Fast & accurate DNA quantification with Lunatic

Importance of the topic

Accurate and rapid quantification of DNA is fundamental for molecular biology, genomics and diagnostic workflows. Traditional fluorescent dye assays involve multiple pipetting and dilution steps that can introduce error, consume valuable sample and delay downstream processes. UV/Vis spectrophotometry offers direct absorbance measurements and purity metrics, but conventional instruments lack the throughput, dynamic range and impurity correction needed for modern high-throughput genomics applications.

Objectives and study overview

This technical note evaluates the performance of Lunatic, a UV/Vis microfluidic reader, for fast and precise DNA quantification. Key aims were to demonstrate:

• The dynamic range and throughput advantages of Lunatic versus standard dye assays
• The ability of Lunatic’s spectral correction and Unmix algorithms to quantify DNA in the presence of common impurities
• A comparison of DNA concentration results from Lunatic, Qubit and PicoGreen in spiked samples

Methodology and instrumentation

Samples of calf thymus DNA were prepared in TE buffer and supplemented with turbidity beads, calf liver RNA or bovine serum albumin to mimic common contaminants. Two system formats were tested:

• Little Lunatic: chip format, 16 samples in 2 minutes
• High Lunatic: plate format, 96 samples in 5 minutes

Each 2 µL sample was analyzed with:

• The A260 dsDNA application and its variant A260–bg(340) for turbidity correction
• Unmix applications (“DNA Mammalian” and “Nucleic Acids (DNA equiv.)”) to deconvolute overlapping absorbance spectra of DNA, RNA, protein and buffer components

Main results and discussion

Baseline-corrected spectra allowed accurate dsDNA quantification even in turbid samples, with Rayleigh scattering removed across 230–750 nm. Unmix algorithms successfully separated DNA from RNA or protein contributions, reporting individual concentrations and purity ratios (A260/A280 ~1.8, A260/A230 >2). In salt-spiked samples (400 mM NaCl), Lunatic measurements remained within 5% of the theoretical 100 ng/µL value, whereas Qubit and PicoGreen underestimated DNA by more than 20%.

Microfluidic plates and chips prevented cross-contamination and evaporation, ensuring reproducibility. The broad dynamic range (1.5–13,750 ng/µL or 0.03–275 OD) and minimal sample requirement (2 µL) streamline workflows by eliminating dye preparation, standard curves and extensive dilutions.

Benefits and practical applications of the method

• High throughput: up to 96 samples in 5 minutes or 16 in 2 minutes
• Wide dynamic range without reagents or dilution steps
• Purity assessment via A260/A280 and A260/A230 ratios plus spectral deconvolution
• Compatibility with impure or “messy” samples containing salts, proteins or phenol
• Minimal sample consumption and no cross-contamination
• Automation-friendly plate format and optional compliance package (21 CFR Part 11)

Future trends and applications

Integration of Lunatic systems with laboratory automation will further accelerate genomics pipelines. Expansion of Unmix algorithms to other biomolecules (RNA-seq, proteins) and integration of AI-driven data analysis could enhance impurity recognition and quantification accuracy. Cloud-based reporting and remote monitoring will support distributed laboratory networks and large-scale biobanking initiatives.

Conclusion

Lunatic provides a robust, high-throughput UV/Vis solution for DNA quantification that overcomes the limitations of fluorescent dye assays. By combining microfluidic sampling, broad dynamic range and advanced spectral deconvolution, it delivers fast, accurate concentration and purity measurements from pure or complex samples with minimal manual effort.

References

• Dragan AI, et al. Characterization of PicoGreen interaction with dsDNA and the origin of its fluorescence enhancement upon binding. Biophys J. 2010;99(9):3010–3019.
• Singer VL, et al. Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation. Anal Biochem. 1997;249(2):228–238.