Advertisement

Journal of Applied Phycology

, Volume 21, Issue 6, pp 657–668 | Cite as

Evaluation of locked nucleic acids for signal enhancement of oligonucleotide probes for microalgae immobilised on solid surfaces

  • Sonja Diercks
  • Christine Gescher
  • Katja Metfies
  • Linda K. Medlin
Article

Abstract

Biosensors and microarrays are powerful tools for species detection and monitoring of microorganisms. A reliable identification of microorganisms with probe-based methods requires highly specific and sensitive probes. The introduction of locked nucleic acid (LNA) promises an enhancement of specificity and sensitivity of molecular probes. In this study, we compared specificity and sensitivity of conventional probes and LNA modified probes in two different solid phase hybridisation methods: sandwich hybridisation on biosensors and on DNA microarrays. In combination with DNA-microarrays, the LNA probes displayed an enhancement of sensitivity, but also gave more false-positive signals. With the biosensor, the LNA probes showed neither signal enhancement nor discrimination of a single mismatch. In all cases, conventional DNA probes showed equal or better results than LNA probes. In conclusion, LNA technology may have great potential in methods that use probes in suspension and in gene expressions studies, but under certain solid surface-hybridisation applications, they do not improve signal intensity.

Keywords

Locked nucleic acids Oligonucleotide probes Microalgae Sandwich hybridisation Microarray 

Notes

Acknowledgements

The authors would like to thank Annick Sawala (University of Durham, United Kingdom) for her assistance in the hybridisation experiments. The LNA probes were designed and provided by Exiqon A/S, Bygstubben 9, 2950 Vedbaek, Denmark and financed by the EU projects ALGADEC and FISH & CHIPS. Christine Gescher and Sonja Diercks were supported by the EU-projects FISH&CHIPS (GOCE-CT-2003-505491) and ALGADEC (COOP-CT-2004-508435-ALGADEC) of the 6th framework program of the European Union and the Alfred Wegener Institute for Polar and Marine Research.

