Restriction Fragment Length Polymorphism

What is Restriction Fragment Length Polymorphisms (RFLPs)?:
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RFLP; the acronym is pronounced "riflip". Polymorphism is any difference in DNA sequence, coding or non-coding, that can be detected between individuals. Is a biotechnological laboratory technique in which DNA regions are digested using restriction endonuclease(s) and subjected to radioactive complementary DNA probes to compare the difference in DNA fragment lengths between individuals Differences are noticed when the length of fragments are not the same, telling us that the restriction enzyme cut the DNA at two unrelated locations.

Genetic variations at the site where a restriction enzyme cuts a piece of DNA, Such variations affect the size of the resulting fragments. These sequences can be used as markers on physical maps and linkage maps. Restriction fragment length polymorphism is the identification of specific restriction enzymes that reveal a pattern difference between the DNA fragment sizes in individual organisms, usually results from a genetic mutation (as an insertion or deletion) and that may be used as a genetic marker.

A Closer Look…

Restriction fragment length polymorphism analysis involves the comparison of different lengths of DNA The first step is to extract a sample of DNA and subject it to the restriction enzyme(s), this enzyme will produce restriction fragments (fragments that will vary in size depending on the DNA of the organism) as seen in figure 1

Figure 1

Next through a process known as “Gel Electrophoresis”, the fragments are separated, each sample forms a characteristic pattern of bands because of the slight variation in each organism’s DNA. The fragments form a band as a electrical current is passed through the gel causing the negatively charged DNA to travel to the positive side of the electrode. The whole process is shown in figure 2.
FIGURE 2

The amount of DNA is usually so large and the bands so numerous that gel electrophoresis is unable to resolve them. Differences in the pattern of fragments must be detected to distinguish DNA from two different sources. The gel is subjected to a chemical that causes the double-stranded DNA to be de-natured into single-stranded DNA as in figure 3

Figure 3

These single-strands are transferred onto special paper or nylon membranes through capillary action known as “southern blotting” named after E.M. Southern who developed it. The single stranded DNA is transferred to the nylon membrane with the aid of an electrical current. The nylon membrane is placed on the gel with a positive electrode behind; since DNA is negatively charged it will be transferred out of the gel and “blotted” on to the nylon membrane, where they will bind.

Figure 4

The nylon membrane containing the single-stranded DNA is immersed in a solution containing radioactive complementary nucleotide probes for specifically chosen regions. These regions can be a mutation in an allele that lead to a specific disease or variable number tandem repeat found in non-coding region of the DNA. Complementary base pairing between the radio active probes and the DNA via hydrogen bonds will occur this is known as “HYBRIDIZATION” as seen in figure 5

Figure 5

The nylon membrane is placed against X-RAY film. The radioactive probes cause exposure of the x-ray film in the area where hybridizations took place. This is called an “AUTORADIOGRAM” the differences in pattern can be detected. Seen on the right of figure 5

Figure 5

How Is It Used?

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RFLPs have provided valuable information in many areas of biology including: Systematics, Genetics and Ecology DNA Fingerprinting Screening human DNA for the presence of potentially deleterious genes

•Systematics, Genetics and Ecology

Phylogentics is the study of evolutionary relationships, this is just one field that has benefited from RFLPs. By comparing the fragment patterns of different species (Fig. 6) Top, an evolutionary biologist can gather information about the possible relatedness of those species. RFLPs are not only used to compare species, but information about intraspecific variation can also be obtained. Such comparisons can be useful to a geneticist trying to determine the genetic make-up of a population or to an ecologist who needs information about the genealogy of a population (Fig 7) Bottom.

•DNA Fingerprinting

Sequences of DNA differ from person to person, but every cell within the same person contains the same sequence of DNA. So, your hair, blood, skin and all of the other cells in your body are exactly the same at the molecular level.This comes in very handy when police are investigating a crime. If a person left a strand of hair, a drop of blood or any other cells at a crime scene, the police will know that that person was there. Criminal investigation also uses RFLPs as part of forensic analysis of the crime scene. They compare RFLP patterns of DNA found on evidence to those of the victim and suspect(s) (Fig. 4). They observe the patterns in the fragments and try to match the suspects DNA with the DNA found at the crime scene. As in figure 8 DNA fingerprinting is based on variations of satellites DNA (non-coding regions), since humans share almost all the same DNA in the coding regions the only variation that occurs is in the noncoding regions. DNA fingerprinting takes advantage of this fact.

Figure 8

• Screening human DNA for the

presence of potentially deleterious genes
RFLPs analyses have become very popular in the field of medicine. Genetic counselling is founded on RFLP analyses and the ability to determine genotypes (Fig.9 ). RFLPs can be used to determine the genotype of potential parents, and following an amniocentesis or human chronic fluid sampling, RFLPs are used to screen for deleterious genotypes in the fetus. For instance, an RFLP has been identified that is associated with the genetic disease sickle cell anaemia. Sickle cell anaemia is caused by a mutation in the alpha-globin gene and results in an abnormal form of haemoglobin.

Figure 9

“Works Cited”
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Restriction fragment length polymorphism Torbert R. Rocheford, assistant professor of corn breeding, Department of Agronom, cited dec 19 2005. < http://www.ag.uiuc.edu/~vista/html_pubs/irspsm91/fragment.html> U. Melcher molecular genetics-RFLP’s Last Updated: 3 September, 2001 cited December 19, 2005, < http://opbs.okstate.edu/~melcher/MG/MGW1/MG11124.html> Tara T. VanToai Seed Biology:Department of Horticulture and Crop Science, The Ohio State University, USES OF DNA TECHNOLOGY IN SEED RESEARCH, < http://www.ag.ohio-state.edu/~seedbio/van1.html > Kimball JW. 1997 May. Restriction Fragment Length Polymorphisms (RFLPs). <http:// www.ultranet.com/~jkimball/BiologyPages/R/RFLPs.html> Neale D, Sederoff R. 1996 Sept. Genome mapping in pines takes shape. National Agricultural Library. < http://www.nalusda.gov/pgdic/Probe/v1n3_4/genome.html> Stokely RD. 1997 May. DNA profiling in a first degree murder case: RFLP analysis. < http://www.biology.arizona.edu/human_bio/activities/stokely/RFLP.html> Department of Biology, Davidson College, Davidson, NC 28036 <http://www.bio.davidson.edu/Courses/Molbio/MolStudents/01anford/molecularpaper1.html > Lebrasseur, Nicole D.. Restriction Fragment Length Polymorphism (Rflp)." < http://www.bookrags.com/sciences/genetics/restriction-fragment-length-polymor-wog.html >