Work

Immuno-Southern blot assay: combined detection of DNA modifications with methylation-responsive restriction Enzymes and antibody screening

Year: 2026

Type: article

Abstract: DNA base modifications influence genome function in both prokaryotes and eukaryotes, yet their detection often relies on specialised and resource-intensive technologies. Here we present immuno-Southern (iSouthern), a simple method that combines two independent detection strategies within a single procedure. First, restriction enzymes with methylation-dependent activity, coupled with gel electrophoresis, generate modification-specific DNA fragmentation patterns. Second, antibody screening of immobilised DNA enables specific detection of modified bases of interest. This dual readout increases confidence in the results, particularly for modifications whose presence remains under debate. We exemplify the method by demonstrating detection of varying levels of N6-methyldeoxyadenosine (6mA) in bacteriophage λ genomic DNA. iSouthern only requires standard laboratory equipment and reagents, making it economical and widely accessible. It can be applied to any DNA modification of interest, whether epigenetic or not, and is well suited for screening samples prior to costly, high-resolution analyses. Beyond research, iSouthern also provides a valuable platform for antibody benchmarking and teaching. These features position iSouthern as a versatile complement to sequencing- and mass spectrometry-based approaches, lowering barriers to the study of DNA modifications.

Source: Epigenetics Reports

DOI: 10.1080/28361512.2026.2627181