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”
∗ 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)
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
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
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
∗ 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
∗ 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
∗ 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
∗ 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.
∗ PCRPCRhttp://www.youtube.com/watch?v=2KoLnIwoZKU&feature=relmfu
Let’s review!Let’s review!
Exploring the Mycobacteriophage Metaproteome: Phage Genomics as
an Educational Platform
Discussion of figures Discussion of figures from the article:from the article:
Complex relationships within highly abundant Mycobacteriophage Phamilies
Complex relationships within highly abundant Mycobacteriophage Phamilies
Complex relationships within highly abundant Mycobacteriophage Phamilies
Representation of Mycobacteriophage Clusters using Splitstree
THANKS FOR YOUR ATTENTION!THANKS FOR YOUR ATTENTION!
QUESTIONS?QUESTIONS?