Title

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

Authors

Authors

M. D. Halpern;J. Ballantyne

Comments

Authors: contact us about adding a copy of your work at STARS@ucf.edu

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

Share

COinS