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Pcr group 2final

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Pcr group 2final

  1. 1. Polymerase Chain Reaction and Primer Design Workshop: “Cluster classification of Mycobacteriophages Isolated from Tropical soils of Puerto Rico” Angélica M. González Pablo González Carolina Montañez Natalia A. Manzano
  2. 2. Polymerase Chain Reaction (PCR)∗ Is an in vitro molecular replication technique used to amplify any specific segment of DNA based on sequence specificity.∗ Used in a wide range of experimental and diagnostic applications.
  3. 3. The Inventor “We are the recipients of scientific method. We can each be a creative and active part of it if we so desire.” Kary Mullis (1983)http://www.420hook.com/?p=12229
  4. 4. Essential components of PCR ∗ Taq Polymerase ∗ DNA Primers ∗ Nucleotide triphosphate ∗ DNA template ∗ ThermocyclerReferences: http://tolweb.org/treehouses/?treehouse_id=472 waynesword.palomar.edu dynamicscience.com.au nature.com
  5. 5. Steps in PCR Denaturation: 95ºCAnnealing: between 50ºC and65ºC. Extension: 72ºC http://www.genes.com/pcr/pcrinfo.html
  6. 6. DNA Primers ∗ A primer is a short synthetic oligonucleotide which is used in many molecular techniques from PCR to DNA sequencing. ∗ They are designed to have a sequence which is the reverse complement of a region of template or target DNA to which we wish the primer to anneal.References: http://bioweb.uwlax.edu
  7. 7. Primer Design ∗ Bioinformatic tools: useful for their design. - Both for known and unknown DNA target sequences.  Example: http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome  Multiple Sequence Alignments (MSAs): determine the most frequent positions of the bases in an unknown target sequence. ∗ Complementarity-based design.References: obiolabs.com filebowl.com dnasoftware.com
  8. 8. Primer Design∗ Considerations: 9 Primers should be 17-28 bases in length c Base composition should be 50-60% (G+C) G+C Primers should end (3) in a G or C, or CG or GC: this C prevents "breathing" of ends and increases efficiency of priming;
  9. 9. ∗ Melting temperatures between 55-80oºC are preferred.∗ 3-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesised preferentially to any other product.
  10. 10. Let’s review!∗ PCRhttp://www.youtube.com/watch?v=2KoLnIwoZKU&feature=relm
  11. 11. Discussion of figures from the article:Exploring the MycobacteriophageMetaproteome: Phage Genomics as an Educational Platform
  12. 12. Complex relationships within highlyabundant Mycobacteriophage Phamilies
  13. 13. Complex relationships within highlyabundant Mycobacteriophage Phamilies
  14. 14. Complex relationships within highlyabundant Mycobacteriophage Phamilies
  15. 15. Representation of Mycobacteriophage Clusters using Splitstree
  16. 16. THANKS FOR YOUR ATTENTION! QUESTIONS?

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