophoretic devices. Anal. Chem. 70, 2303–2310.

    Article  PubMed  CAS  Google Scholar 

  18. Marsh M., Tu O., Dolnik V., Roach D., Solomon N., Bechtol K., Smietana P., Wang L., Li X., Cartwright P., Marks A., Barker D., Harris D., and Bashkin J. (1997) High-throughput DNA sequencing on a capillary array electrophoresis system. J. Capillary Electrophor. 4, 83–89.

    PubMed  CAS  Google Scholar 

  19. Klepárník K., Foret F., Berka J., Goetzinger W., Miller A. W.,and Karger B. L. (1996) The use of elevated column temperature to extend DNA sequencing read lengths in capillary electrophoresis with replaceable polymer matrices. Electrophoresis 17, 1860–1866.

    Article  PubMed  Google Scholar 

  20. van der Schans M. J., Kuypers A. W. H. M., Kloosterman A. D., Janssen H. J. T., and Everaerts F. M. (1997) Comparison of resolution of double-stranded and single-stranded DNA in capillary electrophoresis. J. Chromatogr. 772, 255–264.

    Article  Google Scholar 

  21. Pariat Y. F., Berka J., Heiger D. N., Schmitt T., Vilenchik M., Cohen A. S., Foret F., and Karger B. L. (1993) Separation of DNA fragments by capillary electrophoresis using replaceable linear polyacrylamide matrices. J. Chromatogr. A 652, 57–66.

    Article  PubMed  CAS  Google Scholar 

  22. Leving L., Gordon J. A., and Jencks W. P. (1963) The relationship of structure to the effectiveness of denaturing agents for deoxyribonucleic acid. Biochemistry 2, 168–175.

    Article  Google Scholar 

  23. Cortadas J. and Subirana J. A. (1977) The incomplete denaturation of DNA in N,N-dimethyl formamide. Biochim. Biophys. Acta 476, 203–206.

    PubMed  CAS  Google Scholar 

  24. Blake R. D. and Delcourt S. G. (1996) Thermodynamic effects of formamide on DNA stability. Nucleic Acids Res. 24, 2095–2103.

    Article  PubMed  CAS  Google Scholar 

  25. Lando D. Y., Haroutiunian S. G., Kulba A. M., Dalian E. B., Orioli P., Mangani S., and Akhrem A. A. (1994) Theoretical and experimental study of DNA helix-coil transition in acidic and alkaline medium. J. Biomol. Struct. Dyn. 12, 355–366.

    PubMed  CAS  Google Scholar 

  26. Tajmir-Riahi H. A., Ahmad R., Naoui M.,and Diamantoglou S. (1995) The effect of HCl on the solution structure of calf thymus DNA: a comparative study of DNA denaturation by proton and metal cations using Fourier transform IR difference spectroscopy. Biopolymers 35, 493–501.

    Article  PubMed  CAS  Google Scholar 

  27. Ranhand J. M. (1985) The enrichment of plasmid DNAs, in bacterial cell lysates, using an alkline-pH procedure that does not permanently denature them. Prep. Biochem. 15, 121–123.

    Article  PubMed  CAS  Google Scholar 

  28. Camien M. N. and Warner R. C. (1986) Denaturation of covalently closed circular DNA. Kinetics, comparison of several DNAs, mechanism and ionic effects. J. Biol. Chem. 261, 6026–6033.

    PubMed  CAS  Google Scholar 

  29. Vaghef H., Wisen A. C., and Hellman B. (1996) Demonstration of benyo(a)pyrene-induced DNA damage in mice by alkaline single cell gel electrophoresis: evidence for strand breaks in liver but not in lymphocytes and bone marrow. Pharmacol. Toxicol. 78, 37–43.

    Article  PubMed  CAS  Google Scholar 

  30. Miyamae Y., Iwasaki K., Kinae N., Tsuda S., Murakami M., and Sasaki Y. F. (1997) Detection of DNA lesions induced by chemical mutagens using the single-cell gel electrophoresis (comet) assay. 2. Relationship between DNA migration and alkaline condition. Mutat. Res. 393, 107–113.

    PubMed  CAS  Google Scholar 

  31. Fairbairn D. W., Olive P., and O‘Neil K. L. (1995) The comet assay: a comprehensive review. Mutat. Res. 339, 37–59.

    PubMed  CAS  Google Scholar 

  32. Malá Z., Klepárník K., Havač Z., and Bocek P. (1998) New electrolyte system for single-stranded DNA separations by electrophoresis in uncoated capillaries, in 11th International Symposium on Capillary Electroseparation Techniques, Abstracts volume, Venice, Italy, pp. 70.

    Google Scholar 

  33. Malá Z., Klepárník K., and Bocek P. (1999) Highly alkaline electrolyte for single-stranded DNA separations by electrophoresis in bare silica capillaries. J. Chromatogr. A. 853, 371–379.

