A single nucleotide polymorphism melt curve assay employing an intercalating dye probe fluorescence resonance energy transfer for forensic analysis

    Authors

    M. D. Halpern;J. Ballantyne

    Comments

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    Abbreviated Journal Title

    Anal. Biochem.

    Keywords

    SNP; FRET; Dye; Probe; Multiple; Melt curve analysis; Forensics; POLYMERASE CHAIN-REACTION; UNLABELED PROBE; DNA; HYBRIDIZATION; SNPS; PRODUCT; Biochemical Research Methods; Biochemistry & Molecular Biology; Chemistry, Analytical

    Abstract

    The characterization and use of DNA sequence polymorphisms are an important aspect of forensic analysis. A number of approaches are being explored for single nucleotide polymorphism (SNP) genotyping, but Current detection methods are Subject to limitations that adversely impact their utility for forensic analysis. We have developed a novel method for genotyping both single and Multiple SNPs that uses art intercalating dye and a probe labeled with a single fluorophore to affect a fluorescence energy transfer. Melting Curve analysis is then used to distinguish true alleles from mismatched alleles. We term the new method dye probe fluorescence resonance energy transfer (dpFRET). In the Current work, development proceeded at first with synthetic DNA template testing to establish proof of concept for the chemistry involved, followed by the design of polymerase chain reaction (PCR)-based genomic DNA assays to demonstrate potential forensic applications. The loci chosen for testing included both nuclear (MHC DRB) and mitochondrial DNA (cytochrome b) genes. A preliminary assessment of the sensitivity limits of the technology indicated that dpFRET was capable of accurately genotyping DNA from one single diploid cell equivalent. This technology Could also potentially impact a wide range of nonforensic disciplines to aid in discovery, screening, and association of DNA sequence polymorphisms. (C) 2009 Elsevier Inc. All rights reserved.

    Journal Title

    Analytical Biochemistry

    Volume

    391

    Issue/Number

    1

    Publication Date

    1-1-2009

    Document Type

    Article

    Language

    English

    First Page

    1

    Last Page

    10

    WOS Identifier

    WOS:000267063200001

    ISSN

    0003-2697

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