References

  1. Braasch DA, Corey DR (2001) Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA. Chem Biol 8:1–87CrossRefPubMedGoogle Scholar
  2. Chou L-S, Meadows C, Wittwer CT, Lyon E (2005) Unlabeled oligonucleotide probes modified with locked nucleic acids for improved mismatch discrimination in genotyping by melting analysis. BioTechniques 39:644–647CrossRefPubMedGoogle Scholar
  3. DeRisi JL, Iyer VR, Brown PO (1997) Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278:680–686CrossRefPubMedGoogle Scholar
  4. Diercks S, Metfies K, Medlin LK (2008a) Molecular probe sets for the detection of toxic algae for use in sandwich hybridisation formats. J Plankt Res 30:439–448CrossRefGoogle Scholar
  5. Diercks S, Metfies K, Medlin LK (2008b) Colorimetric detection of the toxic dinoflagellate Alexandrium minutum using sandwich hybridization in a microtiter plate assay. Harmful Algae 7:137–145CrossRefGoogle Scholar
  6. Diercks S, Metfies K, Medlin LK (2008c) Development and adaptation of a multiprobe biosensor for the use in a semi-automated device for the detection of toxic algae. Biosens Bioelectron 23:1527–1533CrossRefPubMedGoogle Scholar
  7. Eppley RW, Holmes RW, Strickland JDH (1967) Sinking rates of marine phytoplankton measured with a fluorometer. J Exp Mar Biol Ecol 1:191–208CrossRefGoogle Scholar
  8. Gagnon S, Bourbeau D, Levesque RC (1996) Secondary structures and features of the 18S, 5.8S and 26S ribosomal RNAs from the apicomplexan parasite Toxoplasma gondii. Gene 173:129–135CrossRefPubMedGoogle Scholar
  9. Gescher C, Metfies K, Medlin LK (2007) The ALEX CHIP-Development of a DNA chip for identification and monitoring of Alexandrium. Harmful Algae 7:485–494CrossRefGoogle Scholar
  10. Gescher C, Metfies K, Frickenhaus S, Knefelkamp B, Wiltshire KH, Medlin LK (2008) Feasibility of assessing the community composition of Prasinophytes at the Helgoland Roads sampling site with a DNA microarray. Appl Env Microbiol 74:5305–5316CrossRefGoogle Scholar
  11. Graves DJ (1999) Powerful tools for genetic analysis come of age. Trends Biotechnol 17:127–134CrossRefPubMedGoogle Scholar
  12. Groben R, Medlin L (2005) In Situ hybridization of phytoplankton using fluorescently labeled rRNA probes. Methods in Enzymology. In: Zimmer EAS, Roalson EH (ed) Molecular Evolution: Producing the Biochemical Data Academic Press, NY, pp 299–310Google Scholar
  13. Groben R, John U, Eller G, Lange M, Medlin LK (2004) Using fluorescently-labelled rRNA probes for hierarchical estimation of phytoplankton diversity—a mini-review. Nova Hedwigia 79:313–320CrossRefGoogle Scholar
  14. Guillou L, Chrétiennot-Dinet M-J, Medlin LK, Claustre H, Loiseaux-De Goer S, Vaulot D (1999) Bolidomonas: A new genus with two species belonging to a new algal class, the Bolidophyceae (Heterokonta). J Phycol 35:368–381CrossRefGoogle Scholar
  15. Howley PM, Israel MA, Law M, Martin MA (1979) A rapid method for detecting and mapping homology between heterologous DNAs. J BiolChem 254:4876–4883Google Scholar
  16. Hummelshoj L, Ryder LP, Madsen HO, Poulsen LK (2005) Locked nucleic acid inhibits amplification of contaminating DNA in real-time PCR. BioTechniques 38:605–610CrossRefPubMedGoogle Scholar
  17. Jacobsen N, Bentzen J, Meldgaard M, Jakobsen MH, Fenger M, Kauppinen S, Skouv J (2002a) LNA-enhanced detection of single nucleotide polymorphisms in the apolipoprotein E. Nucl Acids Res 30:e100CrossRefPubMedGoogle Scholar
  18. Jacobsen N, Fenger M, Bentzen J, Rasmussen SL, Jakobsen MH, Fenstholt J, Skouv J (2002b) Genotyping of the apolipoprotein B R3500Q mutation using immobilized locked nucleic acid capture probes. Clin Chem 48:657–660PubMedGoogle Scholar
  19. Kappel K, Westernhagen HV, Blohm DH (2003) Microarray-based identification of eggs and larvae from fish species common in the North Sea, Dechema Chip-Technology Meeting, Frankfurt, GermanyGoogle Scholar
  20. Kauppinen S, Nielsen PS, Mouritzen P, Nielsen AT, Vissing H, Møller S, Ramsing NB (2003) LNA Microarrays in Genomics. Pharma Genomics 24–32Google Scholar
  21. Keller MD, Selvin RC, Claus W, Guillard RRL (1987) Media for the culture of oceanic ultraphytoplankton. J Phycol 23:633–338Google Scholar
  22. Ki J-S, Han M-S (2006) A low-density oligonucleotide array study for parallel detection of harmful algal species using hybridization of consensus PCR products of LSU rDNA D2 domain. Biosens Bioelectr 21:1812–1821CrossRefGoogle Scholar
  23. Kim C-J, Sako Y (2005) Molecular identification of toxic Alexandrium tamiyavanichii (Dinophyceae) using two DNA probes. Harmful Algae 4:984–991CrossRefGoogle Scholar
  24. Kloosterman WP, Wienholds E, De Bruijn E, Kauppinen S, Plasterk RHA (2006) In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes. Nature Methods 3:27–29CrossRefPubMedGoogle Scholar
