Topic 7, Restriction mapping & recombinant DNA techniques

Topic 7, Restriction mapping & recombinant DNA techniques

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Topic 7—Restriction enzymes, electrophoresis, cloning
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Learning Objectives Be able to define the following terms: cloning, cloned DNA, recombinant DNA, restriction endonuclease (aka restriction enzyme), DNA ligase, restriction map, restriction digest, palindrome, cloning vector, plasmid, mass ladder, sticky ends, gene expression Be able to explain: Need for various components of a plasmid cloning vector How/where restriction endonuclease enzymes cut DNA and be able to identify a palindromic sequence in DNA Rationale for blue/white screening Be able to interpret sizes of molecules on a gel relative to a mass ladder Be able to predict sizes of DNAs after a restriction digest using a restriction map After completing the homework assignment, be able to construct a restriction map based upon single and double digest information of either circular or linear DNA molecules
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Electron micrograph of plasmid DNA Plasmids are small (sometimes large) accessory DNA molecules in bacteria Circular (no ends) and double- stranded under normal conditions Plasmids typically are used to house non-essential genes which may be necessary for bacteria in the future (such as antibiotic resistance in case the cell encounters that antibiotic)
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Endonuclease s: enzymes that cut nucleic acids endo means it cuts in the middle of a molecule; exo means it only cuts at the ends
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Endo=middle cutters Exo=end cutters Endo=cut at specific DNA sequences Exo=cut at random sequences at end of molecule
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Why are they called restriction endonucleases? Because bacteria use these enzymes as a defense mechanism to protect themselves from phage (bacterial viruses). A species of phage can be “restricted” from infecting a species of bacteria due to the presence of a restriction enzyme (phage DNA is cut to pieces and is no longer functional)
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What is a restriction map? A diagram that shows relative positions of various restriction enzyme cut sites in a DNA molecule
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How do you create a restriction map? Cut DNA with a single restriction enzymes and look at fragments produced (run on gel to separate distinct fragments) Cut DNA with that same enzyme and a 2 nd enzyme and look to see which bands remain and which change If a fragment remains the same size after a “double digest”, it must not be cut by the 2 nd enzyme If a fragment is eliminated from a “double digest”, it must be cut by the other enzyme
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Recognition Enzyme Sequence Name How to interpret enzymes Slash shows where cut actually takes place
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sites are palindromes A DNA palindrome reads the same way forwards on the sense strand as it reads backwards on the antisense strand Example palindrome: DNA palindromes concern both strands Is the sequence to the right a palindrome? Would it be cut by a restriction enzyme?
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This document was uploaded on 11/04/2011 for the course MMBIO 240 at BYU.

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Topic 7, Restriction mapping & recombinant DNA techniques

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