Abstract
Hybridization-based assays for the detection of nucleic acids including in situ hybridization are increasingly being utilized in a wide variety of disciplines such as cytogenetics, microbiology, and histology. Generally in situ hybridization assays utilize either cloned genomic probes for the detection of DNA sequences or oligonucleotide probes for the detection of DNA or RNA sequences. Alternately, PNA probes are increasingly being utilized in a variety of in situ hybridization assays [1, 2]. The neutral backbone of the PNA molecule allows for the PNA probes to bind to DNA or RNA under low ionic strength conditions that will either disfavor reannealing of complimentary genomic sequences or are denaturing for RNA secondary structure but are favorable for PNA/DNA or PNA/RNA hybridization [3, 4]. For in situ hybridization assays these unique properties of PNA probes offer significant advantages that allow for the development of fast, simple, and robust assays (Figs. 14.1 and 14.2).
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
Buchardt O, Egholm M, Berg RH, Nielsen PE (1993) Peptide nucleic acids and their potential applications in biotechnology. Trends Biotechnol 11:384–386
Thisted M, Just T, Pluzek KJ, Hyldig-Nielsen JJ, Nielsen KV, Mollerup TA, Stender H, Rasmussen OF, Adelhorst K, Godtfredsen A (1999) Application of peptide nucleic acids probes for in situ hybridization. In: Nielsen PE, Egholm M (eds) Peptide nucleic acids: protocols and applications. Horizon Scientific Press, Wymondham, pp 99–119
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
Egholm M, Buchardt O, Christensen L, Behrens C, Freier SM, Driver DA, Berg RH, Kim SK, Norden B, Nielsen PE (1993) PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen bonding rules. Nature 365:556–568
Oliveira K, Hyldig-Nielsen JJ, Kurtzman C, Stender H (2001) Differentiation between Candida albicans and Candida dubliniensis by fluorescence in situ hybridization using PNA probes. Annual meeting of the American Society of Microbiology, Washington DC, C-387
Oliveira K, Procop G, Wilson D, Coull J, Stender H (2001) Rapid identification of Staphylococcus aureus directly from blood cultures by PNA FISH. Annual interscience conference of antimicrobial agents and chemotherapy, Washington DC, Abstract (submitted)
Stender H, Lund K, Petrsen KH, Rasmussen OF, Hongmanee P, Miorner H, Godtfredsen SE (1999) Fluorescence in situ hybridization assay using peptide nucleic acid probes for differentiation between tuberculous and nontuberculous mycobacterium species in smears of mycobacterium cultures. J Clin Microbiol 37:2760–2765
Stender H, Kurtzman C, Hyldig-Nielsen JJ, Sørensen D, Broomer A, Oliveira K, Perry-O’Keefe H, Sage A, Young B, Coull J (2001) Identification of Brettanomyces (Dekkera bruxellensis) from wine by fluorescence in situ hybridization using peptide nucleic acid probes. Appl Environ Microbiol 67:938–941
Delong EF, Wickham GS, Pace NR (1989) Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science 243:1360–1363
Woese CR (1987) Bacterial evolution. Microbiol Rev 51:221–271
Stefano K, Hyldig-Nielsen JJ (1997) Diagnostic applications of PNA oligomers. In: Diagnostic gene detection & quantification technologies. IBC Library series publication # 948
Trnovsky J, Klimas LL, Stevens S, Gillespie WB, Kazemi B, Hansen DS, Stender H (2011) Evaluation of PNA FISH® for identification of microorganisms in smear-positive urine specimens. J Clin Microbiol, Submitted 19 May 2011
Fazli M, Bjarnsholt T, Kirketerp-Moller K, Jorgenesen A, Andersen CB, Givskov M, Tolker-Nielsen T (2011) Quantitative analysis of the cellular inflammatory response against biofilm bacteria in chronic wounds. Wound Repair Regen 19(3):387–391
Taneja KL, Chavez EA, Coull J, Lansdorp PM (2001) Multicolor fluorescence in situ hybridization with peptide nucleic acid probes for enumeration of specific chromosomes in human cells. Genes Chromosomes Cancer 30:57–63
Chen C, Wu B, Wei T, Egholm M, Strauss WM (2000) Unique chromosome identification and sequence-specific structural analysis with short PNA oligomers. Mamm Genome 11(5):384–391
Pezzlo M (1998) Aerobic bacteriology (chap. 2). In: Isenberg HD (ed) Essential procedures for clinical microbiology. American Society for Microbiology, Washington, DC
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
Stender, H., Williams, B., Coull, J. (2014). PNA Fluorescent In Situ Hybridization (FISH) for Rapid Microbiology and Cytogenetic Analysis. 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_14
Download citation
DOI: https://doi.org/10.1007/978-1-62703-553-8_14
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