DNA Enrichment by Allele-Specific Hybridization (DEASH): A Novel Method for Haplotyping and for Detecting Low-Frequency Base Substitutional Variants and Recombinant DNA Molecules

Abstract

Detecting rare sequence variants in genomic DNA is central to the analysis of de novo mutation and recombination events and the detection of rare pathological mutations in mixed cell populations. Current PCR techniques suffer from noise that limits detection to variants present at a frequency of at least 10-4-10-5 per cell. We nowdescribe an alternative approach that recovers genomic DNA molecules containing a known single-nucleotide variant by hybridization selection using a biotinylated allele-specific oligonucleotide, followed by hybrid capture on streptavidin-coated paramagnetic beads and subsequent analysis by PCR. This technique of DNA enrichment by allele-specific hybridization (DEASH) is fast, effective for all tested single-nucleotide polymorphisms (SNPs), and can recover large (>10 kb) single-stranded molecules. A single round of DEASH is effective in separating haplotypes from genomic DNA and can not only readily detect and validate DNA molecules containing a single base change at a frequency of 10-5 per cell, but can also place these changes within the context of an extended haplotype. This technique offers a newapproach to the analysis of mutation and recombination, and has the potential to detect very rare de novo base substitutions.