Abstract
The unique ability of triplex-forming PNAs to invade the double helix has made it possible to develop a highly specific and sensitive approach for bacterial detection. The method uses short, about 20-bp-long, signature sequences presented as a single copy in the bacterial genome. Bacterial cells are fixed on slides and the PD-loop structure is assembled on the signature site with the help of PNA openers, which includes the circular probe. The sensitivity of the method is achieved via Rolling Circle Amplification (RCA) of the circular probe. The obtained amplicon is detected using short ssDNA decorator probes carrying fluorophores and via standard fluorescent microscopy.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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 363:206–220
Procop GW (2002) In situ hybridization for the detection of infectious agents. Clin Microbiol Newsl 24:121–125
Wagner M, Horn M, Daims H (2003) Fluorescence in situ hybridisation for the identification and characterisation of prokaryotes. Curr Opin Microbiol 6:302–309
Zwirglmaier K (2005) Fluorescence in situ hybridisation – the next generation. FEMS Microbiol Lett 246:151–158
Amann R, Glockner F-O, Neef A (1997) Modern methods in subsurface microbiology: in situ identification of microorganisms with nucleic acid probes. FEMS Microbiol Rev 20:191–200
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 50:75–84
Pernthaler A, Amann R (2004) Simultaneous fluorescence in situ hybridization of mRNA and rRNA in environmental bacteria. Appl Environ Microbiol 70:5426–5433
Wagner M, Schmid M, Juretschko S et al (1998) In situ detection of a virulence factor mRNA and 16S rRNA in Listeria monocytogenes. FEMS Microbiol Lett 160:159–168
Zwirglmaier K, Ludwig W, Schleifer K-H (2004) Recognition of individual genes in a single bacterial cell by fluorescence in situ hybridization: RING-FISH. Mol Microbiol 51:89–96
Smolina I, Kuhn H, Lee C, Frank-Kamenetskii MD (2008) Fluorescence-based detection of short DNA sequences under non-denaturing conditions. Bioorg Med Chem 16:84–93
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 73:2324–2328
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 1:1–7
Nielsen PE, Egholm M, Berg RH, Buchardt O (1991) Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254:1497–1500
Uhlmann E, Peyman A, Breipohl G, Will DW (1998) PNA: synthetic polyamide nucleic acids with unusual binding properties. Angew Chem Int Ed 37:2797–2823
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 95:5516–5520
Demidov VV, Frank-Kamenetskii MD (2004) Two sides of the coin: affinity and specificity of nucliec acid interactions. Trends Biochem Sci 29:62–71
Demidov VV, Kuhn H, Lavrentieva-Smolina IV, Frank-Kamenetskii MD (2001) Peptide nucleic acid-assisted topological labelling of duplex DNA. Methods 23:123–131
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 11:221–225
Nilsson M (2006) Lock and roll: single-molecule genotyping in situ using padlock probes and rolling-circle amplification. Histochem Cell Biol 126:159–164
Zhang D, Wu J, Ye F et al (2006) Amplification of circularizable probes for the detection of target nucleic acids and proteins. Clin Chim Acta 363:61–70
Egholm M, Christensen L, Dueholm KL et al (1995) Efficient pH-independent sequence-specific DNA binding by pseudoisocytosine-containing bis-PNA. Nucleic Acids Res 23:217–222
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Smolina, I.V., Frank-Kamenetskii, M.D. (2014). PNA Openers and Their Applications for Bacterial DNA Diagnostics. In: Nielsen, P., Appella, D. (eds) Peptide Nucleic Acids. Methods in Molecular Biology, vol 1050. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-553-8_10
Download citation
DOI: https://doi.org/10.1007/978-1-62703-553-8_10
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-552-1
Online ISBN: 978-1-62703-553-8
eBook Packages: Springer Protocols