Freezing shortens the lifetime of DNA molecules under tension

Abstract

DNA samples are commonly frozen for storage. However, freezing can compromise the integrity of DNA molecules. Considering the wide applications of DNA molecules in nanotechnology, changes to DNA integrity at the molecular level may cause undesirable outcomes. However, the effects of freezing on DNA integrity have not been fully explored. To investigate the impact of freezing on DNA integrity, samples of frozen and non-frozen bacteriophage lambda DNA were studied using optical tweezers. Tension (5–35 pN) was applied to DNA molecules to mimic mechanical interactions between DNA and other biomolecules. The integrity of the DNA molecules was evaluated by measuring the time taken for single DNA molecules to break under tension. Mean lifetimes were determined by maximum likelihood estimates and variances were obtained through bootstrapping simulations. Under 5 pN of force, the mean lifetime of frozen samples is 44.3 min with 95% confidence interval (CI) between 36.7 min and 53.6 min while the mean lifetime of non-frozen samples is 133.2 min (95% CI: 97.8–190.1 min). Under 15 pN of force, the mean lifetimes are 10.8 min (95% CI: 7.6–12.6 min) and 78.5 min (95% CI: 58.1–108.9 min). The lifetimes of frozen DNA molecules are significantly reduced, implying that freezing compromises DNA integrity. Moreover, we found that the reduced DNA structural integrity cannot be restored using regular ligation process. These results indicate that freezing can alter the structural integrity of the DNA molecules.

Toward Best Practice in Livestock Microbiota Research: A Comprehensive Comparison of Sample Storage and DNA Extraction Strategies

“…Nevertheless, freezing still does not guarantee full flexibility during sampling and transport, and it is often not logistically feasible. Freezing and thawing of bacterial cells might help in opening the cell envelope, making DNA extraction more efficient, yet it can compromise the integrity of the DNA ( Chung et al, 2017 ). Thus, for the second phase of the experiments, we further investigated the effect of commonly used storage approaches on the DNA quality and the shift in the chicken and pig digesta microbial communities.…”

DNA stability: a central design consideration for DNA data storage systems

“…During the freezing and thawing process, ice crystals are formed that generate forces that could lead to breakage of DNA polymers. There is also evidence that freezing makes DNA more susceptible to breakage than non-frozen DNA at similar tensional forces 38 . Several studies have repeatedly frozen and thawed DNA samples, generally observing exponential degradation.…”

Single-Molecule Mechanics in Ligand Concentration Gradient

“…Ligation was carried out with T7 DNA ligase (Thermo Fisher Scientific). Following biotin labeling the DNA samples were stored at 4 • C to prevent degradation caused by freezing [42]. For optical tweezer measurements DNA was used at 30 ng/mL concentration in Tris-NaCl buffer (20 mmol/L Tris-HCl, pH 7.4, 50 mmol/L NaCl) throughout the experiments.…”