How do rflps work

Probe Reagents for Functional Genomics. Genotyping Developing RFLP probes Total DNA is digested with a methylation-sensitive enzyme for example, PstI , thereby enriching the library for single- or low-copy expressed sequences PstI clones are based on the suggestion that expressed genes are not methylated.

The digested DNA is size-fractionated on a preparative agarose gel, and fragments ranging from to bp are excised, eluted and cloned into a plasmid vector for example, pUC Digests of the plasmids are screened to check for inserts.

Southern blots of the inserts can be probed with total sheared DNA to select clones that hybridize to single- and low-copy sequences. 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. Strengths RFLPs are generally found to be moderately polymorphic. In addition to their high genomic abundance and their random distribution, RFLPs have the advantages of showing codominant alleles and having high reproducibility.

Weaknesses The main drawbacks of RFLPs are the requirement of laborious and technically demanding methodological procedures, and high expense. 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.

That sequence difference doesn't necessarily mean that there's a disease associated with it.