1 / 16

Amplifying DNA

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.

sydney-beard
Télécharger la présentation

Amplifying DNA

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Amplifying DNA

  2. 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

  3. 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

  4. 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

  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. 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/

  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). • 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.

More Related