- Restriction fragment length polymorphism (RFLP) is an enzymatic procedure for the separation and identification of desired fragments of DNA.
Principle of RFLP:
Using restriction endonucleases, fragments of DNA are obtained and the desired fragment is detected by using restriction probes. Southern hybridization or southern blotting, using restriction endonucleases for isolation of desired length of DNA fragments is an example of RFLP.
Procedures of RFLP:
- Restriction digest:
- Extraction of DNA fragments after digestion of genomic DNA by restriction endonuclease (RE).
- RE has specific restriction sites on the DNA strand, so it cuts or nicks DNA into fragments. Different sizes of fragments are generated along with the specific desired fragments.
- Gel electrophoresis:
- Polyacrylamide gel electrophoresis or Agarose gel electrophoresis can be used to separate the fragments on the basis of their length i.e. size or molecular weight.
- Different fragments form different bands on the basis of their size.
- The gel with bands is placed in sodium hydroxide (NaOH) solution for denaturation, so that the double stranded DNA fragments become single stranded.
- The single stranded DNA obtained is transferred into charge membrane i.e. Nitrocellulose paper by electro-blotting or capillary blotting.
- Baking and blocking:
- The nitrocellulose paper with transferred DNA is fixed by autoclaving.
- This membrane is then blocked by using bovine serum albumin or casein to prevent binding of labeled probe nonspecifically to the charged membrane.
- Hybridization and visualization:
- The labeled RFLP probe is allowed to base pair with the complementary single-stranded DNA on the nitrocellulose paper, the process called hybridization.
- The RFLP probes are labeled with radioactive isotopes so they form color bands under visualization by autoradiography.
Applications of RFLP:
- It helps in analysis of unique pattern in genome of living organisms for their identification and differentiation.
- The recombination rate in the loci between restriction sites can be determined.
2.Genetic disease analysis:
- After identification of gene for a particular genetic or hereditary disease, that gene can be analyzed among other family members.
3.Detection of mutated gene.
4.DNA finger printing or DNA profiling or DNA typing (forensic test):
- DNA finger printing, one of the techniques for paternity test, criminal identification etc. is based on RFLP.