Oligonucleotide Microarray Technology and its Application to Mycobacterium avium subsp. paratuberculosis Research: A Review

Pribylova, Radka; Králík, Petr; Pavlík, I.

doi:10.1007/s12033-008-9137-5

Cited by 6 publications

(13 citation statements)

References 99 publications

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“…This bacterium is characterized by a very slow growth rate and limited genomic diversity (Stevenson et al, 2009). Numerous methods have been proposed to sub-type Map strains, such as multiplex PCR for IS900 integration loci, IS900 restriction fragment length polymorphism, amplified fragment length polymorphism, pulsed field gel electrophoresis and genotyping microarrays (Motiwala et al, 2006;Pribylova et al, 2009;Stevenson et al, 2009). However, methods based on micro-and minisatellite analyses are the most widely used techniques in this regard (Motiwala et al, 2006;Thibault et al, 2007) because of their relative simplicity and efficacy.…”

Section: Introductionmentioning

confidence: 99%

High-resolution melting for analysis of short sequence repeats in Mycobacterium avium subsp. paratuberculosis

Ricchi

Barbieri

Cammi

et al. 2011

FEMS Microbiol Lett

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Analysis of micro- and minisatellite loci is widely used in sub-typing of Mycobacterium avium subsp. paratuberculosis. Microsatellite (short sequence repeat, SSR) loci have shown highest discriminatory power, but direct sequencing of amplicons is required for correct assignment of the repeat number. We developed an alternative method to sequencing, focusing on the SSR8 locus (constituted by GGT triplets from three to six repeats). The approach is based on asymmetric quantitative PCR, followed by high-resolution melting analysis with unlabelled probes (UP-HRM). Data showed perfect concordance between direct sequencing and UP-HRM, which is faster, simpler and more cost effective.

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Section: Introductionmentioning

confidence: 99%

High-resolution melting for analysis of short sequence repeats in Mycobacterium avium subsp. paratuberculosis

Ricchi

Barbieri

Cammi

et al. 2011

FEMS Microbiol Lett

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“…The quantum yield of each compound was calculated from the observed absorbance and the area of the fluorescence emission band. The fluorescence quantum yield of all nucleosides was determined relative to the quantum yield of quinine sulfate (0.58) 59 or tryptophan (0.14) 59 in H 2 O ( pH 7) or MeOH, respectively, according to eqn (9).…”

Section: Quantum Yield Measurementsmentioning

confidence: 99%

“…3 Currently marketed dyes 4,5 for nucleic acid staining and labeling include intercalating dyes that are incorporated non-covalently to double stranded nucleic acids, 6 minor-groove binding dyes, 7 and large hydrophobic fluorescent dyes 8 that are incorporated covalently to nucleotide (oligonucleotide) positions that do not interfere with base pairing. 8 These dyes are used in various techniques for detecting genetic material in DNA arrays, 9 FISH, 10 gels, 11 in virus particles, 12 and in cells, by fluorescence microscopy, or in electrophoresed gels. 13 Although the above-mentioned fluorescent dyes are useful for nucleic acid detection and quantification, they suffer from limitations regarding the preparation and application of nucleic acid probes.…”

Section: Introductionmentioning

confidence: 99%

“…In addition, the large hydrophobic dye attached to a nucleotide alters the efficiency of enzymatic incorporation into DNA/RNA, resulting in different levels of labeling and prohibits quantification of nucleic acids. 9,14 A two-step protocol involving first the incorporation of a slightly modified nucleotide into nucleic acid, followed by covalent binding of fluorescent dye, also suffers from the limitation that a relatively small number of dye molecules that can be incorporated. 15,16 The limitations and complications of current methodologies applying extrinsic dyes triggered the development of conceptually different fluorescent probes, formed by extension of the natural chromophore.…”

Section: Introductionmentioning

confidence: 99%

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Analogues of uracil nucleosides with intrinsic fluorescence (NIF-analogues): synthesis and photophysical properties

Segal

Fischer

2012

Org. Biomol. Chem.

