Restriction Fragment Length Polymorphism

Restriction fragment length polymorphism (RFLP) is a technique used to distinguish organisms by analyzing and comparing patterns that originate from the cleavage of their DNA.

If two organisms differ in the distance between the cleavage sites of a particular restriction endonuclease, the length of the resulting fragments will be different Rudner R et al. 1994). The similarity of the patterns generated can be used to distinguish species (and even strains) (Amélia Camarinha-Silva et al. 2012).

In this method, DNA fragments are cut enzymatically (by restriction enzymes), sorted by length using gel electrophoresis. This makes them comparable with other DNA samples.

RFLP enables the detection of polymorphisms at the DNA sequence level. Changes in the DNA fragments are mainly due to base exchange mutations in the recognition sites for the enzymes used. However, rearrangements within the DNA, insertions, deletions or duplications can also lead to an RFLP. In the case of simple base substitutions, a polymorphism is always found when bases within a recognition sequence of the restriction enzyme currently used are affected. The loss or new appearance of a recognition sequence associated with these changes is accompanied by the loss or appearance of a new DNA fragment and the lengthening or shortening of other fragments when genomic DNA is cleaved.

The RFLP method is used in forensic science or for paternity tests. It was the first cost-effective method for comparing the relationship between two samples, but is increasingly being replaced by other biochemical methods such as DGGE or TGGE, phospholipid analysis, polymerase chain reaction (sometimes with DNA sequencing), RAPD, STR analysis, SSCP analysis or further developments of RFLP such as AFLP, T-RFLP, ARISA, ARDRA.

A variant of RFLP with rDNA is ribotyping.

A more specialized application is terminal RFLP(T-RFLP). Here, the target genes are amplified in the polymerase chain reaction (PCR) with one (or both) fluorescently labeled primer(s). The amplification products are digested with a restriction enzyme and analyzed by an automated sequencer.

The T-RFLP technique of amplified restriction fragment length polymorphism is now also used to analyze polymorphism in bacterial and mycological samples (Masiga DK et al. 2000; Verrier J et al. 2019). The T-RFLP test has proven to be highly sensitive and specific and enables the rapid detection and direct identification of dermatophytes in medical practice (Verrier J et al. 2019).

1. Amélia Camarinha-Silva et al. (2012) Validating T-RFLP as a sensitive and high-throughput approach to assess bacterial diversity patterns in human anterior nares. FEMS Microbiology Ecology 79: 98-108.
2. Didehdar M et al. (2016) Characterization of clinically important dermatophytes in North of Iran using PCR-RFLP on ITS region. J Mycol Med 26:345-350.
3. Masiga DK et al. (2000) Amplified restriction fragment length polymorphism in parasite genetics. Parasitol Today 16:350-353.
4. Rudner R et al. (1994) Determinations of restriction fragment length polymorphism in bacteria using ribosomal RNA genes. In: Methods in Enzymology. Volume 235:184-196
5. Verrier J et al. (2019) PCR-terminal restriction fragment length polymorphism for direct detection and identification of dermatophytes in veterinary mycology. Med Mycol 57:447-456.