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Methods

Fluorescent Detection and Isolation of DNA Variants Using Stabilized RecA-Coated Oligonucleotides

¹ Department of Biology, Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19711, USA ² NaPro Genomics, Newark, Delaware 19711, USA

Several genome resequencing strategies have been developed to detect genetic variation in populations and correlate diversity with phenotypic consequences. Commonly used methods of detecting single nucleotide polymorphisms (SNPs) use PCR amplification and indirect analysis, which can create template biases and enable user contamination. Here we present a novel assay to detect and isolate DNA variants using stabile nanostructures formed directly on duplex DNA. The assay incorporates the well-established RecA-catalyzed strand invasion process witha novel stabilizing hybridization step. First, short RecA-coated oligonucleotide filaments invade duplex DNA to form a synaptic intermediate or "D-loop." Sequentially, chemically modified oligonucleotide probes anneal to the displaced DNA strand of the complex to form a stable "double D-loop." These joint molecules resist dissociation when both oligonucleotides are completely complementary to the target duplex; however, if the probes are mismatched, the complex is inherently instable and rapidly dissociates. SNPs are identified by detecting the fluorophore assimilated into stable complexes produced by homologous probes compared to unstable differentially labeled mismatched probes. Furthermore, this strategy can be used to isolate specific allelic variants by affinity purification from complex populations. Stabilized double D-Loop intermediates accordingly offer the promise of haplotyping and pharmacogenomic analysis directly in double-stranded DNA samples.

³ Corresponding author.
E-MAIL ekmiec{at}udel.edu; FAX (302) 831-3427.

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