Accurate Crystal Digital PCR™ quantification of a human target gene using the naica® PCR MIX and EvaGreen®
EvaGreen® is a sequence-independent double-stranded DNA intercalating dye with low fluorescence when free in solution and high fluorescence when bound to DNA. Using EvaGreen® on the naica® system enables absolute quantification of a DNA target molecule using a simple primer pair and represents a low-cost solution when simultaneous quantification of multiple individual targets is not required. The naica® PCR MIX is a Polymerase Chain Reaction (PCR) master mix specially formulated to ensure robust partition compatibility and excellent linear Crystal Digital PCR™ quantification optimized for EvaGreen® on the naica® system.
Linear quantification across a 4-log dilution range using the naica™ PCR MIX
The naica™ PCR MIX is specially formulated for Crystal Digital PCR™ detection and quantification with the naica™ system using EvaGreen® dye chemistry. In the presence of varying levels of fluorescence background signal, the naica™ PCR MIX allows reliable quantification of human genomic DNA (hgDNA) across a 4-log dilution range (Figure 1).
Higher EvaGreen® final concentrations allow improved quantification of concentrated targets
In the presence of high concentrations of DNA, the signal from the proportion of EvaGreen® dye bound to the amplicon could become difficult to distinguish from the background signal contributed by the proportion of EvaGreen® dye bound to the template DNA. By increasing the final reaction concentration of EvaGreen® from 1.9 µM to 3.7 µM, the signal to noise ratio was improved (Figure 2). Compared to conventional EvaGreen®-compatible PCR master mixes, the naica® PCR MIX is available at high initial concentrations (5X and 10X), freeing up valuable reaction volume that can be instead occupied by additional sample input and/or increased EvaGreen® final concentrations up to 3.7 µM.
Technical Note Highlights
- The naica® PCR MIX enables an excellent linear Crystal Digital PCR™ detection across 4-log dilutions using EvaGreen® dye chemistry.
- naica® PCR MIX is available in two concentration formats, 5x and 10x concentrations, maximizing the reaction volume that can be occupied by a sample.
- naica® PCR MIX allows EvaGreen® concentration optimization increasing the detection sensitivity and resulting in improved assay separability.