Restriction Fragment Length Polymorphism Analysis

AbstractThis unit describes Restriction Fragment Length Polymorphism (RFLP) analysis, which utilizes restriction endonuclease digestion to identify DNA sequence polymorphisms in genes or DNA regions of interest. When investigating families for inheritance of an RFLP, aliquots of genomic DNA from individual family members are digested to completion with the restriction enzyme known to generate the polymorphism of interest. After size fractionation on an agarose gel, DNA is transferred to a membrane by capillary action in a high‐salt buffer. The gel is first treated with NaOH to denature DNA, and after neutralization, the gel is placed between buffer‐soaked filter paper and a sheet of membrane. Labeled probe is hybridized overnight to the Southern blot. The blot is washed under conditions designed to remove all nonspecifically adherent probe and exposed to X‐ray film. Identified fragment sizes differ among individuals and can be traced from generation to generation.