Genetic Engineering 1 Lecture 18 Pages
The Tools of Molecular Biology
10_01_experiment.DNA.jpg Old fashioned way was to breed for what you wanted 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
10_19_DNA.uptake.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_20_Bacteria.plasmid.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_21_DNA ligase.jpg
10_22_cloned.DNA.frag.jpg Small numbers of transformed bacteria can be grown to large numbers in simple growth media.
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
10_27_2_PCR_amplify.jpg PCR - Polymerase Chain Reaction
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