Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1990 Mar 25;18(6):1481–1487. doi: 10.1093/nar/18.6.1481

The basis of high resolution separation of small DNAs by asymmetric-voltage field inversion electrophoresis and its application to DNA sequencing gels.

B W Birren 1, M I Simon 1, E Lai 1
PMCID: PMC330515  PMID: 2326189

Abstract

We have previously shown that asymmetric-voltage field inversion electrophoresis produces more uniform separation for fragments between 1 and 50 kilobases (kb) than other modes of pulsed field gel electrophoresis. We now report on the basis of this phenomenon. As in conventional electrophoresis, the pulsed field mobility of DNAs between 1 and 50 kb varies with voltage in a size dependent manner. The complex migration pattern obtained with asymmetric-voltage field inversion electrophoresis reflects the difference between the mobilities of each sized fragment under the conditions used for the forward and reverse fields. We have applied this technique to DNA sequencing gels and find improvement in resolution for single-stranded fragments in polyacrylamide gels.

Full text

PDF
1481

Images in this article

1483Image
on p.1483
1483Image
on p.1483
1485Image
on p.1485
1485Image
on p.1485

Selected References

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

  1. Beverley S. M. Characterization of the 'unusual' mobility of large circular DNAs in pulsed field-gradient electrophoresis. Nucleic Acids Res. 1988 Feb 11;16(3):925–939. doi: 10.1093/nar/16.3.925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birren B. W., Lai E., Clark S. M., Hood L., Simon M. I. Optimized conditions for pulsed field gel electrophoretic separations of DNA. Nucleic Acids Res. 1988 Aug 11;16(15):7563–7582. doi: 10.1093/nar/16.15.7563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Birren B. W., Lai E., Hood L., Simon M. I. Pulsed field gel electrophoresis techniques for separating 1- to 50-kilobase DNA fragments. Anal Biochem. 1989 Mar;177(2):282–286. doi: 10.1016/0003-2697(89)90052-3. [DOI] [PubMed] [Google Scholar]
  4. Bostock C. J. Parameters of field inversion gel electrophoresis for the analysis of pox virus genomes. Nucleic Acids Res. 1988 May 25;16(10):4239–4252. doi: 10.1093/nar/16.10.4239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Carle G. F., Olson M. V. Orthogonal-field-alternation gel electrophoresis. Methods Enzymol. 1987;155:468–482. doi: 10.1016/0076-6879(87)55031-5. [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. Clark S. M., Lai E., Birren B. W., Hood L. A novel instrument for separating large DNA molecules with pulsed homogeneous electric fields. Science. 1988 Sep 2;241(4870):1203–1205. doi: 10.1126/science.3045968. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Graham M. Y., Otani T., Boime I., Olson M. V., Carle G. F., Chaplin D. D. Cosmid mapping of the human chorionic gonadotropin beta subunit genes by field-inversion gel electrophoresis. Nucleic Acids Res. 1987 Jun 11;15(11):4437–4448. doi: 10.1093/nar/15.11.4437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Heller C., Pohl F. M. A systematic study of field inversion gel electrophoresis. Nucleic Acids Res. 1989 Aug 11;17(15):5989–6003. doi: 10.1093/nar/17.15.5989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Holzwarth G., Platt K. J., McKee C. B., Whitcomb R. W., Crater G. D. The acceleration of linear DNA during pulsed-field gel electrophoresis. Biopolymers. 1989 Jun;28(6):1043–1058. doi: 10.1002/bip.360280603. [DOI] [PubMed] [Google Scholar]
  13. Lai E., Birren B. W., Clark S. M., Hood L. Relaxation intervals alter the mobility of large DNA molecules in pulsed field gel electrophoresis. Nucleic Acids Res. 1988 Nov 11;16(21):10376–10376. doi: 10.1093/nar/16.21.10376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lai E., Davi N. A., Hood L. E. Effect of electric field switching on the electrophoretic mobility of single-stranded DNA molecules in polyacrylamide gels. Electrophoresis. 1989 Jan;10(1):65–67. doi: 10.1002/elps.1150100116. [DOI] [PubMed] [Google Scholar]
  15. Lasker B. A., Carle G. F., Kobayashi G. S., Medoff G. Comparison of the separation of Candida albicans chromosome-sized DNA by pulsed-field gel electrophoresis techniques. Nucleic Acids Res. 1989 May 25;17(10):3783–3793. doi: 10.1093/nar/17.10.3783. [DOI] [PMC free article] [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. Mickel S., Arena V., Jr, Bauer W. Physical properties and gel electrophoresis behavior of R12-derived plasmid DNAs. Nucleic Acids Res. 1977;4(5):1465–1482. doi: 10.1093/nar/4.5.1465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Olson M. V. Separation of large DNA molecules by pulsed-field gel electrophoresis. A review of the basic phenomenology. J Chromatogr. 1989 May 26;470(2):377–383. doi: 10.1016/s0021-9673(01)83565-2. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Serwer P. Improvements in procedures for electrophoresis in dilute agarose gels. Anal Biochem. 1981 Apr;112(2):351–356. doi: 10.1016/0003-2697(81)90304-3. [DOI] [PubMed] [Google Scholar]
  21. Smith C. L., Matsumoto T., Niwa O., Klco S., Fan J. B., Yanagida M., Cantor C. R. An electrophoretic karyotype for Schizosaccharomyces pombe by pulsed field gel electrophoresis. Nucleic Acids Res. 1987 Jun 11;15(11):4481–4489. doi: 10.1093/nar/15.11.4481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Stellwagen N. C. Effect of pulsed and reversing electric fields on the orientation of linear and supercoiled DNA molecules in agarose gels. Biochemistry. 1988 Aug 23;27(17):6417–6424. doi: 10.1021/bi00417a033. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Waterbury P. G., Lane M. J. Generation of lambda phage concatemers for use as pulsed field electrophoresis size markers. Nucleic Acids Res. 1987 May 11;15(9):3930–3930. doi: 10.1093/nar/15.9.3930. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES