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RFLP relies on changes in DNA—such as a single-nucleotide change, insertion, or deletion—that create or abolish a restriction enzyme recognition site. Digested fragments are separated by agarose gel electrophoresis, transferred by Southern blot, and detected with a labeled probe. The method requires relatively large amounts of non-degraded genomic DNA and can analyze single-locus or multilocus polymorphisms; heterozygosity makes a marker informative. Applications include linkage analysis, selected genetic disorders, VNTR-based forensic profiling and paternity testing, allele-phase determination in pedigrees, and detection of loss of heterozygosity in tumors. RFLP offers specific, co-dominant markers, but its workflow is labor-intensive and slow, uses radioactive probes, and performs poorly with degraded samples. PCR-based methods have largely replaced it in modern laboratories.