Optimization of Diamond Nucleic Acid Dye for Quantitative PCR

Abstract

Here, we evaluate Diamond Nucleic Acid Dye (DD) for use in quantitative PCR (qPCR) applications. Although DD is a commercially available stain for detection of DNA separated by gel electrophoresis, its use as a detection dye in qPCR has yet to be described. To determine if DD can be used in qPCR, we investigated its inhibitory effects on qPCR at concentrations ranging 0.1-2.5×. Serial dilution of DNA was used to determine the efficiency, sensitivity, and linearity of DD-generated qPCR data in comparison to other commonly used fluorescent dyes such as SYBR Green (SG), EvaGreen (EG), and BRYT Green (BG). DD was found to be comparable with other dyes for qPCR applications, with an R2 value >0.9 and an efficiency of 0.83. Mitochondrial DNA (mtDNA) target signals were successfully produced by DD over a DNA dilution range of ~28 ng- 0.28 pg, demonstrating comparable sensitivity to the other dyes investigated. Cq values obtained using DD were lower than those using EG by almost 7 cycles. We conclude that Diamond Nucleic Acid Dye is a cheaper, less toxic alternative for qPCR applications.

Illuminating touch deposits through cellular characterization of hand rinses and body fluids with nucleic acid fluorescence

“…DD is an external groove DNA-binding dye intended for visualization of nucleic acids in gels. Its use in quantitative PCR and microscopy for touch DNA has been reported in the forensic literature [26,5].…”

Microfluidic centrifugation assisted precipitation based DNA quantification

“…Detection of amplified DNA is often based on measurement of turbidity, 12 fluorescence after staining with a detection dye 13 or absorbance. 14 Commercially available instruments for DNA quantification can be broadly divided into three categories: instruments that measure turbidity (e.g.…”

Visualising latent DNA on swabs

“…The study looking at the recovery of cellular material showed that there was a higher percentage of recovery of DNA as a result of swabbing a non-stained fingermark than when applying DD to the fingermark and then collecting with a swab. DD, which is an external binder, binds to the sugar phosphate backbone of DNA, which is negatively charged [7]. It is therefore feasible that the overall negative charge of DNA molecules will be altered by staining, thus reducing the binding between DNA and fibres of swab.…”

“…Diamond ™ Nucleic Acid Dye (DD) (Promega, Madison, WI, USA) has recently been reported to stain DNA in samples such as hair roots and shafts [1,2], saliva [3,4], fingermarks [5] and other forensically relevant items [6]. The DD molecule is an external groove-binder that is able to bind to the backbone of DNA effectively, but it does not bind effectively to RNA and prokaryote supercoiled DNA [1,7]. DD has an excitation maximum at the blue wavelength (494 nm) and emits green fluorescence (558 nm) when bound to DNA.…”