    Article  PubMed  Google Scholar 

  34. Hjertén S. (1985) High-performance electrophoresis. Elimination of electroendoosmosis and solute adsorption. J. Chromatogr. 347, 191–198.

    Article  Google Scholar 

  35. Hjertén S. and Kubo K. (1993) A new type of pH-and detergent-stable coating for elimination of electroendoosmosis and adsorption in (capillary) electrophoresis. Electrophoresis 14, 390–395.

    Article  PubMed  Google Scholar 

  36. Goetzinger W. and Karger B. L. (1996) International Patent Application WO 96/23220.

    Google Scholar 

  37. Coob K. A., Dolník V., and Novotny M. (1990) Electrophoretic separations of proteins in capillaries with hydrolytically stable surface structures. Anal. Chem. 62, 2478–2483.

    Article  Google Scholar 

  38. Liu Y. and Kuhr W. (1999) Separation of double-and single-stranded DNA restriction fragments: Capillary electrophoresis with polymer solution under alkaline conditions. Anal. Chem. 71, 1668–1673.

    Article  PubMed  CAS  Google Scholar 

  39. Liu Y., Fu R., and Gu J. (1996) Epoxy resin coatings for capillary zone electrophoretic separation of basic proteins. J. Chromatogr. A 723, 157–167.

    Article  CAS  Google Scholar 

  40. Fung E. N. and Yeung E. S. (1995) High-speed DNA sequencing by using mixed poly(ethylene oxide) solutions in uncoated capillary columns. Anal. Chem. 67, 1913–1919.

    Article  CAS  Google Scholar 

  41. Preisler J. and Yeung E. S. (1996) Characterization of nonbonded poly(ethylene oxide) coating for capillary electrophoresis via continuous monitoring of electroosmotic flow. Anal. Chem. 68, 2885–2889.

    Article  CAS  Google Scholar 

  42. Towns J. K. and Regnier F. E. (1992) Impact of polycation adsorption on efficiency and electroosmotically driven transport in capillary electrophoresis. Anal. Chem. 64, 473–478.

    Article  Google Scholar 

  43. Yeung K. K. C. and Luci Ch. A. (1997) Suppression of electroosmotic flow and prevention of wall adsorption in capillary zone electrophoresis using zwitterionic surfactants. Anal. Chem. 69, 3435–3441.

    Article  CAS  Google Scholar 

  44. Barron A. E., Soane D. S., and Blanch H. W. (1993) Capillary electrophoresis of DNA in uncross-linked polymer solution. J. Chromatogr. A 652, 3–16.

    Article  PubMed  CAS  Google Scholar 

  45. Barron A. E., Blanch H. W., and Soane D. S. (1994) A transient entanglement coupling mechanism for DNA separation by capillary electrophoresis in ultradilute polymer solution. Electrophoresis 15, 597–615.

    Article  PubMed  CAS  Google Scholar 

  46. Klepárník K., Fanali S., and Bocek P. (1993) Selectivity of separation of DNA fragments by capillary zone electrophoresis in low-melting-point agarose sol. J. Chromatogr. 638, 283–292.

    Article  Google Scholar 

  47. Klepárník K. and Bocek P. (1991) Theoretical background for clinical and biomedical applications of electromigration techniques. J. Chromatogr. 569, 3–42.

    Article  PubMed  Google Scholar 

  48. Iler R. K. (1979) The Chemistry of Silica, Wiley, New York.

    Google Scholar 

  49. Gerald D. and Fasman P. D. (1976) Handbook of Biochemistry and Molecular Biology, CRC Press, Boca Raton, FL.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Analytical Chemistry of the Czech Academy of Sciences, Brno, Czech Republic

    Karel Klepárník, Zdeňvka Malá & Petr Bočvek

Authors
  1. Karel Klepárník
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zdeňvka Malá
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Petr Bočvek
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Australian Genome Research Facility, University of Queensland, Brisbane

    Keith R. Mitchelson

  2. Walter & Eliza Hall Institute, Parkville, Australia

    Keith R. Mitchelson

  3. Biochip Research and Development Center, State Key Laboratory for Biomembrane and Membrane Biotechnology, Tsinghua University, Beijing, China

    Jing Cheng

  4. Aviva Biosciences Corporation, San Diego, CA

    Jing Cheng

Rights and permissions

Reprints and permissions

Copyright information

© 2001 Humana Press Inc.

About this protocol

Cite this protocol

Klepárník, K., Malá, Z., Bočvek, P. (2001). 16 DNA Analysis Under Highly Denaturing Conditions in Bare Fused Silica Capillaries. In: Mitchelson, K.R., Cheng, J. (eds) Capillary Electrophoresis of Nucleic Acids. Methods in Molecular Biology, vol 162. Humana Press. https://doi.org/10.1385/1-59259-055-1:239

Download citation

  • DOI: https://doi.org/10.1385/1-59259-055-1:239

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-779-3

  • Online ISBN: 978-1-59259-055-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation