Quantification of total nucleic acids from unknown sources (DNA eq)

Importance of the Topic

Accurate measurement of total nucleic acids is vital for molecular biology workflows, diagnostic assays, and quality control in research and industrial laboratories. By isolating the UV/Vis spectral contribution of nucleic acids from co-absorbing species, the Lunatic Unmix application enables precise quantification of DNA-equivalent concentrations from diverse and unknown sample sources.

Study Objectives and Overview

This application note demonstrates the use of the Nucleic Acids (DNA equiv.) Unmix application on Lunatic and Little Lunatic platforms. The primary goals are to guide users through app selection, describe the spectral deconvolution workflow, and illustrate how results—nucleic acid concentration, impurities, and background turbidity—are reported and interpreted.

Methodology

Samples are measured by UV/Vis spectroscopy using water as a blank. The Unmix algorithm analyzes the full absorption spectrum to separate three components:

• Nucleic acids (purple profile): calculated from the A260 peak and a dsDNA factor (50 ng/µL per A260).
• Impurities (blue profile): non-nucleic acid absorbers reported in OD260; detailed reporting triggered when predefined thresholds for phenol, salts, detergents, or proteins are exceeded.
• Background (gray profile): turbidity signal subtracted from the raw spectrum; additional detailing provided when background markers like beads, heme, or chlorophyll exceed set thresholds.

The quality of fit (residual relative standard error, RRSE) is displayed as a yellow curve and percentage. A warning icon appears if RRSE exceeds 2.5% or if A260 is below 0.5 OD, indicating potential sample issues.

Instrumentation Used

• Lunatic high-throughput spectrophotometer system
• Little Lunatic compact UV/Vis spectrophotometer

Key Results and Discussion

The Unmix app successfully deconvolutes total nucleic acid content from complex UV/Vis spectra. It provides DNA-equivalent concentration values in ng/µL, flags impurity and background contributions, and quantifies contaminants only when they exceed diagnostic thresholds. The interactive results screen displays spectral profiles, concentration metrics, and quality warnings to support rapid sample assessment.

Benefits and Practical Applications

• Label-free, high-throughput quantification of total nucleic acids without specialized reagents.
• Automatic impurity and turbidity detection improve sample quality assessment.
• Multi-format reporting (HTML, XML, TXT, CSV, XLSX, PDF) integrates seamlessly with laboratory information management systems.

Future Trends and Opportunities

Advancements may include expanded spectral reference libraries for distinguishing RNA subclasses, integration with automated sample handlers, enhanced algorithms for low-concentration sensitivity, and miniaturized field-deployable spectrophotometers.

Conclusion

The Lunatic Unmix Nucleic Acids (DNA equiv.) application offers a robust, user-friendly solution for accurate quantification of total nucleic acids from unknown sample matrices. Its spectral deconvolution approach, automated impurity detection, and flexible reporting make it a valuable tool for research, QA/QC, and diagnostics laboratories.

References

Unchained Labs. Quantification of Total Nucleic Acids from Unknown Sources (DNA eq), Application Note Rev B, 2020.