General rules For Designing A primer @Gene.Editor18

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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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