Restriction fragment length polymorphism

Restriction fragment length polymorphism (RFLP) is a technique invented in 1984 by the English scientist Alec Jeffreys during research into hereditary diseases. It is used for the analysis of unique patterns in DNA fragments in order to genetically differentiate between organisms – these patterns are called Variable Number of Tandem Repeats (VNTRs).

Genetic polymorphism is defined as the inherited genetic differences among individuals in over 1% of normal population. The RFLP technique exploits these differences in DNA sequences to recognize and study both intraspecies and interspecies variation.

1. Faculty of Biosciences, Institute of Biosciences and Technology, Shri Ramswaroop Memorial University By Abhinav Baranwal Roll No. 202110902120035 BSc. (H) Biotechnology 1 st Year Submitted To Dr. Nishu Mittal Restriction Fragment Length Polymorphism R F L P

2. Restriction fragment length polymorphism (RFLP) is a technique invented in 1984 by the English scientist Alec Jeffreys during research into hereditary diseases. It is used for the analysis of unique patterns in DNA fragments in order to genetically differentiate between organisms – these patterns are called Variable Number of Tandem Repeats (VNTRs). Genetic polymorphism is defined as the inherited genetic differences among individuals in over 1% of normal population. The RFLP technique exploits these differences in DNA sequences to recognize and study both intraspecies and interspecies variation.

3. RFLP is an enzymatic procedure for separation and identification of desired fragments of DNA. Using Restriction Endonuclease enzymes fragments of DNA is obtained and the desired fragment is detected by using restriction probes. May be used to differentiated two organism by analysis of patterns derived from cleavage of their DNA.

4. Step I: Restriction digest Extraction of desired fragments of DNA using restriction endonuclease (RE). The enzyme RE has specific restriction site on the DNA, so it cut DNA into fragments. Different size of fragments are generated along with the specific desired fragments. Step II: Gel electrophoresis The digested fragment are run in polyacrylamide gel electrophoresis or Agarose gel electrophoresis to separate the fragments on the basis of length or size or molecular weight. Different size of fragments form different bands.

5. Step IV: Blotting The single stranded DNA obtained are transferred into charge membrane i.e. Nitrocellulose paper by the process called capillary blotting or electro-blotting. Step III: Denaturation The gel is placed in sodium hydroxide (NaOH) solution for denaturation so that single stranded DNA are formed. Step V: Baking and blocking The nitrocellulose paper transferred with DNA is fixed by autoclaving. Then the membrane is blocked by using bovine serum albumin or casein to prevent binding of labelled probe nonspecifically to the charged membrane.

6. Step VI: Hybridization and visualization The labelled RFLP probe is hybridized with DNA on the nitrocellulose paper. The RFLP probes are complimentary as well as labelled with radioactive isotopes so they form color band under visualization by autoradiography.

7. To determine the status of genetic diseases such as Cystic Fibrosis in an individual. To determine or confirm the source of a DNA sample such as in paternity tests or criminal investigations. In genetic mapping to determine recombination rates that show the genetic distance between the loci. To identify a carrier of a disease-causing mutation in a family.

8. https://www.nlm.nih.gov/visibleproofs/education/dna/rflp.pdf http://www.bio.davidson.edu/courses/genomics/method/rflp.html http://www.kau.edu.sa/Files/0002923/Files/18591_Restriction%20Fragment%20Length%20Polymorphism.pdf http://cdn.intechopen.com/pdfs-wm/35104.pdf http://www.dnaforensics.com/DNAFingerprinting.aspx