Bio 1510 - 17 - Bio 1510 Basic Life Mechanisms Fall 2008 Dr...

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1 Bio – 1510 Basic Life Mechanisms Fall 2008 Dr. Rasheeda Zafar Chapter # 17 Biotechnology
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2 DNA Manipulation DNA Manipulation Restriction endonucleases Restriction endonucleases Cleave DNA at specific sites Used for physical mapping Creation of recombinant DNA recombinant DNA Recognize DNA sequences called restriction restriction sites sites Two types of restriction enzymes (RE) Type l Type ll
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3 DNA Manipulation DNA Manipulation Type l RE Cut near restriction site Rarely used in DNA manipulation Type ll RE Cut at the restriction site Sites are pallindromes pallindromes Both strands have same sequence when read 5’ to 3’ Produced staggered cuts (sticky ends) Overhanging ends DNA ligase can join two fragments DNA ligase can join two fragments
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4
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5 Gel Electrophoresis Separates DNA fragments by size The gel (agarose or polyacrylamide) is subjected to an electrical field The DNA, which is negatively-charged, migrates towards the positive pole The larger the DNA fragment, the slower it will move through the gel matrix DNA is visualized using fluorescent dyes
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Gel Electrophoresis 6
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Transformation Transformation 7 Transformation Transformation Introduction of DNA from an outside source into a cell Natural transformation occurs in many species not in E. coli Artificial transformation Artificial transformation Techniques have been developed to introduce foreign DNA into E. coli
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Molecular Cloning Molecular Cloning 8 Molecular cloning (gene cloning) Molecular cloning (gene cloning) Isolation of a specific DNA sequence (usually protein-encoding) Clone Clone genetically identical copy Most common host E.coli Vector Plasmid Phage Artificial Chromosomes The toolkit for DNA cloning includes The toolkit for DNA cloning includes restriction enzymes, DNA vectors and host restriction enzymes, DNA vectors and host organisms organisms
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Vectors
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