Published online 15 September 2004
Nucleic Acids Research, Vol. 32 No. 16 © Oxford University Press 2004; all rights reserved
Improving specificity of DNA hybridization-based methods
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117871, Russia
* To whom correspondence should be addressed. Tel: +7 095 3306329; Fax: +7 095 3306538; Email: anton{at}humgen.siobc.ras.ru
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
+AY688954–AY688956
Received July 21, 2004; Revised and Accepted August 19, 2004
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 chimeric 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 technique presented here is of universal usefulness and can be applied to both cDNA and genomic DNA subtractions 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.
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