Quantification of purified PCR samples

Importance of the Topic

PCR-based workflows rely on precise measurements of amplicon yield to ensure efficient downstream applications such as sequencing, cloning, or quantitative analyses. Traditional absorbance readings may overestimate target concentration by including co-absorbing impurities or scattering artifacts.

Objectives and Study Overview

This work evaluates an Unmix algorithm implemented on the Unchained Labs Lunatic and Little Lunatic UV/Vis spectrophotometers to isolate the specific DNA amplicon signal from background and impurity contributions, enabling accurate concentration determination of purified PCR products.

Methodology and Instrumentation Used

• Sample type: purified PCR products processed with various commercial cleanup kits.
• Instrumentation: Lunatic and Little Lunatic spectrophotometers equipped with the Purified PCR Unmix application.
• Analytical approach: Deconvolution of UV/Vis spectra into amplicon, impurity, and turbidity components using a dsDNA concentration factor.
• Pure water blanks are used for background calibration; no additional user input is required beyond sample names.

Main Results and Discussion

• Spectral profiles for amplicon, impurities and background were successfully resolved, and the residual fitting error (RRSE) below 2.5% ensures high-quality quantification.
• Low OD260 samples (<0.5) or those with high residue trigger reporting of total nucleic acids instead of DNA-specific readings.
• Reporting formats include HTML, XML, TXT, CSV on all systems, plus XLSX and PDF on the Lunatic.
• A comparative study showed Unmix quantification closely matched fluorescence-based measurements, while raw A260 values were consistently higher due to non-target contributions.
• Fluorescence assays calibrated with lambda DNA underestimate concentrations of fragments under 1000 bp, whereas the Unmix method remains agnostic to fragment length.

Benefits and Practical Applications

• Label-free, rapid quantification of PCR amplicons with minimal user interaction.
• Elimination of background and impurity interference for reliable concentration measurements.
• Flexible reporting outputs to fit diverse laboratory workflows.

Future Trends and Applications

• Integration into high-throughput and automated platforms.
• Extension of spectral deconvolution libraries to other nucleic acid types and complex matrices.
• Enhanced software algorithms for improved sensitivity and broader application range.

Conclusion

The Unmix Purified PCR application on Lunatic platforms provides accurate, impurity-free quantification of PCR products, matching fluorescence-based methods while offering streamlined spectral analysis and diverse reporting capabilities.

References

• Choppee Bortz PD, Wamhoff BR (2011) PLoS ONE 6(10): e26015. doi:10.1371/journal.pone.0026015