Directional cloning of DNA Fragments with Heterologus Protruding Termini, Circularization, Method for CLoning, Generated Plasmid, Recombinant Molecules

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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 >> Directional cloning of DNA Fragments with Heterologus Protruding Termini

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Directional cloning of DNA Fragments with Heterologus Protruding Termini

This is the most efficient method for cloning, and any other method should be tried only when this method can not be used. In this method, the fragments to be cloned are generated by digestion with two different restriction enzymes.

Similar heterologous termini are generated in the plasmid vector by removing a small fragment due to cleavage with two different enzymes. In this method. since the two termini are not compatible.

Self ligation of either the linearized vector or the foreign DNA fragment leading to circularization is eliminated. Moreover, chimaeric DNA molecules will contain each only a single copy of the insert DNA oriented in a defined direction. Recombinant molecules with two or three copies of insert will be very rare (this is unlike the case with cloning of DNA fragments with homologous cohesive ends).

As an illustration of directional cloning, pUCl9 can be digested with BamHI and HindIIIand the resulting large fragment can be purified, removing the small fragment. The vector can then be ligated (using T4 ligase) to a segment of foreign DNA that contains termini compatible with those generated in the vector due to BamH I and Hind III. The steps involved in this directional cloning

Cloning of a DNA fragment in a plasmid vector by cleavage of both vector DNA and the foreign DNA by two different enzymes, producing non-compatible termini

Cloning of DNA Fragement in A Plasmid Vector by Cleavage of both Vector DNA and THe Foreign DNA by Two Different Enzymes, Producing Non-Compatiable termini

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