INTRO TO MOLECULAR GENETICS

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INTRO TO MOLECULAR GENETICS

• Restriction enzymes
• Mapping
• Cloning
• PCR
• Sequencing
• Genetic engineering

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A restriction enzyme cuts DNA at a specific
sequence
(Bacteria are safe because their DNA is
methylated (with a CH3 group) at these sites.)
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RFLPs were an early form of genetic marker
http//www.mun.ca/biology/scarr/RFLP_test_for_rece
ssive_trait.html
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Click here to see a map of Chromosome 6 in
humans http//www.ncbi.nlm.nih.gov/mapview/maps
.cgi?taxid9606chr6 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.
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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. If clone into expression
vector can make gene product. Can also clone
genomic DNA, which will include non-coding
regions.
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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

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• PCR repeats 3 steps
• Denaturation
• Primer annealing
• Extension
• At each repetition, the number
• of copies of DNA doubles.
• Its fun and easy!

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DNA sequencing Requires A mix of dNTPs
and ddNTPs Polymerase A labeled
primer Generates A mix of fragments. The
length of each fragment indicates which ddNTP
was added, and therefore the base at
that position.
Normal (deoxy) dNTP (extends strand)
Di-deoxy nucleotide ddNTP (terminates extension)
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We use automated sequencing here at Colby.
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What do we do with this new information? Genetic
testing Improved understanding of phenotype
treatments Information on relatedness of
populations and species Genetic engineering?
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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.
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Genetic engineering is widespread in
agriculture. 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
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.
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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.ht
ml Interesting Purdue website
http//www.agriculture.purdue.edu/agbiotech/onthet
able.html