Single-nucleotide polymorphism discovery by targeted DNA photocleavage
- *Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125; and †Applied Biosystems, Foster City, CA 94404
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Contributed by Jacqueline K. Barton, August 20, 2004
Abstract
Single-nucleotide polymorphisms are the largest source of genetic variation in humans. We report a method for the discovery of single-nucleotide polymorphisms within genomic DNA. Pooled genomic samples are amplified, denatured, and annealed to generate mismatches at polymorphic DNA sites. Upon photoactivation, these DNA mismatches are then cleaved site-specifically by using a small molecular probe, a bulky metallointercalator, Rhchrysi or Rhphzi. Fluorescent labeling of the cleaved products and separation by capillary electrophoresis permits rapid identification with single-base resolution of the single-nucleotide polymorphism site. This method is remarkably sensitive and minor allele frequencies as low as 5% can be readily detected.
Footnotes
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↵ ‡ To whom correspondence should be addressed. E-mail: jkbarton{at}caltech.edu.
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Abbreviations: SNP, single-nucleotide polymorphism; phzi, 3,4-benzo[a]phenazine quinone diimine; chrysi, 9,10-chrysene quinone diimine; phi, phenanthrenequinone diimine; TNF, tumor necrosis factor.
- Copyright © 2004, The National Academy of Sciences
