RESUMO
In a recent study, we showed how novel stem-loop DNA probes on dendron-modified aldehyde substrates could be used to detect synthetic nucleic acid targets without amplification. In this article, we demonstrate the application of stem-loop DNA probes as arrays for the detection of specific families and genera of methane-producing bacteria from sludge samples harvested from an anaerobic digester using 16S ribosomal RNA (rRNA) signatures. Specific 16S rRNA could be detected in samples that had 0.2ng/µl total sludge RNA without any target amplification.
Assuntos
Methanosarcinales/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Esgotos/microbiologia , Sequência de Bases , Methanosarcinales/isolamento & purificação , RNA Bacteriano/isolamento & purificação , RNA Ribossômico 16S/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
Specificity and sensitivity are important factors affecting DNA microarrays. Stem-loop DNA probes (SLPs) can be more specific in their recognition of target sequences than linear DNA probes, but unless they are carefully designed, surface interactions can disrupt the native stem-loop structure. In this study, we show how dendron-modified surfaces with well-defined, uniform spacing of aldehyde chemical functionalities offer an ideal substrate to immobilize SLPs and use them to detect nucleic acid targets. The mesospacing provided by the dendron-modified surfaces produces a solution-like environment that allows the SLPs to detect target nucleic acids at concentrations as low as 1pM in concentration.
