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Chapter 20 Notes: DNA Technology. Understanding & Manipulating Genomes 1995: sequencing of the first complete genome (bacteria) 2003: sequencing of the

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  • Chapter 20 Notes: DNA Technology

  • Understanding & Manipulating Genomes1995: sequencing of the first complete genome (bacteria)2003: sequencing of the Human Genome mostly completedThese accomplishments depended on new technology:Recombinant DNA: DNA from 2 sources (often 2 species) are combined in vitro into the same DNA moleculeCalled Genetic engineering: direct manipulation of genes for practical purposes

  • DNA technology has launched a revolution in the area of: BIOTECHNOLOGY: the use of living organisms or their components to do practical tasks-microorganisms to make wine/cheese

    -selective breeding of livestock

    -production of antibiotics-agriculture-criminal law

  • **Practical goal of biotech = improvement of human health and food production

  • Ch 20 looks at:Main techniques for manipulating DNAHow genomes are analyzed & compared at the DNA levelPractical applications of DNA technology (including social & ethical issues)

  • Toolkit for DNA technology involves:

    -DNA vectors-host organisms- restriction enzymes

  • VECTORS = carriers for moving DNA from test tubes back into cells

    -bacterial plasmids (small, circular DNA molecules that replicate within bacterial cells)

    -viruses

  • HOST ORGANISMS:bacteria are commonly used as hosts in genetic engineering because:

    1)DNA can easily be isolated from & reintroduced into bacterial cells;

    2) bacterial cultures grow quickly, rapidly replicating any foreign genes they carry.

  • RESTRICTION ENZYMES = enzymes that recognize and cut short, specific nucleotide sequences (called restriction sites)-in nature, these enzymes protect the bacterial cell from other organisms by cutting up their foreign DNA

  • Restriction Enzymes (cont.)most restriction sequences are symmetrical in that the same sequence of 4-8 nucleotides is found on both strands, but run in opposite directions

    restriction enzymes usually cut phosphodiester bonds of both strands in a staggered manner producing single stranded sticky ends

  • Restriction Enzymes (cont.)sticky ends of restriction fragments are used in the lab to join DNA pieces from different sources (complementary base pairing) *RECOMBINANT DNA

    unions of different DNA sources can be made permanent by adding DNA ligase enzyme (form covalent bonds between bases)

  • DNA Technologies:1)Cloning

    2) DNA fingerprinting (profiling)

    3) Microarray

    4) Gene therapy

  • Steps Involved in Cloning a Human Gene:1) Isolate human gene to clone (ex: insulin);2) Isolate plasmid from bacterial cell; 3) cut both DNA samples with the same restriction enzyme to open up bacterial plasmid & create sticky ends on both samples; 4) Mix the cut plasmids and human DNA genes & seal with DNA ligase;plasmidHuman gene

  • Cloning a Human Gene (cont.)5) Insert recombinant DNA plasmid back into bacterial cell;

    6) As bacterial cell reproduces, it makes copies of the desired gene;-grow cells on a petri dish

    7) Identify cell clones carrying the gene of interest.-HOW? Which ones took up the gene & are making insulin?*Add a 2nd gene besides insulin; add one for antibiotic resistance & then grow bacteria on that antibiotic

  • LE 20-4_3Isolate plasmid DNAand human DNA.Cut both DNA samples withthe same restriction enzyme.Mix the DNAs; they join by base pairing.The products are recombinant plasmidsand many nonrecombinant plasmids.Bacterial celllacZ gene(lactosebreakdown)HumancellRestrictionsiteampR gene(ampicillinresistance)BacterialplasmidGene ofinterestStickyendsHuman DNAfragmentsRecombinant DNA plasmidsIntroduce the DNA into bacterial cellsthat have a mutation in their own lacZgene.RecombinantbacteriaPlate the bacteria on agarcontaining ampicillin and X-gal.Incubate until colonies grow.Colony carrying non-recombinant plasmidwith intact lacZ geneColony carryingrecombinantplasmid withdisrupted lacZ geneBacterialclone

  • Why can bacteria produce insulin through recombinant DNA technology? The genetic code is universal!!!!

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