  25. Kongsbak L (2002) LNA: fine-tuning of primers and probes. LNA 01:1Google Scholar
  26. Koshkin AA, Rajwanshi VK, Wengel J (1998a) Novel convenient syntheses of LNA [2.2.1]bicyclo nucleosides. Tet Lett 39:4381–4384CrossRefGoogle Scholar
  27. Koshkin AA, Singh SK, Nielsen P, Rajwanshi VK, Kumar R, Meldgaard M, Olsen CE, Wengel J (1998b) LNA (locked nucleic acids): synthesis of the adenine, cytosine, guanine, 5–methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition. Tetrahedron 54:3607–3630CrossRefGoogle Scholar
  28. Kubota K, Ohashi A, Imachi H, Harada H (2006) Improved in situ hybridization efficiency with locked-nucleic-acid-incorporated DNA probes. Appl Env Microbiol 72:5311–5317CrossRefGoogle Scholar
  29. Lehner A, Loy A, Behr T, Gaenge H, Ludwig W, Wagner M, Schleifer K-H (2005) Oligonucleotide microarray for identification of Enterococcus species. FEMS Microbiol Lett 246:133–142CrossRefPubMedGoogle Scholar
  30. Lim EL, Amaral LA, Caron DA, Delong EF (1993) Application of rRNA-based probes for observing marine nanoplanktonic protists. Appl Env Microbiol 59:1647–1655Google Scholar
  31. Lockhart DJ (1996) Expression monitoring by hybridization to high-density oligonucleotide arrays. Nature Biotechnology 14:1675–1680CrossRefPubMedGoogle Scholar
  32. Loy A, Lehner A, Lee N, Adamczyk J, Meier H, Ernst J, Schleifer K-H, Wagner M (2002) Oligonucleotide microarray for 16S rRNA gene-based detection of all recognized lineages of Sulfate-reducing prokaryotes in the environment. Appl Env Microbiol 68:5064–5081CrossRefGoogle Scholar
  33. Loy A, Schulz C, Lucker S, Schopfer-Wendels A, Stoecker K, Baranyi C, Lehner A, Wagner M (2005) 16S rRNA Gene-based oligonucleotide microarray for environmental monitoring of the betaproteobacterial Order “Rhodocyclales”. Appl Env Microbiol 71:1373–1386CrossRefGoogle Scholar
  34. Medlin LK, Elwood HJ, Stickel S, Sogin ML (1988) The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene 71:491–499CrossRefPubMedGoogle Scholar
  35. Medlin LK, Metfies K, Mehl H, Wiltshire KH, Valentin K (2006) Picoeukaryotic plankton diversity at the Helgoland time series site as assessed by three molecular methods. Microb Ecol 52:53–71CrossRefPubMedGoogle Scholar
  36. Metfies K, Medlin LK (2004) DNA microchips for phytoplankton: the fluorescent wave of the future. Nova Hedwigia 79:321–327CrossRefGoogle Scholar
  37. Metfies K, Medlin LK (2005) Ribosomal RNA probes and microarrays: Their potential use in assessing microbial biodiversity. In Zimmer EA Roalson EH (eds) Methods in Enzymology, Molecular Evolution: Producing the Biochemical Data Academic, NY, pp 258–278Google Scholar
  38. Metfies K, Medlin LK (2008) Feasibility of transferring fluorescent in situ hybridization probes to an 18S rRNA gene Phylochip and mapping of signal intensities. Appl Env Microbiol 74:2814–2821CrossRefGoogle Scholar
  39. Metfies K, Huljic S, Lange M, Medlin LK (2005) Electrochemical detection of the toxic dinoflagellate Alexandrium ostenfeldii with a DNA-biosensor. Biosens Bioelect 20:1349–1357CrossRefGoogle Scholar
  40. Metfies K, Berzano M, Gualerzi C, Muyzer G, Medlin LK (2006) Meeting report: molecular ecology workshop. Detection of microbial biodiversity in environmental samples, Camerino, Italy, September 19–21, 2005. Protist 157:247–250CrossRefPubMedGoogle Scholar
  41. Metfies K, Berzano M, Mayer C, Roosken P, Gualerzi C, Medlin LK, Muyzer G (2007) An optimised protocol for the identification of diatoms, flagellated algae and pathogenic protozoa with phylochips. Mol Ecol Notes 7:925–936CrossRefGoogle Scholar
  42. Metfies K, Borsutzki P, Gescher C, Medlin LK, Frickenhaus S (2008) PhylochipAnalyzer—A program for analyzing hierachical probe-sets. Molec Ecol Res 8:99–102CrossRefGoogle Scholar
  43. Montresor M, John U, Beran A, Medlin LK (2004) Alexandrium tamutum sp. nov. (Dinophyceae): a new nontoxic species in the genus Alexandrium. J Phycol 40:398–411CrossRefGoogle Scholar
  44. Nielsen PS, Kauppinen S (2002) The use of LNA Oligonucleotide Microarrays provides superior sensitivity and specificity in expression profiling. LNA 17:1–3CrossRefGoogle Scholar
  45. Niemeyer CM, Blohm D (1999) DNA Microarrays. Angewandte Chemie International Edition, 38:2865–2869Google Scholar
  46. Obika S, Nanbu D, Hari Y, Andoh J-I, Morio K-I, Doi T, Imanishi T (1998) Stability and structural features of the duplexes containing nucleoside analogues with a fixed N-type conformation, 2′-O,4′-C-methyleneribonucleosides. Tet Lett 39:5401–5404CrossRefGoogle Scholar
  47. Parekh-Olmedo H, Drury M, Kmiec EB (2002) Targeted nucleotide exchange in Saccharomyces cerevisiae directed by short oligonucleotides containing locked nucleic acids. Chem Biol 9:1073–1084CrossRefPubMedGoogle Scholar
