Learn About RFLP Analyses

What is RFLP?

Performing a RFLP Analyses

How are RFLP Analyses Used?

What is RFLP?

RFLP (pronounced "rif-lip") stands for Restriction Fragment Length Polymorphism. The restriction fragments mentioned in the name are sections of a DNA sequence which have been cut by a restriction enzyme. (Click, to learn more about restriction enzymes and even more). The lengths of these fragments are polymorphic because certain differences in DNA strands such as "base substitutions, additions, deletions and other sequence rearrangements" can change the size of the restriction fragments by affecting the number and distribution of restriction sites (University of Nebraska-Lincoln 1997). Therefore, researchers have developed a method of comparing DNA samples based on this differential cleavage of DNA by restriction enzymes.

Performing a RFLP Analyses

To perform a RFLP analysis, the DNA must first be extracted from the sample. Because RFLPs are used on DNA samples collected from places like crime scenes and fossil records, this can be an interesting procedure. The DNA must then be cut with one or more restriction enzyme. Each resulting restriction fragment library is placed into the well of an agarose gel. The subsequent electrophoresis separates the restriction fragments by size. The shorter fragments are able to travel more quickly down the gel than the larger ones. Because the fragments are different lengths, each DNA sample is spread over the gel in certain banding pattern. The final step is to visualize the DNA. In some cases, this constitutes running the DNA with ethidium bromide in the agarose gel and/or the buffer. This allows the researcher to see the fragment bands because the ethidium bromide complexes with the DNA and fluoresces under UV light. In many situations, however, there are so many DNA fragments that the use of ethidium bromide would result in an entire lane fluorescing, instead of a few distinct bands. Therefore, a radioactive complementary DNA probe is developed to find and bind to a specific DNA sequence. (For more on probing a RFLP gel, chick here.) An autoradiograph is then made by exposing X-ray film to the radioactive probe. When the film is developed, the targeted DNA bans can be seen.

How is it used?

So now that you understand what a RFLP is and how to perform an analysis, don't you what to know what can be done with this technology? In fact, there are several scientific areas that use RFLP analyses.

Systematics, Genetics and Ecology:

Phylogenetics, the study of evolutionary relationships, is just one field that has benefited from the advent of RFLP technologies. By comparing the fragment patterns of different species (Fig. 1), an evolutionary biologist can gather information about the possible relatedness of those species.

Fig. 1. RFLP gel showing the banding differences between two closely related species, Soybean Cyst Nematode and Sugar Beet Cyst Nematode (University of Nebraska-Lincoln 1997).

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 2).

Fig. 2. An RFLP Map of a Loblolly Pine Population (Neale and Sederoff R. 1996)


RFLPs analyses have become very popular in the field of medicine. Genetic counseling is founded on RFLP analyses and the ability to determine genotypes (Fig. 3). RFLPs can be used to determine the genotype of potential parents, and following an amniocentesis or human chorionic fluid sampling, RFLPs are used to screen for deleterious genotypes in the fetus. One such test is available for sickle-cell anemia (Hushey 1996).

Fig. 3. A restriction site diagram (lower right) showing the different fragment sizes (yellow) of the dominant beta-A chain and the recessive beta-S sequence. A pedigree diagram (upper left) depicting the genotype of children from parents that are heterozygous for sickle-cell anemia allele (red). In the lower left are the results of the RFLP analysis which has two bands for the heterozygous parents and child 2, a thick band of beta-S for the homozygous recessive, sickle-cell afflicted, child 1 and a thick band of beta-A for the homozygous dominant bands child 3 (Kimball 1997).


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). The probability that some DNA in the evidence comes from the suspect is related to the number of bands in the evidence lanes that are also found in the suspect's pattern, but cannot be accounted for in the victim's RFLP.

Fig. 4. An example of a RFLP analysis performed for forensic purposes. Lanes 1,2,7,11 and 15 have molecular weight markers. Evidence is in lanes 9 and 12. The victim's DNA is in lane 4 and suspects' in 5 and 6 (Stokely 1997).

In one famous case, the results of tests similar to this one were presented as evidence. Click here for the results and see if you think he did it.

I hope that this brief synopsis of RFLP and its uses have made you fall in love with RFLP. So if you really want to know more check out these additional links on RFLP methods and types of RFLP Polymorphisms.

Works Cited

Hushey RJ. 1996 May. Genotype determination using RFLP's and a gene probe. <http://wsrv.clas.virginia.edu/~rjh9u/hbsrflp.html> Accessed 1998 17 Feb.

Johns Hopkins Medical Institute. 1994 Aug. Mapping and Sequencing the Human Genome. <http://www.bis.med.jhmi.edu/Dan/DOE/prim2.html>

Kimball JW. 1997 May. Restriction Fragment Length Polymorphisms (RFLPs). <http://www.ultranet.com/~jkimball/BiologyPages/R/RFLPs.html> Accessed 1998 17 Feb.

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> Accesssed 1998 17 Feb.

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> Accessed 1998 17 Feb.

University of Nebraska-Lincoln, Institute of Agriculture and Natural Resources. 1997 Nov. Restriction Fragment Length Polymorphism (RFLP). <http://ianrwww.unl.edu/ianr/plntpath/nematode/its_id/pcr_rflp.htm> Accessed 1998 17 Feb.

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