RFLP/ PCR-RFLP

PCR-RFLP

   Restriction fragment length polymorphism (RFLP) is a technique invented in 1984 by the English scientist Alec Jeffreys during research into hereditary diseases. It is used for the analysis of unique patterns in DNA fragments in order to genetically differentiate between organisms – these patterns are called Variable Number of Tandem Repeats (VNTRs). Restriction Fragment Length Polymorphism (RFLP) is a technique in which organisms may be differentiated by analysis of patterns derived from cleavage of their DNA. If two organisms differ in the distance between sites of cleavage of a particular restriction endonuclease, the length of the fragments produced will differ when the DNA is digested with a restriction enzyme. The similarity of the patterns generated can be used to differentiate species (and even strains) from one another. Polymorphisms are inherited differences found among the individuals in more than 1% of normal population. The RFLP technique exploits these differences in DNA sequences to recognize and study both intraspecies and interspecies variation.

Principle

Restriction endonucleases are enzymes that cut lengthy DNA into short pieces. Each restriction endonuclease targets different nucleotide sequences in a DNA strand and therefore cuts at different sites. The distance between the cleavage sites of a certain restriction endonuclease differs between individuals. Hence, the length of the DNA fragments produced by a restriction endonuclease will differ across both individual organisms and species.

DNA Extraction

To begin with, DNA is extracted from blood, saliva or other samples and purified.

PCR

Isolation of sufficient DNA for RFLP analysis is time-consuming and labor intensive. However, PCR using specific primers can be used to amplify very small amounts of DNA, usually in 2-3 hours, to the levels required for RFLP analysis. Therefore, more samples can be analyzed in a shorter time.

DNA Fragmentation/ Production of Restriction Fragments

The purified DNA is digested using restriction endonucleases. The recognition sites of these enzymes are generally 4 to 6 base pairs in length.  The shorter the sequence recognized, the greater the number of fragments generated from digestion. The shorter the sequence recognized, the greater the number of fragments generated from digestion.

Gel Electrophoresis

The samples of DNA that have been treated with restriction enzymes are placed in separate lanes on a slab of electrophoretic gel across which is placed an electric field. The fragments migrate to wards the positive electrode, the smaller fragments moving faster than the larger fragments, thus separating the DNA samples in to distinct bands.

Visualization of Bands

The gel is treated with luminescent dyes in order to make the DNA bands visible.

Applications of RFLP:

RFLPs can be used in many different settings to accomplish different objectives.

RFLPs can be used in paternity cases or criminal cases to determine the source of a DNA sample. (i.e. it has forensic applications). RFLPs can be used determine the disease status of an individual. (e.g. it can be used in the detection of mutations particularly known muations). RFLPs can be used to measure recombination rates which can lead to a genetic map with the distance between RFLP loci measured in centiMorgans.