Optimization of Diamond Nucleic Acid Dye for quantitative PCR

Area of Science:

• Molecular Biology
• Biochemistry

Background:

• Diamond Nucleic Acid Dye (DD) is a DNA stain used in gel electrophoresis.
• Its application as a detection dye in quantitative PCR (qPCR) remains underexplored.

Purpose of the Study:

• To evaluate Diamond Nucleic Acid Dye (DD) for quantitative PCR (qPCR) applications.
• To compare DD's performance against established qPCR dyes like SYBR Green, EvaGreen, and BRYT Green.

Main Methods:

• Investigated inhibitory effects of DD on qPCR across various concentrations (0.1-2.5×).
• Assessed qPCR efficiency, sensitivity, and linearity using serial DNA dilutions with DD.
• Compared DD-derived data with that from SYBR Green (SG), EvaGreen (EG), and BRYT Green (BG).

Main Results:

• DD demonstrated comparable performance to SG, EG, and BG in qPCR, achieving an R2 value >0.9 and efficiency of 0.83.
• Successfully detected mitochondrial DNA (mtDNA) signals across a wide DNA dilution range (~28 ng to 0.28 pg), showing sensitivity similar to other dyes.
• DD yielded lower Cq values than EvaGreen (EG) by nearly 7 cycles.

Conclusions:

• Diamond Nucleic Acid Dye (DD) is a suitable and effective dye for quantitative PCR (qPCR).
• DD presents a more economical and environmentally friendly alternative to existing qPCR dyes.