Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

Sílvia Fontenete, Barros Joana, Madureira Pedro, Figueiredo Céu, Jesper Wengel, Azevedo Nuno Filipe

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

In the past few years, several researchers have focused their attention on nucleic acid mimics due to the increasing necessity of developing a more robust recognition of DNA or RNA sequences. Fluorescence in situ hybridization (FISH) is an example of a method where the use of these novel nucleic acid monomers might be crucial to the success of the analysis. To achieve the expected accuracy in detection, FISH probes should have high binding affinity towards their complementary strands and discriminate effectively the noncomplementary strands. In this study, we investigate the effect of different chemical modifications in fluorescent probes on their ability to successfully detect the complementary target and discriminate the mismatched base pairs by FISH. To our knowledge, this paper presents the first study where this analysis is performed with different types of FISH probes directly in biological targets, Helicobacter pylori and Helicobacter acinonychis. This is also the first study where unlocked nucleic acids (UNA) were used as chemistry modification in oligonucleotides for FISH methodologies. The effectiveness in detecting the specific target and in mismatch discrimination appears to be improved using locked nucleic acids (LNA)/2'-O-methyl RNA (2'OMe) or peptide nucleic acid (PNA) in comparison to LNA/DNA, LNA/UNA, or DNA probes. Further, the use of LNA modifications together with 2'OMe monomers allowed the use of shorter fluorescent probes and increased the range of hybridization temperatures at which FISH would work.

Original languageEnglish
JournalApplied Microbiology and Biotechnology
Volume99
Issue number9
Pages (from-to)3961-3969
ISSN0175-7598
DOIs
Publication statusPublished - May 2015

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Nucleic Acids
Fluorescent Dyes
RNA
Peptide Nucleic Acids
Helicobacter pylori
Base Pairing
Research Personnel
DNA

Cite this

Fontenete, Sílvia ; Joana, Barros ; Pedro, Madureira ; Céu, Figueiredo ; Wengel, Jesper ; Filipe, Azevedo Nuno. / Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids. In: Applied Microbiology and Biotechnology. 2015 ; Vol. 99, No. 9. pp. 3961-3969.
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Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids. / Fontenete, Sílvia; Joana, Barros; Pedro, Madureira; Céu, Figueiredo; Wengel, Jesper; Filipe, Azevedo Nuno.

In: Applied Microbiology and Biotechnology, Vol. 99, No. 9, 05.2015, p. 3961-3969.

Research output: Contribution to journalJournal articleResearchpeer-review

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T1 - Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

AU - Fontenete, Sílvia

AU - Joana, Barros

AU - Pedro, Madureira

AU - Céu, Figueiredo

AU - Wengel, Jesper

AU - Filipe, Azevedo Nuno

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