Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1989 Apr 25;17(8):2889–2895. doi: 10.1093/nar/17.8.2889

Determination of microbial genome sizes by two-dimensional denaturing gradient gel electrophoresis.

S K Poddar 1, J Maniloff 1
PMCID: PMC317699  PMID: 2786189

Abstract

In two-dimensional denaturing gradient gel electrophoresis, DNA is digested with a restriction endonuclease and the resulting DNA fragments are separated as a function of size by conventional agarose gel electrophoresis. Following this first dimension electrophoresis, the fragment distribution is placed at the top of a denaturing gradient slab gel and electrophoresis is carried out parallel to the gradient direction. This second dimension separation is a complex function of the base sequence of each fragment. Analysis of the DNA fragment distribution as a function of fragment size allows the DNA size to be calculated. This method has been applied to calculate three microbial genome sizes: Mycoplasma capricolum, 724 kb; Acholeplasma laidlawii, 1646 kb; and Hemophilus influenzae, 1833 kb.

Full text

PDF
2889

Images in this article

Selected References

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

  1. Askaa G., Christiansen C., Erno H. Bovine mycoplasmas: genome size and base composition of DNA. J Gen Microbiol. 1973 Apr;75(2):283–286. doi: 10.1099/00221287-75-2-283. [DOI] [PubMed] [Google Scholar]
  2. Bak A. L., Black F. T., Christiansen C., Freundt E. A. Genome size of mycoplasmal DNA. Nature. 1969 Dec 20;224(5225):1209–1210. doi: 10.1038/2241209a0. [DOI] [PubMed] [Google Scholar]
  3. Bautsch W. Rapid physical mapping of the Mycoplasma mobile genome by two-dimensional field inversion gel electrophoresis techniques. Nucleic Acids Res. 1988 Dec 23;16(24):11461–11467. doi: 10.1093/nar/16.24.11461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bode H. R., Morowitz H. J. Size and structure of the Mycoplasma hominis H39 chromosome. J Mol Biol. 1967 Jan 28;23(2):191–199. doi: 10.1016/s0022-2836(67)80026-3. [DOI] [PubMed] [Google Scholar]
  5. Fischer S. G., Lerman L. S. Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis. Cell. 1979 Jan;16(1):191–200. doi: 10.1016/0092-8674(79)90200-9. [DOI] [PubMed] [Google Scholar]
  6. Fischer S. G., Lerman L. S. Two-dimensional electrophoretic separation of restriction enzyme fragments of DNA. Methods Enzymol. 1979;68:183–191. doi: 10.1016/0076-6879(79)68013-8. [DOI] [PubMed] [Google Scholar]
  7. Haberer K., Klotz G., Maniloff J., Kleinschmidt A. K. Structural and biological properties of mycoplasmavirus MVL3: an unusual virus-procaryote interaction. J Virol. 1979 Oct;32(1):268–275. doi: 10.1128/jvi.32.1.268-275.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. LePecq J. B., Paoletti C. A fluorescent complex between ethidium bromide and nucleic acids. Physical-chemical characterization. J Mol Biol. 1967 Jul 14;27(1):87–106. doi: 10.1016/0022-2836(67)90353-1. [DOI] [PubMed] [Google Scholar]
  9. Lee J. J., Smith H. O. Sizing of the Haemophilus influenzae Rd genome by pulsed-field agarose gel electrophoresis. J Bacteriol. 1988 Sep;170(9):4402–4405. doi: 10.1128/jb.170.9.4402-4405.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lerman L. S., Fischer S. G., Hurley I., Silverstein K., Lumelsky N. Sequence-determined DNA separations. Annu Rev Biophys Bioeng. 1984;13:399–423. doi: 10.1146/annurev.bb.13.060184.002151. [DOI] [PubMed] [Google Scholar]
  11. Maniloff J. Anomalous values of Mycoplasma genomes sizes determined by pulse-field gel electrophoresis. Nucleic Acids Res. 1989 Feb 11;17(3):1268–1268. doi: 10.1093/nar/17.3.1268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. McClelland M., Jones R., Patel Y., Nelson M. Restriction endonucleases for pulsed field mapping of bacterial genomes. Nucleic Acids Res. 1987 Aug 11;15(15):5985–6005. doi: 10.1093/nar/15.15.5985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Poddar S. K., Maniloff J. Chromosome analysis by two-dimensional fingerprinting. Gene. 1986;49(1):93–102. doi: 10.1016/0378-1119(86)90388-4. [DOI] [PubMed] [Google Scholar]
  14. Pyle L. E., Corcoran L. N., Cocks B. G., Bergemann A. D., Whitley J. C., Finch L. R. Pulsed-field electrophoresis indicates larger-than-expected sizes for mycoplasma genomes. Nucleic Acids Res. 1988 Jul 11;16(13):6015–6025. doi: 10.1093/nar/16.13.6015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Pyle L. E., Finch L. R. A physical map of the genome of Mycoplasma mycoides subspecies mycoides Y with some functional loci. Nucleic Acids Res. 1988 Jul 11;16(13):6027–6039. doi: 10.1093/nar/16.13.6027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rogers M. J., Simmons J., Walker R. T., Weisburg W. G., Woese C. R., Tanner R. S., Robinson I. M., Stahl D. A., Olsen G., Leach R. H. Construction of the mycoplasma evolutionary tree from 5S rRNA sequence data. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1160–1164. doi: 10.1073/pnas.82.4.1160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Wenzel R., Herrmann R. Physical mapping of the Mycoplasma pneumoniae genome. Nucleic Acids Res. 1988 Sep 12;16(17):8323–8336. doi: 10.1093/nar/16.17.8323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Wilcox K. W., Smith H. O. Isolation and characterization of mutants of Haemophilus influenzae deficient in an adenosine 5'-triphosphate-dependent deoxyribonuclease activity. J Bacteriol. 1975 May;122(2):443–453. doi: 10.1128/jb.122.2.443-453.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Yee T., Inouye M. Two-dimensional DNA electrophoresis applied to the study of DNA methylation and the analysis of genome size in Myxococcus xanthus. J Mol Biol. 1982 Jan 15;154(2):181–196. doi: 10.1016/0022-2836(82)90059-6. [DOI] [PubMed] [Google Scholar]

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

RESOURCES