Journal Article

Comparison of DeNovix, NanoDrop and Qubit for DNA quantification and impurity detection of bacterial DNA extracts

Versmessen, Nick, Negash, Abel Abera, Vaneechoutte, Mario, Vandekerckhove, Marjolein, Hulpiau, Paco, Van Simaey, Leen, Cools, Piet

2024

Overview

Accurate DNA quantification is key for downstream application including library preparations for whole genome sequencing (WGS) and the quantification of standards for quantitative PCR. Two commonly used technologies for nucleic acid quantification are based on spectrometry, such as NanoDrop, and fluorometry, such as Qubit. The DS–11+ Series spectrophotometer/fluorometer (DeNovix) is a UV spectrophotometry-based instrument and is a relatively new spectrophotometric method but has not yet been compared to established platforms. Here, we compared three DNA quantification platforms, including two UV spectrophotometry-based techniques (DeNovix and NanoDrop) and one fluorometry-based approach (Qubit). We used genomic prokaryotic DNA extracted from Streptococcus pneumoniae using a Roche DNA extraction kit. We also evaluated purity assessment and effect of a single freeze-thaw cycle. Spectrophotometry-based methods reported 3 to 4-fold higher mean DNA concentrations compared to Qubit, both before and after freezing. The ratio of DNA concentrations assessed by spectrophotometry on the one hand, and Qubit on the other hand, was function of the A 260/280 . In case DNA was pure (A 260/280 between 1.7 and 2.0), the ratio DeNovix or Nanodrop vs. Qubit was close or equal to 2, while this ratio showed an incline for DNA with increasing A 260/280 values > 2.0. The A 260/280 and A 260/230 purity ratios exhibited negligible variation across spectrophotometric methods and freezing conditions. The comparison of DNA concentrations from before and after freezing revealed no statistically significant disparities for each technique. DeNovix exhibited the highest Spearman correlation coefficient (0.999), followed by NanoDrop (0.81), and Qubit (0.77). In summary, there is no difference between DeNovix and NanoDrop in estimated gDNA concentrations of S . pneumoniae , and the spectrophotometry methods estimated close or equal to 2 times higher concentrations compared to Qubit for pure DNA.

• Publisher: Public Library of Science
• Subject: Bacteria / Bacterial genetics / Bacterial Lysates / Bacterial pneumonia / Biology and Life Sciences / Comparative analysis / Computer and Information Sciences / Correlation coefficient / Correlation coefficients / Deoxyribonucleic acid / DNA / DNA sequencing / DNA, Bacterial - analysis / DNA, Bacterial - genetics / DNA, Bacterial - isolation & purification / Engineering and Technology / Fluorimetry / Fluorometers / Fluorometry / Fluorometry - methods / Freeze thaw cycles / Freeze-thawing / Freezing / Gene sequencing / Genetic testing / Genomes / Genomics / Medicine and Health Sciences / Nucleic acids / Nucleotide sequencing / Physical Sciences / Platforms / Pneumonia / Protozoa / Purity / Ratios / Regression analysis / Research and analysis methods / Science / Scientific imaging / Spectrometry / Spectrophotometers / Spectrophotometry / Spectrophotometry - methods / Spectrophotometry, Ultraviolet - methods / Statistical analysis / Streptococcus infections / Streptococcus pneumoniae / Streptococcus pneumoniae - genetics / Streptococcus pneumoniae - isolation & purification / Technology application / Whole genome sequencing