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Genetics6 - 105 Recombinant DNA and Biotechnology Chapter...

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Co In E Recombinant DNA and Biotechnology Chapter 16 Gel analysis of restriction fragments Southern blot analysis Cleaving and Rejoining DNA Cloning DNA into Cells Biotechnology: Applications of DNA Manipulation 105
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Co In E DNA Gel Electrophoresis and Hybridization Movie Animation 16-1
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Co In E Example of RE = EcoRI RE are named after their source EcoRI = from a strain of E. coli Cuts DNA at specific sequence = palindrome 5’…GAATTC…3’ …G AATTC… 3’…CTTAAG…5’ …CTTAA G… RE cut DNA into fragments Fragments can be joined by 2nd type of enzyme = DNA ligase Scientist can use these enzymes to splice together any 2 DNAs
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Co In E
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Co In E Figure 16.8 Cutting and Splicing DNA 113 Recombinant DNA Sticky ends
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Co In E Insert gene of interest into a vector Then transfect it into bacteria Why bacteria? Easy to grow and manipulate. Much of their molecular biology is known. They grow very fast so can amplify gene of interest very quickly Bacteria contain plasmids = small circular chromosomes that are easily manipulated to carry recombinant DNA into cells Vector must have same restriction site as DNA fragment Reporter gene = usually antibiotic resistance gene, so all non transfected cells will die Recombinant DNA technology
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Co In E Getting New Genes into Cells DNA can be incorporated into the host cell by a vector , which should have four characteristics: – The ability to replicate independently in the host cell – A recognition sequence for a restriction enzyme, permitting it to form recombinant DNA – A reporter gene that will announce its presence in the host cell – A small size in comparison to host chromosomes 114
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Co In E Figure 16.9 Vectors for Carrying DNA into Cells 115
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Co In E DNA fragments used in cloning are obtained from : • Random fragments of chromosomes maintained as gene libraries Complementary DNA obtained by reverse transcription from mRNA Artificial synthesis or mutation of DNA Gene libraries = collections of DNA fragments
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Co In E Figure 16.11 Constructing a Gene Library
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