RESUMO
DNA nanoswitches can be designed to detect unlabelled nucleic acid targets and have been shown to discriminate between targets which differ in the identity of only one base. This paper demonstrates that the fluorescent base analogue 2-aminopurine (AP) can be used to discriminate between nanoswitches with and without targets and to discriminate between matched and mismatched targets. In particular, we have used both steady-state and time-resolved fluorescence spectroscopy to determine differences in AP environment at the branchpoint of nanoswitches assembled using complementary targets and targets which incorporate single base mismatches.
Assuntos
2-Aminopurina/química , Pareamento Incorreto de Bases , DNA/química , Sondas de DNA/química , Transferência Ressonante de Energia de Fluorescência , Nucleotídeos/genética , Espectrometria de Fluorescência/métodosRESUMO
We present a new type of DNA switch, based on the Holliday junction, that uses a combination of binding and conformational switching to enable specific label-free detection of DNA and RNA. We show that a single RNA oligonucleotide species can be detected in a complex mixture of extracted cellular RNA and demonstrate that by exploiting different aspects of the switch characteristics we can achieve 30-fold discrimination between single-nucleotide mismatches in a DNA oligonucleotide.
Assuntos
Técnicas Biossensoriais/métodos , DNA/análise , RNA/análise , Técnicas Biossensoriais/instrumentação , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos/análise , Oligonucleotídeos/análise , Sensibilidade e EspecificidadeRESUMO
This work reports how the use of a standard integrated circuit (IC) fabrication process can improve the potential of silicon nitride layers as substrates for microarray technology. It has been shown that chemical mechanical polishing (CMP) substantially improves the fluorescent intensity of positive control gene and test gene microarray spots on both low-pressure chemical vapor deposition (LPCVD) and plasma-enhanced chemical vapor deposition (PECVD) silicon nitride films, while maintaining a low fluorescent background. This results in the improved discrimination of low expressing genes. The results for the PECVD silicon nitride, which has been previously reported as unsuitable for microarray spotting, are particularly significant for future devices that hope to incorporate microelectronic control and analysis circuitry, due to the film's use as a final passivating layer.