RFLP and the DNA Analysis Applications
Restriction Fragment Length Polymorphism (RFLP) is a molecular method of genetic analysis that allows individuals to be identified based on unique patterns of restriction enzyme cutting in specific regions of DNA.
Also referred to as RFLP Analysis, the technique takes advantage of the polymorphisms in individual people's genetic codes. Even though all members of a species have essentially the same genetic makeup, these slight differences account for variations in phenotype, such as appearance or metabolism, between individuals.
RFLP: Analysis Technique and Applications
RFLP analysis technique involves cutting a particular region of DNA with known variability, with restriction enzymes, then separating the DNA fragments by agarose gel electrophoresis and determining the number of fragments and relative sizes.
A restriction enzyme is an enzyme, a protein molecule, that cuts DNA at restriction sites. In essence, the DNA sample is broken up and digested by the restriction enzymes. The resulting fragments are separated according to their lengths, and the pattern of fragment sizes will differ for each individual tested.
The full RFLP process requires probe labeling, DNA fragmentation, electrophoresis, blotting, hybridization, washing, and autoradiography. The detected RFLP is visualized using X-ray film in an autoradiography, where DNA fragments can be viewed and analyzed after they are separated from one another by electrophoresis.
Some of the applications for RFLP analysis include:
- DNA Fingerprinting. Forensic scientists may use RFLP analysis to identify suspects based on evidence samples collected at scenes of crimes.
- Paternity. RFLP is also used in the determination of paternity or for tracing ancestry.
- Genetic Diversity. The technique can be used in studying evolution and migration of wildlife, studying breeding patterns in animal populations and the detection and diagnosis of certain diseases.
The technique of using RFLP detection of variation in genomes is a vital tool in genome mapping and genetic disease analysis. If the location of a particular disease gene is being sought in a certain chromosome, then researchers would analyze the DNA of members of a family with the disease, then look for similar patterns of inheritance in RFLP alleles.
Once a disease gene is localized, conducting RFLP analysis on other family members could reveal a carrier of the mutant genes or signal overall disease risk. It is important to note that the RFLP technique is not widely used now that newer, more robust techniques are used for DNA analysis in forensic science and several other fields.
Unfortunately, the RFLP analysis technique is tedious and slow. Aside from requiring a large amount of sample DNA--the sample would usually need to be about the size of a quarter, which is relatively large for DNA samples--the process, from probe labeling to washing and autoradiography, can take up to a full month to complete.
Results from the Human Genome Project has pretty much replaced the need for RFLP. The project allowed for determining the entire sequence of the DNA found in human cells, the human genome, and for identifying all of the genes in the human genome.