Restriction fragment length polymorphisms, or RFLPs, are differences between individuals in the lengths of DNA fragments cut by enzymes. Restriction enzymes are proteins that cut DNA at short, specific sequences called restriction sites. After cutting a segment of DNA with restriction enzymes, researchers can examine the fragments using a laboratory method called gel electrophoresis, which separates DNA fragments based on their size.
If two individuals have differences in their DNA sequences at particular restriction sites, the restriction enzymes will cut their DNA into fragments of different lengths. There may also be differences in the number of DNA fragments observed between two or more individuals. Gentaur RFLP analysis can be used as a form of genetic testing to see if an individual carries a mutant gene for a disease that runs in their family.
Restriction endonucleases are enzymes that cut long DNA into short pieces. Each restriction endonuclease targets different nucleotide sequences in a DNA strand and therefore cuts at different sites. The distance between the cleavage sites of a given restriction endonuclease differs between individuals. Therefore, the length of the DNA fragments produced by a restriction endonuclease will differ between individual organisms and species.
How does it work?
The RFLP is carried out through a series of steps that are briefly described below:
- Extraction of DNA
To begin with, DNA is extracted from blood, saliva, or other samples and purified.
- DNA fragmentation
The purified DNA is digested using restriction endonucleases. The recognition sites of these enzymes are generally 4 to 6 base pairs in length. The shorter the recognized sequence, the greater the number of fragments generated from digestion.
For example, if there is a short GAGC sequence that occurs repeatedly in a DNA sample. The restriction endonuclease that recognizes the GAGC sequence cuts the DNA at each repeat of the GAGC pattern. If one sample repeats the GAGC sequence 4 times while another sample repeats it 2 times, the length of the fragments generated by the enzyme for the two samples will be different.
- Gel electrophoresis
Restriction fragments produced during DNA fragmentation are analyzed by gel electrophoresis. The fragments are negatively charged and can be easily separated by electrophoresis, which separates molecules based on their size and charge. The fragmented DNA samples are placed in the chamber containing the electrophoretic gel and two electrodes. When an electric field is applied, the fragments migrate towards the positive electrode. Smaller fragments move faster through the gel, leaving larger ones behind, and therefore the DNA samples separate into distinct bands on the gel.
- Band Visualization
The gel is treated with luminescent dyes to make the DNA bands visible.
RFLP has been used for various genetic analysis applications since its invention.
Some of these key RFLP applications are listed below:
- Determine the status of genetic diseases such as Cystic Fibrosis in an individual.
- To determine or confirm the origin of a DNA sample, such as in paternity tests or criminal investigations.
- In genetic mapping to determine recombination rates showing the genetic distance between loci.
- To identify a carrier of a disease-causing mutation in a family.
1. Since its invention, RFLP has been a widely used genome analysis technique in forensic science, medicine, and genetic studies. However, it has become almost obsolete with the advent of relatively simple and less expensive DNA profiling technologies, such as polymerase chain reaction (PCR).
2. The RFLP procedure requires numerous steps and takes weeks to obtain results, while techniques such as PCR can amplify target DNA sequences in a few hours.
3. In addition, RFLP requires a large DNA sample, the isolation of which can be a laborious and time-consuming process. In contrast, PCR can amplify minute amounts of DNA in a matter of hours.
4. Due to numerous reasons like these, the PCR technique has largely replaced RFLP in most applications that require DNA sequencings, such as paternity testing or forensic sample analysis.
5. Furthermore, the identification of single nucleotide polymorphisms in the Human Genome Project has nearly replaced the need for RFLP in disease state analysis.