Intercalator Dotaz Zobrazit nápovědu
Analysis of PCR fragments for applications, such as screening of nucleotide polymorphisms, detection of somatic mutations, or quantification of reverse-transcription PCR products, becomes central in clinical research as well as preventive testing, diagnostic screening, and pharmacogenomic genotyping. A variety of CE techniques, utilizing great potential of multicapillary-array sequencers, is now commonly applied in prevention, diagnosis, and treatment of a wide range of genetic diseases (cancer, cardiovascular, and neurodegenerative diseases, etc.). Costs of fluorescently labeled primers is often a major factor in large-scale projects requiring mutation analysis in hundreds or thousands of samples. In the present paper we introduce a simple approach of detecting unlabeled DNA fragments through intercalation without a need for adding intercalator to the separation polymer matrix. The dye is only added to the anode reservoir, and mixing with the separated DNA fragments takes place upon its migration opposite to the direction of the CE separation. Using two common intercalating dyes (ethidium bromide and SYBR Green II) we present this method as a tool for routine PCR detection and quantification.
A simple and elegant method for inhibition of non-templated nucleotide addition by DNA polymerases and for following DNA 3'-heterogeneity in enzymatic DNA synthesis by primer extension (PEX) is described. When template bearing ortho-twisted intercalating nucleic acid (ortho-TINA) at the 5'-end is used, non-templated nucleotide addition is reduced in both the A- and B-family DNA polymerases (KOD XL, KOD (exo-), Bst 2.0, Therminator, Deep Vent (exo-) and Taq). Formation of a single oligonucleotide product was observed with ortho-TINA modified template and KOD XL, KOD (exo-), Bst 2.0, Deep Vent (exo-) and Taq DNA polymerases. This approach can be applied to the synthesis of both unmodified and base-modified oligonucleotides.
- MeSH
- biotin chemie MeSH
- DNA-dependentní DNA-polymerasy chemie genetika MeSH
- DNA chemie genetika MeSH
- Geobacillus stearothermophilus enzymologie MeSH
- interkalátory chemie MeSH
- pyreny chemie MeSH
- Pyrococcus MeSH
- Thermococcus enzymologie MeSH
- Thermus enzymologie MeSH
- tritylové sloučeniny chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The short oligodeoxynucleotide (ODN) probes are suitable for good discrimination of point mutations. However, the probes suffer from low melting temperatures. In this work, the strategy of using acridine-4-carboxamide intercalators to improve thermal stabilisation is investigated. The study of large series of acridines revealed that optimal stabilisation is achieved upon decoration of acridine by secondary carboxamide carrying sterically not demanding basic function bound through a two-carbon linker. Two highly active intercalators were attached to short probes (13 or 18 bases; designed as a part of HFE gene) by click chemistry into positions 7 and/or 13 and proved to increase the melting temperate (Tm) of the duplex by almost 8°C for the best combination. The acridines interact with both single- and double-stranded DNAs with substantially preferred interaction for the latter. The study of interaction suggested higher affinity of the acridines toward the GC- than AT-rich sequences. Good discrimination of two point mutations was shown in practical application with HFE gene (wild type, H63D C > G and S65C A > C mutations). Acridine itself can also serve as a fluorophore and also allows discrimination of the fully matched sequences from those with point mutations in probes labelled only with acridine.
The present study was performed to examine the affinity of Escherichia coli mismatch repair (MMR) protein MutS for DNA damaged by an intercalating compound. We examined the binding properties of this protein with various DNA substrates containing a single centrally located adduct of ruthenium(II) arene complexes [(eta(6)-arene)Ru(II)(en)Cl][PF(6)] [arene is tetrahydroanthracene (THA) or p-cymene (CYM); en is ethylenediamine]. These two complexes were chosen as representatives of two different classes of monofunctional ruthenium(II) arene compounds which differ in DNA-binding modes: one that involves combined coordination to G N7 along with noncovalent, hydrophobic interactions, such as partial arene intercalation (tricyclic-ring Ru-THA), and the other that binds to DNA only via coordination to G N7 and does not interact with double-helical DNA by intercalation (monoring Ru-CYM). Using electrophoretic mobility shift assays, we examined the binding properties of MutS protein with various DNA duplexes (homoduplexes or mismatched duplexes) containing a single centrally located adduct of ruthenium(II) arene compounds. We have shown that presence of the ruthenium(II) arene adducts decreases the affinity of MutS for ruthenated DNA duplexes that either have a regular sequence or contain a mismatch and that intercalation of the arene contributes considerably to this inhibitory effect. Since MutS initiates MMR by recognizing DNA lesions, the results of the present work support the view that DNA damage due to intercalation is removed from DNA by a mechanism(s) other than MMR.
