Understanding PCR: The Basics and Applications in Genetic Research

Amplifying DNA

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

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

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

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

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.

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

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.

“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

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

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”

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

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

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 DNA Sequencing View the animation at http://www.dnalc.org/resources/animations/cycseq.html (may require Flash player or Shockwave player) http://www.scq.ubc.ca/genome-projects-uncovering-the-blueprints-of-biology/

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). • 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.

Understanding PCR: The Basics and Applications in Genetic Research