BB lecture 11-23-09 biotech

BB lecture 11-23-09 biotech - Chapter 20 (pp. 396-410)...

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Chapter 20 (pp. 396-410) Biotechnology I Learning objectives: Describe the natural function of restriction enzymes and explain how they are used in recombinant DNA technology Outline the procedures for cloning a eukaryotic gene in a bacterial plasmid Describe the polymerase chain reaction (PCR) Know the basis for detecting specific DNA sequences using the Southern blot method terms to know include…plasmid, restriction enzyme, sticky end, cDNA, nucleic acid probe, transformation, gel electrophoresis, ,…
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Plasmids are small circles of DNA found naturally in the cells of some bacteria. A plasmid can replicate itself as well as any other DNA that has been added to it (by insertion). Plasmids make excellent cloning vectors structures that can carry DNA from the cells of one organism into the cells of another .
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Recombinant DNA , DNA from two different sources are combined into one DNA molecule Cloning, making multiple identical copies
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Recombinant DNA and cloning – Basic Steps 1. Vector and gene-source DNA are isolated 2. DNA is inserted into the vector 3. Recombinant plasmids are mixed with bacteria 4. The bacteria are plated and incubated 5. Screening identifies those cells that have taken up the recombinant DNA
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Restriction enzymes are used to isolate the gene and the vector Bacterial restriction enzymes cut DNA molecules at sites with a specific nucleotide sequence (the cuts are “restricted” to specific sites) A restriction enzyme cuts DNA into fragments Staggered cuts produce fragments with “sticky ends” these bond with complementary “sticky ends” of other fragments DNA ligase is used to join restriction fragments together
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Fig. 20-3-3 Restriction site DNA Sticky end Restriction enzyme cuts sugar-phosphate backbones. 5 3 3 5 1 One possible combination Recombinant DNA molecule DNA ligase seals strands. 3 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. 2
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Fig. 20-2a DNA of chromosome Cell containing gene of interest Gene inserted into plasmid Plasmid put into bacterial cell Recombinant DNA ( plasmid ) Recombinant bacterium Bacterial chromosome Bacterium Gene of interest Plasmid 2 1 2
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Fig. 20-4-4 Bacterial cell Bacterial plasmid lacZ gene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends amp R gene TECHNIQUE Recombinant plasmids Nonrecombinant plasmid Bacteria carrying plasmids RESULTS Colony carrying non- recombinant plasmid with intact lacZ gene One of many bacterial clones Colony carrying recombinant plasmid with disrupted lacZ gene Grow in agar plates…. each cell forms a colony
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The plasmid has two genes: one for antibiotic resistance , and
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This note was uploaded on 01/20/2010 for the course BIO 50415 taught by Professor Batterton during the Spring '09 term at University of Texas at Austin.

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BB lecture 11-23-09 biotech - Chapter 20 (pp. 396-410)...

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