pcr group 2final
TRANSCRIPT
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Polymerase Chain Reaction Polymerase Chain Reaction and Primer Designand Primer Design
Angélica M. GonzálezPablo González
Carolina MontañezNatalia A. Manzano
Workshop: “Cluster classification of Mycobacteriophages Isolated from Tropical soils of Puerto Rico”
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∗ 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.
Polymerase Chain Reaction (PCR)Polymerase Chain Reaction (PCR)
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The InventorThe 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
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Essential components of PCREssential components of PCR
∗ Taq Polymerase∗ DNA Primers∗ Nucleotide
triphosphate∗ DNA template∗ Thermocycler
References: http://tolweb.org/treehouses/?treehouse_id=472 waynesword.palomar.edu dynamicscience.com.au nature.com
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http://www.genes.com/pcr/pcrinfo.html
Steps in PCRSteps in PCR
DenaturationDenaturation: 95ºC: 95ºC
AnnealingAnnealing: between 50ºC and : between 50ºC and 65ºC.65ºC.
ExtensionExtension: 72ºC: 72ºC
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∗ A primer 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 complementthe reverse complement of a
region of template or target DNA to which we wish the primer to anneal.
DNA PrimersDNA Primers
References: http://bioweb.uwlax.edu
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∗ 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.
Primer DesignPrimer Design
References: obiolabs.com filebowl.com dnasoftware.com
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∗ Considerations:
9 Primers should be 17-28 bases in lengthlength
c Base composition should be 50-60% (G+CG+C)
Primers should end should end (3') in a G or Cin a G or C, or CG or GC: this prevents "breathing" of ends and increases efficiency of priming;
Primer DesignPrimer Design
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∗ Melting temperatures Melting temperatures between 55-80oºC are preferred.
∗ 3'-ends of primers should not be complementary 3'-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesised preferentially to any other product.
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∗ PCRPCRhttp://www.youtube.com/watch?v=2KoLnIwoZKU&feature=relmfu
Let’s review!Let’s review!
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Exploring the Mycobacteriophage Metaproteome: Phage Genomics as
an Educational Platform
Discussion of figures Discussion of figures from the article:from the article:
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Complex relationships within highly abundant Mycobacteriophage Phamilies
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Complex relationships within highly abundant Mycobacteriophage Phamilies
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Complex relationships within highly abundant Mycobacteriophage Phamilies
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Representation of Mycobacteriophage Clusters using Splitstree
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THANKS FOR YOUR ATTENTION!THANKS FOR YOUR ATTENTION!
QUESTIONS?QUESTIONS?