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

Abstract

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 with a 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.

Regulation of targeted gene repair by intrinsic cellular processes

“…Once bound and annealed, the ssODN forms a three‐stranded heteroduplex with the target site. Overexpression of genes such as RAD52, RAD54, RECA, and RAD51, all of which are involved in promoting the pairing process, has been shown to enhance gene correction 29–35. Furthermore, the presence of homologous recombination repair (HRR) proteins enhances repair in bacteria, yeast, and mammalian systems,29, 31, 32, 36–39 supporting a role for conventional DNA pairing activity in the ODN‐mediated repair reaction.…”

Marking of specific sequences in double-stranded DNA molecules—SNP detection and direct observation

“…Attempts have been made to make the structure stable enough to withstand various subsequent manipulations (Fujiwara et al 1998a,b;Potaman et al 2002;Roulon et al 2002). Recently, Rice et al (2004) reported that a D-loop formed with RecA protein is stabilized by converting it to a stable double D-loop.Using a different approach, we have devised a method to stably mark specific sequences in double-stranded DNA molecules by converting a RecA-mediated triple-stranded structure to a stable one by use of a second deoxyoligonucleotide (a splint oligonucleotide), to which the first probing oligonucleotide is aligned to covalently ligate itself. The resultant regional multi-(possibly quintuple) strand structure is stable and easily detectable by various means, including observation by scanning probe microscopy such as atomic force microscopy (AFM).…”

“…Attempts have been made to make the structure stable enough to withstand various subsequent manipulations (Fujiwara et al 1998a,b;Potaman et al 2002;Roulon et al 2002). Recently, Rice et al (2004) reported that a D-loop formed with RecA protein is stabilized by converting it to a stable double D-loop.…”