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Restriction fragment length polymorphism is a technique to study the large number of disorders which are in genetics. The restriction map identifies the uncommon chromosome which is separated from one another by distance along with nucleic acid.If the difference between the DNA in the population is rare, then it is termed mutation and if it is common, it is called polymorphism.
A restriction fragment is a pattern obtained from one person and then that pattern is compared to another pattern.Restriction fragment length is used as a genetic marker, which is used to follow the inheritance of DNA through families.
There are proteins called restriction enzymes that are cut into small DNA fragments, particular sequences known as restriction sites.
Example
After the segments have been made of DNA with the help of restriction enzymes, the researchers examine the segrigated part according to their size. If each of them have differences in their respective DNA sequences at specific sites, so the restriction enzymes will separate their DNA into smaller length of the fragment for research. There is also a difference in the number of DNA fragments observed in two or more than two individuals.
Restriction fragment length polymorphism was invented in the year 1984 by an English scientist Alec Jeffreys who at that time was doing his research on hereditary diseases.
Mechanism of Restriction Fragment Length Polymorphism
- DNA Extraction: DNA can be collected in the form of blood, saliva, hair strand, or any other way.
- DNA Fragmentation: After the collection,the sample is purified, then the DNA is dissolved using a restriction site. The base of all thr enzymes by recognition sites are commonly base pairs(4 to 6) in length.
The fragments that are generated by digestion is based on sequence recognition.
Example- If the sequence of GAGC are repeated 4 times by the sample, while the other sample repeats 2times, the fragmets generated by the enzyme for two separate samples will be different.
- Gel Electrophoresis: Gel electrophoresis are used to analyze those restriction fragments that areproduced in the time of DMA fragmentation.
After being negatively charged, the fragments get smoothlysegregated by electrophoresis thatis separated depends on their chargeand size.
After the separation, the fragmented DNAs are placed inside the chamber which contains the two electrodes and electrophoretic gel.
When the electric waves are applied to the field, the fragments start to move to the direction of positive electrode.By the gel, the smaller fragments travels faster because of their size. Which leaves the bigger one behind, by this process the DNAs are splited into two different bands.
- Visualization of bands: After all this process, To study the DNA. The luminescent dyesare used to highlight the bands to make them visible.
Application of Restriction Fragment Length Polymorphism
- To study the diseases related to genetics like cystic fibrosis and diabin a person
- For the confirmation of the DNA sample for paternity tests, forensic tests, and criminal investigation.
- To identify hereditary diseases coursing through the family.
Advantages of Restriction Fragment Length
- High reliability, Produced by specific sites and known enzymes which makes the result constant over time and location.
- Co-dominance makes the investigators able to distinguish heterozygotes from homozygotes.
Disadvantages of Restriction Fragment Length
- Time-consuming and more working hours.
- Specific mutation recognition because of cut sites.
- Low restriction fragment length polymorphism maker, difficult to detect it is only detectable by radioisotope which limits the application.
Conclusion
it is to be concluded that RFLP (Restriction Fragment Length Polymorphism) is a molecular technique that uses restriction enzymes to identify variations in homologous DNA sequences. The genomic DNA is fragmented and isolated with a restriction enzyme. The DNA fragments produced by the restriction enzyme are separated by gel electrophoresis, once they get segregated, the DNA fragments in the gel get denatured using an alkali, the alkali breaks the hydrogen bond between the DNA strengths. As a result of which DNA in the gel now becomes single-stranded. These fragments are transferred to a membrane by applying pressure. The next step to this is to detect fragments present in the membrane using a prob.
