1. Amplifying DNA

2. The Power of PCR http:// www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553 View the animation at http://www.dnalc.org/resources/animations/pcr.htmlhttp://www.dnalc.org/resources/animations/pcr.html (may require Flash player or Shockwave player)

3. PCR Ingredients 1. DNA “template”Your purified DNA sample 2. DNA PolymeraseSpecial DNA polymerase enzyme that is heat stable 3. Deoxynucleotides (dNTPs) Building blocks of DNA 4. PrimersSmall pieces of DNA that match the flanks of your gene or DNA region of interest 5. Buffer and waterEnvironment necessary for DNA Polymerase to work; mimics conditions in nucleus

4. Agarose Gel Electrophoresis Molecular Weight Standard (DNA of Known Sizes) 1 2 3 4 5 6 Samples of DNA 2000 bp 1000 bp 750 bp Lanes:

5. Agarose Gel Electrophoresis 1. Prepare agarose gel 2. Prepare your sample 3. Load your sample on the gel 4. Run gel 5. Stain & view gel http://oceanexplorer.noaa.gov/explorations/03bio/background/molecular/media/gel_plate.html

6. Genetic Researchers Developed Primers for Barcoding Pool COI-2: mammals, and insects Pool COI-3: fish Ivanova et al. 2007. Universal primer cocktails for fish barcoding. Mol Ecol Notes.

7. Fish DNAbarcode Primers Primer mix: 2 forward, 2 reverse 5’- TGTAAAACGACGGCCAGTCAACCAACCACAAAGACATTGGCAC-3’ 5’- TGTAAAACGACGGCCAGTCGACTAATCATAAAGATATCGGCAC-3’ 5’- CAGGAAACAGCTATGACACTTCAGGGTGACCGAAGAATCAGAA-3’ 5’- CAGGAAACAGCTATGACACCTCAGGGTGTCCGAARAAYCARAA-3’

8. Bioinformatics The type of computer sciences that aids biological researchers. Using informatics to decipher and elucidate the information entailed in biological molecules, structures, organisms and populations.

9. “Electronic PCR” Searching databases for sequences Using primer sequences as search terms No amplification Common database: NCBI’s GenBank – National Center for Biotechnology Information Common search tool: BLAST – Basic Local Alignment Search Tool

10. Exercise A Identify the targets of our barcoding primers (Day 1, Tuesday) Determine length of amplicon DNA (Day 2, Wednesday)

11. SNPs Polymorphism: A genetic locus that exists in different forms Pointmutation: A change in a single nucleotide (alteration, deletion or insertion) SNP: Single nucleotide polymorphism – A pointmutation that occurs in at least 0.5% of the population Haplotype: A bunch of SNPs that are connected in one strand of DNA – SNPs that do not separate by crossover form a “Haplotype”

12. Exercise B Find differences in DNA (SNPs) Extract haplotypes Construct haplotype phylogenetic tree

13. Exercises C & D Conduct Exercise B electronically Compare and contrast results from C & D with those from B

14. DNA Sequencing http://www.scq.ubc.ca/genome-projects-uncovering-the-blueprints-of-biology/ 1. DNA “template” Your PCR fragment, purified 2. Taq Polymerase Heat-stable DNA polymerase 3. Deoxynucleotides (dNTPs) and Dideoxynucleotides Building blocks of DNA; regular and altered 4. Primers Specific for your gene of interest 5. Buffer and water View the animation at http://www.dnalc.org/resources/animations/cycseq.html (may require Flash player or Shockwave player)

15. Homework Work through Study questions Register for your own account on DNA Subway (Google it) Review Bioinformatics readings (Binder) Review all 3 barcoding-related papers (Pre-readings) Bonus: Work through the HHMI Seashell Phylogeny exercise using DNA – download Word doc from www.hhmi.org/biointeractive/activities/shells/Shell-DNA-0-6.docx). www.hhmi.org/biointeractive/activities/shells/Shell-DNA-0-6.docx – If you don’t wish to install the software indicated in the document you could use BioServers/Sequence Server or, alternatively, the Blue Line in DNA Subway to conduct alignments and generate phylogenetic trees.