primer design 2013.pdf
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Primer DesignDESIGN OF OLIGONUCLEOTIDE PRIMERS FORBASIC PCRJ E R E M Y G . V I C E N C I O
D E PA R T M E N T O F B I O L O G Y
C O L L E G E O F A R T S A N D S C I E N C E S
U N I V E R S I T Y O F T H E P H I L I P P I N E S M A N I L A
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Before you start designing primers
find and use the right resources!o What are the primers for?
o General purposeamplification?
o SNPs detection/validation
o Methylation study?
o Real-time PCR?
o Microarray probes?
o Degenerate PCR?
o Multiplex PCR?
o What do you have to begin
with?
o Single DNA/protein
sequence?
o Multiple DNA/protein
sequence files?
o
GenBank ID/Gene ID/GeneSymbol/rsSNP ID?
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After you have designed your primers
Consider a second opinion!o Several different software are available for designing
primers
o Various software may differ significantly in:
o Concrete and overall approaches
o Designing criteria and default settings
o Comprehensiveness
o Usability, accessibility, and speed
o Consider a second opinion when
o You are new to such a design task/application
o You dont have a lot of confidence in the initial result
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Question 1What is the chief goal of primer design?
A. Specificity
B. Sensitivity
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Selecting PCR Primerso Analysis of the target gene for potential primingsites
o free of homopolymeric tracts
o have no obvious tendency to form secondarystructures
o not self-complementary
o have no significant homology with other sequences
on either strand of the target genome
o Creation of lists of possible forward and reverseprimers
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Selecting PCR Primerso Selection of well-matched pairs of forward and
reverse primerso similar in G+C content
o must generate an amplified product of the
appropriate size and base composition
o Refining the length and/or placement of theoligonucleotides
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Properties of OligonucleotidesThat Influence the Efficiency ofAmplification
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Base Compositiono G+C content should be somewhere between 40%
and 60%, with an even distribution of all fourbases along the length of the primero avoid polypurine tracts or polypyrimidine tracts
o avoid long runs of a single base (max 4 bases)
o avoid dinucleotide repeats (max 4 repeats)
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Lengtho Primer length determines the specificity and significantly
affects its annealing to the template
o Too short low specificity, resulting in non-specific
amplificationo Too long decrease the template-binding efficiency at normal
annealing temperature due to the higher probability offorming secondary structures such as hairpins
o
Optimal primer lengtho 18-25 nucleotides long for general applications
o 30-35 nucleotides for multiplex PCR
o Members of a primer pair should not differ in length by >3 bp.
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Lengtho Optimal amplicon size
o 300-1000 bp for general applications, avoid >3 kb
o
50-150 bp for real-time PCR, avoid >400 bp
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Repeated and Self-
Complementary Sequenceso No inverted repeat sequences or self-
complementary sequences >3 bp in length
should be present.
o If primers can anneal to themselves, or anneal to
each other than anneal to the template, the PCR
efficiency will be decreased dramatically. They
shall be avoided.
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Secondary structures
Hairpino Formed via intra-molecular
interactions
o
Negatively affect primer-template binding, lead to pooror no amplification
o Acceptable G (free energyrequired to break the structure):>-2 kcal/mol for 3end hairpin;>-3 kcal/mol for internal hairpin
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Secondary structures
Self-dimer (homodimer)o Formed by inter-
molecular interactions
between the two same
primers
o Acceptable G: >-5kcal/mol for 3 end self-
dimer; >-6 kcal/mol forinternal self-dimer
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Complementarity between
members of a primer pairo The 3 terminal sequences of one primer should
not be able to bind to any other site on the other
primer.
o Formation of primer dimers can be reduced by
o careful primer design
o use of hot start or touchdown PCR
o use of specially formulated DNA polymerases
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Secondary structures
Cross-dimer (heterodimer)o Formed by inter-
molecular interactionsbetween the sense and
antisense primers
o Acceptable G: >-5kcal/mol for 3 end
cross-dimer; >-6kcal/mol for internalcross-dimer
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What is a primer dimer?o An unwanted extension
product
o Results from primers
annealing tothemselves, or eachother, at 3 ends
o Extended primers areno longer available toprime target for PCR
atcggactatcga
gctatacttatggcca
atcggactatcgatatgaataccgga
tagcctgatagctatacttatggcca
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Melting Temperatures (T
m)o Tm is the temperature at which 50% of the DNA
duplex dissociates to become single-stranded
o Determined by primer length, base composition, and
concentration
o Also affected by the salt concentration of the PCR reaction
mix
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Melting Temperatures (T
m)o Wallace rule: Tm (in C) = 2(A+T)+4(G+C)
o can be used to calculate the melting temperature for perfect
duplexes 15-20 nucleotides in length in solvents of high ionic
strength (e.g., 1 M NaCl):
o Baldino algorithmTm (in C) = 81.5C + 16.6 (log10[K+]) + 0.41(%[G+C]) (675/n)where n is the number of bases in the oligonucleotideo predicts reasonably well the melting temperature of
oligonucleotides, 14-70 nucleotides in length, in cation
concentrations of 0.4 M or less:
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Melting Temperatures (T
m)o Should not differ by >5C between members of a
primer pair
o The Tm of the amplified product should not differfrom the Tm values of the primer pairs by >10C
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Melting Temperatures (T
m)o Optimal Tm
o 52C to 60C
o Tm
above 65C should be avoided because of the
potential for secondary annealing
o Higher Tm is recommended for amplifying high GC
content targets
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3 Terminio If possible, the 3 base of each primer should be
G or C.
o However, primers with a .NNCG or .NNGC
sequence at their 3 termini are not recommended
since this promotes the formation of hairpin
structures and may generate primer dimers.
o GC Clamp refers to the presence of G or Cwithin the last 5 bases from the 3 end of primers
o Essential for preventing mis-priming and enhancing
specific primer-template binding
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Adding restriction sites, bacteriophagepromoters, and other sequences to the
5 termini of primerso In general, the presence of additional sequences
does not significantly affect annealing of the
oligonucleotide to its target DNA.
o Restriction sites: the primer should be extendedby at least three additional nucleotides beyond
the recognition sequence of the restriction
enzyme.
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Placement of priming siteso Various constraints on the placement of priming
sites:
o mutations
o restriction sites
o coding sequences
o microsatellites
o cis-acting elements
o Use forward and reverse primers that bind todifferent exons when designing primers for useon cDNA templates.
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Cross-homologyo Cross homology may become a problem when PCR template
is genomic DNA or consists of mixed gene fragments.
o BLASTing PCR primers against the NCBI non-redundant
sequence database is a common way to avoid designingprimers that may amplify non-targeted homologous regions.
o Use primers spanning intron-exon boundaries to avoid non-specific amplification of gDNA due to cDNA contamination.
o Use primers spanning exon-exon boundaries to avoid non-specific amplification cDNA due to gDNA contamination.
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Annealing temperature (Ta)
o Ta vs. Tmo Ta is determined by the Tm of both primers and amplicons:
optimal Ta = 0.3 x Tm (primer) + 0.7 x Tm (amplicon) 25o General rule: Ta is 5C lower than Tmo Higher Ta enhances specific amplification but may reduce
yield
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Test yourunderstandingEVALUATE WHETHER TH E FOLLOWING P RIMERSMEET THE GUIDELINES FOR EACH SPECIFIEDCRITERION
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Question 2
o Length
5 ACT GCC TCG ACT ACT TAC GC 3
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Question 3
o Length
Forward: 5 GCT TCA ACG GAC CAT TGC 3Reverse: 5 CTT ACG ACT TCC ACT TCC GCA C 3
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Question 4
o Base composition
5 GCCACGTCGCGCATGCGC 3
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Question 5
o Base Composition
5 ACACACACTTTTGCC 3
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Question 6
o Self-complementary sequences
5 GCTTCACGGATCTGAAGC 3
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Question 7
o Hairpins
5 ACGCTCTCCACGAGTCACGC 3
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Question 8
o Homodimers
5 GCGTTAGGCACGAATCACGC 3
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Question 9
o Heterodimers
Forward:5 GCGTTAGGCACGAATCACGC 3
Reverse:
5 GCGTCAGAACCGTACGAGCG 3
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Question 10
o Melting Temperature
5 ATTGAATCTACTACTTACGC 3Tm: 50.7C
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Question 11
o Melting Temperature
Forward: 5 GATTTAAGCACGAATCACGC 3Tm: 54.7C
Reverse: 5 TAGTCAGCATCGTATGAGCG 3Tm: 58.0C
Amplified Product: 851 bp, Tm: 64.0C
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Question 12
o 3 Terminus
5 GATTTAAGCACGAATCACGC 3
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Computer-Assisted Designof Oligonucleotide Primers
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Primer3
o Web-based tool for primer design
http://frodo.wi.mit.edu/o Example gene: GFP5 Green Fluorescent Protein
o GenBank: U87973.1
http://frodo.wi.mit.edu/http://frodo.wi.mit.edu/ -
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Primer3
Enter sequence
Pick Primers
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Primer Size
Primer Tm
Complementarity
Primer3 Advanced Controls
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Primer3 Output
Details:-Start
-Length-Tm-% GC-Sequence
Where they bind:
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Primer3 Output
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Primer Evaluation
o OligoAnalyzer 3.1 (web-based)
http://sg.idtdna.com/analyzer/Applications/OligoAnalyzer/
http://sg.idtdna.com/analyzer/Applications/OligoAnalyzer/http://sg.idtdna.com/analyzer/Applications/OligoAnalyzer/ -
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OligoAnalyzer 3.1: Analyze
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OligoAnalyzer 3.1: Hairpin
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OligoAnalyzer 3.1: Homodimer
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OligoAnalyzer 3.1: Heterodimer
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AmplifX
o Standalone desktop software
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AmplifX
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AmplifX
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OligoAnalyzer 1.5
o Standalone desktop software
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OligoAnalyzer 1.5
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OligoExplorer 1.5
o Standalone desktop software
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OligoExplorer 1.5
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Primer-BLAST
o Web-based tool
o Provides primers specific to the PCR template
sequence.
o Includes primer pair specificity checking against a
selected database.
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Primer-BLAST