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.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Base Pair Mismatch / genetics
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Base Sequence
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Cattle
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DNA / genetics*
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DNA / radiation effects*
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Electrophoresis, Capillary
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Gene Frequency / genetics
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Genetic Techniques
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Humans
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Molecular Sequence Data
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Organometallic Compounds / chemistry
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Organometallic Compounds / radiation effects
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Photolysis
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Plasmids / genetics
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Polymorphism, Single Nucleotide / genetics*
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Polymorphism, Single Nucleotide / radiation effects
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Promoter Regions, Genetic / genetics
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Promoter Regions, Genetic / radiation effects
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Ruthenium Compounds / chemistry
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Ruthenium Compounds / radiation effects
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Templates, Genetic
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Tumor Necrosis Factor-alpha / genetics
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Tumor Necrosis Factor-alpha / radiation effects
Substances
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Organometallic Compounds
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Ruthenium Compounds
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Tumor Necrosis Factor-alpha
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DNA