Title

A Single Nucleotide Polymorphism Melt Curve Assay Employing An Intercalating Dye Probe Fluorescence Resonance Energy Transfer For Forensic Analysis

Keywords

Dye; Forensics; FRET; Melt curve analysis; Multiple; Probe; SNP

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 an 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. © 2009 Elsevier Inc. All rights reserved.

Publication Date

8-1-2009

Publication Title

Analytical Biochemistry

Volume

391

Issue

1

Number of Pages

1-10

Document Type

Article

Personal Identifier

scopus

DOI Link

https://doi.org/10.1016/j.ab.2009.05.003

Socpus ID

67349104997 (Scopus)

Source API URL

https://api.elsevier.com/content/abstract/scopus_id/67349104997

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