Restriction Fragment Length Polymorphism (RFLP) Overview
- Restriction Using the method of fragment length polymorphism (RFLP), it is possible to distinguish between different species by examining patterns left over from the cleavage of their DNA.
- It is a technique that exploits variations between homologous DNA sequences.
- A restriction fragment length polymorphism is comprised of alternative alleles associated with restriction fragments of different sizes. Simply explained, RFLP refers to variations in the length of restriction DNA fragments amongst individuals of the same species.
- As part of a procedure known as a restriction digest, a restriction enzyme that can detect and cut DNA whenever a certain short sequence occurs can fragment a sample of DNA in order to find these restriction fragment length polymorphisms.
- The resultant DNA fragments are then placed on a membrane with the Southern blot method after being length-separated by agarose gel electrophoresis. The membrane is then hybridised with a tagged DNA probe to determine the length of the matching DNA fragments.
- An RFLP occurs when a recognized fragment’s length varies between individuals.
- Although mostly outdated now as a result of the development of low-cost DNA sequencing technologies, the initial technique for DNA profiling was RFLP analysis, which was sufficiently affordable to be applied widely.
Principle of Restriction Fragment Length Polymorphism (RFLP)
The length of the fragments produced when a restriction enzyme breaks down DNA varies if two organisms have different distances between the sites of cleavage of a given restriction endonuclease. The similarities and differences in the resulting patterns can be exploited to distinguish across species (and even strains).
Steps Involved in Restriction Fragment Length Polymorphism (RFLP)
- Isolating the target’s DNA is the initial step in this procedure.
- Following DNA extraction from the sample, restriction digestion is carried out using restriction enzymes.
- The DNA is subsequently sorted depending on size using gel electrophoresis on the digested DNA sample. Numerous DNA fragments with minute length variations are created.
- The double-stranded DNA is subsequently single-stranded by subjecting the gel to a reagent.
- Southern blotting, in which DNA is transferred from gel to nylon membrane, comes next.
- The radioactive complementary nucleotide probes are then dissolved in a solution, where they interact with the nylon membrane’s DNA to form hybrids,
- The membrane is subsequently subjected to an X-ray film that is developed into an autoradiogram using hybridised radioactive probes.
- RFLP analysis is employed to find pattern differences and validate polymorphisms.
Applications of Restriction Fragment Length Polymorphism (RFLP)
- In the past, RFLP analysis was a crucial technique for paternity testing, illness risk assessment, localisation of genes for genetic diseases, and genome mapping.
- RFLP may be used in a variety of situations to accomplish a range of objectives:
- To identify the origin of a DNA sample in criminal or paternity proceedings. It has forensic applications, so to speak.
- Establishing a person’s illness or condition. (For instance, it may be used to find mutations.)
- to assess recombination rates, which may be used to create a genetic map using the separation of RFLP loci.
- while describing the breeding habits or genetic diversity of animal groups.
- For mapping the chromosomes of humans, mice, maize, tomatoes, rice, etc., RFLP has been created.
Advantages of Restriction Fragment Length Polymorphism (RFLP)
- The main advantage of RFLP analysis over PCR-based methods is that no prior sequence information or oligonucleotide synthesis is required.
- Results are mostly based on reliable genotypic features rather than phenotypes.
- Based on RFLP Genetic Marker
- Given that RFLP is a co-dominant marker, it can calculate heterozygosity.
- A very helpful research in genomic DNA sequence is RFLP.
- It is a highly reliable approach that is easily portable between laboratories.
Limitations of Restriction Polymorphism in Fragment Length (RFLP)
- It needs a significant amount of sample DNA.
- No possibility of automation
- Low polymorphism levels in some species
- Few loci are found in each experiment.
- It requires a good probe library.
- requiring a comprehensive procedure that includes DNA fragmentation, probe labelling, electrophoresis, blotting, hybridization, washing, and autoradiography.