Sensitive quantiﬁcation of DNA in a sample is of vital importance to applications such as forensic DNA analysis where low amounts of DNA may be present. Similarly, accurate DNA quantiﬁcation is paramount for Next Generation Sequencing (NGS), where precise amounts of DNA are required to enable this powerful tool in the investigation of genetic phenomena involved in mutations that lead to cancer or disease. Fluorescent quantiﬁcation methods have proven accuracy and excellent sensitivity. An analysis of cyanine dyes indicated that AccuBlueTM NextGen from Biotium exhibits the best sensitivity available1.
Here we show the performance of DNA quantiﬁcation using AccuBlueTM NextGen reagent with detection by the CLARIOstar microplate reader. The results indicate that this combination of reader and reagents provides excellent sensitivity that extends to detection in 384-well plates.
Detection of low concentrations of DNA is achieved with AccuBlueTM NextGen by using a dye with low intrinsic ﬂuorescence in the absence of nucleic acids but a large ﬂuorescence enhancement in the presence of DNA. In addition, the use of a patented enhancer technology to further suppress background ﬂuorescence results in an even lower limit of detection (Figure 1).
Materials & Methods
- AccuBlueTM NextGen dsDNA Quantiﬁcation Kit (Biotium #31060)
- CLARIOstar microplate reader (BMG LABTECH)
- Black, 96-well plates (Costar)
- Black, 384-well plates (Corning)
Standard curves were prepared using 1X AccuBlueTM NextGen Buffer and High Sensitivity dsDNA Standard (10 ng/μl), supplied with the kit. For tests using 96-well plates, dsDNA dilutions were prepared ranging from 3000 to 1 pg. To show assay performance in 384-well plates dsDNA dilutions ranged from 750 to 0.25 pg.
Assays using 96 well plates were performed as described in the instructions from Biotium.
Assays using 384-well plates were performed as described with the following modiﬁcations. For the triplicate samples and buffer blank 50 μl of working solution was added to wells followed by 2.5 μl of varying concentrations of dsDNA for a standard curve or buffer for blanks. Following incubation plates were read using CLARIOstar test protocols with the following settings:
|Optic settings||Fluorescence intensity, top optic
|Focus height and gain adjusted prior to reading|
||Settling time: 0.2 s (96 well); 0.1 s (384 well)|
|40 flashes per well|
Results & Discussion
A previous comparison of ﬂuorescent cyanine dyes indicated that AccuBlueTM NextGen was linear from 5 to 5000 pg1. Our results (Figure 2) indicate linear detection from 1 to 3000 pg in agreement with the kit literature.
Figure 3 examines the same data set presented in Figure 2 but employs only the 6 lowest concentrations of DNA used. This provides a better appreciation of assay linearity at low concentrations.
Furthermore, previous results indicated that AccuBlueTM NextGen had a limit of detection (LOD) of 50 pg1 using the formula:
LOD = xbl + ksbl
Where xbl is the mean of blank, σbl is the standard deviation of the blank, and k is numerical conﬁdence factor chosen to be 3. Using the same formula we calculated the LOD to be nearly 4 pg for our 96 well assay.
Next, we sought to investigate the suitability to measure this same assay in 384 well plates. Again, in this scenario, we saw that the assay was linear across the entire tested range from 750 to 0.25 pg (Figure 4). Finally, we were able to calculate the LOD for this 384 well assay to be 0.309 pg.
The CLARIOstar plate reader has the sensitivity needed to enable optimal performance of the AccuBlueTM NextGen dsDNA Quantiﬁcation Kit. We obtained excellent linearity across all DNA concentrations tested and observed LOD values that are better than previously reported. Performance extends to 384 well plates where calculated LOD is even better than in 96-well plates. Thus this combination allows for excellent sensitivity in the quantiﬁcation of DNA and the ability to conserve samples when needed.
- Bruijns, B.B. et al. (2016). Fluorescent cyanine dyes for the quantiﬁcation of low amounts of dsDNA. Anal. Biochem. 511, 74-79.