Fabrication of Renewable RNA Microarrays and Methods
Background: Numerous methods and devices are available to detect and/or quantify target DNA in a test sample. In almost all of these methods, detection and/or quantification are performed utilizing a detectable label such as radiolabels, fluorescent and luminescent labels. Methodologies employing labels are costly and laborious. RNA microarrays for sensitive target DNA detection not requiring labels have been described. The major issue with creating RNA microarrays is that the attachment of probe RNA molecules to the chip monolayer involves multiple expensive and laborious processing steps that can lead to RNA degradation. Furthermore, each chip can only be used once for DNA detection, and chips cannot be regenerated for repeat use. Technology: Researchers at the University of California have developed a novel method utilizing RNA-DNA surface ligation chemistry to create "renewable" RNA microarrays from DNA microarrays. The RNA microarrays can be used together with RNase H for ultrasensitive detection of target DNA by enzymatically amplified surface plasmon resonance imaging (SPRI).
Specifically, the invention uses a process by which RNA probe molecules are covalently attached to single stranded anchor DNA which had been chemically attached to a microarray chip. Single stranded test sample DNA is then exposed to the microarray and specific RNA-DNA heteroduplexes are formed by hybridization. Amplification occurs by releasing the hybridized target DNA from the RNA-DNA heteroduplex by RNase H mediated hydrolysis of the RNA strand. This cycle is repeated until all RNA probe molecules are destroyed by a very small number of input DNA target molecules, and the corresponding change in reflectivity is measured by SPRI.
The invention can in principle be used with any DNA microarray. Fabricating the RNA microarray in-situ using unlabeled RNA means that there is minimal ex-situ handling or chemical exposure that may cause RNA degradation. Reduced RNA handling improves the reproducibility of the arrays, and there is no need to utilize costly, chemically-modified RNA. Due to a spacer effect of the anchor DNA, enzymatic activity of RNAse H is drastically increased allowing for assays to be performed rapidly. The RNA microarray surface is "renewable" and can be regenerated in situ after RNase H hydrolysis. Thus, an array can be reused where previously a new RNA array had be fabricated for each DNA detection assay. Application: This invention can be utilized in a variety of bioresearch areas including diagnostics, gene expression analysis, biowarfare detection and viral identification.
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