Analytical Profile of Coleus Forskohlii | Forskolin .pptx
Primer Designing Event.ppt
1. In-Silico Primer Designing
Dr. Shahzad Bashir Grewal (PhD Biochemistry)
Associate Head of Department, Minhaj University, Lahore
Instructor
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2. • A primer is a short, single-stranded DNA
sequence used in the polymerase chain
reaction (PCR) technique.
• Primers is used to hybridize with the
sample DNA and define the region of the
DNA that will be amplified.
• Primers are also referred to as
oligonucleotides.
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Primers
3. • Polymerase Chain Reaction (PCR) is widely
held as one of the most important inventions of
the 20th century in molecular biology
• Small amounts of the genetic material can now
be amplified to be able to a identify, manipulate
DNA, detect infectious organisms, detect genetic
variations, and numerous other tasks
PCR (Polymerase Chain Reaction)
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4. Inventor of PCR Method
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• Dr. Kary B. Mullis
(1944-2019)
• American Biochemist
• The Nobel Prize in
Chemistry 1993
• For his invention of the
polymerase chain reaction (PCR) method
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5. • Denaturation (94oC): First, the genetic material
is denatured, converting the double stranded
DNA molecules to single strands
• Annealing (60oC): The primers are then
annealed to the complementary regions of the
single stranded molecules
• Extension (72oC): They are extended by the
action of the DNA polymerase
PCR : Three Steps
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10. PICME labs : Your Research Partner
Design Considerations
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11. • Optimal length of PCR primers is 18-24 bp
• This length is long enough for adequate specificity
and short enough for primers to bind easily to the
template at the annealing temperature
1. Primer Length
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12. • This is the temperature at which 50% of the
primer and its complement are hybridized
• Primers with Tm in the range of 52-58oC
generally produce the best results
• Primers with melting temperatures above 65oC
have a tendency for secondary annealing
2. Primer Melting Temperature
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13. • The GC content of the sequence gives a fair
indication of the primer Tm
• “Itakura's empirical rule” makes a quick and dirty
estimate of the Tm of an oligonucleotide:
Tm = 2 (A+T) + 4 (G+C)
2. Primer Melting Temperature
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14. • The primer melting temperature is the estimate
of the DNA-DNA hybrid stability and critical in
determining the annealing temperature
– Too high Ta will produce insufficient primer-template
hybridization resulting in low PCR product yield
– Too low Ta may possibly lead to non-specific
products caused by a high number of base pair
mismatches
3. Primer Annealing Temperature
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15. • The GC content (the number of G's and C's
in the primer as a percentage of the total
bases) of primer should be 40-60%
4. GC Content
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16. • The presence of G or C bases within the last five
bases from the 3' end of primers (GC clamp)
helps promote specific binding at the 3' end due
to the stronger bonding of G and C bases
– More than 3 G's or C's should be avoided in
the last 5 bases at the 3' end of the primer
5. GC Clamp
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17. i) Hairpins:
Primer self-complementarity (ability to form
secondary structures such as hairpins.
6. Primer Secondary Structures
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18. ii) Primer Dimer:
Primer-dimers are caused when two primers with
the same or different sequences hybridise.
6. Primer Secondary Structures
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19. • A repeat is a di-nucleotide occurring many times
consecutively
• For example: ATATATAT
– maximum acceptable number of di-nucleotide
repeats is 4
7. Repeats
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20. • Primers with long runs of a single base should
generally be avoided
• For example, AGCGGGGGATGGGG has runs
of base 'G' of value 5 and 4
– maximum number of runs accepted is 4 bp
8. Runs
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21. • It is the maximum ΔG value of the five bases
from the 3' end
– An unstable 3' end (less negative ΔG) will
result in less false priming
9. 3' End Stability
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22. • The stability of the template secondary structures
depends largely on their free energy and melting
temperatures(Tm)
• Consideration of template secondary structures is
important in designing primers, especially in
qPCR
• If primers are designed on a secondary structures
which is stable even above the annealing
temperatures, the primers are unable to bind to
the template
10. Avoid Template Secondary Structure
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23. • Hence, it is important to design primers in the
regions of the templates that do not form stable
secondary structures
10. Avoid Template Secondary Structure
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24. • Primers designed for a sequence must not
amplify other genes in the mixture
• Commonly, primers are designed and then
BLASTed to test the specificity
11. Avoid Cross Homology
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25. PICME labs : Your Research Partner
Parameters for
Primer Pair Design
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26. • The amplicon length is dictated by the
experimental goals. For qPCR, the target length
is closer to 100 bp and for standard PCR, it is
near 500 bp
– Product length = (Position of antisense
primer-Position of sense primer) + 1
1. Amplicon Length
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27. • Primer can be located near the 5' end, the 3' end
or any where within specified length
• Generally, the sequence close to the 3' end is
known with greater confidence and hence
preferred most frequently
2. Product Position
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28. • Melting Temperature (Tm) is the temperature
at which one half of the DNA duplex will
dissociate and become single stranded
3. Tm of Product
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29. • The formula of Rychlik is most respected
Ta Opt = 0.3 x(Tm of primer) + 0.7 x(Tm of product)
- 14.9
– where
Tm of primer: the melting temperature of
primer-template pair
Tm of product: the melting temperature of the
PCR product
4. Optimum Annealing Temperature
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30. • The two primers of a primer pair should have
closely matched melting temperatures for
maximizing PCR product yield
– The difference of 5oC or more can lead no
amplification
5. Primer Pair Tm Mismatch Calculation
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31. PICME labs : Your Research Partner
Primer Design Using Software
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32. • A number of primer design tools are available
that can assist in PCR primer design for new
and experienced users alike
• These tools may reduce the cost and time
involved in experimentation by lowering the
chances of failed experimentation
Primer Design Using Software
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