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Announcements 1. Lab reports (X-linked cross) due today - start of lecture 2. Pick up lab overview 12 - read and answer pre-lab questions, due at start of lab (instead of quiz!). 3. On 3x5 card - write 1-2 specific topics you would like “reviewed” before the exam or questions you have 4. Exam 3 will cover material through end of today’s lecture 5. Problem set 7 available for practice - not graded; answers posted Monday. Review of Last Lecture I. Origins of mutation - 1 spontaneous, 4 chemical, and 2 environmental II. Mechanisms of DNA repair Learning Check Although mutations are generally considered deleterious and to be avoided in the “real world”, they are sometimes intentionally introduced into organisms in research laboratories. (1) Why? You’ve learned of 2 ways to introduce mutations in lab organisms: using EMS (an alkylating agent) and using sitedirected mutagenesis. (2) If you are studying a biological process in which no specific genes have yet been identified, which of the above tools could you use and what might you learn? (3) If you are studying a process in which a specific gene is known to be involved, which tool might you use and what specific information could you acquire? Outline of Lecture 30 I. Transposable elements II. Recombinant DNA - restriction enzymes III. Vectors IV. Cloning: using restriction enzymes and vectors together V. Practice problems I. Transposable Elements • Also called Transposons or “Jumping Genes”; can move within the genome. • Present in all organisms; well-studied in bacteria, maize, flies. • Discovered in Maize Mendel’s wrinkled Phenotype in Peas Also Caused by Transposon Transposons in Humans • Alu family of short interspersed elements (SINEs) – Moderately repetitive DNA – 500,000 copies of 200-300 bp repeats • Medical example: in a male child with hemophilia, a transposon (LINE) jumped into the gene on X chromosome responsible for hemophilia – Not present on either X chromosome of mother – Present on chromosome 22 of mother – This mobile element may have “moved” from chr. 22 to X chr. in the precursor cells of the mother’s egg II. Uses of Recombinant DNA Technology • • • • • • • • Basic biomedical science Basic ecological/evolutionary biology Applied microbiology Plant genetic engineering Transgenic animals Human Genome Project Medical biotechnology Forensic science Recombinant DNA refers to a new combination of DNA molecules not found together naturally Cutting with Restriction Enzymes: “Sticky” or Cohesive Ends Cutting and Pasting (Annealing and Ligation) of Sticky Ends Digest with EcoRI Complementary base-pairing DNA ligase Agarose Gel stained with Ethidium Bromide, Visualized By UV MW plasmid Nicked Circular - May be higher or lower Linear Accurate Supercoiled More compact, so runs faster Accurate Gel Mobility of DNA fragments Depends on Complete Cutting Joining Blunt Ends III. Vectors - EM of Small Plasmid DNA Plasmids are vectors, molecular tools for carrying DNA of interest. Other vectors include bacteriophage, cosmids, etc. pUC18 Plasmid DNA fragment up to 5 kbp can insert Origin of replication IV. Cloning with a Plasmid Vector Recombinant DNA Transformation Selection for cells carrying recombinant plasmids by plating cells on media with antibiotic. Learning Check When you are doing a transformation in lab, you might add your recombinant DNA to competent E. coli, incubate on ice to allow DNA to adhere to the cell wall, heatshock, let cells recover in liquid media, and plate out on antibiotic-containing media. List all of the controls that are necessary in order for you to interpret your results the next day. ie. if no cells/colonies grow on your plates, what will you conclude? How will you determine what part of the expt. went wrong? Insertion into a Plasmid can be Detected by Disruption of -gal • Only bacteria which have taken up plasmid grow on ampicillin. • Blue-white selection: – white colonies have insert – blue colonies have no insert • To see blue color, add IPTG (an inducer of -galactosidase expression) and Xgal substrate. Restriction Mapping problems *Note: fragments are linearized to start with. * Figure 18.23 Restriction Mapping (cont’d) Model 1 Model 2 • Same logic can be used on a circular DNA in homework. Analyze each lane from left to right and any other information given. Redraw the plasmid each time for each step you solve. • Make alternative hypotheses and test them against the data. • Check that total # of fragments = total size of plasmid. There could be two same-sized fragments in one gel band. Learning Check Linear DNA fragment Marker EcoRI BamHI EcoRI/BamHI 15 10 9 8 6 7 5 8 6 5 4 2 What is the restriction map of this cloned DNA fragment, showing the locations of the restriction sites and relative distance between sites?