Primer design for both conventional and real-time PCR Analysis of gel image by Image J

1. Primer design for PCR
Dr. Thoria Donia
Lecturer of Biochemistry, Faculty of Science,
Tanta University
thoria_donia@science.tanta.edu.eg
1

2. Primers
• Have different sequences
• Are complementary to the sequences on
the opposite strands of the template
DNA.
• flank the segment of target DNA that is
to be amplified.
2

3. Requirements for conventional PCR
• 1-A good starting template
• 2- Taq polymerase
• 3-Buffer solution
• 4- Designing primers
3

4. Resources for General Purpose PCR Primer Design
 Primer3
 Primer3Plus
 PrimerZ
 PerlPrimer
 Vector NTI Advantage 10
 IDT Primer quest tool

5. Let’sDO PCR
for PARK2

6. DNA Isolation

7. To check integrity of DNA
• By agarose gel electrophoresis
• Determination of the concentration of the DNA
The fact: 1 absorbance unit equates to 50 µg/ml
of DNA :
•
• Contaminants can be identified by scanning UV
spectrophotometry from 200 nm to 300 nm. A
ratio of 260nm: 280nm of approximately 1.8
indicates that the sample is free of protein
contamination, which absorbs strongly at 280
nm.
7

8. • A260/A230
 (2.0-2.2).
 Lower -----it may indicate the presence of
contaminants (Guanidine isothiocyanate and /or
phenol), which absorb at 230 nm.

9. Instruments used in RNA or DNA
analysis

10. Typical spectral pattern for Nucleic Acid

11. PCR

12. Gel Electrophoresis

13. PCR Purification

14. • PCR product is produced in
sufficient quantities for
subsequent experimental
analysis.
14

15. PCR
(Replication of DNA in vitro)
• Can generate billions of copies of DNA in few
hours,
• ng of DNA can be amplified to get mg of DNA.
• is based on in vitro enzymatic amplification of
a specific target DNA sequence in a cyclic
process using two oligonucleotides (Primers).
15

16. The PCR reaction cannot be
completed without the primers?
• The DNA primer used in the amplification
provide 3′ end to the Taq DNA
polymerase for initiating the
amplification.
• Once the Primer: DNA junction is
recognised by the Taq DNA polymerase,
it starts adding dTNPs to the DNA strand
and synthesize the new DNA strand.
16

17. What is PCR primer?
• PCR primers are short single-stranded
DNA sequences which help in the
amplification of DNA during PCR
reaction.”
17

18. Applications of PCR
1. Detection of infectious diseases (PCR can be
used to detect the presence of a viral genome in
a sample of blood).
2. Detection of Variations and Mutations in Genes
3. Detects people with inherited disorders and
carriers.
4. Track presence or absence of DNA abnormalities
characteristic to cancer.
5. Prenatal diagnosis of genetic disorders.
18

19. PCR can be used to detect the presence of a viral
genome in a sample of blood
19

20. PCR reaction completes in three steps
20

21. Amplification rate of PCR
21

22. Primers guarantee the successfulness of a PCR
Specificity?
Proper
annealing to
the template?

23. Specificity and efficiency of primers
are important in primers design
• Specificity ?? mispriming will occur
when primers are poorly
designed……….leads to nonspecific
amplification of sequences found in
the template pool.
• Efficiency ?? An efficient primer pair
will produce a twofold increase in
amplicon for each cycle of the PCR. 23

24. General rules for primer design
for conventional PCR
24

25. Melting temperature (Tm)
 The temperature at which 50% of the DNA duplex dissociates
to become single stranded
 50% of the primer and its complement are hybridized
 Tm=2(A+T)+4(G+C).
 Optimal melting temperature 50°C-- 60°C
 Tm above 65°C should be generally avoided because of the
potential for secondary annealing (hairpin, self dimer,
cross dimer)
 Higher Tm (75°C-- 80°C) is recommended for amplifying
high GC content targets.
 The difference in two primers melting temperature
should be 3 degree maximum.

26. • If you know melting temperature you can
calculate annealing temperature.
• Annealing temperature =Tm+/- 5 degree
• OR
• Annealing temperature = Tm of forward
primer+ Tm of reverse primer /2
26

27. Annealing temperature:
• A temperature at which primer can bind to its
complementary sequence.
• An ideal annealing temperature of the primer
is ranging between 56ºC to 65ºC.
• 5 degree lower than Tm
27

28. General rules for primer design
Specificity
 Determined by primer length as well as sequence
 The primer specificity is dependent on the nature of
the template used in the PCR reaction.

