Restriction fragment length polymorphism (RFLP)
Restriction fragment length polymorphism (RFLP) is a molecular biology technique that identifies variations in the lengths of DNA fragments generated by cutting DNA with restriction enzymes. This method has significant applications in gene mapping, forensic science, and genetic identification. RFLPs serve as markers that can reveal individual differences in DNA sequences, making them useful for determining paternity, assessing disease status, and providing forensic evidence in criminal investigations.
The technique was first applied in forensic contexts in 1985 by geneticist Alec Jeffreys, who utilized it to establish DNA fingerprints from regions in the human genome characterized by variable numbers of tandem repeats (VNTRs). The process involves isolating DNA, using restriction enzymes to cut the DNA, and then separating the fragments by size through gel electrophoresis. The fragments are subsequently transferred to a membrane, where labeled DNA probes can hybridize to the VNTR regions, allowing for detection of the fragment sizes that vary among individuals.
Although RFLPs were pivotal in the development of DNA profiling, their use has waned with the advent of polymerase chain reaction (PCR) techniques, which can amplify smaller DNA samples and are effective even with degraded DNA. Today, PCR methods are more commonly employed in forensic analysis due to their efficiency and adaptability.
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Restriction fragment length polymorphism (RFLP)
DEFINITION: Variations in the lengths of pieces of DNA cut with restriction enzymes.
SIGNIFICANCE: Restriction fragment length polymorphisms are used as markers in gene mapping and to identify individuals in DNA fingerprinting. They are used to determine disease status, to test for paternity, and as forensic evidence to identify the sources of DNA samples.
Restriction fragment length polymorphisms (RFLPs) were the first (deoxyribonucleic acid) typing to be used as forensic evidence. English geneticist Alec Jeffreys and his colleagues identified DNA fingerprints for forensic application in 1985. They examined regions of the human genome called minisatellites, which are made of a DNA sequence that is tandemly (end-to-end) repeated hundreds of times. These are also called variable number of tandem repeats (VNTRs). Individuals vary in the numbers of tandem repeats of that DNA sequence they have in their genomes.
In detecting these RFLPs, DNA is isolated from cells. A restriction enzyme (which makes sequence-specific cuts in DNA) is used to cut outside the repeating sequence. DNA is separated based on size through the use of gel electrophoresis. The DNA fragments are transferred from the gel to a membrane (called a Southern blot). A single-stranded, labeled DNA probe complementary to the repeating sequence is hybridized with the DNA on the membrane. The probe base-pairs with its complementary sequences on the membrane. This labeled probe allows the VNTR regions to be detected among all the DNA in the genome. The size of the fragment detected varies depending on the number of tandem repeats the individual has. This is length polymophism—length variation from individual to individual.
Samples of blood, semen, saliva, or other biological materials are often collected at crime scenes for later DNA analysis. As such a sample ages, dries, or is subjected to high temperatures, the DNA may degrade (break into smaller pieces). To use RFLPs, intact DNA of 20,000 to 25,000 base pairs is required. If DNA degraded to smaller sizes than this is used, a restriction fragment might not be generated, which could cause the false exclusion of a suspect. The minimum amount of DNA that is needed for detection using a radioactive or chemiluminescent probe is 10 to 50 nanograms.
RFLPs were used in the first forensic DNA profiling. As many as fifteen different loci could be examined. The Combined DNA Index System (CODIS), the system developed by the Federal Bureau of Investigation (FBI) for tracking DNA profiles, keeps DNA information from convicted felons for five RFLP loci.
The use of RFLPs for DNA fingerprinting is increasingly being replaced by polymerase chain reaction (PCR) methods, which amplify DNA. With PCR, many copies of short pieces of DNA can be made rapidly, and variations in sequences copied can be used to create a DNA profile. Because PCR can be done on degraded DNA, this newer method has widely replaced RFLPs in forensic applications.
Bibliography
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