LAB3.NEW - Recombinant DNA Session 3: Restriction digestion...

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Recombinant DNA Session 3: Restriction digestion and agarose gel electrophoresis There are many enzymes that act on DNA. Some modify the DNA but most are nucleases , enzymes that digest and or degrade the DNA. Nucleases can be divided into two types exonucleases and endonucleases . The exonuclease is a nuclease that degrades DNA, proceeding from a free end or a double-stranded break. In contrast, the endonuclease cuts DNA within an intact double stranded region. Some endonucleases are non-specific, nicking the DNA everywhere. However, there is a large family of endonucleases that only cut at specific (and often rare) sequences. These are called restriction endoncleases . Restriction endonucleases (REs) are believed to protect the bacterial genome against foreign DNA. Since the sequences they recognize are seldom found within the host’s genome, they can be used to safely degrade foreign DNA with little effect on the host. For the molecular biologist, there hasn’t been a more providential discovery than the restriction endonuclease. Because of their inherent specificity, REs can be used to cut DNA into fragments of manageable size which can be isolated and studied. Restriction endonucleases have been purified from various bacteria and each recognize short, specific, DNA sequences. For example, the restriction enzyme EcoR1 recognizes the sequence GAATTC and will cut between the G and A (G AATTC) while the restriction enzyme HindIII recognizes AAGCTT and cuts between the A's (A AGCTT). Both of these enzymes produce irregular cuts leaving short single- stranded ends (or overhangs,) and these overhangs can be spliced back together. This has made it possible for the molecular biologist to produce recombinant molecules , designer DNA and is vital for the “cloning” of genes. But an enzyme that can cut up DNA is not something any organism wants to have around unchecked. Whether a given restriction endonucleases is able to cut DNA depends not only on the existence of the specific short DNA sequence that the particular enzyme recognizes but also on the state of methylation of that recognition sequence in the DNA to be cut. A methylase adds a methyl group to either an adenine or a cytosine in the DNA but like REs , methylases recognizes specific sequences. Dam methylase, for example, is structurally similar to EcoR1 and recognizes the same sequence. The presence of a large methyl group in the EcoR1 site effectively blocks the restriction endonuclease. Thus, dam methylase protects the site from
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LAB3.NEW - Recombinant DNA Session 3: Restriction digestion...

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