Skip to main content
SpringerLink
Log in

Capillary electrophoresis ribosomal RNA single-stranded conformation polymorphism: a new approach for characterization of low-diversity microbial communities

  • Original Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Capillary electrophoresis (CE) has been the principle system for nucleic acid analysis since the early 1990s due to its inherent advantages such as fast analysis time, high resolution and efficiency, minimal sample requirement, high detection sensitivity, and automation. In the past few decades, microbial community fingerprinting methods such as terminal restriction fragment length polymorphism and single-stranded conformation polymorphism (SSCP) have migrated to CE to utilize its advantages over conventional slab gel electrophoresis. Recently, a gel-based direct rRNA fingerprint method was demonstrated. Different from other existing microbial community characterization approaches, this novel approach is polymerase chain reaction free and capable of providing information on the relative abundance of rRNA from individual phylotypes in low-diversity samples. As a gel-based method, it has a long analysis time and relatively large reagent and sample requirements. Here, we addressed these limitations by transferring the RNA fingerprint approach to the CE platform. Analysis time significantly improved from 24 h to 60 min, and the use of a fluorescently labeled hybridization probe as the detection strategy decreased the sample requirement by ten-fold. The combination of fast analysis time, low sample requirement, and sensitive fluorescence detection makes CE-RNA-SSCP an appealing new approach for characterizing low-diversity microbial communities.

Schematic representation of direct rRNA microbial community characterization workflow using CE-LIF

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Editorial (2010) Nat Rev Microbiol 8:384

    Google Scholar 

  2. Felske A, Rheims H, Wolterink A, Stackebrandt E, Akkermans AD (1997) Microbiol 143:2983–2989

    Article  CAS  Google Scholar 

  3. Weller R, Weller JW, Ward DM (1991) Appl Environ Microbiol 57:1146–1151

    CAS  Google Scholar 

  4. Ward DM, Weller R, Bateson MM (1990) Nature 345:63–65

    Article  CAS  Google Scholar 

  5. Wagner R (1994) Arch Microbiol 161:100–109

    Article  CAS  Google Scholar 

  6. Ren JC, Ulvik A, Ueland PM, Refsum H (1997) Anal Biochem 245:79–84

    Article  CAS  Google Scholar 

  7. Andersen PS, Jespersgaard C, Vuust J, Christiansen M, Larsen LA (2003) Hum Mutat 21:116–122

    Article  CAS  Google Scholar 

  8. Skeidsvoll J, Ueland PM (1996) Electrophoresis 17:1512–1517

    Article  CAS  Google Scholar 

  9. Sigmon J, Larcom LL (1996) Electrophoresis 17:1524–1527

    Article  CAS  Google Scholar 

  10. Liang D, Zhang J, Chu B (2003) Electrophoresis 24:3348–3355

    Article  CAS  Google Scholar 

  11. Shin GW, Cho YS, Hwang HS, Park JH, Jung GY (2008) Anal Biochem 383:31–37

    Article  CAS  Google Scholar 

  12. Widjojoatmodjo MN, Fluit AC, Verhoef J (1995) J Clin Microbiol 33:2601–2606

    CAS  Google Scholar 

  13. Zinger L, Gury J, Giraud F, Krivobok S, Gielly L, Taberlet P, Geremia RA (2007) Microb Ecol 54:203–216

    Article  CAS  Google Scholar 

  14. Hiibel SR, Pruden A, Crimi B, Reardon KF (2010) J Microbiol Meth 83:286–290

    Article  CAS  Google Scholar 

  15. Zemb O, Lee M, Low A, Manefield M (2010) Appl Microbiol Biotechnol 88:319–325

    Article  CAS  Google Scholar 

  16. Duthoit F, Tessier L, Montel MC (2005) J Appl Microbiol 98:1198–1208

    Article  CAS  Google Scholar 

  17. Brinkmann N, Martens R, Tebbe CC (2008) Appl Environ Microbiol 74:7189–7196

    Article  CAS  Google Scholar 

  18. Whiteley AS, Thomson B, Lueders T, Manefield M (2007) Nat Protoc 2:838–844

    Article  CAS  Google Scholar 

  19. Gutierrez-Zamora M-L, Zemb O, Manefield M (2011) Microb Ecol 62:177–187

    Article  CAS  Google Scholar 

  20. Han F, Lillard SJ (2002) Anal Biochem 302:136–143

    Article  CAS  Google Scholar 

  21. Imbeaud S, Graudens E, Boulanger V, Barlet X, Zaborski P, Eveno E, Mueller O, Schroeder A, Auffray C (2005) Nucleic Acids Res 33:1–12

