This document discusses primer design for PCR. It defines a primer as a short DNA sequence that provides a starting point for DNA synthesis. It notes that DNA polymerase can only add nucleotides to an existing DNA strand. The document then covers criteria for primer design, including amplicon size, primer length and GC content, melting temperature, primer pair matching, annealing temperature, and 3' terminal properties. It also discusses potential problems like self-complementarity and provides examples of using software to design and analyze primers.

1. PRIMER
DESIGNING

2. Presenters
Fatima Khalid
Barira Zahid
BS Bioinformatics 6th Semester
University of Agriculture Faisalabad

3.  A primer is a short strand of oligonucleotides
that serves as a starting point for DNA
synthesis.
 Because DNA polymerases, can only add
new nucleotides to an existing strand of DNA.
What is Primer ?

5. Sequence from which
to choose the primer
PCR reaction
parameters
Primer selection rules
PRIMER
DESIGN

6.  Size of amplicon
 Primer length
 G-C content
 Primer melting temperature
 Primer pair Tm match
 Primer annealing temperature
 3’– terminal properties
Primer Design Criteria

7.  The amplicon length is dictated by the
experimental goals. For standard PCR, it is
near 500 bp.
o Size of Amplicon:

8.  It is generally accepted that the optimal length
of PCR primers is 18-22 bp.
o Primer Length:

9.  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%.
o G-C Content:

10.  Primers with melting temperatures in the
range of 52-58oC generally produce the best
results.
 Tm is directly proportional to GC content.
Formula for calculating the Tm of primer:
 Tm = 4(G+C) + 2(A+T)
o Primer Melting Temperature:

11.  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.
o Primer Pair Tm Match:

12.  The primer melting temperature is critical in
determining the annealing temperature.
 Ta = 0.3 x Tm(primer) + 0.7 x Tm (product) – 14.9
where,
Tm(primer) = Melting Temperature of the primers
Tm(product) = Melting temperature of the product
o Primer Annealing Temperature:

13.  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.
o 3’- Terminal properties:

14.  Homo-complementary (self-complementary)
 Hetero-complementary
Common problem in primer designing:

15.  Self complementarity of regions within a primer
can lead to the formation of hairpin structures,
where the primer folds on itself.
• Homo-complementary:

16.  When there are complimentary bases present
between the forward and reverse primer, so these
primers combines themselves instead of DNA
sequence.
• Hetero-complementary:

17.  Delta-G represents the quantity of energy needed to
fully break a secondary DNA structure. The lower dG
values, the higher the quantity of energy needed if a
secondary structure is formed.
 This is very important in PCR reaction because in that
cases, the energy is given for temperature.
 If quantity of energy needed to break the dimer is
higher, you need higher temperatures, which can
disturb the reaction.
 In general terms, values of dG more positives than - 9
kcal/mol are acceptable.
Delta-G value:

18. 5’-ACGATGATAG-3’
5’-ACGATGATAGACCCAGTTCGAGTCAGTTAC-3’
3’-TGCTACTATCTGGGTCAAGCTCAGTCAATG-5’
It will code for 3’ to 5’ strand
Forward Primer Designing:

19. 5’-ACGATGATAGACCCAGTTCGAGTCAGTTAC-3’
3’-TGCTACTATCTGGGTCAAGCTCAGTCAATG-5’
3’-CAGTCAATG-5’
It will code for 5’ to 3’ strand
Reverse Primer Designing:

20. Software for PRIMER
DESIGNING and PRIMER
ANALYSIS

21. Primer 3:
o Select a sequence you want to design the
primer for and copy it.
o Open the primer3 software and paste
your sequence in the sequence field.

23. The field to insert
the sequence.

24. Paste the sequence in the field and select the
primer or primers you want to produce.

25. There are default general primer picking
conditions but they can be changed.

26. After selecting the general primer conditions
select the “Pick Primer” option.

27. Our primers are displayed along with their properties
and sequences.

28. The points where these primers will attach are shown
with <<<,>>> signs.

29. Other options for primers are also given at the end
along with there properties.

30.  Software use to analyze the primer, its properties
like;
Hairpin
Self-Dimer
Hetero-Dimer
OligoAnalyzer:

31. Paste forward primer CAG CCT GAG TGC AAA ATT CA

32. Paste reverse primer GGA GAC AAG AGC CCA CAG AG

33. Result of Primer Analysis:

34. Hairpin:

35. Hairpin:

36. Self-Dimer:

37. Hetero-dimer:

38. Hetero-dimer: