Restriction Fragment Length Polymorphism (RFLP) Analysis

Graphic for Restriction Fragment Length Polymorphism
Figure 7 - Graphic by CSS, Inc.

RFLP analysis is used to detect mutations in DNA sequences that are recognized by a specific restriction enzyme. The test DNA may not typically be the target of restriction enzyme activity - remember restriction enzymes are used only by bacteria, and the test DNA may be from an animal or plant. However, because the base sequences targeted by restriction enzymes commonly and frequently occur throughout every genome, comparing the number and placement of these sites is an effective method to screen for individual and species DNA differences.

To perform RFLP analysis, DNA samples are digested with a restriction enzyme (for example, EcoR1, from the bacterium Escherichia coli) . In the Figure 7A, DNA (1) has three EcoR1 restriction sites, so incubating (digesting) the DNA with EcoR1 cuts out two short pieces. DNA (2) has a mutation at one of the restriction sites, therefore only one piece will be cut out. To visualize the number and size of the digestion products, all the DNA is separated by electrophoresis, transferred to a membrane by the Southern blot method, and probed with a labeled piece of DNA that will hybridize to the original DNA, as well as any smaller pieces of the same sequence (Figure 7B).

The sensitivity or resolution of an RFLP is dictated by the number of bases in the recognition site and the size of the test DNA. Generally, researchers will first isolate a larger piece of DNA (several thousand bases) and use RFLP analysis as a starting point to screen for sequence differences. For example, RFLP analysis is commonly used to compare mitochondrial DNA of closely related species to help determine evolutionary relationships, followed by DNA sequencing for fine-tuning relationships within each lineage.

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