intro to molecular genetics restriction enzymes mapping cloning pcr sequencing genetic engineering
TRANSCRIPT
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.)
http://www.mun.ca/biology/scarr/RFLP_test_for_recessive_trait.html
RFLPs were an early form of genetic marker
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 expressedin 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:
1. Denaturation
2. Primer annealing
3. Extension
At each repetition, the numberof copies of DNA doubles.
It’s fun and easy!
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Normal (deoxy) dNTP(extends strand)
Di-deoxy nucleotide ddNTP(terminates extension)
DNA sequencing
Requires:A mix of dNTPs and
ddNTPsPolymeraseA labeled primer
Generates:
A mix of fragments.The length of each fragment indicates which ddNTP was added, andtherefore the base at thatposition.
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We use
automated
sequencing
here at Colby.
What do we do with this new information?
Genetic testing
Improved understanding of phenotype & treatments
Information on relatedness of populations and species
Genetic engineering?
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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both viral and human sequences.
genes enter host cell. stranded DNA
version of introduced genes.
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.
Examples of genetically engineered crops:(the FDA has completed consultations on these crops* )
Soybean -- herbicide resistanceCorn -- resistance to herbivory by insects; herbicide resistanceSugar beet -- herbicide resistance (the final product contains no DNA or protein)
Alfalfa -- herbicide resistanceWheat -- herbicide resistanceRice -- herbicide resistanceCanteloupe -- delayed ripening due to reduced ethyleneTomato -- resistance to herbivory by insects; delayed ripeningPotato -- resistance to virus and beetlesSquash -- resistance to virusesPapaya -- resistance to viruses
*http://vm.cfsan.fda.gov/%7Elrd/biocon.htmlInteresting Purdue website: http://www.agriculture.purdue.edu/agbiotech/onthetable.html