Advertisement

Application of PNA Openers for Fluorescence-Based Detection of Bacterial DNA

  • Irina Smolina
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1039)

Abstract

Peptide nucleic acid (PNA) openers have the unique ability to invade double-stranded DNA with high efficiency and sequence specificity, making it possible to detect short (about 20 bp), single-copy bacterial DNA sequences. PNA openers bind to a target signature site on one strand of bacterial DNA, leaving the other strand open for hybridization with a circularizable oligonucleotide probe. The assembled complex serves as a template for rolling circle amplification. The obtained amplicon is decorated with short, single-stranded DNA probes carrying fluorophores and detected via fluorescence microscopy.

Key words

Peptide nucleic acid (PNA) Padlock probe Rolling circle amplification (RCA) Bacterial detection Fluorescence in situ hybridization (FISH) 

References

  1. 1.
    Mothershed EA, Whitney AM (2006) Nucleic acid-based methods for the detection of bacterial pathogens: present and future considerations for the clinical laboratory. Clin Chim Acta 363:206–220PubMedCrossRefGoogle Scholar
  2. 2.
    Procop GW (2002) In situ hybridization for the detection of infectious agents. Clin Microbiol Newsl 24:121–125CrossRefGoogle Scholar
  3. 3.
    Wagner M, Horn M, Daims H (2003) Fluorescence in situ hybridization for the identification and characterization of prokaryotes. Curr Opin Microbiol 6:302–309PubMedCrossRefGoogle Scholar
  4. 4.
    Zwirglmaier K (2005) Fluorescence in situ hybridization—the next generation. FEMS Microbiol Lett 246:151–158PubMedCrossRefGoogle Scholar
  5. 5.
    Amann R, Glockner FO, Neef A (1997) Modern methods in subsurface microbiology: in situ identification of microorganisms with nucleic acid probes. FEMS Microbiol Rev 20:191–200CrossRefGoogle Scholar
  6. 6.
    Bakermans C, Madsen EL (2002) Detection in coal tar waste-contaminated groundwater of mRNA transcripts related to naphthalene dioxygenase by fluorescent in situ hybridization with tyramide signal amplification. J Microbiol Methods 50:75–84PubMedCrossRefGoogle Scholar
  7. 7.
    Pernthaler A, Amann R (2004) Simultaneous fluorescence in situ hybridization of mRNA and rRNA in environmental bacteria. Appl Environ Microbiol 70:5426–5433PubMedCrossRefGoogle Scholar
  8. 8.
    Wagner M, Schmid M, Juretschko S, Trebesius KH, Bubert A, Goebel W et al (1998) In situ detection of a virulence factor mRNA and 16S rRNA in Listeria monocytogenes. FEMS Microbiol Lett 160:159–168PubMedCrossRefGoogle Scholar
  9. 9.
    Zwirglmaier K, Ludwig W, Schleifer KH (2004) Recognition of individual genes in a single bacterial cell by fluorescence in situ hybridization: RING-FISH. Mol Microbiol 51:89–96PubMedCrossRefGoogle Scholar
  10. 10.
    Smolina I, Kuhn H, Lee C, Frank-Kamenetskii MD (2008) Fluorescence-based detection of short DNA sequences under non-denaturing conditions. Bioorg Med Chem 16:84–93PubMedCrossRefGoogle Scholar
  11. 11.
    Smolina I, Lee C, Frank-Kamenetskii MD (2007) Detection of low-copy-number genomic DNA sequences in individual bacterial cells by using peptide nucleic acid-assisted rolling circle amplification and fluorescence in situ hybridization. Appl Environ Microbiol 73:2324–2328PubMedCrossRefGoogle Scholar
  12. 12.
    Smolina IV, Miller NS, Frank-Kamenetskii MD (2010) PNA-based microbial pathogen identification and resistance marker detection: an accurate, isothermal rapid assay based on genome-specific features. Artif DNA PNA XNA 1:1–7PubMedCrossRefGoogle Scholar
  13. 13.
    Nielsen PE, Egholm M, Berg RH, Buchardt O (1991) Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254:1497–1500PubMedCrossRefGoogle Scholar
  14. 14.
    Uhlmann E, Peyman A, Breipohl G, Will DW (1998) PNA: synthetic polyamide nucleic acids with unusual binding properties. Angew Chem Int Ed 37:2797–2823CrossRefGoogle Scholar
  15. 15.
    Bukanov NO, Demidov VV, Nielsen PE, Frank-Kamenetskii MD (1998) PD-loop: a complex of duplex DNA with an oligonucleotide. Proc Natl Acad Sci U S A 95:5516–5520PubMedCrossRefGoogle Scholar
  16. 16.
    Demidov VV, Frank-Kamenetskii MD (2004) Two sides of the coin: affinity and specificity of nucleic acid interactions. Trends Biochem Sci 29:62–71PubMedCrossRefGoogle Scholar
  17. 17.
    Demidov VV, Kuhn H, Lavrentieva-Smolina IV, Frank-Kamenetskii MD (2001) Peptide nucleic acid-assisted topological labelling of duplex DNA. Methods 23:123–131PubMedCrossRefGoogle Scholar
  18. 18.
    Kuhn H, Demidov VV, Frank-Kamenetskii MD (2000) An earring for the double helix: assembly of topological links comprising duplex DNA and a circular oligodeoxynucleotide. J Biomol Struct Dyn 17(Supp 1):221–225PubMedCrossRefGoogle Scholar
  19. 19.
    Nilsson M (2006) Lock and roll: single-molecule genotyping in situ using padlock probes and rolling-circle amplification. Histochem Cell Biol 126:159–164PubMedCrossRefGoogle Scholar
  20. 20.
    Zhang D, Wu J, Ye F, Feng T, Lee I, Yin B (2006) Amplification of circularizable probes for the detection of target nucleic acids and proteins. Clin Chim Acta 363:61–70PubMedCrossRefGoogle Scholar
  21. 21.
    Egholm M, Christensen L, Dueholm KL, Buchardt O, Coull J, Nielsen PE (1995) Efficient pH-independent sequence-specific DNA binding by pseudoisocytosine-containing bis-PNA. Nucleic Acids Res 23:217–222PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Irina Smolina
    • 1
  1. 1.Department of Biomedical EngineeringBoston UniversityBostonUSA

Personalised recommendations