RESUMO
A novel template-dependent extension based isothermal amplification (TEIA) system with high single-base discrimination capability is developed, where the interference caused by non-specific reaction in isothermal strand displacement amplification (SDA) technique is substantially avoided via using a functionalized template probe, showing potential value in the development and application of SDA based detection devices.
Assuntos
DNA/análise , Técnicas de Amplificação de Ácido Nucleico , Pareamento Incorreto de Bases , DNA/metabolismo , Humanos , Sondas de Oligonucleotídeos/química , Sondas de Oligonucleotídeos/metabolismo , Polimorfismo de Nucleotídeo Único , Espectrometria de FluorescênciaRESUMO
The development of autonomous DNA machines and their use for specific sensing purpose have recently attracted considerable research attention. In existing autonomous machines, the target recognition process and signal transduction are separated from each other. This results in misunderstanding of the operation behavior, and the assay capability is compromised when serving as a sensing tool. In this communication, the integrated signal transduction-based autonomous aptameric machine, in which the recognition element and signal reporters are integrated into a DNA strand, is developed. This new machine can execute the in situ amplification of target binding-induced signal. The authentic operation behavior of autonomous DNA machine is discovered: the machine's products directly hybridize to the "track" rather than to the signaling probes. Along this line, the machine is employed to detect the cocaine in a more straightforward fashion, and improved assay characteristics (for example, the dynamic response range is widened by more than 500-fold) are achieved. Our efforts not only clarify the concept described in traditional autonomous DNA machines but also have made technological advancements that are expected to be especially valuable in designing nucleic acid-based machines employed in basic research and medical diagnosis.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/instrumentação , Cocaína/análise , Detecção do Abuso de Substâncias/instrumentação , Sequência de Bases , Fluorescência , Sensibilidade e Especificidade , TransdutoresRESUMO
A simple and universal quencher-free molecular beacon (MB) with low background fluorescence is developed based on an intermolecular G-quadruplex signaling probe. Unlike previous fluorescent MB strategies, it can function without any fluorophore and quencher modifications on its hairpin sequence.
