Group testing to annihilate pairs applied to DNA cross-hybridization elimination using SYBR Green I

A group testing (or pooling) method for DNA strands that identifies at least one strand in a pair of cross-hybridized oligonucleotides is given. This pooling method can be extended to any population of objects where certain pairs together produce an observable function or signal. Pairs of objects may work together to produce an undesirable result or a detrimental function. If just a single element of such a pair is identified and eliminated, then the undesirable function of that pair is destroyed. In particular, the ability to ensure that a set DNA probes do not yield undesired cross-hybridizations is important when these probes and/or their complements are used in the production of a hybridization signal that is intended to convey information. Here we report a "proof of principle" method, similar to those used to screen DNA libraries, that screens pools of probes for unwanted cross-hybridization events and identifies the offending probes. In the reported experiment, a cross-hybridized duplex in a pool of probes is detected by using the fluorescent dye SYBR Green I. This dye is known to produce greater fluorescence when bound to duplex DNA as opposed to single-stranded DNA. The method described here is sensitive, fast, and simple.