Part 2: Unmix nucleic acid quality control with Lunatic
Blog
This blog is part 2 of a 3-part series exploring genomics with UV/Vis and Lunatic. Part 1 covers how UV/Vis is a first confidence check that keeps genomics workflows moving. Part 2 goes into unique Lunatic applications that simplify and accelerate DNA QC for sequencing. Part 3 follows Lunatic through real-world next-gen sequencing workflows.
Nucleic acid quality for the modern world
Researchers and health professionals are putting genomics to work in new and exciting ways every day by diagnosing diseases, powering cancer screening and treatment, and making gene therapies real. As genomics and next-gen sequencing become standard practice, samples now come from a wider range of sources, collection sites, and purification methods. With so much potential for variability, sample quality control has become a critical checkpoint in keeping downstream assays running smoothly.
Nucleic acid quality control for sequencing has been relying on the same metrics for more than 30 years: purity ratios from UV/Vis absorbance spectra. An A260/A230 ratio is often used to assess whether there are contaminating chemical components, like residual guanidine, phenol, or excess EDTA. The A260/A280 ratio helps determine nucleic acid purity. Pure DNA usually has a ratio of ~1.8, RNA usually has ~2.0, and lower values indicate either phenol or protein contamination. These ratios may be tried-and-tested, but they only tell you if a DNA or RNA sample is pure, not the actual concentration nor the identity of the impurities.
As we discussed in Part 1 of this series, Lunatic provides the standard A260/A280 and A260/A230 absorbance ratios as part of its nucleic acid measurements for quick quality checks, but it also has a more sophisticated way to investigate nucleic acid purity: Unmix.
Digging up nucleic acid dirt with Unmix
Mix blue and yellow dyes, and their absorbance spectra add together to make the solution look green. Biomolecules can muddy a spectrum the same way. DNA, RNA, proteins and other sample components contribute overlapping UV signals, making complex samples tough to quantify. Unmix taps into Lunatic’s full-spectrum UV/Vis data to quantify DNA or RNA in the presence of common impurities and reveal more about sample quality than absorbance ratios alone.
Each Unmix application fits the measured spectrum against a library of reference spectra of common impurities for a specific sample type. In the example below, the incompletely purified DNA contains residual protein. A single-wavelength measurement cannot deconvolute those overlapping signals, but Unmix can. It identifies each contribution, quantifies the DNA, and flags the protein contamination.
Unmix even tackles the turbidity common in nucleic acid samples. Despite being the result of light scattering, rather than true absorbance, it reduces light transmission and shows up in the measured spectrum. Unmix adds a wavelength-specific turbidity profile to the fit for a more reliable result. With concentration, impurity, and turbidity data in one read, Unmix gives you a faster way to troubleshoot variable nucleic acid samples before they trip up sequencing.
A tale of two impurities
Nucleic acid purification can leave behind UV-absorbing impurities like EDTA, which is commonly used in elution buffers to chelate Mg2+ and Ca2+ and inhibit DNases. At high enough concentrations, EDTA can interfere with polymerases needed for sequencing. Unmix flags the excess EDTA while still quantifying the DNA, helping scientists decide whether there is enough DNA to continue or additional purification is needed. In this example, EDTA was 5.5 times higher than normal. Unmix measured 97.1 ng/µL DNA while single-wavelength analysis overestimated the concentration at 109.5 ng/µL and missed the contamination.
Bead-based DNA purification delivers high recovery and works well with automation, but bead carry-over can also interfere with sequencing. Unmix detects this contamination as excess turbidity before downstream steps. In this example, Unmix measured 109 ng/µL DNA while single-wavelength analysis reported 132 ng/µL.
Conclusion
Lunatic’s Unmix deconvolutes complex UV/Vis spectra to precisely quantify nucleic acids, even when co-absorbing impurities are present. It also identifies common impurities and shows their severity, enabling scientists to quickly decide whether a sample is ready to move forward or requires repurification or further troubleshooting.
Lunatic
Lunatic is a UV/Vis spectrophotometer designed for accurate, microvolume quantification of proteins and nucleic acids. It delivers the capabilities of a general-purpose UV/Vis spectrophotometer, optimized for high-throughput, low-volume UV/Vis absorbance data from just 2 µL per sample across up to 96 wells of an SBS format plate. It precisely quantifies proteins, DNA, and RNA in minutes - revealing both concentration and purity without dilutions or extra prep