RESUMEN
We report a simple method for fluorescence detection of single-nucleotide alterations in a long target DNA, which is based on the formation of a three-way-junction-structured cholic-acid-binding DNA aptamer by the hybridization of the target with binary DNA probes. The new method was successfully exploited for SNP genotyping of human CYP2C19 gene.
Asunto(s)
Aptámeros de Nucleótidos , Sondas de ADN , ADN/química , Polimorfismo de Nucleótido Simple , Ácido Cólico/química , Citocromo P-450 CYP2C19/genética , Técnicas de Genotipaje , Humanos , Hibridación de Ácido NucleicoRESUMEN
We developed a light-up fluorophore-DNA aptamer pair for label-free aptamer sensors that fluoresce upon binding to the analyte. A 42mer DNA aptamer binding to the environment-sensitive fluorophore, dapoxyl, which increased the fluorescence by more than 700-fold upon binding, was successfully used to construct aptamer sensors by fusion with analyte-binding DNA aptamers.
Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , ADN/química , Fluorescencia , Luz , Espectrometría de FluorescenciaRESUMEN
To remove lipopolysaccharide (LPS) from pure water, we developed polymer hydrogels that selectively recognize LPS. A molecular imprinting technique was used to prepare the polymer hydrogels. We prepared the polymer hydrogels with LPS-binding sites by using acryloyllysine and acryloylphenylalanine as functional monomers and used lipid A as a template because it is the biologically active part of LPS and contains two phosphate groups. Co-existence of n-octane during the polymerization process was highly effective in promoting the formation of LPS-accessible sites on the surface of the hydrogels. Both an electrostatic and a hydrophobic interaction between the lipid A portion of LPS and the recognition site of the imprinted hydrogel are necessary for LPS recognition. The adsorption isotherm of LPS to the lipid A-imprinted hydrogels was Langmuir-type; the saturated adsorption capacity and the adsorption constant, calculated by applying an equation for Langmuir-type adsorption isotherms, were 1.0 × 10(-11)mol/cm(2) and 2.5 × 10(5)M(-1), respectively. The imprinted hydrogels selectively recognized toxic LPS in a competition experiment in which two other kinds of LPS with similar chemical structures to that of the LPS of E. coli (toxic LPS) were adsorbed to the lipid A-imprinted hydrogels.
Asunto(s)
Hidrogeles/química , Lípido A/química , Lipopolisacáridos/aislamiento & purificación , Impresión Molecular , Polímeros/química , Purificación del Agua/métodos , Adsorción , Sitios de Unión , Bacterias Gramnegativas/aislamiento & purificación , Lisina/análogos & derivados , Octanos/química , Fenilalanina/análogos & derivados , Microbiología del AguaRESUMEN
A simple and rapid method for the detection of single nucleotide polymorphisms (SNPs) is essential for the development of personalized medicine because SNPs correlate with some diseases and side effects of some drugs. Here we report a new method for the fluorescent detection of single nucleotide mutations that is based on the formation of cholic-acid binding DNA aptamers which form fully matched three-way junctions.
