This presentation discusses primer design for PCR. It begins with an overview of PCR and what primers are. It then outlines several general rules for effective primer design, such as having a length of 18-24 base pairs, a melting temperature of 52-60°C, limiting runs and repeats, and avoiding hairpins and dimers. The presentation also notes that primers should be specific to the target sequence and provides resources for designing primers, such as Primer3 and Vector NTI Advantage. The goal is to explain the basic principles of primer design to optimize PCR amplification.

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Muhammad Khurram Shahzad
Founder Of #Gene.Editor Page
Member Of Pakistan Biotechnology Student Society
Founder OF #Gene editor Group on Facebook
https://www.facebook.com/gene.editor18/

4. Basic Over view Of this Presentation :
• Discuss about PCR
• Some Basic Information About Primer
• Discuss some basic rule of primer designing
• Also discuss some tools which we can use for
designing a primer
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5. What is PCR?
• PCR is a polymerase reaction which is used to
amplify the DNA .
• Most vital creations of the twentieth century
in molecular biology.
• Little amount of a genetic material can be
amplified by a PCR to identify and manipulate
DNA as well as detect infectious organisms,
including the viruses that cause, hepatitis,
AIDS, tuberculosis.
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6. PCR
• It can also be used to detect genetic
variations, including mutations in human
genes and many other tasks.
• So There is a different Steps in PCR :
• Denaturation
• Annealing
• Extension
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7. Polymerase Chain reaction :
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8. What is primer ?
• Primer is a short oligonucleotide sequence
which consist of 18-28bp
• Used in many molecular techniques ranging
from PCR (polymerase chain reaction) to DNA
sequencing.
• Primers are designed to sequence the region
of a template which are to be annealed.
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9. Primer
• Primer are essential for the DNA amplification
• For the purpose of detection
• Cloning and
• Sequencing
• That’s why it is important to know the primer
designing
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10. Primer
• Primer that is designed impact the entire DNA
amplification process
• DNA polymerases, Enzyme that catalyze
replication of DNA only initiate replication
process by adding nucleotide to primers.
• It is also important to choose the right primer
for a successful DNA amplification.
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11. Primer
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12. General rules for primer designing
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.
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13. Continue
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
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14. General rules for primer Designing
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 mixture
 Working approximation: Tm=2(A+T)+4(G+C) (suitable only for
18mer or shorter).
• Melting Temperature Tm (K) = {ΔH/ ΔS + R ln(C)}
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15. Continue
 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
 Desirable Tm difference < 5°C between the primer pair
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16. General rules for primer designing
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
 To improve specificity of the primers it is necessary to avoid
regions of homology. Primers designed for a sequence must not
amplify other genes in the mixture. Commonly, primers are
designed and then BLASTed to test the specificity
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17. General rules for primer designing
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
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18. Continue
 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;
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19. 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.
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20. Annealing Temperature And Other
consideration
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
Primer location on template
 Dictated by the purpose of the experiment
 For detection purpose, section towards 3’ end may be preferred.
When using composite primers
 Initial calculations and considerations should emphasize on the
template-specific part of the primers
 Consider nested PCR
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21. Resource for primer designing
Primer3
Primer3Plus
PrimerZ
PerlPrimer
Vector NTI Advantage 10
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22. Video
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