IN THE NAME OF GOD
PCR Primer Design
Lecturer: Dr. Farkhondeh Poursina
Definition
 PCR primer design
is the creation of
short nucleotide
sequences for use
in amplifying a
specific region of
DNA.
Polymerase chain reaction (PCR)
 Developed in 1985 by Kary Mullis
 Amplifies a single or a few copies of a piece of DNA across several
orders of magnitude, generating thousands to millions of copies of a
particular DNA Sequence.
 PCR is now a common and often indispensable technique used in
medical and biological research labs for a variety of applications.
Examples
PCR primers are designed to:
Highly conserved DNA regions
Protein-coding regions with low
degeneracy
More conserved regions that flank variable
regions
Applications
Primer design is used for:
Finding new genes
Developing new identification tools
 Optimizing PCRs



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Cloning
Sequencing,
DNA-based phylogeny,
functional analysis of genes etc….
PCR buffer
dNTP Mix
Thermostable DNA polymerase
Primers
Template
DDW
…
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Primer Design Criteria
Primer Specificity
Target: Conserved nucleotide or protein regions
Primer length
If the length is too short, it is difficult to design gene-specific
primers and choose optimal annealing temperature. On the other
hand, longer primers are more likely to form secondary structures
that result in decreased PCR efficiency or promote primer dimer
formation Usually 18 - 24 bases
,,
Base composition
G+C content should be between 40% and 60%, with an even
distribution of all four bases along the length of the primer.
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End with 1-2 GC pairs, if possible,Primer 3’ end
stability
No inter- or intra-primer interactions
Check with databases for specificity
Cycling conditions and buffer concentrations should be
adjusted for each primer pair (see PCR
troubleshooting)
Avoiding base run.
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Avoid sequence secondary structures
Avoid complementary at 3` end of primers
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Primer melting temperature (Tm):

Tm: is the temperature at which half the DNA strands are
single stranded and half are double-stranded.

The melting temperature (Tm) is the most important factor in
determining the optimal PCR annealing temperature (Ta).

Calculated Tm values of members of a primer pair should not
differ by >5°C.
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Wallace rule:
Tm = 4 * (G + C) + 2 * (A + T)
Bolton and McCarthy:
Tm = 81.5 + 16.6 * Log [I] + 0.41 * (%GC) – 600/L
The nearest neighbor method (Santalucia et.al, 1998):
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
Δ G (Gibbs free energy) is the most important factor
that is to be taken into consideration in primer
designing.

Δ G definition: The Gibbs Free Energy G is the
measure of the amount of work that can be
extracted from a process operating at a constant
pressure. It is the measure of the spontaneity of
the reaction.
Rules for Primer designing…….

Primer secondary structures: Presence of the primer secondary
structures adversely affect primer template annealing and thus the
amplification. They greatly reduce the availability of primers to the
reaction.

Hairpins: A 3' end hairpin with a ΔG of -2 kcal/mol and an internal
hairpin with a ΔG of -3 kcal/mol is tolerated generally.


Larger negative value for ΔG indicates stable, undesirable
hairpins. Presence of hairpins at the 3' end most adversely
affects the reaction.
Self Dimer : A 3' end self dimer with a ΔG of -5 kcal/mol and an
internal self dimer with a ΔG of -6 kcal/mol is tolerated generally.
Rules for Primer designing…….
 Cross Dimer: Optimally a 3' end cross dimer with a ΔG of -5 kcal/mol and
an internal cross dimer with a ΔG of -6 kcal/mol is tolerated generally.
 3' End Stability : It is the maximum ΔG value (-8.5 kCal/mol is the default,
however a value of -10 to -12kCal/mol is tolerated) of the five bases from
the 3’.
 An unstable 3' end (less negative ΔG) will result in less false priming.
Primers with pentamer ΔG more stable than -12.0 kCal/mol (more
negative) have a tendency to false prime and are more likely to form
hairpins and self dimers.
Primer Design Softwares
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Tool name
URL
CODEHOP
http://blocks.fhcrc.org/codehop.html
Gene Fisher
http://bibiserv.techfak.uni-bielefeld.de/genefisher/
DoPrimer
http://doprimer.interactiva.de/
Primer3
http://frodo.wi.mit.edu/primer3/
Primer Selection
Http://alces.med.umn.edu/rawprimer.html
Web Primer
http://genome.www2.stanford.edu/cgi.bin/SGD/web.primer
PCR designer
http://cedar.genetics.ston.ac.uk/public_html/primer.html
Primo pro 3.4
http://www.changbioscience.com/primo.html
Primo Degenerate
http://www.changbioscience.com/primo/primod.html
3.4
PCR Primer Design
http://pga.mgh.harvard.edu/serviet/org.mgh.proteome.primer
The Primer
http://www.med.jhu.edu/medcenter/primer/primer.cgi
Generator
EPRIMERS
http://bioweb.pasteur.fr/seqanal/interfaces/eprimer3.html
PRIMO
http://bioweb.pasteur.fr/seqanal/interfaces/eprimo.html3
PrimerQuest
http://www.idtdna.com/biotools/primer_quest/primer_quest.asp
MethPrimer
http://itsa.uscf/~uralab/methprimer/index1.html
Rawprimer
http://alces.med.umn.edu/rawprimer.html
MEDUSA
http://www.cgr.ki.se/cgr/MEDUSA/
The Primer Prim’er
http://www.nmr.cabm.rutgers.edu/bioinformatics/primer_primer_proj
Project
ect/primer.html
GAP
http://promoter.ics.uci.edu/primers/
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Software name
Primerselect
Description
Analyses a template DNA sequence and chooses primer pairs for PCR and
primers for DNA sequencing
DANSIS Max
DANASIS Max is a fully integrated program that includes a wide range of
standard sequence analysis features.
Primer Primer 5
Primer design for windows and power macintosh.
Primer Primer:
Comprehensive primer design for windows and Power Macintosh.
NetPrimer
Comprehensive analysis of individual primers and primer pairs.
Array Designer 2
For fast, effective design of specific oligos or PCR primer pairs for microarrays.
AlleleID 7
Design molecular beacons and TaqMan probes for robust amplification and
fluorescence in real time PCR.
GenomePRIDE 1.0
Primer design for DNA-arrays/chips.
Fast PCR
Software for Microsoft Windows has specific. Ready-to-use template for many
PCR and sequencing applications; standard and long PCR inverse PCR.
Degenerate PCR directly on amino acid sequence. Multiplex PCR.
OLIGO 7
Primer Analysis Software for Mac and Windows.
Primer Designer 4
Will find optimal primers in target regions of DNA or protein molecules, amplify
leatures in molecules, or create products of a specified length.
GPRIME
Software for primer design.
Sarani Gold
Genome Oligo Designer is a Software for automatic large scale design of optimal
oligonucleotide probes for microarray experiments.
PCR Help
Primer and template design and analysis.
Genorama chip Design
Genorama Chip Design Software is a complete set of programs required for
Software
genotyping chip design.The programs can also be bought separately.
Primer Designer
The Primer Designer features a powerful, yet extremely simple, real-time interface
to allow the rapid identification of theoretical ideal primers for your PCR
reactions.
Primer Primer
Automatic design tools for PCR. Sequencing or hybridization probes, degenerate
primer design, restriction, Nested/Multiplex primer design, restriction enzyme
analysis and more.
PreimerDesign
DOS-program to choose primer for PCR or oligonucleotide probes.
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Gene of interest
Gene expression ( mRNA)
Microbial agents detection
Mutation Detection
Quantification
Allelic discrimination
…
Disease
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