The Procedure (Genetics & Inherited Conditions)
Restriction fragment length polymorphism (RFLP) analysis is a method for distinguishing individuals and analyzing relatedness, based on genetic differences. RFLP analysis relies on small DNA sequence differences that lead to the loss or gain of restriction enzyme sites in a chromosome or to the change in size of a DNA fragment bracketed by restriction enzyme sites. These sequence differences lead to a different pattern of bands on a gel (reminiscent of a bar code) that varies from individual to individual.
RFLP analysis starts with the isolation of DNA. Typically, DNA isolation requires the use of detergents, protein denaturants, RNA degrading enzymes, and alcohol precipitation to separate the DNA from the other cellular components. This DNA could be isolated from a blood sample provided by an individual, from evidence left at the scene of a crime, or from other sources of cells or tissues.
The purified DNA is then digested with a molecular “scissors” called a restriction enzyme. Restriction enzymes recognize and cut precise sequences, typically six base pairs in length. If one base pair is changed in that recognition sequence, the enzyme will not cut the DNA at that point. However, if a sequence that is not recognized by a restriction enzyme is altered by mutation, so that it now is recognized, the DNA will be cleaved at that point. In other cases, the DNA sequences recognized by the restriction enzymes themselves...
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Applications (Genetics & Inherited Conditions)
One of the earliest uses of this technique in clinical medicine was in the prenatal diagnosis of sickle-cell disease. Previous work had shown that many individuals with the disease had a mutation in their DNA that eliminated a restriction enzyme site in a gene encoding a hemoglobin protein. This information was used to develop a diagnostic RFLP procedure. A section of the hemoglobin gene is used as a probe. The size of restriction enzyme fragments identified is different in individuals who have sickle-cell disease (and therefore have two mutant alleles) compared with individuals who carry either one mutant allele or have two unmutated hemoglobin alleles. This method allowed for the identification of affected fetuses using DNA from cells isolated from amniotic fluid (a much simpler and safer procedure than the previous method of diagnosis, which required isolating fetal red blood cells).
Another widely reported use of RFLP analysis has been in forensic science. RFLP methods have been critical in helping to identify criminals, and these methods have also helped exonerate innocent people. The first application of RFLP in forensic analysis was in the case of the murders of two young girls in England, in 1983 and 1986. Initially, a seventeen-year-old boy confessed to the murders. RFLP analysis, using DNA from the crime scene, indicated that he was not the murderer. After extensive investigation, including RFLP analysis of DNA from...
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Further Reading (Genetics & Inherited Conditions)
Allison, Lizabeth A. “Recombinant DNA Technology and Molecular Cloning.” In Fundamental Molecular Biology. Malden, Mass.: Blackwell, 2007. Includes information about RFLP.
Chang, J. C., and Y. W. Kan. “A Sensitive New Prenatal Test for Sickle-Cell Anemia.” New England Journal of Medicine 307, no. 1 (July 1, 1982): 30-32. A short and readable scientific paper describing one of the first applications of RFLP analysis to clinical medicine. The same issue of the journal also has another, somewhat more detailed, article on the same topic by S. H. Orkin et al.
Guilfoile, P. A Photographic Atlas for the Molecular Biology Laboratory. Englewood, Colo.: Morton, 2000. An illustrated guide to molecular biology techniques, including a substantial illustrated section on RFLP analysis.
Hartl, D. L., and Elizabeth W. Jones. “Types of DNA Markers Present in Genomic DNA.” In Genetics: Analysis of Genes and Genomes. 7th ed. Sudbury, Mass.: Jones and Bartlett, 2009. This excellent introductory genetics textbook devotes a section of chapter 2 to a discussion of RFLP within the broader context of DNA structure and genetic variation.
Jeffreys, A., V. Wilson, and S. L. Thein. “Individual-Specific Fingerprints of Human DNA.” Nature 316, no. 6023 (July 4-10, 1985): 76-79. A technical article that describes some of the background information that led to the use of RFLP...
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Web Sites of Interest (Genetics & Inherited Conditions)
Kimball’s Biology Pages. http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RFLPs.html. John Kimball, a retired Harvard University biology professor, includes a page about restriction fragment length polymorphisms in his online cell biology text.
NCBI, Restriction Fragment Length Polymorphism (RFLP). http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechRFLP.shtml. The National Center for Biotechnology Information has prepared this explanation of RFLP.
RFLP Method: Restriction Fragment Length Polymorphism. http://www.bio.davidson.edu/COURSES/genomics/method/RFLP.html. An explanation of RFLP is featured in this site designed for a microbiology course at Davidson College.
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