DNA molecules within chromosomes undergo constant, dynamic changes yet maintain the integrity of the primary DNA sequence. DNA replication, adjustment of helical density, resolution of catenenes, repair of DNA damage, and homologous recombination each involve breakage and religation of the phosphate backbone of the double helix. Although the analysis of dsDNA breaks is facile, the analysis of ssDNA nicks is not. The principal impediment is that conventional, one-dimensional electrophoresis methods cannot readily detect ssDNA nicks in the context of dsDNA breaks. We therefore developed a two dimensional (native/denaturing) gel electrophoresis approach to map the positions of ssDNA nicks. Analysis of cohesive ends of lambda phage DNA, UV-nicked DNA molecules, and DNA treated with ssDNA nicking endonuclease N-BbvcIB revealed that the method can detect and map with precision the positions of ssDNA nicks. Titration experiments revealed the ability to detect and quantitate nicked DNA molecules present at a frequency of 1% of total DNA molecules. This method can be used both to scan rapidly through large regions of the genome of interest and to map with high-resolution the location of ssDNA nicks in populations of dsDNA molecules. It is of utility for the analysis of ssDNA nicks involved in a variety of chromosomal processes.
Wahls, Wayne P.; DeWall, K. Mark; and Davidson, Mari K.
"Mapping of ssDNA Nicks within dsDNA Genomes by Two-dimensional Gel Electrophoresis,"
Journal of the Arkansas Academy of Science: Vol. 59
, Article 25.
Available at: http://scholarworks.uark.edu/jaas/vol59/iss1/25