Fast & accurate DNA quantification with Lunatic

Significance of the topic

Accurate DNA quantification is a critical step in molecular biology workflows from next-generation sequencing preparation to diagnostic assays. Traditional dye-based methods require multiple dilutions, standard curves and extra reagents, increasing the risk of error, sample waste and delays. A streamlined approach that combines speed, sensitivity and purity assessment can greatly enhance throughput and data quality in research and clinical laboratories.

Objectives and study overview

This technical note evaluates Lunatic, a UV/Vis microvolume reader designed for rapid, high-throughput DNA measurement without dyes or extensive preparation. Key goals include

• Comparing classic UV absorbance at 260 nm with Lunatic’s built-in scatter correction and Unmix algorithms
• Assessing performance in the presence of common impurities such as beads, RNA, protein and salts
• Benchmarking against fluorescence-based assays (Qubit and PicoGreen)

Methodology and instrumentation

Samples of calf thymus DNA in TE buffer were prepared at ~100 ng/µL and spiked with either polystyrene beads, calf liver RNA, bovine serum albumin or salts (NaCl, EDTA). Measurements used 2 µL per sample loaded into SBS-compatible Lunatic plates or Little Lunatic chips. Lunatic records full spectra from 230 to 750 nm and applies Rayleigh scatter correction for A260 dsDNA quantification or proprietary Unmix deconvolution to separate nucleic acids from co-absorbing impurities. Results were compared with Qubit dsDNA BR and PicoGreen assays on a Qubit fluorometer.

Main results and discussion

Lunatic’s classic A260 method with wavelength-specific scatter correction provided accurate dsDNA concentrations and reliable A260/A280 and A260/A230 purity ratios, even in turbid samples. The Unmix algorithms successfully deconvoluted overlapping spectra, quantifying DNA in mixtures with RNA or protein and reporting impurity contributions. Fluorescence assays under-reported DNA when high salt was present, while Lunatic measurements remained stable. Throughput reached 16 samples in 2 minutes on Little Lunatic and 96 samples in 5 minutes on the standard system without cross-contamination or evaporation.

Benefits and practical applications

• Eliminates the need for dye preparation and standard curves, reducing hands-on time and error
• Handles clean to complex samples with reliable concentration and purity metrics
• Requires only 2 µL of sample, conserving precious material
• High throughput for core facilities and automated workflows
• Optional compliance package supports regulated environments

Future trends and potential applications

Integrating full-spectrum UV/Vis readers with laboratory automation will accelerate genomics pipelines. Advances in spectral deconvolution algorithms may expand quantification to include RNA-only or small RNA species. Combining purity assessment and nucleic acid sizing on a single platform could further streamline preparative workflows for sequencing and diagnostic assays.

Conclusion

Lunatic offers a robust, reagent-free method for rapid and accurate DNA quantification across a wide dynamic range. Its microvolume UV/Vis spectroscopy with scatter correction and Unmix deconvolution overcomes limitations of dye-based assays, providing concentration and purity data in minutes and supporting high-throughput genomic applications.

References

1. Dragan AI, Pavlovic M, McGivney JB, Casas-Fernandez JC, Crane RH, First EA. Characterization of PicoGreen interaction with dsDNA and the origin of fluorescence enhancement upon binding. Biophys J. 2010;99(9):3010–3019.
2. Singer VL, Jones LJ, Yue S, Haugland RP. Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation. Anal Biochem. 1997;249(2):228–238.