Cutting and Joining DNA Molecules - Enabling Techniques

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Chapter: Pharmaceutical Microbiology : Recombinant DNA Technology

DNA isolated from any type of cell can be fragmented using restriction endonucleases. These are enzymes produced by microorganisms which cut foreign DNA and can restrict the proliferation of infecting viruses.


CUTTING AND JOINING DNA MOLECULES

 

DNA isolated from any type of cell can be fragmented using restriction endonucleases. These are enzymes produced by microorganisms which cut foreign DNA and can restrict the proliferation of infecting viruses. Some of these enzymes cut at specific points known as restriction sites which are palindromic sequences (complementary sequences with identical nucleotide sequences when read in the 5′ to 3′ direction) of various lengths. For example, EcoRI (Escherichia colirestriction enzyme I) has specificity for the sequence GAATTC and hydrolyses the G-A phosphodiester bond. Enzymes which recognize and cut at restriction sites of 4–8 base pairs (bp) are particularly useful, as the probability of a site appearing in a random DNA fragment is inversely proportional to its length.

 

There are two different types of DNA ends that can be generated using restriction enzymes: cohesive or sticky and blunt ends (Table 25.1). DNA fragments obtained by restriction enzyme digestion can be covalently joined together using the enzyme DNA ligase. There is a limitation, however, with regards to the type of ends this enzyme is able to bond together. Only blunt ends generated by some restriction enzymes (e.g. DraI and EcoRV) or compatible sticky ends generated by either the same restriction enzymes (e.g. Eco RI) or by enzymes that generate complementary overhanging ends (e.g. Sau3AI and BamHI) will be bonded by the DNA ligase (Table 25.1).

 


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