RFLP (Restriction Fragment Length Polymerization)

                 

               RFLP (Restriction Fragment Length Polymerization)

 

 

Principle: -

Restriction fragment length polymerization (RFLP) technique in which organism may be differentiated by analysis of pattern derived from cleavages of this DNA

-        If two organism differ in this distance between sites of cleavage of particular restriction endonucleases, the length of the fragment produced wise differ when the DNA is digested with a restriction enzyme.

-        The similarity of the pattern generated can be used to differentiated species (and even strain) from one other.

 

Introduction: -

Alc Jeffery in 1985 discovered the technique called restriction fragment length polymerization (RFLP)

-        RFLP is technique that exploits variation in homologous DNA sequences.

-        It refers to a difference between sample of homologous DNA molecule that come from differing location of restriction enzymes sites and to related laboratory technique locating of restriction enzyme sites, and to a related laboratory technique by which these segments can be illustrated

-        RFLP analysis was the first DNA profiling technique inexpensive enough to see widespread application

 

Procedure: -

It uses various produces like PCR, digestion of DNA with restriction enzyme endonuclease, Agarose gel electrophoresis, radiolabeling of DNA with probes, southern blotting, Autoradiography.

 

1] PCR: -

to analyze the DNAs with the keep of RFLP it requires large amount of DNA for a better detection. Therefore, the DNA to be analyzed can be amplified with the help of PCR i.e. Polymerase chain reaction and multiple copies of desired DNA can be obtained to a level required for RFLP.

 

2] Restriction endonuclease: -

These are the enzyme which can cleave the DNA at a specific site present in the recognition sequence. This sequence may be 6-8 bp long and it is α palindromic sequence on the both the strand of DNA in a opposite direction. This enzyme is obtained from certain microorganism as these enzymes play a major role in the cell defense mechanism which bacteriophage enter the cell of these bacteria certain enzyme are produced with recognize some specific sequence on the phage DNA to cleave and destroy these DNA. Therefore, the entry of those phage is restricted. Therefore, these enzymes are called as Restriction enzyme/endonuclease.

 

3] Gel electrophoresis: -

The DNA fragment obtained the digestion with restriction enzyme can be separated on the basis of this length by gel electrophoresis. The fragment length solely depend on the frequency of recognition sequence and the sequence can be present on the DNA the various sites to the DNA is cleaved at every palindromic sequence and fragment are made.

 

4] Southern blotting: -

It is a technique by which the band obtained on agarose gel can be recorded permanently by transforming the band from gel to the membrane. A nylon/nitrocellular membrane is used for these purposes. Membrane is kept the agarose gel and top of it dry papers and heavy weight is pest. Due to the weight applied band start containing out of the gel and with the liquid part also start coming. The bands are trapped in the membrane and the liquid is soaked by the dry papers. This way the position of band on the membrane is exactly same as that on the gel.

 

 

5] Autoradiography: -

Radio labelled (⁸²P, ³³P,³⁵S and ³H) probe i.e. the ssDNA having ability to hybridize with the complimentary sequence are added on the membrane and allowed there to hybridize with the DN present on the bands under string out condition. After some time repeated washing is done to remove unbound probes and the membrane is held against the X-ray film, Radioactive compound in the bound probes emit rays which are captured by X-ray, and black-White image is formed when the image is developed the location of probes where they bind to the DNA on membrane is obtained.