Claire Corlett

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SABER-FISH Enabling the sensitive and multiplexed detection of nucleic acids within thick tissues

SABER-FISH Enabling the sensitive and multiplexed detection of nucleic acids within thick tissues

SABER first uses the Primer Exchange Reaction method to synthesize in the test tube a longer concatemer of identical short sequences shown here in red with the help of a catalytic self-folding DNA hairpin structure, a primer, and a DNA polymerase enzyme. The primer end of the resulting PER generated concatemer strand works with a handle that is complimentary to the target sequence within the genomic DNA or one of the many RNA molecules of a cell. In this example, three adjacent target sequences in a single molecule are simultaneously bound by three PER concatemers. The PER concatemers provide scaffolds onto which in the next step, multiple fluorescence imagers with short DNA sequences that are complimentary to the concatemer repeats can be assembled. This boosted fluorescence signal can even be further amplified by synthesizing branch concatemers with secondary and higher-order branches that are initiated at the internal sites of already existing PER concatemers. Once attached to DNA or RNA target molecules, these can incorporate a significantly higher number of fluorescent imagers and provide greater sensitivities. SABER amplified FISH analysis can be used in a tunable fashion to visualize a combination of DNA and RNA target inside the cell using fluorescence imagers that emit light at different wavelengths corresponding to different colors. The number of DNA and RNA targets, however, can be significantly increased by performing a technique known as DNA exchange, in which one set of fluorescence imagers bound to PER concameters at a series of DNA and RNA target sites is captured under the microscope, washed out of the sample, and replaced by another set of fluorescent imagers that binds the PER concameters at different DNA and RNA target sites. This exchange reaction can be repeated multiple times.

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