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?