Primer Design
Ameer Effat M. Elfarash
Dept. of Genetics
Fac. of Agriculture, Assiut Univ.
amir_effat@yahoo.com
PCR buffer
dNTP Mix
Taq DNA polymerase
Primers
Template
DDW
Very-Brief PCR Reminder
PCR is a method to amplify large quantities of a
DNA covering a specific sequence.
What is a primer?
A primer is a short synthetic oligonucleotide which is used
in many molecular techniques from PCR to DNA
sequencing. These primers are designed to have a
sequence which is the reverse complement of a region of
template or target DNA to which we wish the primer to
anneal.
Why Are Primers Important?
Primers are what gives PCR its
SPECIFICITY!!!
Good primer design: PCR works great.
Bad primer design: PCR works terrible.
Good Primer’s Characteristic
Primer length
5..TCAACTTAGCATGATCGGGTAGTAGCTTGACTGTACAACTCAG
18-24 bp for general applications
General rules for primer design
Primer length determines the specificity and
significantly affect its annealing to the template
Too short -- low specificity, resulting in non-specific
amplification
Too long -- decrease the template-binding efficiency at
normal annealing temperature due to the higher
probability of forming secondary structures such as
hairpins.
Base Composition
• Usually, average (G+C) content around 50-60% will give
us the right melting/annealing temperature for ordinary PCR
reactions, and will give appropriate hybridization stability.
5 GTGGATGTGGTGTCGATGGC 3
5’ 3’
Max 3’end stability
It’s critical that the stability at 3’ end be high
5 GTGGATGTGGTGTCGATGGC 3
5’
Primer melting temperature (Tm):
The melting temperature (Tm) is the most important
factor in determining the optimal PCR annealing
temperature (Ta).
Melting Tm between 50-70 C are preferred
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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Annealing temperatures
37 – 60oC
gradient
Primer Pair Matching
Primers work in pairs – forward primer and reverse
primer. Since they are used in the same PCR reaction, it
shall be ensured that the PCR condition is suitable for
both of them.
One critical feature is their annealing temperatures,
which shall be compatible with each other. The
maximum difference allowed is 3 C. The closer their
Tanneal are, the better.
5 CTGATCAAGTCGATGGCTTG 3 Fw 59 C
5 GATGGAGAGGCTTGACTGC 3 Rv 58 C
Current Oligo, 20-mer [68]:
Avoid
Current+ Oligo: the most stable 3'-dimer: 2 bp, -1.9 kcal/mol
5' C CAGTC GTTACA AACTGA C A 3'
:
::: ||
3' ACA G T C AAACAT TGCTG ACC 5'
Current- Oligo: no 3'-terminal dimer formation
A. Avoid hairpin and stem-loop formation
Current+ Oligo: the most stable dimer overall: 4 bp, -4.8 kcal/mol
5' CC A G T C GTTAC AAACTG ACA 3'
|||| :
::: :
::::
:::
3' A CAG T C A A ACATT GCTGAC C 5'
Hairpin: ²G = -0.7 kcal/mol, Loop = 8 nt, Tm = 41°
5' CC A G T C GTT
|||| A
3' A CAG T C A A ACA
Avoid complementary at 3` end of primers
when is a “primer” a primer?
5’ 3’
5’
3’
5’ 3’
3’
5’
PCR primers are designed to:
For Cloning a special sequence
Full length
Gene of interest
Gene expression ( mRNA)
Microbial agents detection
Mutation Detection
For Detection
Quantification
100 - 500 bp
Allelic discrimination
…
Disease
Random
??
Random primers
Example: RAPD-PCR
RAPD = Random Amplified Polymorphic DNA
5`-TCG GCG GTT C-3`
Universal Primers
Primers can be designed to amplify only one product.
Primers can also be designed to amplify multiple products. We call
such primers “universal primers”. For example, design primers to
amplify all HPV genes.
Strategy:
1. Align groups of sequences you want to amplify.
2. Find the most conservative regions at 5’ end and at 3’ end.
3. Design forward primer at the 5’ conservative region.
4. Design reverse primer at the 3’ conservative regions.
5. Matching forward and reverse primers to find the best pair.
6. Ensure uniqueness in all template sequences.
7. Ensure uniqueness in possible contaminant sources.
Cloning Overview
Four main steps in cloning:
•Insert synthesis
•Restriction enzyme digest
•Ligation
•Transformation
Insert
(your gene)
+
Plasmid Functional
(vector) construct
5’
3’
PCR
RE
Ligation of the Insert into the Vector
•Ligation covalently attaches the vector and the insert via
a phosphodiester bond (5’phosphate and 3’ hydroxyl of the
next base)
Restriction enzymes (NEB)
oligo % cleavage
sequence 2h 20h
BamHI CGGATCCG 10 25
CGGGATCCCG >90 >90
CGCGGATCCGCG >90 >90
EcoRI GGAATTCC >90 >90
CGGAATTCCG >90 >90
CCGGAATTCCGG >90 >90
HindIII CAAGCTTG 0 0
CCAAGCTTGG 0 0
CCCAAGCTTGGG 10 75
NcoI CCCATGGG 0 0
CATGCCATGGCATG 50 75
NdeI GGGTTTCATATGAAACCC 0 0
GGAATTCCATATGGAATTCC 75 >90
Site-directed mutagenesis
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/
Software name Description
Primerselect 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.
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PreimerDesign DOS-program to choose primer for PCR or oligonucleotide probes.
Primer3
Primer3 Output
