DNA fragments are separated by agarose gel electrophoresis and are detected by subsequent Southern blot hybridization to a labelled DNA probe. Labeling of the probe may be performed with a radioactive isotope or with alternative non-radioactive stains, such as digoxigenin or fluorescein.
The locus specific RFLP probes consist of a homologous sequence of a specific chromosomal region. Probes are generated through the construction of genomic or complementary DNA cDNA libraries and therefore may be composed of a specific sequence of unknown identity genomic DNA or part of the sequence of a functional gene exons only, cDNA.
RFLP probes are maintained as clones in suitable bacterial vectors that conveniently allow the isolation of the DNA fragments they hold. Probes from related species may be used heterologous probes. DNA sequence variation affecting the absence or presence of recognition sites of restriction enzymes, and insertions and deletions within two adjacent restriction sites, form the basis of length polymorphisms. RFLPs can be used determine the disease status of an individual.
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. Each organism inherits its DNA from its parents. Since DNA is replicated with each generation, any given sequence can be passed on to the next generation.
A target sequence is any segment of DNA that bind to a probe by forming complementary base pairs. 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. 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.
RFLP produces a series of bands when a Southern blot is performed with a particular combination of restriction enzyme and probe sequence. When this segment of DNA is cut by EcoR I, three restriction fragments are produced, but only one contains the target sequence which can be bound by the complementary probe sequence purple.
When we examine one copy from Jack and one copy from Jill, we see that they are identical:. When we examine their second copies of this RFLP, we see that they are not identical. Jack 2 lacks an EcoR I restriction site that Jill has 1. Therefore, when Jack and Jill have their DNA subject to RFLP analysis, they will have one band in common and one band that does not match the other's in molecular weight:.
The results are shown below:. In this case, it appears that Jack could be the father, since Payle inherited the You have authorized LearnCasting of your reading list in Scitable. Do you want to LearnCast this session? This article has been posted to your Facebook page via Scitable LearnCast. Change LearnCast Settings.
0コメント