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RFLP is a variation in homologous DNA sequences, which can be cut using restriction enzymes. Restriction enzymes fragment DNA at known sites of variability, which are then analyzed by gel electrophoresis. RFLP analysis is used in various fields, including clinical and medical purposes, and can provide useful information for tracing human ancestors and studying wildlife migration and evolution.
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 sections of DNA have greater variability than others, and sections with a high degree of variability are useful in the analysis of restriction fragment length polymorphism.
The restriction fragment length polymorphism is based on the activity of restriction endonucleases. This class of enzymes is found in many bacterial species. In a bacterium, the role of restriction endonucleases is to provide protection against incoming foreign DNA, 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 sequence of DNA base pairs and acts to cleave the DNA molecule at any point it recognizes the sequence. Bacteria protect their DNA from breakage with a distinctive pattern of DNA methylation that prevents the binding of restriction endonucleases. More than 800 different restriction endonucleases have been discovered, which together recognize more than 100 unique nucleotide sequences. Most of the recognized sequences are between four and six base pairs in length.
Restriction enzymes are used in biotechnology to analyze the length polymorphism of restriction fragments for a variety of reasons. Enzymes are used to cut lengths of DNA in certain sequences so that differences between individuals can be examined. Solutions of fragmented DNA, cut at certain known nucleotide sequences, are hybridized with probes designed to detect predefined DNA sequences. The samples are then analyzed by gel electrophoresis. This technique produces a gel with multiple bands of dark staining, each representing a different DNA fragment. By examining the staining pattern, scientists can determine the pattern of RFLPs present in a sample.
Polymorphisms occur extensively throughout the human genome and analysis of these has shown that different groups of people tend to share RFLPs in common. Some types of RFLP are common to people of different ethnic groups or geographic origins, for example, and the linkage exists even within family groups. This variation exists throughout the animal kingdom, making it a useful research tool not only for tracing human ancestors 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 testing, hereditary disease diagnosis, forensic science, and genotyping. The analysis of RFLP patterns over a whole genome is the basis of DNA fingerprinting, the technique behind all these different uses.
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