DNAFiberAnalysen

DNAFiberAnalysen, or DNA fiber analysis, refers to microscopy-based methods that study individual DNA molecules stretched into elongated fibers on a solid substrate. By spreading single DNA molecules into linear fibers and labeling them with fluorescent probes, researchers visualize features along the molecule with high physical resolution. The main approaches include molecular combing and related fiber-stretching techniques, often combined with fluorescent in situ hybridization (FISH) or sequential nucleotide labeling.

In replication assays, cells are labeled sequentially with nucleotide analogs such as IdU and CldU. After spreading the fibers, antibodies reveal colored tracks that indicate replication dynamics. By measuring track lengths and spacing, researchers estimate replication fork speed, origin firing, and fork progression or stalling. Fiber maps can also locate specific sequences using gene-specific probes.

Applications include studying replication dynamics under normal and stressed conditions, DNA repair pathways, and structural variation, as well as mapping replication timing on a coarse scale. The method supports basic cell biology and translational research, including assessment of drug effects on DNA replication.

Limitations include low throughput, labor-intensive sample preparation, and potential artifacts from stretching and drying. Resolution typically covers kilobase-scale features per fiber, and it is not a genome-wide sequencing method. Data interpretation can be subjective, requiring controls and standardized analysis.

History and development: DNA fiber analysis emerged in the late 1990s and early 2000s to study replication and genome structure with physical maps. Since then, improvements in combing methods, labeling strategies, and image analysis have expanded its applicability to diverse organisms and cell types.