Single-Nuclear DNA Instability Analyses by Means of Single-Cell Pulsed- Field Gel Electrophoresis - Technical Problems of the Comet Assay and Their Solutions for Quantitative Measurements

Abstract

Throughout the past decade, the neutral and alkaline comet assays, which lyses cells with high salt in the absence of proteolysis, have been the most widely used methods to observe single- or double-strand breaks (SSB, DSB) in a single nucleus. These methods evaluate the degree of DNA damage based on the amount of granular fragments discharged from the origin, the so-called “comet tail,” but not of long chain DNA fibers. Meanwhile, a novel, single-cell pulsed-field gel electrophoresis (SCPFGE) method, which digested cells with trypsin, observed a different course of DNA fragmentation: first, a few large fibrous fragments were derived from a bundle of long chain fibers, then the cleavages were advanced until finally almost all the DNA was shredded to granular fragments. Some nuclear DNA binding components were tolerant for high salt and high alkalinity, but were degraded by trypsin digestion. A lack of trypsin digestion causes false negative results in both SCPFGE and comet assays. Also, most DNA fibers were still fixed with components, and the comet tail did not reflect a total amount of granular fragments, but rather those that released components. Repair of DSB is intrinsically difficult as compared to other DNA lesions, and the critical threshold is extremely low.DNA fibers that have already been shredded to numerous granular fragments may be irreparable as a result. Although 100 - 300 mmol/L NaOH was commonly used in the alkaline comet assay, the naked DNA fibers persisted in 10 mmol/L NaOH after trypsin digestion, but were shredded to granular fragments by 30 mmol/L. Neogenesis of the granular fragments by high pH did not clarify the mechanism of such a result; that is, it was unknown whether it was due to dissociation of hydrogen bonds, strand breaks through alkaline labile sites, artifactual DSB, or a combination of actions. Optimum conditions for the comet assay need to be defined to achieve quantitative measurements. DNA instability analyses by means SCPFGE is likely to serve as a fundamental step in single-cell genomics to determine the competence of the cell population provided for DNA amplification methods and is likely to play an important role in ensuring the safety of clinical regenerative medicine.