Improving specificity of DNA hybridization-based methods

Methods based on DNA reassociation in solution with the subsequent PCR amplification of certain hybrid molecules, such as coincidence cloning and subtractive hybridization, all suffer from a common imperfection: cross-hybridization between various types of paralogous repetitive DNA fragments. Although the situation can be slightly improved by the addition of repeat-specific competitor DNA into the hybridization mixture, the cross-hybridization outcome is a significant number of background chi-meric clones in resulting DNA libraries. In order to overcome this challenge, we developed a technique called mispaired DNA rejection (MDR), which utilizes a treatment of resulting reassociated DNA with mismatch-specific nucleases. We examined the MDR efficiency using cross-hybridization of complex, whole genomic mixtures derived from human and chimpanzee genomes, digested with frequent-cutter restriction enzyme. We show here that both single-stranded DNA-specific and mismatched double-stranded DNA-specific nucleases can be used for MDR separately or in combination, reducing the background level from 60 to 4 % or lower. The tech-nique presented here is of universal usefulness and can be applied to both cDNA and genomic DNA sub-tractions of very complex DNA mixtures. MDR is also useful for the genome-wide recovery of highly conserved DNA sequences, as we demonstrate by comparing human and pygmy marmoset genomes