Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1996 Dec 1;24(23):4759–4767. doi: 10.1093/nar/24.23.4759

Real-time imaging of the reorientation mechanisms of YOYO-labelled DNA molecules during 90 degrees and 120 degrees pulsed field gel electrophoresis.

S Gurrieri 1, S B Smith 1, K S Wells 1, I D Johnson 1, C Bustamante 1
PMCID: PMC146294  PMID: 8972863

Abstract

Pulsed field gel electrophoresis (PFGE) techniques have been developed to overcome the limitations of conventional electrophoresis and to increase the separation to DNA chromosomes of few megabase pairs in size. Despite of the large success of these techniques, the various separation protocols employed for PFGE experiments have been determined empirically. However, a deep understanding of the molecular mechanisms of motion responsible for DNA separation becomes necessary for the rational optimization of these techniques. This paper shows the first clear observations of individual molecules of DNA during the reorientation process in 90 degrees PFGE and 120 degrees PFGE. Real-time visualization of the DNA dynamics during PFGE was possible with the use of an epi-illumination fluorescence microscope specifically equipped to run these experiments and by staining the DNA with YOYO-1 (1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-meth yl -2,3-dihydro-(benzo-1,3-oxazole)-2-methyl-idene]-quinolinium tetraiodide). This dye forms a very stable, highly fluorescent complex with double-stranded DNA and dramatically improves the quality of the DNA images. The results of computer simulations used to reproduce the molecular mechanisms of motion as well as the DNA separation features are also discussed.

Full Text

The Full Text of this article is available as a PDF (851.1 KB).

