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Modification of Cut Ends - The 3'-ends of DNA strands always carry a free hydroxyl (-OH) group, while their 5'-ends always bear a phosphate group. Often the ends produced by restriction enzymes have to be modified for further manipulation of the fragments; some of the modifications are summarised below.
1. Removal of the 5'-phosphate group of vector DNA by alkaline phosphatase treatment in order to prevent vector circularization during DNA insert integration.
2. Addition of a phosphate group to a free 5'-hydroxyl group by T4 polynucleotide kinase.
3. Removal of the protruding ends by digestion with, say, S1 nuclease; this enzyme digests both 3'- and 5'-protruding ends.

4. Filling in of the protruding ends by extending the recessed (shorter) strand with, say, Klenow fragment of E. coli DNA polymerase I. (Both the strategies 3 and 4 generate blunt ends which can be ligated by T4 polynucleotide ligase.)

5. Synthesis of single-stranded tails (protruding ends) at the 3'-ends of blunt ended fragments by the enzyme terminal deoxynucleotidyl transferase; this is called tailing. This reaction can be used to generate protruding ends of defined sequence, e.g., poly-A tails on the 3'­ends of the DNA insert and poly- T tails on the 3'-ends of the vector; the protruding ends of the DNA insert and the vector will, therefore, base pair under annealing conditions.

6. Linker and/or adaptor molecules can be joined to the cut ends. Linkers are short, chemically synthesized, self complementary, double stranded oligonucleotides, which contain within them one or more restriction endonuclease sites, e.g., linker 5' -CCGAA TTC­GG (only one strand of the linker is shown here) contains one EcoRI site.

Linkers are joined with blunt ended DNA fragments; cleavage of the linker with the appropriate restriction enzyme creates suitable cohesive protruding ends Linkers create cohesive ends Oft blunt ended DNA fragments, and on fragments having unmatched or undefined sequences in their protruding ends. In the latter situation, the DNA fragments are first made blunt-ended, following which the selected linkers are ligated to them by T 4 ligase.

7. Adaptors are short, chemically synthesized- DNA double strands, which can be used to link the ends of two DNA molecules that have different sequences at their ends. There are different kinds of adaptors suited for different purposes.

For example, a conversion adaptor is used to join a DNA fragment or insert cut with one restriction enzyme, say, EcoRI, with a vector opened with another enzyme, e.g., BamHI. These adaptors have the recognition sequences of different endonucleases at their ends.

For example, the conversion adaptor has recognition sequence for BamHI at one end and that for EcoRI at the other. This adaptor can be used to convert the cohesive end generated by BamH1 to one produced by EcoRI or vice versa.
 

 
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