Transcript Lecture 19 POWERPOINT here

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