1. INTRO TO MOLECULAR GENETICS Restriction enzymes Mapping Cloning PCR Sequencing Genetic engineering

2. A restriction enzyme cuts DNA at a specific sequence (Bacteria are safe because their DNA is methylated (with a CH3 group) at these sites.)

3. http://www.mun.ca/biology/scarr/RFLP_test_for_recessive_trait.html RFLPs were an early form of genetic marker

4. 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.

5. 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.

6. 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

7. PCR repeats 3 steps: 1.Denaturation 2.Primer annealing 3.Extension At each repetition, the number of copies of DNA doubles. It’s fun and easy!

8. Normal (deoxy) dNTP (extends strand) Di-deoxy nucleotide ddNTP (terminates extension) 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.

9. We use automated sequencing here at Colby.

10. What do we do with this new information? Genetic testing Improved understanding of phenotype & treatments Information on relatedness of populations and species Genetic engineering?

11. 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. both viral and human sequences. genes enter host cell. stranded DNA version of introduced genes.

12. 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.

13. 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