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PCR Primer Design

This document discusses guidelines for designing primers for PCR. It explains that primers are short DNA sequences used to initiate DNA synthesis during PCR. Key factors in primer design include length of 18-24 bp, melting temperature of 52-60°C, GC content of 45-55%, avoidance of repeats or runs, and secondary structures. Proper primer design is important for specificity and efficiency of DNA amplification.

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Aiman Arshad
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129 views13 pages

PCR Primer Design

This document discusses guidelines for designing primers for PCR. It explains that primers are short DNA sequences used to initiate DNA synthesis during PCR. Key factors in primer design include length of 18-24 bp, melting temperature of 52-60°C, GC content of 45-55%, avoidance of repeats or runs, and secondary structures. Proper primer design is important for specificity and efficiency of DNA amplification.

Uploaded by

Aiman Arshad
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPT, PDF, TXT or read online on Scribd
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Designing

PCR
Primers
PCR: the technology that changed the world we knew
 The Polymerase Chain Reaction (PCR) revolutionized life
sciences as it provides a sensitive, reliable, efficient, and
convenient means of amplifying relatively large quantities of
DNA
 Invented in 1983 by Kary Mullis, who won a Nobel Prize 1993
 The technique was made possible by the discovery of Taq
polymerase, the DNA polymerase that is used by the bacterium
Thermus aquaticus, discovered in hot springs.
 The primary materials used in PCR:
- DNA nucleotides: the building blocks for the new DNA
- Template DNA: the DNA sequence that you want to amplify
- Primers: single-stranded short DNA (16--50 nucleotides
long) that are complementary to a short region on either end of
the template DNA
- DNA polymerase: a heat stable enzyme that catalyzes the
synthesis of new DNA
Primers dictate the successfulness of a PCR

Specificity?

Proper
annealing to
the template?
General rules for primer design
-- Primer and amplicon length
 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.
 Optimal primer length
 18-24 bp for general applications
 30-35 bp for multiplex PCR
 Optimal amplicon size
 300-1000 bp for general application, avoid > 3 kb
 50-150 bp for real-time PCR, avoid > 400 bp
General rules for primer design
-- Melting temperature (Tm)
 Tm is the temperature at which 50% of the DNA duplex
dissociates to become single stranded
 Determined by primer length, base composition and concentration.
 Also affected by the salt concentration of the PCR reaction mix
 Working approximation: Tm=2(A+T)+4(G+C) (suitable only for 18mer
or shorter).
 Optimal melting temperature
 52°C-- 60°C
 Tm above 65°C should be generally avoided because of the potential
for secondary annealing.
 Higher Tm (75°C-- 80°C) is recommended for amplifying high GC
content targets.
 Primer pair Tm mismatch
 Significant primer pair Tm mismatch can lead to poor amplification
General rules for primer design
-- Specificity and cross homology
 Specificity
 Determined primarily by primer length as well as sequence
 The adequacy of primer specificity is dependent on the nature of the
template used in the PCR reaction.
 Cross homology
 Cross homology may become a problem when PCR template is genomic
DNA or consists of mixed gene fragments.
 Primers containing highly repetitive sequence are prone to generate non-
specific amplicons when amplifying genomic DNA.
 Avoid non-specific amplification
 BLASTing PCR primers against NCBI non-redundant sequence
database is a common way to avoid designing primers that may amplify
non-targeted homologous regions.
 Primers spanning intron-exon boundaries to avoid non-specific
amplification of gDNA due to cDNA contamination.
 Primers spanning exon-exon boundaries to avoid non-specific
amplification cDNA due to gDNA contamination.
General rules for primer design
-- GC content; repeats and runs
 Primer G/C content
 Optimal G/C content: 45-55%
 Common G/C content range: 40-60%

 Runs (single base stretches)


 Long runs increases mis-priming (non-specific annealing)
potential
 The maximum acceptable number of runs is 4 bp

 Repeats (consecutive di-nucleotide)


 Repeats increases mis-priming potential
 The maximum acceptable number of repeats is 4 di-
nucleotide
General rules for primer design
-- Primer secondary structures
 Hairpins
 Formed via intra-molecular interactions
 Negatively affect primer-template binding, leading to poor or no
amplification
 Acceptable ΔG (free energy required to break the structure): >-2
kcal/mol for 3’end hairpin; >-3 kcal/mol for internal hairpin;
 Self-Dimer (homodimer)
 Formed by inter-molecular interactions between the two same primers
 Acceptable ΔG: >-5 kcal/mol for 3’end self-dimer; >-6 kcal/mol for
internal self-dimer;
 Cross-Dimer (heterodimer)
 Formed by inter-molecular interactions between the sense and antisense
primers
 Acceptable ΔG: >-5 kcal/mol for 3’end cross-dimer; >-6 kcal/mol for
internal cross-dimer;
General rules for primer design
-- GC clamp and max 3’ end stability
 GC clamp
 Refers to the presence of G or C within the last 4 bases from
the 3’ end of primers
 Essential for preventing mis-priming and enhancing specific
primer-template binding
 Avoid >3 G’s or C’s near the 3’ end
 Max 3’end stability
 Refers to the maximum ΔG of the 5 bases from the 3’end of
primers.
 While higher 3’end stability improves priming efficiency,
too higher stability could negatively affect specificity
because of 3’-terminal partial hybridization induced non-
specific extension.
 Avoid ΔG < -9.
General rules for primer design
-- Annealing temperatures

 Ta (Annealing temperature) vs. Tm


 Ta is determined by the Tm of both primers and amplicons:

optimal Ta=0.3 x Tm(primer)+0.7 x Tm(product)-25


 General rule: Ta is 5°C lower than Tm
 Higher Ta enhances specific amplification but may lower yields
 Crucial in detecting polymorphisms
Resources for General Purpose PCR Primer Design

 Primer3
 Primer3Plus
 PrimerZ
 PerlPrimer
 Vector NTI Advantage 10
Primer Design Resources for Real-time PCR

 NCBI Probe Database


 RTPrimerDB
 Primer Bank
 qPrimerDepot
 PCR-QPPD
 PerlPrimer
 QuantPrime
Resources for PCR Primer or Oligo Analysis

 AutoDimer
 IDT OligoAnalyzer 3.0
 PUNS
 NCBI BLAST
 UCSC In-Silico PCR

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