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Home >>Molecular Probes and Gene Libraries >> Detection of RFLPs - Restriction Fragment Length Polymorphism
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Detection of RFLPs - Restriction Fragment Length Polymorphism -  A RFLP can be demonstrated using the following steps:
(i) Extract and purify DNA from several individuals which may differ in some respects among themselves.
(ii) Digest DNA samples with a restriction endonuclease.
(iii) Subject each DNA digest to gel electrophoresis separately, but on the same gel slab.
(iv) The DNA fragments of different lengths resulting from digestion can be separated by gel electrophoresis, but the differences in the distribution patterns of fragments in several DNA samples due to digestion by several endonucleases can not be detected directly. This is because the number of fragments is large and the range in size is rather continuous, such that they form a continuous smear on the gel. Electrophoresis is, therefore, followed by the following steps.

(v) Fragments are transferred from gel to nitrocellulose filters using the technique of Southern blotting.

(vi)The filters with small fragments, fully or partially homologous to the probe, can be detected by autoradiography after hybridization.

Since DNA clones having repetitive DNA may hybridize with many fragments, they will give a smear on autoradiography. Therefore, unique DNA sequences are generally used as probes for detecting RFLPs. The DNA fragments hybridizing with the probe, can be visualized by autoradiography, and will be called RFLPs at, the phenotypic level.

In, results of a hypothetical experiment are shown where RFLPs are detected by using four plants (which differ by point mutations as well as by insertions) and three enzymes individually and in combination.

 

  

 
 

 
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