Fluorescent detection and isolation of DNA variants using stabilized RecA-coated oligonucleotides.
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.
Rice
Michael C
MC
Department of Biology, Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19711, USA.
Heckman
Brandy M
BM
Liu
Yi
Y
Kmiec
Eric B
EB
D004260:DNA Repair / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis*; D004270:DNA, Circular / Q000032:analysis; D005456:Fluorescent Dyes / Q000032:analysis*; D014644:Genetic Variation / Q000235:genetics*; D036103:Nanotechnology; D009693:Nucleic Acid Hybridization; D009696:Nucleic Acids / Q000235:genetics / Q000378:metabolism; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D011938:Rec A Recombinases / Q000378:metabolism*; D017422:Sequence Analysis, DNA / Q000379:methods / Q000639:trends; D013194:Staining and Labeling; D013696:Temperature
D004269:DNA, Bacterial; D004270:DNA, Circular; D005456:Fluorescent Dyes; D009696:Nucleic Acids; D009841:Oligonucleotides; D011938:Rec A Recombinases