    Article  Google Scholar 

  22. Uyeno Y, Sekiguchi Y, Sunaga A, Yoshida H, Kamagata Y (2004) Appl Environ Microbiol 70:3650–3663

    Article  CAS  Google Scholar 

  23. Persat A, Chivukula RR, Mendell JT, Santiago JG (2010) Anal Chem 82:9631–9635

    Article  CAS  Google Scholar 

  24. Nishimura A, Tsuhako M (2000) Chem Pharm Bull 48:774–778

    Article  CAS  Google Scholar 

  25. Goldsmith JG, Ntuen EC, Goldsmith EC (2007) Anal Biochem 360:23–29

    Article  CAS  Google Scholar 

  26. Benesova-Minarikova L, Fantova L, Minarik M (2005) Electrophoresis 26:4064–4069

    Article  CAS  Google Scholar 

  27. Frank JA, Reich CI, Sharma S, Weisbaum JS, Wilson BA, Olsen GJ (2008) Appl Environ Microbiol 74:2461–2470

    Article  CAS  Google Scholar 

  28. Mao D-P, Zhou Q, Chen C-Y, Quan Z-X (2012) BMC Microbiol 12:66

    Article  Google Scholar 

  29. Soergel DAW, Dey N, Knight R, Brenner SE (2012) ISME J 6:1440–1444

    Article  CAS  Google Scholar 

  30. Lafontaine DL, Tollervey D (2006) Ribosomal RNA. eLS. doi:10.1038/npg.els.0003832

  31. Yip SP, Hopkinson DA, Whitehouse DB (1999) Biotechniques 27:20–22, 24

    CAS  Google Scholar 

  32. Chiari M, Riva S, Gelain A, Vitale A, Turati E (1997) J Chromatogr A 781:347–355

    Article  CAS  Google Scholar 

  33. Rosenblum BB, Oaks F, Menchen S, Johnson B (1997) Nucleic Acids Res 25:3925–3929

    Article  CAS  Google Scholar 

  34. Heller C (2001) Electrophoresis 22:629–643

    Article  CAS  Google Scholar 

  35. Saevels J, Van Schepdael A, Hoogmartens J (1999) Anal Biochem 266:93–101

    Article  CAS  Google Scholar 

  36. Glavač D, Dean M (1993) Hum Mutat 2:404–414

    Article  Google Scholar 

  37. MacGregor BJ, Amann R (2006) Syst Appl Microbiol 29:661–670

    Article  CAS  Google Scholar 

  38. Todorov TI, de Carmejane O, Walter NG, Morris MD (2001) Electrophoresis 22:2442–2447

    Article  CAS  Google Scholar 

  39. Andersen PS, Jespersgaard C, Vuust J, Christiansen M, Larsen LA (2003) Hum Mutat 21:455–465

    Article  CAS  Google Scholar 

  40. Jacob F, Monod J (1961) J Mol Biol 3:318–356

    Article  CAS  Google Scholar 

  41. Bremer H, Dennis P (1996) In: Neidhart F (ed) Escherichia coli and Salmonella: cellular and molecular biology, 2nd edn. ASM Press, Washington, DC

    Google Scholar 

  42. Kerkhof L, Kemp P (1999) FEMS Microbiol Ecol 30:253–260

    Article  CAS  Google Scholar 

  43. Jacobson A, Gillespie D (1968) J Bacteriol 95:1030–1039

    CAS  Google Scholar 

  44. Siegele DA, Kolter R (1992) J Bacteriol 174:345

    CAS  Google Scholar 

  45. Aviv M, Giladi H, Oppenheim AB, Glaser G (1996) FEMS Microbiol Lett 140:71–76

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This study was supported by the Australian Antarctic Division through Australia Antarctic science program grant. The authors acknowledge the University of Tasmania for Tasmania Postgraduate Research Scholarship awarded to YHN. MCB would like to thank the Australian Research Council for funding and provision of a QEII Fellowship (DP0984745), and MM would like to thank the Australian Research Council for funding and provision of a Future Fellowship (FT100100078).

Author information

Authors and Affiliations

  1. Australian Centre for Research on Separation Science (ACROSS), School of Chemistry, University of Tasmania, Private bag 75, Hobart, TAS, 7001, Australia

    Yi H. Nai & Michael C. Breadmore

  2. Tasmanian Institute of Agriculture (TIA), University of Tasmania, Private bag 98, Hobart, TAS, 7001, Australia

    Shane M. Powell

  3. Centre for Marine Bioinnovation, (CMB), School of Biotechnology and Biomolecular Science, University of New South Wales, Sydney, NSW, 2025, Australia

    Oliver Zemb, Maria-Luisa Gutierrez-Zamora & Mike Manefield

Authors
  1. Yi H. Nai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Oliver Zemb
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Maria-Luisa Gutierrez-Zamora
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mike Manefield
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shane M. Powell
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michael C. Breadmore
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michael C. Breadmore.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 100 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nai, Y.H., Zemb, O., Gutierrez-Zamora, ML. et al. Capillary electrophoresis ribosomal RNA single-stranded conformation polymorphism: a new approach for characterization of low-diversity microbial communities. Anal Bioanal Chem 404, 1897–1906 (2012). https://doi.org/10.1007/s00216-012-6268-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-012-6268-0

Keywords

Search

Navigation