Construction of a Restriction Map, Individual Enzymes, DNA Digestion, Original DNA, Reciprocal Digests, Original DNA

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Chimeric DNA Molecular Probes and Gene Libraries Chimeric DNA Molecular Probes and Gene Libraries Introduction Restriction Enzymes for Cloning Technique of Restriction Mapping Restriction Clevage and Gel Electrophoresis Construction of a Restriction Map Use of Partial Digests end Labelling and Hybridization in Restriction Mapping Construction of Chimeric DNA Cloning of Plasmid Vectors Directional Cloning of DNA Fragments with Heterologus Protruding Termini Adding Poly dA at 3' Ends of DNA Clone Blunt End Ligation by T4 DNA Ligase Cloning in Phage λ and Cosmid Vectors Cloning in phage λ Vectors Cloning in Cosmid Vectors Hosts for Cloning Vectors Bacteria as Hosts Eukaryotes as Hosts Molecular Probes Preparation of Probes Genomic DNA Probes

Home >> Biotechnology and Genomics >> Chimeric DNA Molecular Probes and Gene Libraries >> Construction of a Restriction Map

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Construction of a restriction map

The data on DNA digestion by more than one endonucleases as discussed above can be utilized to arrange the sites of cleavage in a defined order. This can be done by several methods and one of them is illustrated. This method involves successive digests with two individual enzymes, where we extract each fragment produced in the individual digests with either enzyme A or enzyme B and then cleave it with the other enzyme.

The original DNA sample is also digested by a mixture of both the enzymes to confirm the results of individual successive digests. we have shown results of such an exercise. When the fragment A-21O0 is obtained and digested with enzyme B, it is cut into two fragments of 1900 bp and 200 bp.

Fragment B-2500 (obtained from individual digest with enzyme B) is similarly cut by enzyme A into two fragments of 1900 bp and 600 bp, suggesting of A-2100 and B- 2500, in the region of fragment of 1900 bp which is obtained by one cut with enzymes A and B (double digest). It may be that in reciprocal digests (A followed by B and B followed by A) and double digests, same fragments are available. Using this information, we can field the overlapping regions in A and B degests and find out the sites of cleavage by A and B.

Reconstruction of restriction map showing restriction sites of enzymes A and B in two overlapping fragments

A restriction map, in the form of linear arrangement of cleavage sites

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