Restriction Fragment Length Polymorphism. Arielle Weir. The Uses. Polymorphism, which is in both coding and noncoding parts, is a difference in DNA that can be analyzed between two different origins. Two individuals of the same species have the same genome, but different alleles.
Restriction Fragment Length Polymorphism
The sample of DNA is first cut up using restriction endonucleases. The sample must be quite large, to have enough to compare. This can be blood the size of a quarter, semen the size of a dime, etc. This creates many fragments of the DNA.
The fragments are pulled through gel using gel electrophoresis. Since there are so many fragments, they cannot be clearly identified. They appear as long smears. The whole gel is placed in a chemical that separates the DNA into single strands.
The DNA fragments are moved from the gel to a nylon membrane. This is achieved through a process called Southern blotting. Since DNA is negatively charged, when a positive electrode is put behind the nylon, the DNA moves towards it and binds.
Once the DNA has bound to the nylon membrane, the whole thing is dipped into a solution with radioactive complimentary nucleotide probes for parts that have been chosen to be compared, such as certain alleles that may have a mutation or parts that will not be the same in another person. These probes will bind to the complimentary sequence of DNA with hydrogen bonds. This is called hybridization.
An X ray film is then put on top of the nylon for two to three weeks. The radioactive probes make the film expose, creating patterns in the film. This is called an autoradiogram. The patterns that are created are unique to every individual. These can now be compared to find matches.