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

Importance of the Topic

Accurate measurement of total nucleic acids extracted from diverse biological sources is essential for applications ranging from PCR and next-generation sequencing to clinical diagnostics and industrial quality control. Spectral unmixing in UV/Vis analysis enables precise discrimination of nucleic acid signals from co-absorbing contaminants.

Objectives and Overview

This application note describes the implementation of the Nucleic Acids (DNA equiv.) Unmix application on Lunatic and Little Lunatic spectrophotometers. The goal is to isolate the UV/Vis spectral fraction corresponding to total nucleic acids and calculate concentration in DNA equivalents without prior knowledge of sample origin.

Methodology and Instrumentation

The Unmix algorithm performs spectral deconvolution by fitting measured absorption spectra to predefined component profiles. Key components include:

• Nucleic acids (DNA equivalent profile, A260)
• Impurities (UV-absorbing non-nucleic acid molecules)
• Background turbidity (scattering profile)

The systems require only selection of the Nucleic Acids (DNA equiv.) application and a pure water blank. Instrumentation:

• Lunatic UV/Vis spectrophotometer
• Little Lunatic UV/Vis spectrophotometer

Main Results and Discussion

The application produces a component-separated spectrum showing nucleic acid (purple), impurities (blue) and background (gray). Concentration is calculated by multiplying the isolated A260 peak by the dsDNA factor (50 ng/µL per A260 unit). A residual or quality of fit (RRSE) value indicates the percentage of unassigned spectrum. Samples with RRSE above 2.5% or A260 below 0.5 trigger a warning and may lack detailed impurity/background reporting. Thresholds for impurity and background components ensure additional reporting when exceeded.

Benefits and Practical Applications

• Rapid, label-free quantification with minimal user input
• Applicable to mixed DNA, RNA, degraded fragments and various source types
• Automated multi-format reporting (HTML, XML, TXT, CSV, XLSX, PDF)
• High-throughput capability for research, QC/QC and industrial labs

Future Trends and Applications

Integration with laboratory information management systems (LIMS) and machine learning-driven spectral analysis could enhance automation and expand analyte coverage. Development of additional Unmix modules for proteins, metabolites or complex matrices may broaden platform utility.

Conclusion

The Nucleic Acids (DNA equiv.) Unmix application on Lunatic systems offers a streamlined, robust approach to quantify total nucleic acids in unknown samples. By isolating nucleic acid signals from impurities and background, it delivers reliable concentration data and comprehensive quality metrics with minimal user intervention.

Reference

• Unchained Labs. Quantification of total nucleic acids from unknown sources (DNA equiv.), Application Note Rev B, 2020.