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Sequence-Specific DNA Assay
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Molecular Devices

Measurement of specific DNA sequences can be achieved using the Threshold® System. Biotinylated and fluoresceinated oligonucleotide probes specific to the target sequence to be measured are used with the Immuno Ligand Assay (ILA) kit components. The probes are chosen so that they both anneal to the same strand of the target, adjacent to each other. DNA samples to be measured are digested with an appropriate restriction enzyme to liberate the target sequence on a small DNA fragment. The digested DNA is denatured in the presence of an excess of the biotinylated and fluoresceinated probes. The probes and denatured target are allowed to anneal, generating probe-target hybrids. These probe-target hybrids are captured on the biotinylated Threshold sticks using the ILA capture reagent (streptavidin) which binds to the biotinylated probe. The captured probe-target hybrids are then detected and quantitated using the ILA enzyme reagent (antifluorescein- urease conjugate) which binds to the fluoresceinated probe. This application note is intended as a guide and does not represent a validation of this assay, nor necessarily the optimal performance parameters for all probe and target DNA combinations.

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