Sensitive and specific quantitation of biomolecules can be performed using fluorescence methods. Fluorophores can be described as molecules that absorb light at one wavelength (i.e. excitation wavelength) and subsequently emit light at another wavelength (i.e. emission wavelength).
It is possible to manipulate some fluorophore structures to fluoresce only if they are attached to a particular target molecule, for example, double-stranded DNA.
In fluorescence assays, this binding specificity is used to determine a direct relationship between the concentration of the biomolecule of interest in solution and the level of fluorescence emitted by a sample. A fluorophore can be mixed in a sample of known concentration to measure the relative fluorescent units (RFU). The relationship between the RFU and the concentration can be plotted and utilized as a standard curve.
Then, the emission of the same fluorophore, attached to unknown samples, can be plotted against this standard curve to identify the concentration of the sample. The binding specificity of the assays implies that the impact of contaminants (such as degraded nucleic acids or protein) included in the sample on quantitative accuracy is reduced.
Concentration Versus Total Mass
Optimization of fluorescent assays is performed over a core assay range of the total mass of the sample, defined by the fluorophore’s spectral properties and the chemistry of the assay. This range can be extended for certain assays by altering the volume of the sample added to the assay. To obtain reproducible and reliable results, it is essential for the total mass of the sample mixed to be within the range prescribed for that assay kit.
Reviewing the specifications based on common nomenclature is crucial when comparing concentration ranges reported for kits developed by various manufacturers. Three types of specifications may be specified:
- Initial sample concentration
- Concentration after being diluted in the assay tube
- Total mass of target biomolecule in the assay tube
Figure 1. Effect of sample volume on total mass of DNA in an assay. Image Credit: DeNovix Inc.
Example: Minimum Kit Concentration
While using 10 μL of the lowest initial sample concentration covered by a kit with a specified lower limit of 10 pg/μL, the result is an absolute mass of 100 pg in 200 μL. This can be represented as 0.5 pg/μL after dilution in the assay tube.
Lower initial sample concentrations can be quantified by increasing the volume (for example, 20 μL of a 5 pg/μL sample) in case the total mass per assay tube is not less than the lower specification equivalent.
Example: Maximum Kit Concentration
While using 10 μL of the highest initial sample with a specified upper limit of 25 ng/μL, the result is an absolute dsDNA mass of 250 ng in 200 μL. This can be represented as 1.25 ng/μL after dilution in the assay tube.
Higher initial sample concentrations can be quantified by reducing the volume (for example, 1 μL of a 250 ng/μL sample) in case the total mass per assay tube is not more than the upper specification equivalent.
DeNovix dsDNA Assay Kits
DeNovix fluorescence assays use three different fluorescence assays kits to enable dsDNA quantitation over an initial sample concentration range of 0.5 pg/µL to 4000 ng/µL. A simple mix-and-measure protocol is followed by each kit, with a simple, two-point standard curve.
- The DeNovix dsDNA Broad Range Assay is best suited for the quantification of sample concentrations in the range of 0.1 to 2000 ng/μL, with an extended upper range of 4000 ng/μL.
- The DeNovix dsDNA High Sensitivity Assay is used to quantify sample concentrations in the range of 5 pg/μL to 250 ng/μL.
- The DeNovix dsDNA Ultra High Sensitivity Assay has been improved to offer unrivaled quantitation sensitivity with a sample concentration range of 0.5 to 300 pg/mL. Precise and specific quantitation could be advantageous to single-cell applications, laser capture dissection, circulating tumor cell analysis, ChIP-Seq, and other techniques that rely on low input amounts of DNA.
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Figure 2. Measurement ranges of DeNovix dsDNA Fluorescence Quantification Kits. Image Credit: DeNovix Inc.
Key Features of DeNovix Assays
- Enhanced to work with DeNovix instruments and compatible with readers with suitable emission and excitation specifications
- Widest possible measurement range—Fewer reruns or dilutions needed than other commonly used assays (refer to Figure 3)
- Compatible with microplate readers—Come in 1000 assay kit size for high-throughput applications
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Figure 3. Comparison of assay measurement ranges of DeNovix and Qubit™ dsDNA Fluorescence Quantification Kits. Image Credit: DeNovix Inc.
Conclusion
The DeNovix dsDNA Fluorescence Quantification Assays offer the widest dynamic range and highest sensitivity available at present. Three highly specific assay kits are provided by DeNovix, covering a broad array of sample concentrations.
DeNovix kits can be used for accurate and selective measurement of dsDNA even when common contaminants like ssDNA, RNA, or protein are present. To realize the optimum sample QC procedure, the specificity of fluorescence can be combined with the purity measurements of absorbance on DeNovix’s DS-11 FX range.
DS 11 Series | Spectrophotometer | Fluorometer
Other Resources
About DeNovix, Inc..jpg)
DeNovix Inc. is an instrumentation company that designs, manufactures and sells laboratory equipment to meet the demands of today’s evolving life science technologies. Our focus is on providing innovative products and outstanding customer support. DeNovix is equipped with the financial, commercial and technical resources to deliver breakthrough products for your research success.
DeNovix offers the DS-11 Series Spectrophotometer/Fluorometer which combines fluorescence analysis and 1uL UV-Vis in the same instrument. Coupled with our new suite of dsDNA Fluorescence assays, DeNovix instruments provide a wider quantification range than any other instrument.
DeNovix instruments are found in life science research labs worldwide. Each instrument is a stand-alone system controlled by a built-in Android™ operating system (no PC). Labs love the smart-phone-like operation, impressive performance and the flexible connectivity of the instrument. Learn more about DeNovix Instruments and how they can benefit your lab.
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