BIOTECHNOLOGY - Inshyna N.M. - 2009

CHAPTER 1. GENETIC ENGINEERING

Gene Isolation

Working with isolated genes is crucial for understanding the MOLECULAR MECHANISMS OF individual Gene functioning and interconnected genetic systems. Such research makes it possible to determine gene sizes, isolate them in pure form, and identify structural elements vital for their function.

The first step in gene isolation involves cutting DNA into small fragments using restriction Enzymes (Restriction Endonucleases). These enzymes cleave the polynucleotide chain at specific sites known as restriction sites. Typically, restriction sites are palindromic sequences characterized by twofold Symmetry (The nucleotide sequences are identical in the 5'→3' direction). Over 200 unique DNA sequences recognized by specific restriction enzymes are currently known. Restriction enzymes do not make a straight cut across both DNA strands, but rather a staggered one, resulting in The formation of "sticky" ends that can easily join together.

For example,

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Mixing two DNA fragments with identical "sticky" ends causes them to anneal. These linear "sticky" ends are held together by Hydrogen Bonds. To covalently seal the nicks, the enzyme T₄ bacteriophage DNA ligase is used (catalyzing the Formation of the 3',5'-phosphodiester bond).

Restriction enzymes allow both chromosomal DNA and plasmid DNA to be converted into a linear form, after which their fragments can be joined together.