Detection of latent DNA using intercalating dyes

Author: Alicia Haines

Haines, Alicia, 2017 Detection of latent DNA using intercalating dyes, Flinders University, School of Biological Sciences

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This thesis outlines the use of DNA binding dyes that can target the collection of latent DNA deposited by touch at crime scenes. A range of dyes are available that bind to DNA at high specificity for laboratory-based applications but rarely applied to in situ detection. This work on a surface-based application of dyes for latent DNA detection has not been investigated previously.

Six common nucleic acid-binding dyes were selected due to their increase in fluorescence when in the presence of double stranded-DNA (SYBR® Green I, Diamond™ Nucleic Acid Dye, GelGreen™, GelRed™, EvaGreen™ and Redsafe™); four of the six dyes are permeable to cell membranes. The fluorescence from dye/DNA complex was detected using a high intensity light source, the Polilight® (PL500), an excitation wavelength of 490 nm and emission observed/recorded through interference filters centred at 530 nm or 550 nm depending on the dye emission. Some biological samples such as hair and skin were visualized under a fluorescent microscope (Nikon Optiphot) using a B2A filter cube.

Detection of DNA was observed within different biological samples such as saliva, skin, blood and hair which make it possible to select samples that are more likely to produce STR profiles after direct amplification. The properties of nucleic acid binding dyes were reviewed within Chapter 1 detailing the use of dyes in various applications along with the chemistry and common modes of interaction with DNA. Chapter 2 looks at the properties of common DNA binding dyes used with gel electrophoresis and determine their sensitivity in a gel medium. Chapter 3 outlines the effects the dyes had on DNA extraction, amplification, quantification and the amplification and detection of STR’s. Diamond™ dye (DD) was found to have the least effect of DNA extraction; GelGreen™ (GG) had the least effect on STR detection and amplification; GelRed (GR) quenched the fluorescent signal in the quantification of DNA (Qubit®) and has no STR amplification or detection. Chapter 4 discussed the results of the in situ detection of DNA within biological samples and as a surface based application. DD appeared to be the ideal dye for detection as it had a lower background signal compared with other dyes and had the highest DNA fluorescent enhancement after SYBR Green. Chapter 5 reviews DD along with other dyes for the detection of DNA within hair follicles; DD shows the staining of nuclei with limited background fluorescence and also showed successful direct STR amplification. Chapter 6 outlines the use of DD for quantitative PCR allowing for a more cost effective alternative to SG.

The outcome of this work is a novel means to detect DNA in situ within biological samples and on surfaces that make the screening of samples more efficient and successful. The investigation so far has concluded that EvaGreen™ and Diamond™ dye are the optimum dyes for this novel application based on their properties of binding and limited interactions with downstream forensic applications such as DNA extraction, amplification and STR typing. The use of these dyes as a screening methodology for tape-lifts would be a future application of this work to provide a way to selectively choose areas that have a higher DNA content for analysis.

Keywords: DNA Binding Dyes; EvaGreen; Latent DNA; SYBR Green

Subject: Biological Sciences thesis

Thesis type: Doctor of Philosophy
Completed: 2017
School: School of Biological Sciences
Supervisor: Prof. Adrian Linacre