Shot Gun Method,Restriction Enzyme,Genomic DNA,DNA Fragments,Chromatography

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	Shot Gun Method - The direct method of obtaining an insert is to digest the total genomic DNA with restriction enzyme. This procedure has the advantage of reproducibility. The same set of fragments is produced each time a specific enzyme cleaves a particular DNA. If the endonuclease yields cohesive ends that match those on the vector, cloning is carried out directly or after enrichment of the population of fragments for the desired insert. When the digested genome is large, few well separated DNA fragments are obtained. Rather, a continuous smear of DNA fragments of all possible sizes appear.
For example, if human genomic DNA (3 x 1O9bp) is subjected to RE digestion which has a six base pair recognition site then the RE will recognize a recognition site for every 4096 (46) bp on average and can yield about 7 x 105 unique fragments. The resulting sets of fragments yield a continuous spread of DNA molecules of all possible sizes. Electrophoresis and high performance reverse phase liquid chromatography are widely used to resolve the fragments based on size. However neither method yields pure DNA fragments from complex mixtures. Preparations are usually contaminated with many fragments of similar size or elution properties. Nevertheless, even minor enrichment for the desired fragment can significantly decrease the problem associated with finding the desired DNA segment in a large collection of clones.