A restriction fragment length polymorphism, or RFLP, is a variation in homologous DNA sequences. Polymorphisms are the variations in homologous DNA that occur between individuals, while the term restriction refers to the fact that small sections of DNA can be cut from a large piece using restriction enzymes. Some DNA sections have more variability than others, and sections with a high degree of variability are useful in restriction fragment length polymorphism analysis.
Restriction fragment length polymorphism relies on the activity of restriction endonucleases. This class of enzyme is found in many bacterial species. In a bacterium, the role of restriction endonucleases is to provide protection against foreign DNA that gains access, by breaking it into pieces. In molecular research, restriction enzymes can be used to fragment DNA at known sites of variability so that the resulting fragments can be analyzed.
Each type of enzyme recognizes a specific DNA base pair sequence, and acts to cleave the DNA molecule at any point where it recognizes the sequence. Bacteria protect their own DNA from being broken with a distinctive pattern of DNA methylation that prevents restriction endonucleases from binding. More than 800 different restriction endonucleases have been discovered, which together recognize more than 100 unique nucleotide sequences. Most recognized sequences are between four and six base pairs in length.
Restriction enzymes are used in biotechnology to analyze restriction fragment length polymorphism for a variety of reasons. The enzymes are used to cut DNA lengths at certain sequences so that the differences between individuals can be examined. Solutions of fragmented DNA, cut at certain known nucleotide sequences, are hybridized with probes designed to detect pre-defined DNA sequences. The samples are then analyzed using gel electrophoresis. This technique produces a gel with multiple bands of dark staining, each of which represents a different DNA fragment. By examining the pattern of staining, scientists can determine the pattern of RFLPs present in a sample.
Polymorphisms occur widely throughout the human genome, and analysis of these has proven that different groups of people tend to share RFLPs in common. Certain RFLP types are common to people of different ethnic groups or geographical origins, for example, and linkage also exists within familial groups. This variation exists throughout the animal kingdom, making it a useful research tool not only for tracing human ancestry, but also for studying wildlife migration and evolution.
Analysis of restriction fragment length polymorphisms can provide useful information for clinical and medical purposes. Restriction fragment length polymorphism probes are used in paternity tests, in diagnosis of hereditary diseases, in forensic science, and in genotyping. Analyzing patterns of RFLPs across an entire genome is the basis of DNA fingerprinting, the technique which is the underpinning of all of these different uses.
