Polymerase Chain Polymerase Chain Reaction and Primer Reaction and Primer

DesignDesign

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 Polymerase Chain Reaction (PCR)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 Essential components of PCRof 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 Steps in PCRPCR

DenaturationDenaturation: : 95ºC95ºCDenaturationDenaturation: : 95ºC95ºC

AnnealingAnnealing: between : between 50ºC and 65ºC.50ºC and 65ºC.AnnealingAnnealing: between : between 50ºC and 65ºC.50ºC and 65ºC.

ExtensionExtension: 72ºC: 72ºCExtensionExtension: 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 the reverse

complementcomplement 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:

1. Primers should be 17-28 bases in lengthlength

1. 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

4.4. Melting temperatures Melting temperatures between 55-80oºC are preferred.

5. 3'-ends of primers should not be 3'-ends of primers should not be complementary 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 Discussion of figures from the figures from the

article: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 THANKS FOR YOUR ATTENTION!ATTENTION!

QUESTIONQUESTIONS?S?