PCR Primer DesignPCR Primer Design
Lecturer: Dr. Farkhondeh PoursinaLecturer: Dr. Farkhondeh Poursina
DefinitionDefinition
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.
ExamplesExamples
PCR primers are designed to:
Highly conserved DNA regionsProtein-coding regions with low
degeneracyMore conserved regions that flank variable
regions
ApplicationsApplications
Primer design is used for:
Finding new genesDeveloping new identification tools
Optimizing PCRsCloning Sequencing, DNA-based phylogeny, functional analysis of genes etc….
Primer Specificity
Target: Conserved nucleotide or protein regions
Primer lengthIf 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 compositionG+C content should be between 40% and 60%, with an even
distribution of all four bases along the length of the primer.9
End with 1-2 GC pairs, if possible,Primer 3’ end stability
No inter- or intra-primer interactions Check with databases for specificityCycling conditions and buffer concentrations should be
adjusted for each primer pair (see PCR troubleshooting)
Avoiding base run.
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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):
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.
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.
Rules for Primer designing…….
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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
3.4
http://www.changbioscience.com/primo/primod.html
PCR Primer Design http://pga.mgh.harvard.edu/serviet/org.mgh.proteome.primer
The Primer
Generator
http://www.med.jhu.edu/medcenter/primer/primer.cgi
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
Project
http://www.nmr.cabm.rutgers.edu/bioinformatics/primer_primer_proj
ect/primer.html
GAP http://promoter.ics.uci.edu/primers/
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Description Software name
Analyses a template DNA sequence and chooses primer pairs for PCR and
primers for DNA sequencing
Primerselect
DANASIS Max is a fully integrated program that includes a wide range of
standard sequence analysis features.
DANSIS Max
Primer design for windows and power macintosh. Primer Primer 5
Comprehensive primer design for windows and Power Macintosh. Primer Primer:
Comprehensive analysis of individual primers and primer pairs. NetPrimer
For fast, effective design of specific oligos or PCR primer pairs for microarrays. Array Designer 2
Design molecular beacons and TaqMan probes for robust amplification and
fluorescence in real time PCR.
AlleleID 7
Primer design for DNA-arrays/chips. GenomePRIDE 1.0
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.
Fast PCR
Primer Analysis Software for Mac and Windows. OLIGO 7
Will find optimal primers in target regions of DNA or protein molecules, amplify
leatures in molecules, or create products of a specified length.
Primer Designer 4
Software for primer design. GPRIME
Genome Oligo Designer is a Software for automatic large scale design of optimal
oligonucleotide probes for microarray experiments.
Sarani Gold
Primer and template design and analysis. PCR Help
Genorama Chip Design Software is a complete set of programs required for
genotyping chip design.The programs can also be bought separately.
Genorama chip Design
Software
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 Designer
Automatic design tools for PCR. Sequencing or hybridization probes, degenerate
primer design, restriction, Nested/Multiplex primer design, restriction enzyme
analysis and more.
Primer Primer
DOS-program to choose primer for PCR or oligonucleotide probes. PreimerDesign
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Gene of interest
Mutation Detection
Allelic discrimination
Gene expression ( mRNA)
Microbial agents detection
Quantification
… Disease
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