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Uridine cannot be utilized as fluorescent probe due to its extremely low quantum yield. For improving the uracil fluorescence characteristics we extended the natural chromophore at the C5 position by coupling substituted aromatic rings directly or via an alkenyl or alkynyl linker to create fluorophores. Extension of the uracil base was achieved by treating 5-I-uridine with the appropriate boronic acid under the Suzuki coupling conditions. Analogues containing an alkynyl linker were obtained from 5-I-uridine and the suitable boronic acid in a Sonogashira coupling reaction. The uracil fluorescent analogues proposed here were designed to satisfy the following requirements: a minimal chemical modification at a position not involved in base-pairing, resulting in relatively long absorption and emission wavelengths and high quantum yield. 5-((4-Methoxy-phenyl)-trans-vinyl)-2'-deoxy-uridine, 6b, was found to be a promising fluorescent probe. Probe 6b exhibits a quantum yield that is 3000-fold larger than that of the natural chromophore (Φ 0.12), maximum emission (478 nm) which is 170 nm red shifted as compared to uridine, and a Stokes shift of 143 nm. In addition, since probe 6b adopts the anti conformation and S sugar puckering favored by B-DNA, it makes a promising nucleoside analogue to be incorporated in an oligonucleotide probe for detection of genetic material.

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“…An important aspect of target validation is to identify genes that are essential under all growth conditions [105]. This is of significance as bacterial cells continuously monitor the availability of nutrients and/or the presence of exogenous stress and have developed complex regulatory networks that enable quick responses [263,264]. These responses include differential expression of transcriptional regulators that leads to modulation of gene expression allowing the bacteria to adapt to the various stimuli [263].…”

Section: Discussionmentioning

confidence: 99%

ParA: A Novel Target for Anti-Tubercular Drug Discovery

Nisa¹

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<p>Tuberculosis (Tb) continues to be one of the world's greatest challenges in the public health arena. The current treatment for Tb entails a long duration of therapy making adherence to the whole course difficult. This has given rise to drug resistant strains of Mycobacterium tuberculosis which are posing a significant threat to Tb control strategies. To counteract this problem, there is an urgent need to develop alternative anti-tuberculous drugs which target processes that are critical for the growth and/or survival of this microbe. To identify such targets in M. tuberculosis, I used comparative genomics and mutagenesis data to identify conserved essential genes as viable targets for the development of broad-spectrum antibiotics. In addition, I validated the essentiality of three cell division genes in Mycobacterium smegmatis using conditional antisense RNA expression under different culture conditions. Furthermore, I performed high-throughput screens (HTS) using a differential susceptibility assay against one of the validated targets to identify its cognate inhibitor(s). Lastly, I developed a novel biochemical assay of the target to validate the specificity of the inhibitors identified in the HTS and evaluated the potency of the inhibitors against M. tuberculosis. This study identified 261 conserved putative essential genes as broad-spectrum targets. I hypothesized that antisense RNA expression of such genes will lead to its down-regulation and thereby affect the viability of the cells if these genes are essential. I also hypothesized that an essential gene will be required under all culture conditions. One gene, parA, demonstrated that it was essential under various culture conditions. This gene encodes for a protein which contain the conserved Walker A motif thus I theorized that it may posses ATPase activity. The results illustrated that the M. tuberculosis ParA protein possesses ATPase activity. This biochemical activity was used to validate two specific inhibitors of ParA, phenoxybenzamine and octoclothepin, which were identified in the cell-based HTS. Kinetic studies suggest that phenoxybenzamine is a mixed inhibitor while octoclothepin is a competitive inhibitor of ParA. This data is also supported by in silico docking. Both these compounds show low minimum inhibitory concentrations in M. smegmatis under nitrogen starvation conditions. In summary, this thesis illustrates that ParA is a viable target for anti-tubercular drugs. It demonstrates that ParA is an ATPase which has the potential to bind competitive and non-competitive inhibitors that can be exploited to target cell division in M. tuberculosis. Finally, this study presents phenoxybenzamine and octoclothepin as inhibitors of ParA. In conclusion, these compounds can either be developed to increase potency or be used as reference structures to screen for more potent inhibitors of the enzyme.</p>

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Oligonucleotide Microarray Technology and its Application to Mycobacterium avium subsp. paratuberculosis Research: A Review

Cited by 6 publications

References 99 publications

High-resolution melting for analysis of short sequence repeats in Mycobacterium avium subsp. paratuberculosis

High-resolution melting for analysis of short sequence repeats in Mycobacterium avium subsp. paratuberculosis

Analogues of uracil nucleosides with intrinsic fluorescence (NIF-analogues): synthesis and photophysical properties

ParA: A Novel Target for Anti-Tubercular Drug Discovery

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