Rflp presentation
- 2. Restriction Fragment Length Polymorphism (RFLP) Group Members: Ashfaq Ahmad (08) Pirzad (91) Basit Ali (38) Muhammad Saleh
- 3. What is RFLP ? Restriction Fragment Length Polymorphism is a variation in the length of a DNA fragment produced by a specific restriction enzyme acting on a DNA from different individuals that usually results from a genetic mutation. If two organisms differ in the distance between site of cleavage of a particular restriction endonuclease, the length of the fragments produced will be different when the DNA is digested with a restriction enzyme. RFLP analysis is the detection of the change in the length of the restriction fragments.
- 4. Continuous A restriction enzyme cuts the DNA molecule at every occurrence of a particular sequence, called restriction site. For Example, HindIII enzyme cuts at AAGCTT. If we apply a restriction enzyme on DNA, it is cut every occurrence of the restriction site into a million restriction fragments each a few thousands nucleotides long.
- 5. Continuous Any mutation or polymorphism of a single nucleotide may destroy (AAGCTT for HindIII) and change the length of the fragment. The term polymorphism refers to the slight differences between individuals, in base pair sequences of genes or A polymorphism is a clinically harmless DNA variation that does not affect the phenotype. So RFLP analysis is a technique which is used to detect the change in the length of the restriction fragments.
- 6. RFLP Technology RFLP detection depends on the possibility of comparing band profiles generated after restriction enzyme digestion of target DNA. These differences in fragment lengths can be seen after gel electrophoresis, hybridization and visualization. The basic steps involved are as follows: 1. Isolation of DNA 2. Restriction Digestion & Gel Electrophoresis 3. DNA transfer by Southern blotting 4. DNA hybridization
- 7. (1) Isolation of DNA Isolating DNA is the first step for many DNA-based technologies. DNA is found either in nuclear chromosomes or in organelles (mitochondria and chloroplast). To extract DNA from its location, several laboratory procedures are needed to break the cell wall and nuclear membrane, and so appropriately separate the DNA from other cell components. When doing so, care must be taken to ensure the process does not damage the DNA molecule and that it is recovered in the form of a long thread.
- 8. (2) Restriction Digestion & Gel Electrophoresis Extracted DNA is digested with specific, carefully chosen, restriction enzymes. Each restriction enzyme, under suitable conditions, will recognize and cut DNA resulting in a restriction fragments of different lengths. The thousands of restriction fragments produced are commonly separated by Gel Electrophoresis on agarose gels.
- 9. Continuous For visualization of DNA bands on the gel, it is stained with ethidium bromide. But staining alone cannot detect polymorphisms. Hybridization must therefore be used to detect specific fragments.
- 10. (3) DNA transfer by Southern blotting DNA transfer is called “Southern blotting”, after E.M. Southern (1975), who invented the technique. In this method, the gel is first denatured in a basic solution of NaOH and placed in a tray. A porous Nylon or nitrocellulose membrane is laid over the gel. All the DNA restriction fragments in the gel transfer as single strands by capillary action to the membrane. All fragments retain the same pattern on the membrane as on the gel.
- 11. (4) DNA Hybridization The membrane with the target DNA is exposed to the DNA probe (radioactively labelled). On the basis of availability and complementarity, hybridization will occur. The DNA probe is a singe-stranded molecule, conveniently labelled, using any standard method (e.g. a radioisotope), and hybridize with the target DNA, which is stuck to the membrane.
- 12. Continuous Thus the DNA probe binds with the sequences that are complementary to it among the thousands or millions of undetected fragments that migrate through the gel. Desired fragments maybe detected after simultaneous exposure of the hybridized probe to X-rays, which on exposure appear as black spots on the X-ray film.
- 14. Applications of RFLP The important applications of RFLP are: 1. RFLP can be used paternity cases or criminal cases to determine the source of a DNA sample (i.e. it has forensic applications). 2. RFLP can be used to detect mutations.
- 15. Advantages of RFLP Some of the advantages of RFLP are as follow: 1. It is a simple process. 2. It is an accurate process. 3. No sequence information required.
- 16. Disadvantages of RFLP Some of the disadvantages of RFLP are as follow: 1. It is an expensive process. 2. It is slow and time consuming process. 3. Labour intensive. 4. Requires very large amount of DNA. 5. It is difficult to automate.