RFLP produces a series of bands when a Southern blot is performed with a particular combination of restriction enzyme and probe sequence.įor example, let's follow a particular RFLP that is defined by the restriction enzyme EcoR I and the target sequence of 20 bases GCATGCATGCATGCATGCAT. Separate aliquots of the PCR amplicon are digested with each of up to six different restriction enzymes to produce a species-specific RFLP profile. When a probe base pairs to its target, the investigator can detect this binding and know where the target sequence is since the probe is detectable. Restricted labeled amplicon was used to generate terminal restriction fragment lengths on an ABI 310 sequencer to determine molecular identity of colonizing. A probe is a sequence of single-stranded DNA that has been tagged with radioactivity or an enzyme so that the probe can be detected. A target sequence is any segment of DNA that bind to a probe by forming complementary base pairs. An RFLP is a sequence of DNA that has a restriction site on each end with a " target" sequence in between. Since DNA is replicated with each generation, any given sequence can be passed on to the next generation. The primary conditions of choice were the annealing. On this web page, you can see how RFLPs are produced and then three examples of applying RFLP analysis: paternity, disease status, and genetic mapping.Įach organism inherits its DNA from its parents. Outline of the Terminal Restriction Fragment Length Polymorphism (t-RFLP) analysis procedure. RFLPs can be used to measure recombination rates which can lead to a genetic map with the distance between RFLP loci measured in centiMorgans. RFLPs can be used determine the disease status of an individual. RFLPs can be used in paternity cases or criminal cases to determine the source of a DNA sample. RFLPs can be used in many different settings to accomplish different objectives. RFLP (often pronounced "rif lip", as if it were a word) is a method used by molecular biologists to follow a particular sequence of DNA as it is passed on to other cells. RFLP Method - Restriction Fragment Length Polymorphism The four SNPs (C5178A, A10398G, G13708A, and C13928G) in the mtDNA coding region were detected using PCR-RFLP analysis.
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