Angle modulated two-dimensional single cell pulsed-field gel electrophoresis for detecting early symptoms of DNA fragmentation in human sperm nuclei

Abstract

We here developed a novel angle-modulated two-dimensional single cell pulsed-field gel electrophoresis (2D-SCPFGE). Variations in current-application-time and rotation angle generated different alignments of DNA fibers and segments. After the first run, the specimen was turned by 150° (2D-SCPFGE-0–150) to detect naturally occurring the earliest stage of DNA fragmentation or 75° (2D-SCPFGE-0–75) to analyze artificially induced cleavage. The former revealed that a part of long chain fibers remained at the origin and long segments were still tangled in the bundle of elongated fibers after the first run. The latter visualized the dose-dependent cleavage of DNA by EcoR1. Multicycle 2D-SCPFGE was useful for generating 2D-alignments of single nuclear DNA fibers, which is the first step for visualization of single-strand breaks on stretched fibers. To date, many articles have accepted the pathogenetic significances of DNA fragmentation in human sperm for male infertility and congenital anomaly. It is necessary to perform multivariate analyses of not only earliest-stage DNA fragmentation but also other types of damage, including single-strand breaks, in sequential DNA fibers. 2D-SCPFGE is the fundamental tool for understanding single nuclear DNA damages.