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Highly Selective Isolation of Unknown Mutations in Diverse DNA Fragments: Toward New Multiplex Screening in Cancer¹

Dana Farber Cancer Institute, Adult Oncology and Radiation Oncology [S. C., B. D. P., Y. Z., G. M., G. M. M.] and Molecular Diagnostics Laboratory [E. A. F.], Harvard Medical School, Boston, Massachusetts 02115, and University of California School of Dentistry, Los Angeles, California 90095-1668 [S. T.]

Cancer research would greatly benefit from technologies that allow simultaneous screening of several unknown gene mutations. Lack of such methods currently hampers the large-scale detection of genetic alterations in complex DNA samples. We present a novel mismatch-capture methodology for the highly efficient isolation and amplification of mutation-containing DNA from diverse nucleic acid fragments of unknown sequence. To demonstrate the potential of this method, heteroduplexes with a single A/G mismatch are formed via cross-hybridization of mutant (TG) and wild-type DNA-fragment populations. Aldehydes are uniquely introduced at the position of mismatched adenines via the Escherichia coli glycosylase, MutY. Subsequent treatment with a biotinylated hydroxylamine results in highly specific and covalent biotinylation of the site of mismatch. For PCR amplification, synthetic linkers are then ligated to the DNA fragments. Biotinylated DNA is then isolated and PCR amplified. Mutation-containing DNA fragments can subsequently be sequenced to identify type and position of mutation. This method correctly detects a single TG transversion introduced into a 7-kb plasmid containing full-length cDNA from the p53 gene. In the presence of a high excess wild-type DNA (1:1000 mutant:normal plasmids) or in the presence of diverse DNA fragment sizes, the DNA fragments containing the mutation are readily detectable and can be isolated and amplified. The present Aldehyde-Linker-Based Ultrasensitive Mismatch Scanning has a current limit of detection of one base substitution in 7 Mb of DNA and increases the limit for unknown mutation scanning by two to three orders of magnitude. Homozygous and heterozygous p53 regions (GT, exon 4) from genomic DNA are also examined, and correct identification of mutations is demonstrated. This method should allow large-scale detection of genetic alterations in cancer samples without any assumption as to the genes of interest.

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