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Genetic Engineering 1 Lecture 18 Pages 323 - 340 The Tools of Molecular Biology Old fashioned way was to breed for what you wanted 10_01_experiment.DNA .jpg Mendel did it! 10_02_cell_sorter.jpg Once you have the cells what next? • Issue was to examine the DNA in a consistent manner • Best method is to use restriction enzymes – Come mainly from bacteria – Use individually or in a mix 10_04_Restrict.nuclease.jpg What do you do with these digested fragments of DNA? • Isolate those that you want to work on • How? – Best method is to use gel electrophoresis • Agarose • Polyacrylamide 10_05_gel.electrophor.jpg Then what do you do with this piece of DNA? • Clone • Sequence – Rely on the use of dideoxy nucleotides 10_07_1_enzym.dideoxy.jpg 10_07_2_enzym.dideoxy.jpg 10_08_DNA.sequencing .jpg 10_09_Shotgun.sequenc.jpg 10_10_Repetit.sequence.jpg 10_11_BAC.clones.jpg 10_12_de_renaturation.jpg 10_13_hybridization.jpg Blotting Purpose to make a permanent record of the results of a gel electrophoresis. The compass • Southern blots - DNA • Northern blots - RNA • Western blots - Proteins 10_14_1_Southrn.blotting.jpg 10_14_2_Southrn.blotting.jpg 10_15_DNA.microarrays .jpg Cloning and growing • One can use the techniques of cell biology to manufacture artificial and real products, be they genes, proteins, or organisms • If you want to insert some DNA into another molecule then the best place to start is to use the same restriction enzyme to cut both - so they have the same ends. 10_18_ DNA.in.vitro.jpg Bacteria have the ability to ‘ingest’ DNA from their environment naturally. This property makes them able to change their properties very quickly - and dangerous to us. 10_19_DNA.uptake.jpg Bacteria are able to also pass between themselves, other small pieces of DNA known as plasmids. We can make use of plasmids to carry our test DNA into bacteria as shown on the next side… 10_20_Bacteria.plasmid.jpg 10_21_DNA ligase.jpg Small numbers of transformed bacteria can be grown to large numbers in simple growth media. 10_22_cloned.DNA.frag.jpg 10_23_genomic.library.jpg Genomic libraries of fragments of all human genes can be made by this technique. One can buy these libraries and use them to isolate any gene and grow that for experimental purposes. One can find the right cell using the technique on the next slide… 10_24_hybridization.jpg 10_25_cDNA.jpg Another technique is to use the mRNA from a cell to make DNA in the reverse direction. These DNA molecules represent just the genes that were active at the time the mRNA was recovered from the cell. 10_26_Genomic_cDNA.jpg 10_27_1_PCR_amplify.jpg PCR - Polymerase Chain Reaction 10_27_2_PCR_amplify.jpg 10_28_PCR_clones.jpg 10_29_PCR_viral.jpg 10_30_1_PCR_forensic.jpg 10_30_2_PCR_forensic.jpg 10_31_SerialDNA.clone.jpg 10_32_expressionvector.jpg 10_33_gene_protein.jpg 10_34_Reporter.genes.jpg 10_35_GFP.jpg 10_36_mutagenesis.jpg 10_37_engineered.org.jpg 10_38_ES.cells.jpg 10_39_Transgenic.mice.jpg 10_40_Transgenic.plant.jpg