Selected References

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

  1. Akerman B., Tuite E. Single- and double-strand photocleavage of DNA by YO, YOYO and TOTO. Nucleic Acids Res. 1996 Mar 15;24(6):1080–1090. doi: 10.1093/nar/24.6.1080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bancroft I., Wolk C. P. Pulsed homogeneous orthogonal field gel electrophoresis (PHOGE). Nucleic Acids Res. 1988 Aug 11;16(15):7405–7418. doi: 10.1093/nar/16.15.7405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bustamante C., Gurrieri S., Smith S. B. Towards a molecular description of pulsed-field gel electrophoresis. Trends Biotechnol. 1993 Jan;11(1):23–30. doi: 10.1016/0167-7799(93)90071-G. [DOI] [PubMed] [Google Scholar]
  4. Carle G. F., Frank M., Olson M. V. Electrophoretic separations of large DNA molecules by periodic inversion of the electric field. Science. 1986 Apr 4;232(4746):65–68. doi: 10.1126/science.3952500. [DOI] [PubMed] [Google Scholar]
  5. Carle G. F., Olson M. V. Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis. Nucleic Acids Res. 1984 Jul 25;12(14):5647–5664. doi: 10.1093/nar/12.14.5647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carlsson C., Larsson A., Jonsson M. Influence of optical probing with YOYO on the electrophoretic behavior of the DNA molecule. Electrophoresis. 1996 Apr;17(4):642–651. doi: 10.1002/elps.1150170404. [DOI] [PubMed] [Google Scholar]
  7. Chu G., Vollrath D., Davis R. W. Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science. 1986 Dec 19;234(4783):1582–1585. doi: 10.1126/science.3538420. [DOI] [PubMed] [Google Scholar]
  8. Deutsch JM. Dynamics of pulsed-field electrophoresis. Phys Rev Lett. 1987 Sep 14;59(11):1255–1258. doi: 10.1103/PhysRevLett.59.1255. [DOI] [PubMed] [Google Scholar]
  9. FREIFELDER D., DAVISON P. F., GEIDUSCHEK E. P. Damage by visible light to the acridine orange--DNA complex. Biophys J. 1961 May;1:389–400. doi: 10.1016/s0006-3495(61)86897-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fangman W. L. Separation of very large DNA molecules by gel electrophoresis. Nucleic Acids Res. 1978 Mar;5(3):653–665. doi: 10.1093/nar/5.3.653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gardiner K. Pulsed field gel electrophoresis. Anal Chem. 1991 Apr 1;63(7):658–665. doi: 10.1021/ac00007a003. [DOI] [PubMed] [Google Scholar]
  12. Glazer A. N., Rye H. S. Stable dye-DNA intercalation complexes as reagents for high-sensitivity fluorescence detection. Nature. 1992 Oct 29;359(6398):859–861. doi: 10.1038/359859a0. [DOI] [PubMed] [Google Scholar]
  13. Gurrieri S., Rizzarelli E., Beach D., Bustamante C. Imaging of kinked configurations of DNA molecules undergoing orthogonal field alternating gel electrophoresis by fluorescence microscopy. Biochemistry. 1990 Apr 3;29(13):3396–3401. doi: 10.1021/bi00465a036. [DOI] [PubMed] [Google Scholar]
  14. Hervet H., Bean C. P. Electrophoretic mobility of lambda phage HIND III and HAE III DNA fragments in agarose gels: a detailed study. Biopolymers. 1987 May;26(5):727–742. doi: 10.1002/bip.360260512. [DOI] [PubMed] [Google Scholar]
  15. Lai E., Birren B. W., Clark S. M., Simon M. I., Hood L. Pulsed field gel electrophoresis. Biotechniques. 1989 Jan;7(1):34–42. [PubMed] [Google Scholar]
  16. McDonell M. W., Simon M. N., Studier F. W. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. doi: 10.1016/s0022-2836(77)80102-2. [DOI] [PubMed] [Google Scholar]
  17. Orbach M. J., Vollrath D., Davis R. W., Yanofsky C. An electrophoretic karyotype of Neurospora crassa. Mol Cell Biol. 1988 Apr;8(4):1469–1473. doi: 10.1128/mcb.8.4.1469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rampino N. J. Information concerning the mechanism of electrophoretic DNA separation provided by quantitative video-epifluorescence microscopy. Biopolymers. 1991 Jul;31(8):1009–1016. doi: 10.1002/bip.360310810. [DOI] [PubMed] [Google Scholar]
  19. Rye H. S., Yue S., Quesada M. A., Haugland R. P., Mathies R. A., Glazer A. N. Picogram detection of stable dye-DNA intercalation complexes with two-color laser-excited confocal fluorescence gel scanner. Methods Enzymol. 1993;217:414–431. doi: 10.1016/0076-6879(93)17080-o. [DOI] [PubMed] [Google Scholar]
  20. Rye H. S., Yue S., Wemmer D. E., Quesada M. A., Haugland R. P., Mathies R. A., Glazer A. N. Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications. Nucleic Acids Res. 1992 Jun 11;20(11):2803–2812. doi: 10.1093/nar/20.11.2803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schwartz D. C., Cantor C. R. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell. 1984 May;37(1):67–75. doi: 10.1016/0092-8674(84)90301-5. [DOI] [PubMed] [Google Scholar]
  22. Schwartz D. C., Koval M. Conformational dynamics of individual DNA molecules during gel electrophoresis. Nature. 1989 Apr 6;338(6215):520–522. doi: 10.1038/338520a0. [DOI] [PubMed] [Google Scholar]
  23. Smith S. B., Aldridge P. K., Callis J. B. Observation of individual DNA molecules undergoing gel electrophoresis. Science. 1989 Jan 13;243(4888):203–206. doi: 10.1126/science.2911733. [DOI] [PubMed] [Google Scholar]
  24. Smith S. B., Heller C., Bustamante C. Model and computer simulations of the motion of DNA molecules during pulse field gel electrophoresis. Biochemistry. 1991 May 28;30(21):5264–5274. doi: 10.1021/bi00235a021. [DOI] [PubMed] [Google Scholar]
  25. Southern E. M., Anand R., Brown W. R., Fletcher D. S. A model for the separation of large DNA molecules by crossed field gel electrophoresis. Nucleic Acids Res. 1987 Aug 11;15(15):5925–5943. doi: 10.1093/nar/15.15.5925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stellwagen N. C. Effect of the electric field on the apparent mobility of large DNA fragments in agarose gels. Biopolymers. 1985 Dec;24(12):2243–2255. doi: 10.1002/bip.360241207. [DOI] [PubMed] [Google Scholar]
  27. Vollrath D., Davis R. W. Resolution of DNA molecules greater than 5 megabases by contour-clamped homogeneous electric fields. Nucleic Acids Res. 1987 Oct 12;15(19):7865–7876. doi: 10.1093/nar/15.19.7865. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES