The DNase Assay Kit (Fluorescence) is a fluorescent-probe DNase activity assay used to detect DNase contamination in environments and materials during scientific research and production. It measures activity down to 1.25×10⁻⁶ U/μL — 8× more sensitive than leading competitor kits.

As mRNA synthesis and biopharmaceutical production scale up, trace DNase contamination on lab surfaces, consumables, or in reagents can silently degrade fragile DNA and RNA materials. The DNase Assay Kit (Fluorescence) uses a fluorophore-labeled DNA probe: when DNase is present, the probe is cleaved, separating the fluorophore from its quencher and producing a fluorescent signal proportional to enzyme activity. Compared with UV spectrophotometry or gel electrophoresis, this DNase detection kit offers far higher sensitivity, faster turnaround, and reliable quantitative results — making it a standard tool for plasmid, mRNA, and process-impurity safety testing.

Background

In recent years, the application of mRNA synthesis technology has become more and more extensive, and the requirements for quality control in the process of mRNA vaccine and drug research and production are also increasing. Deoxyribonucleases (DNases) and ribonucleases (RNases) are ubiquitous enzymes that are present in both the environment and many biological materials. They can degrade DNA or RNA, affecting many research and production processes involving DNA and RNA. Due to the fragility of mRNA molecules, it is extremely important to inhibit or eliminate nuclease activity in research and production environments.

To this end, we need a fast and sensitive method to detect nucleases in the environment (such as laboratory benches, pipette tips, storage bags, reactors and other consumables) and materials (such as antibodies, enzymes, buffers), etc. activity, determine the presence of DNases or RNases contamination and quantitatively assess the extent of contamination.

For defining pollution-free methods, there are no clear regulations in the "USP43", "EP10", "Chinese Pharmacopeia 2020 Edition" and other documents. The limit of quantification of nucleic acid hydrolysis-UV spectrophotometry is only 0. 01U/μL, which is not suitable for the detection of micro nuclease' s activity.

The nucleic acid hydrolysis-gel electrophoresis method is greatly affected by the subjective judgment of the experimenter and cannot be accurately quantified. In addition, the long operation time also reduces the measurement throughput. High performance liquid chromatography (HPLC) and electrochemical methods are time-consuming and labor-intensive, and are limited by equipment. In this regard, some researchers [1] proposed a fluorescent probe method, which not only has high sensitivity and fast detection speed, but also can achieve quantitative detection of nuclease activity. The DNase/RNase fluorescent probe detection kit is one of the best choices.

Detection Method

The method of DNase and RNase Assay kits are the same. Fluorophore-labeled DNA probes or RNA probes are designed and mixed with the test sample.

When the sample does not contain DNases or RNases activity, the probe is stable, the distance between the fluorophore and the quencher group is relatively small, no fluorescent signal is generated due to the principle of fluorescence resonance energy transfer.

When the sample contains DNases or RNases activity, the probe is degraded, the fluorophore and the quencher group move away from each other, resulting in a gradually enhanced fluorescent signal.

The rate of increase in fluorescence signal is positively correlated with the number and activity of the enzyme.

Detection method is shown as following:

Fig. 1 Schematic diagram of the principle of the fluorescent probe method.

Figure 2. Schematic diagram of the DNase/RNase detection kit reaction curve.

Product Pictures

DNASE ASSAY KIT (FLUORESCENCE)

Deoxyribonucleases (DNases) are ubiquitous enzymes present in both the environment and many biological materials. They degrade DNA and can affect any research or production process that depends on intact DNA or RNA — which is especially critical given how fragile mRNA molecules are. A DNase Assay Kit is a laboratory tool used to detect and quantify DNase activity in environments (lab benches, pipette tips, storage bags, reactors, consumables) and materials (antibodies, enzymes, buffers), confirming whether DNase contamination is present and how severe it is.

Neither the USP43, EP10, nor the Chinese Pharmacopoeia (2020 Edition) specify a clear regulatory method for defining "nuclease-free." The traditional nucleic acid hydrolysis–UV spectrophotometry method has a quantification limit of only 0.01 U/μL, too insensitive for trace nuclease detection, while gel electrophoresis is subjective, hard to quantify precisely, and slow. HPLC and electrochemical methods are accurate but time-consuming, labor-intensive, and equipment-limited. The fluorescent probe method addressed by this kit was developed to solve exactly this gap: high sensitivity, fast detection, and reliable quantification in one assay.

Application

The DNase Assay Kit (Fluorescence) is used to detect DNase contamination in the environment and materials in scientific research and production processes — including plasmid DNA production, mRNA manufacturing, and other DNA/RNA-dependent biopharmaceutical workflows. The assay detects and quantifies DNase in buffers, consumables, purified IVT product, and mRNA drug substance.

Characteristics of Products

High Sensitivity: Limit of Detection is as low as 1. 25×10^-6 U/uL (DNase I), which is only 1/8 of the best competitor kit.
High precision: intra-batch CV≤10%, inter-batch CV≤15%.

Good stability: valid for one year at -20°C. Once the probe solution is prepared, split and stored at -20°C.

Precision and Stability of Hyzmes DNase Assay Kit

Sensitivity higher than gel electrophoresis method

The Hzymes DNase Assay kit was in comparison with the nucleic acid hydrolysis-gel electrophoresis method, the same low-concentration enzyme samples were measured as shown in the figure:

Hyzmes DNase Assay kit measures 3x10°° U/uL DNase |,   which can be clearly distinguished from 0.

Sensitivity: Hzymes DNase Assay kit VS competitor kit

Compared with one of the best competitor kits (fluorescence), the limit of detection of Hzymes DNase Assay kit is only 1/8 of that of the competitor kit.  

When the sample concentration is 6. 25×10^-6 U/μL (low-value sample), the signal intensity of the competitor's DNase detection kit is indistinguishable from the negative control.  

When the sample concentration is 6. 25×10^-6 U/μL (low-value sample), the signal intensity of the Hzymes DNase detection kit is clearly distinguishable from the negative control.  

When the sample concentration of DNase I is from 1×10^-6 to 10×10^-6 U/μL (low-value sample), the Signal-to-noise (S/N) values of the competitor's DNase detection kit is close to 1, however the signal intensity of Hzymes' DNase Assay Kit is positive correlated to the concentration of DNase I.

Composition of the Hzymes DNase Assay Kit

Note: The standard is DNase I. The unit for DNase I activity is defined as the amount of enzyme that completely degrades 1μg of pBR322 DNA in DNase I reaction buffer at 37°C for 10 minutes [1]; one DNase I activity unit is equivalent at 0. 3 Kunitz units [2].

Equipment and consumables required

Fluorescence microplate reader (including ex/em=485/525nm wavelength).
DNase & RNase-free pipettes and tips.
DNase & RNase-free EP tube.
DNase & RNase-free black non-transparent 96-well plate.

Preparation before experiment (check the user guide for more details)

01. Take out the DNase Assay kit and equilibrate to room temperature (18~25°C), shake and mix the components then centrifuge immediately
02. Centrifuge the DNA probe, add TE buffer to dissolve the powder. Aliquot this DNA probe storage solution; Store at-25~15°C away from light and avoid repeated freezing and thawing.
03. Set the instrument parameters: Shaking plate 10~15s before detecting; Excitation wavelength λ Ex=485nm; Emission wavelength λEm=525nm; Temperature 37°C; Endpoint mode.
04. Set the gain to auto-scale if possible, if the microplate reader supports kinetic mode, it is recommended to use the kinetic detection mode.
05. Qualitative detection: select four wells on a 96-well plate: 2μL of DNase I standard solution is diluted with standard dilution Buffer and DNase & RNase-free water to 2×10< sup> -5< /sup> U/μL. Take 80μL as the positive control; 80μL of DNase & RNase-free water as the negative control, 80 μL samples were prepared in duplicate (if insufficient, dilute to 80 μL with DNase & RNase-free water). Add 10 μL DNA probe working solution and 10 μL 10×reaction solution to each well. Read the fluorescence signal value RFU0 immediately; Inculate the plate in the dark at 37°C for 30min, then read the value RFU30 immediately.

If RFU30 (sample to be tested) ≥ 2 × RFU0 (sample to be tested), it is considered that the sample to be tested is contaminated with DNase.

06. Quantitative detection: Take 2μL of DNase I standard solution and dilute it with standard dilution buffer and DNase-free water to 1×10< sup> -5< /sup> U/μL, 5×10< sup> -6< /sup> U/μL, 2. 5×10< sup> -6< /sup> U/μL, 1. 25×10< sup> -6< /sup> U/μL as standards; DNase & RNase-free water as negative control; follow the same procedures of the qualitative detection, calculate ΔRFU=RFU30-RFU0, with ΔRFU as the ordinate, and the standard DNase I concentration as the abscissa (perform a linear fit, find the fitting equation y=x+b, put ΔRFU (sample to be tested) into the equation as y, identify x, multiplied by the value of sample pre-dilution, it is the approximate concentration value of the sample to be tested. )

Q&A

What is the reaction temperature and time of the DNase Assay kit?

Incubate the reaction system at a constant temperature of 37°C for 30 minutes.

When could false positive or false negative results or inaccurate quantitative results occur?

Gel buffers, high concentrations of viscous substances, surfactants, and dark solutions may interfere with fluorophore luminescence. Substances that inhibit DNase activity — high ionic strength solutions (e.g. 5M NaCl, 20x SSC, 3M sodium acetate), buffers with pH below 4 or above 9, chaotropic agents, detergents, or protein-denaturing solutions such as SDS, guanidine thiocyanate, urea, or EDTA — can also affect results. Solutions that chemically destabilize the DNA probe (pH > 9, strong acids/bases, bleach) are a further source of error.

Why use the standard dilution buffer instead of DNase & RNase-free water when preparing standard solutions?

The standard is relatively stable in the standard dilution buffer even after serial dilution. If diluted with water instead, the standard's activity may drift during serial dilution, causing deviation.

How do you adjust the appropriate microplate reader gain value?

Select the auto-gain option if available. Otherwise, set a middle value within the instrument's gain range, then adjust based on the positive control's fluorescence signal — reduce gain if the signal exceeds the instrument's upper limit, or increase it if the signal is far below that limit.

Why might a seriously contaminated sample lead to a false negative result?

Contamination is judged by RFU30 ≥ 2×RFU0. In a heavily contaminated sample, the reaction can proceed so quickly that a very high RFU0 is already measured at time zero, making RFU30 appear to be less than twice RFU0 even though contamination is present. Diluting the sample with DNase & RNase-free water resolves this.

If the negative control's RFU value is not 0, does that mean it is contaminated?

Not necessarily. The negative control can show a low baseline RFU signal that does not increase significantly as the reaction proceeds. It is considered uncontaminated as long as RFU30 stays below twice RFU0.

Product details

Hzymes offers a companion RNase Assay Kit (Fluorescence) built on the same fluorescent-probe detection principle, with a limit of detection as low as 0.313 pg/mL (≈1.56×10⁻⁹ U/μL) for RNase A — also 1/8 that of the best competitor kit.