  48. Peplies J, Glockner FO, Amann R (2003) Optimization strategies for DNA microarray-based detection of bacteria with 16S rRNA-targeting oligonucleotide probes. Appl Env Microbiol 69:1397–1407CrossRefGoogle Scholar
  49. Peplies J, Glockner FO, Amann R, Ludwig W (2004a) Comparative sequence analysis and oligonucleotide probe design based on 23S rRNA genes of alpha proteobacteria from North Sea bacterioplankton. Syst Appl Microbiol 27:573–580CrossRefPubMedGoogle Scholar
  50. Peplies J, Lau SCK, Pernthaler J, Amann R, Glockner FO (2004b) Application and validation of DNA microarrays for the 16S rRNA-based analysis of marine bacterioplankton. Env Microbiol 6:638–645CrossRefGoogle Scholar
  51. Peplies J, Lachmund C, Glockner FO, Manz W (2006) A DNA microarray platform based on direct detection of rRNA for characterization of freshwater sediment-related prokaryotic communities. Appl Env Microbiol 72:4829–4838CrossRefGoogle Scholar
  52. Scholin CA, Herzog M, Sogin M, Anderson DM (1994) Identification of group- and strain-specific genetic markers for the globally distributed Alexandrium (Dinophyceae). 2. Sequence analysis of a fragment of the LSU rRNA gene. J Phycol 30:999–1011CrossRefGoogle Scholar
  53. Scholin CA, Buck KR, Britschgi T, Cangelosi G, Chavez FP (1996) Identification of Pseudo-nitzschia australis (Bacillariophyceae) using rRNA-targeted probes in whole cell and sandwich hybridization formats. Phycologia 35:190–197CrossRefGoogle Scholar
  54. Scholin CA, Miller P, Buck KR, Chavez FP, Harris P, Haydock P, Howard J, Cangelosi G (1997) Detection and quantification of Pseudo nitzschia australis in cultured and natural populations using LNA rRNA-targeted probes. Limnol Oceanogr 42:1265–1272CrossRefGoogle Scholar
  55. Silahtaroglu AN, Tommerup N, Vissing H (2003) FISHing with locked nucleic acids (LNA): evaluation of different LNA/DNA mixmers. Molec Cellular Probes 17:165–169CrossRefGoogle Scholar
  56. Silahtaroglu AN, Pfundheller HM, Koshkin AA, Tommerup N, Kauppinen S (2004) LNA-modified oligonucleotides are highly efficient as FISH probes. Cytogenetic and Genome Res 107:32–37CrossRefGoogle Scholar
  57. Simon N, Campbell E, Ornolfsdottir E, Groben R, Guillou L, Lange M, Medlin LK (2000) Oligonucleotide probes for the identification of three algal groups by dot blot and fluorescent whole-cell hybridization. J Eukaryot Microbiol 47:76–84CrossRefPubMedGoogle Scholar
  58. Singh SK, Koshkin AA, Wengel J, Nielsen P (1998) LNA (locked nucleic acids): synthesis and high-affinity nucleic acid recognition. Chem Comm 4:455–456CrossRefGoogle Scholar
  59. Sun Z, Zhou L, Zeng H, Chen Z, Zhu H (2007) Multiplex locked nucleic acid probes for analysis of hepatitis B virus mutants using real-time PCR. Genomics 89:151–159CrossRefPubMedGoogle Scholar
  60. Tyrrell JV, Connell LB, Scholin CA (2002) Monitoring for Heterosigma akashiwo using a sandwich hybridization assay. Harmful Algae 1:205–214CrossRefGoogle Scholar
  61. Ugozzoli LA, Latorra D, Pucket R, Arar K, Hamby K (2004) Real-time genotyping with oligonucleotide probes containing locked nucleic acids. Anal Biochem 324:143–152CrossRefPubMedGoogle Scholar
  62. Vester B, Wengel J (2004) LNA (Locked Nucleic Acid): high-affinity targeting of complementary RNA and DNA. Biochem 43:13233–13241CrossRefGoogle Scholar
  63. Vester B, Lundberg LB, Sørensen MD, Babu BR, Douthwaite S, Wengel J (2004) Improved RNA cleavage by LNAzyme derivatives of DNAzymes. Biochem Soc Trans 32:37–40CrossRefPubMedGoogle Scholar
  64. Vester B, Hansen L, Bo Lundberg L, Babu BR, Sorensen M, Wengel J, Douthwaite S (2006) Locked nucleoside analogues expand the potential of DNAzymes to cleave structured RNA targets. BMC Mol Biol 7:19CrossRefPubMedGoogle Scholar
  65. Wienholds E, Kloosterman WP, Miska E, Alvarez-Saavedra E, Berezikov E, De Bruijn E, Horvitz HR, Kauppinen S, Plasterk RHA (2005) MicroRNA expression in zebrafish embryonic development. Science 309:310–311CrossRefPubMedGoogle Scholar
  66. Ye RW, Wang T, Bedzyk L, Croker KM (2001) Applications of DNA microarrays in microbial systems. J Microbiol Meth 47:257–272CrossRefGoogle Scholar
  67. You M, Moreira BG, Behlke MA, Owczarzy R (2006) Design of LNA probes that improve mismatch discrimination. Nucleic Acids Res 34:e60CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Sonja Diercks
    • 1
  • Christine Gescher
    • 1
  • Katja Metfies
    • 2
  • Linda K. Medlin
    • 3
  1. 1.Alfred Wegener Institute for Polar and Marine ResearchBremerhavenGermany
  2. 2.GKSS Research CentreGeesthachtGermany
  3. 3.Observatoire Océanologique de Banyuls sur Mer, Laboratoire AragoBanyuls sur MerFrance

Personalised recommendations