How is rflp performed

Restriction fragment length polymorphism mapping in conifers and applications to forest genetics and tree improvement Neale, D. Canadian Journal of Forest Research, Go directly to: Content Search box Breadcrumb. 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.

So a single base difference between two people could result in either the presence or absence of that restriction site.

So then, if you isolate that piece of DNA surrounding that site from two people, from one of them it will be cut by the enzyme and the other one it won't. And that results in a polymorphism, or difference between those two people. We typically see these, or we monitor these, by isolating the DNA, cutting it with that bacterial restriction enzyme, and running it on a gel using electrophoresis. In one person, without the enzyme site you'll see one band, and the person that has the enzyme site, you'll see two bands, representing the two cleaved products.

So these differences in nucleic acid sequences and restriction enzyme binding sites just mean that there's a difference in the sequence between those two people.

However, it is possible that another man with similar RFLP pattern could be as well. To be certain, several more RFLP loci would be tested. It would be highly unlikely that two men other than identical twins would share multiple RFLP patterns and so paternity could be confirmed. Therefore, it is very probable that Payle's father is not Jack, though it is possible that Payle carries a new mutation at this locus and a different sized band was produced.

What could you do as an investigator to be more certain that Jack was not the father of Payle? In this example, we want to know if a person carries any cystic fibrosis CF alleles and if so, how many.

Because CF is a recessive disease, anyonne with CF must be homozygous for disease alleles. From pedigree information, we can often determine who in this family is a carrier. RFLPs are known for CF and so it would be easy to determine if a person were homozygous wild-type wt , heterozygous "carrier", or homozygous disease alleles and thus have CF. For couples expecting a child, it would be simple to test both parents and make a prediction about the eventual disease status of their fetus.

For example, if both parents were homozygous wt , then all of their children would also be homozygous wt :. However, if both parents were heterozygous, they could have children with any of the three genotypes, though heterozygous children would be twice as likely as either of the homozygous genotypes. With increasing genomic sequence information, increasing numbers of genetic disease can be predicted from RFLP analyses.

To calculate the genetic distance between to loci, you need to be able to observe recombination. Traditionally, this was performed by observing phenotypes but with RFLP analysis, it is possible to measure the genetic distance between two RFLP loci whether they are a part of genes or not.