RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) - PROCEDURE & APPLICATIONS - EASY NOTES

-The property of molecules to exist in more than one form is known as polymorphism, and it is a normal variation and doesn’t have any harmful effect.

-The existence of two or more types of restriction fragment patterns is called RFLP.

-It is a procedure for the separation and identification of desired fragments of DNA

-Restriction endonuclease Enzyme is used for the separation of DNA fragments

-Restriction probes are used for identification of desired fragments

-The southern blotting technique is an example of RFLP.

Step 1

-Restriction endonuclease enzyme cuts DNA at specific restriction sites into fragments

-Different size of fragments are generated along with specific desire fragments

Step 2

-Separation of fragments based on length, size, or molecular weight is done by running the pieces on polyacrylamide gel electrophoresis for agarose gel electrophoresis.

-Different bands are formed by different size of fragments

Step 3

-Denaturation is done by subjecting the gel to sodium hydroxide solution resulting in the formation of single-stranded DNA

Step 4

-Transfer of single-stranded DNA onto nitrocellulose paper is done by a process called capillary blotting or electroblotting

 Step 5

-Autoclaving is done to fix the DNA onto nitrocellulose paper.

Step 6

-The radioactive labeled RFLP Probe is hybridized with DNA on nitrocellulose paper.

-It forms color bands which are visualized by autoradiography

CLINICAL APPLICATIONS

-Useful in diagnosing disputed parenthood -DNA fingerprinting  

-Useful in human population genetic and comparison of the genetic makeup of related species

-Useful to detect mutated gene

-Useful in genome mapping

- Useful in genetic disease analysis