Download Agrobacterium

INTRO TO MOLECULAR GENETICS
•Restriction enzymes
•Mapping
•Cloning
•PCR
•Sequencing
•Genetic engineering
A restriction enzyme cuts DNA at a specific sequence
(Bacteria are safe because their DNA is methylated (with a CH3 group) at these sites.)
RFLPs were an early form of genetic marker
http://www.mun.ca/biology/scarr/RFLP_test_for_recessive_trait.html
Click here to see a map of Chromosome 6 in humans:
http://www.ncbi.nlm.nih.gov/mapview/maps.cgi?taxid=9606&chr=6
Various kinds of information were used to make these maps.
Chromosome 6 contains about 1500 genes.
You can see more of them if you zoom in on the right-most map.
Molecular Cloning -make many copies of certain strands
of DNA
If use messanger RNA, as here,
will clone DNA that is expressed
in a certain tissue.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
If clone into “expression vector”
can make gene product.
Can also clone “genomic DNA”,
which will include non-coding
regions.
Polymerase Chain Reaction (PCR)
Efficient way to make many copies of a piece of DNA.
Has replaced cloning for some applications.
You will need:
•Double stranded DNA (the template)
•Primers (two ~ 20 bp single-stranded oligonucleotides that are
complementary to the template, spanning the region of interest.)
•DNA polymerase, preferably from a hot-spring bacterium
•dNTPs (dioxynucleotide triphosphates A, C, G, and T)
•Buffer to run the reaction in
PCR repeats 3 steps:
1. Denaturation
2. Primer annealing
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
3. Extension
At each repetition, the number
of copies of DNA doubles.
It’s fun and easy!
DNA sequencing
Normal (deoxy) dNTP
(extends strand)
Di-deoxy nucleotide ddNTP
(terminates extension)
Requires:
A mix of dNTPs and
ddNTPs
Polymerase
A labeled primer
Generates:
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
A mix of fragments.
The length of each
fragment indicates which
ddNTP was added, and
therefore the base at that
position.
We use
automated
sequencing
here at Colby.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
What do we do with this new information?
Genetic testing
Improved understanding of phenotype & treatments
Information on relatedness of populations and species
Genetic engineering?
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
genes enter
host cell.
both viral and human
sequences
.
stranded DNA
version of
introduced genes.
Retroviruses can carry the desired DNA into human cells.
These viruses carry reverse transcriptase, which uses RNA as a template to make DNA.
Sometimes, this fragment will be incorporated into the host (human) genome.
Problems:
Viruses are scary and germy!
Immune system response can be serious.
The fragment could happen to land in the middle of a functional gene.
More feasible: implant stem cells that have undergone genetic engineering.
Genetic engineering is widespread in agriculture.
Agrobacterium infects many plants.
This bacterium contains a plasmid,
an extra-chromosomal piece of DNA.
A section of the plasmid is
incorporated into the chromosomes
of the cells (virus-like), causing them
to grow and divide rapidly.
Humans can replace these “tumor”
genes with genes of our choice.
A descriptive animation of cell culture from Agrobacterium transformation:
http://www.agriculture.purdue.edu/agbiotech/images/leafdisk1.html
A descriptive animation of the gene gun:
http://www.agriculture.purdue.edu/agbiotech/images/Genegun1.html
Examples of genetically engineered crops:
(the FDA has completed consultations on these crops* )
Soybean -- herbicide resistance
Corn -- resistance to herbivory by insects; herbicide
resistance
Sugar beet -- herbicide resistance (the final product contains no DNA or protein)
Alfalfa -- herbicide resistance
Wheat -- herbicide resistance
Rice -- herbicide resistance
Canteloupe -- delayed ripening due to reduced ethylene
Tomato -- resistance to herbivory by insects; delayed ripening
Potato -- resistance to virus and beetles
Squash -- resistance to viruses
Papaya -- resistance to viruses
*http://vm.cfsan.fda.gov/%7Elrd/biocon.html
Interesting Purdue website: http://www.agriculture.purdue.edu/agbiotech/onthetable.html