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module:DNA: Structure and function

page:Engineering DNA

A variety of techniques can be used to extract, modify or even create genes. In the following example, recombinant DNA is made by inserting DNA from one organism into DNA from another organism.

The first step involves isolating a gene within DNA. For example, the gene for human insulin can be isolated for insertion into another organism. A chemical process such as the 'restriction enzyme technique' is one method of separating the gene from the rest of the DNA.

Restriction enzymes are bacterial proteins that act like chemical scissors, which target and cut out parts of the DNA molecule. Certain types of restriction enzymes have evolved to cut DNA only at specific sequences of nucleotides, which are typically palindromic regions, 4 to 7 bases long.

The most commonly used restriction enzyme, called EcoRI because it was isolated from the bacterium E. coli, will only cut DNA where the base sequence reads GAATTC. It is palindromic because the DNA mirror sequence of GAATTC is CTTAAG. Because restriction enzymes are so specific, genes from different sources that have been isolated using the same restriction enzyme will have similar cut-ends and can be easily 'stuck back together'.

The next step involves inserting the gene into a new organism such as another cell with the help of a carrier agent. Plasmids are often used as agents. A plasmid is a ring of DNA found in bacteria, capable of replicating itself. Plasmids are able to enter living cells - the bacterial world's natural method of moving genes around. Extracting plasmids is often the first step in genetic engineering.

The same restriction enzyme used to isolate the gene is used to cut open the plasmid DNA. The cut-open ends of the plasmid are complementary to ends on the isolated gene. The gene slots neatly into the gap in the plasmid and is 'glued' in place with the help of a ligation enzyme called DNA ligase.

Using restriction and ligation procedures, a new loop of bacterial plasmid DNA carrying desired genes or genes of interest has now been formed. The plasmid spliced with the isolated gene is known as a recombinant or transformed plasmid.

Recombinant plasmids may be mixed with or returned to bacterial cells, some of which take up the plasmids (and the ability coded by the introduced gene) and incorporate them into their DNA. Bacteria divide about every twenty minutes, and their DNA (including the inserted gene) replicates every time the cell divides. This natural replication process can lead to billions of copies of the gene being produced in a matter of hours.



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