Analysis of Interstrand and Intrastrand DNA Crosslinks with Two-Dimensional Strandness-Dependent Electrophoresis (2D-SDE)
Two-Dimensional Strandness-Dependent Electrophoresis (2D-SDE) is a technique for nucleic acid analysis to assess quality of samples, efficiency of molecular procedures and DNA damage. In the first dimension electrophoresis, nucleic acid fragments are separated based on length and strandness (dsDNA, ssDNA). In the second dimension the nucleic acid fragments are separated only based on length. After 2D separation, different arcs can be seen representing strandness and lengths of the nucleic acids in the original sample.
We tested if 2D-SDE could be used to detect DNA crosslinks. Patients with Fanconi anemia (FA) are extremely susceptible to DNA crosslinking agents. Human genomic DNA in solution and fibroblast cell cultures with mutations in FANCA and FANCD1 genes were treated with crosslinking agents. The DNA was analysed with 2D-SDE and compared to analysis with the comet assay, a versatile technique for DNA damage detection.
Increased amount of DNA migrating behind normal dsDNA was observed representing molecules with interstrand crosslinks that prevent full denaturation of dsDNA. Intrastrand crosslinks causing bending of nucleic acids and ssDNA migrating in front of dsDNA were observed. Repair efficiency, measured with 2D-SDE, was lower in the FA cell types compared to the wild-type cells. The comet assay detected only interstrand crosslinks in an indirect manner after treatment with an extra genotoxic insult.
2D-SDE has potential use in research, for FA diagnosis, and in chemosensitivity testing as the cytotoxicity of many cancer medications depends on their DNA crosslinking ability.