- MeSH
- adukty DNA chemie účinky léků MeSH
- anthraceny chemie MeSH
- chybné párování bází účinky léků MeSH
- DNA chemie účinky léků MeSH
- Escherichia coli chemie MeSH
- ethylendiaminy chemie MeSH
- financování organizované MeSH
- interkalátory farmakologie chemie MeSH
- molekulární struktura MeSH
- monoterpeny chemie MeSH
- organokovové sloučeniny farmakologie chemie MeSH
- poškození DNA MeSH
- ruthenium chemie MeSH
- sekvence nukleotidů MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- vazebný protein MutS opravné syntézy DNA chemie účinky léků MeSH
- vztahy mezi strukturou a aktivitou MeSH
This paper concerns the formation of biofilm in bacteria of the genus Arcobacter. A multiplex polymerase chain reaction (PCR) method was introduced and optimized for detecting biofilm while using the intercalating dyes ethidium monoazide (EMA) and propidium monoazide (PMA), first for analysis of strains of the genus Arcobacter from a collection, and then applied to samples of prepared biofilms. The results of the study indicate considerable variability among species of bacteria within the genus Arcobacter. The EMA-PMA PCR method can distinguish viable cells from dead cells and is therefore suitable for determining the viability of cells.
- MeSH
- azidy chemie MeSH
- biofilmy * MeSH
- Campylobacter genetika izolace a purifikace fyziologie MeSH
- interkalátory chemie MeSH
- mikrobiální viabilita * MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- propidium analogy a deriváty chemie MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
Despite the existence of a wealth of structural and theoretical data relating to palindromic sequences in the genome, the mechanism of cruciform formation in the presence of anthracyclines in miscellaneous biological processes is still poorly understood. Generally, DNA intercalators influence the DNA superhelicity, which plays a key role in the cruciform formation in DNA molecules. The potential of DNA intercalating ligands on the stabilization/destabilization of cruciform in DNA is discussed. Here, the indirect impact of anthracyclines to cell developing and surviving is analyzed for the first time. Primarily, the anthracycline modifies the helical properties of DNA and the overall DNA structure, and secondarily alters any cruciform-dependent processes, mainly DNA replication and transcription.
Selective binding of the wild type tumor suppressor protein p53 to negatively and positively supercoiled (sc) DNA was studied using intercalative drugs chloroquine (CQ), ethidium bromide, acridine derivatives and doxorubicin as a modulators of the level of DNA supercoiling. The p53 was found to lose gradually its preferential binding to negatively scDNA with increasing concentrations of intercalators until the DNA negative superhelix turns were relaxed. Formation of positive superhelices (due to further increasing intercalator concentrations) rendered the circular duplex DNA to be preferentially bound by the p53 again. CQ at concentrations modulating the closed circular DNA topology did not prevent the p53 from recognizing a specific target sequence within topologically unconstrained linear DNA. Experiments with DNA topoisomer distributions differing in their superhelix densities revealed the p53 to bind selectively DNA molecules possessing higher number of negative or positive superturns. Possible modes of the p53 binding to the negatively or positively supercoiled DNA and tentative biological consequences are discussed.
- MeSH
- akridiny chemie farmakologie MeSH
- chlorochin chemie farmakologie MeSH
- doxorubicin chemie farmakologie MeSH
- interkalátory chemie farmakologie MeSH
- kompetitivní vazba MeSH
- konformace nukleové kyseliny účinky léků MeSH
- lidé MeSH
- nádorový supresorový protein p53 chemie metabolismus MeSH
- superhelikální DNA chemie účinky léků metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
