Genetic Engineering 1

Lecture 18Pages 323 - 340

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