Lab 4 - Lab 3: RESTRICTION ENZYMES - OVERVIEW Introduction...

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1 Lab 3: RESTRICTION ENZYMES - OVERVIEW Introduction Recombinant DNA technology or “gene cloning,” as it is frequently called, often involves insertion of selected DNA sequences from a variety of sources into plasmids which are then transformed into bacteria. The resultant recombinant bacterial strain may now express the plasmid genes as well as whatever foreign gene(s) that was inserted. This process would not be possible without accurate and predictable ways of cutting the DNA of the plasmid and the foreign source. Restriction endonuclease enzymes provide this service. Restriction endonucleases, often called restriction enzymes, cut DNA within the molecule by hydrolyzing the phosphodiester bonds between the nucleotides. Restriction enzymes were discovered in bacteria and there are now more than 1200 known restriction enzyme types. These enzymes are named using a simple system. EcoRI, for example, was isolated from E. coli and was the first enzyme isolated from a particular strain, hence the designation of I. HaeIII was isolated from Haemophilus aegyptius . HindIII was isolated from Haemophilus influenzae , and was the third enzyme discovered in a particular strain. Usually, restriction enzymes only cut the DNA at or near a very specific nucleotide sequence known as a recognition site. This “restrictive” nature of these enzymes allows molecular biologists to pin-point exactly where the DNA is to be cut. In many cases, the recognition site of a restriction enzyme consists of a palindromic sequence where the order of the bases is read the same on each side of the helix. The two sides of the helix are antiparallel and the recognition sequence reads from 5’ to 3’ on either side of the helix. For example the recognition site for EcoRI is as follows; GAATTC CTTAAG Some restriction enzymes cut both sides of the helix at the same point. HaeIII is such an enzyme and the fragments produced are “blunt” ended. Many restriction enzymes make assymetric cuts in the helix. EcoRI cuts DNA at the recognition site in the following fashion; G AATTC CTTAA G. This type of assymetric cut leaves a single stranded sequence hanging on either 5’ or 3’ end. Such a trailing end is often called a “sticky” end since it can bond by hydrogen bonding to a complementary single stranded sequence. The sticky ends make it rather easy to insert DNA into a plasmid. If the plasmid and DNA to be inserted are cut with the same restriction enzyme, the sticky ends will match and will anneal using DNA ligase enzyme. The result is a recombinant plasmid with a foreign fragment incorporated. Cleavage of DNA by a restriction enzyme is called digestion and is usually carried out at 37˚C in a small volume (e.g., 20 μl) of buffer and salt. Although restriction enzymes are active over a fairly broad range of conditions, optimal conditions have been determined for each restriction enzyme using a buffer which typically contains 10-100mM Tris-HCl pH 8.0, a salt (usually 0-150 mM NaCl), and 10 mM of Magnesium Chloride.
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This note was uploaded on 04/09/2011 for the course CHEM 4461 taught by Professor Max during the Spring '08 term at Lamar University.

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Lab 4 - Lab 3: RESTRICTION ENZYMES - OVERVIEW Introduction...

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