We propose to adapt novel, entropy-driven DNA circuitry that has only recently been developed to pressing needs in point-of-care diagnostics. The metastable DNA circuits will be activated by hybridization to a specific mRNA allele, and will undergo a series of strand exchange amplification reactions, either linear or exponential, culminating in the production of a deoxyribozyme peroxidase that can generate an easy-to-read colorimetric signal. We will use a novel optimization technique, sequential injection analysis, to find reaction conditions that give robust signal-to-noise ratios under a variety of sample and environmental conditions. We will apply the optimized DNA circuitry to the detection of rifampicin resistant RNA polymerase alleles in M. tuberculosis in sputum samples obtained from Afghanistan. These experiments should set the stage for the development of a one-tube, colorimetric assay for point-of-care diagnosis of drug resistant M. tuberculosis infection.
The impact of marrying novel assay techniques, novel optimization methods, and realistic applications in the field cannot be underestimated for advancing options for public health monitoring and maintenance. The selfsame DNA circuitry can be readily redesigned for the detection of other mRNAs and alleles, and should therefore find great use in first world clinical settings, as well.
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