29. 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%.
 Repeats
 A repeat is a nucleotide sequence (a dinucleotide) that is repeated
(e.g. TCTCTCTCTC) should be avoided because they promote mispriming.
 maximum number should be 4 di‐nucleotides.
 Runs
 Runs are repeated nucleotides (e.g. TAAAAAGC has a 5 bp run of Adenine).
Runs should also be avoided because they are prone to mispriming. The
maximum run should be no more than 3–4 bp.
 At 3´ end C or G should be present

30. • Another requirements for PCR primers
• forward and reverse primers of similar length
and GC content.
• The Tm of both primers should be similar to
• 1-each other
• 2- the primer-binding sites at the ends of the
fragment to be amplified to achieve an optimal
annealing temperature and amplification.
• 3´-end complementarity between primers and
self complementarity within primers must be
avoided because it may increase primer-dimer
formation and reduce PCR efficiency.
30

31. Common problems in primer design
31

32. Length of the primers:
• Primers of 18 to 23 nucleotide long
• If the primer is too short, annealing
temperature becomes lower and it reduces
amplification capacity.
• Long primers -----the annealing temperature is
too high----------leads to non-specific binding.
32

33. Steps to design primers for
conventional PCR
33

34. • 1-Copy FASTA format of sequence from NCBI
nucleotide database and paste it in primer 3
plus
34

35. FASTA format
• Simple database format to store sequence
data.
• The FASTA format consists of
• 1- A single header line that starts with the
character >.
• 2-followed (without a blank) by an identifier
and optionally a short description.
• 3-The subsequent lines contain the sequence
information
35

36. FASTA format of sequence
36

37. NCBI home page
37

38. 38

39. Use filters to select the right variant
for gene of interest
• 1- Species---Animals for example
• 2- mRNA
• 3- Ref seq
• 4- Organism------Homo sapiens
39

40. 40

41. 41

42. • Now select the options for forward primer and
reverse primer and never select hybridization
probe as we run simple PCR
42

43. • do not click on any of the boxes because all
the information is automatically or by default
set by the software.
43

44. Adjust general settings as following
44

45. • In the next step as shown in the figure, click
on the “Pick primer” button and wait for the
result.
45

46. Product size
• 300-1000 bp for general application and
according to the sequence you want to
ampilify.
46

47. The settings are as following
47

48. Click pick primers and about 5 pairs
will appear in this format
48

49. The colors shows the annealing site
of primer to your sequence.
49

50. To check if primers are ok or not
• Use oligo calc to check for self complementary
• Add sequence click calculate reverse
complement strand will appear then click
check for self complementary
50

51. 51

52. Oligo calc is mainly used for
• for reporting DNA and RNA single-stranded
and double-stranded properties, including
molecular weight, solution concentration,
melting temperature, estimated absorbance
coefficients, inter-molecular self-
complementarity estimation and intra-
molecular hairpin loop formation.
52

53. Oligocalc link
• http://biotools.nubic.northwestern.edu/Oligo
Calc.html
53

54. Add forward primer then calculate and
check for self complementarity
54

55. Copy and paste reverse primer then
reverse complement and calculate
55

56. Copy and paste reverse primer then
reverse complement and calculate
56

57. If primer is ok you will get these
results
57

58. To check for self dimer
Primer-dimer estimation
Multiple Primer Analyzer | Thermo Fisher Scientific
58

59. Results that are given by analyzer
59

60. To add sequence of primers as following
• >forward
• Add forward primer sequence
• > reverse
• Add reverse primer sequence
60

61. To enter the sequence
61

62. Continue
62

63. Avoid non-specific amplification
BLASTing PCR primers against NCBI non-
redundant sequence database or Ref
sequence
In blasting you just add forward and reverse
primers, detect organism and ref sequence
mRNA
63

64. Insilico PCR to check specificity of the
primer
• Using Primer blast
•
64

65. Add the sequence, reverse and forward primers
detect homo sapiens then get primers
65

66. 66

67. Primer blast result
67

68. How to pick the right primers for
sequence of interest
• Bring the DNA sequence of interest from NCBI
• https://www.ncbi.nlm.nih.gov/pubmed/
68

69. 69

70. Step 1: Obtain Sequence in FASTA
Format
For example human myoglobin
For designing primers
Choose one with accession number containing
NM
Variant means there are more than isoforms on
genome.
70

71. F11 for full screen view
then print screen and paste in word or powerpoint
page
71

72. 72

73. From the previous page
• Click exons or coding sequence, they will be
selected in the sequence
• You choose the sequence according to the aim
of your experiment .
73

74. Click fasta and to save fasta sequence
follow the next steps
74

75. 75

76. Select and copy the sequence in word
or primer 3 plus
76

77. 77

78. Click general settings or advanced
settings
78

79. Write sequence id and paste the
sequence
79

80. • You can exclude some regions on excluded
regions and detect the included region
• Check left and right primers.
80

81. In general settings
• Product size 700-1000
• The product size should include flanking
sequences around the sequence 50-100 bp
especially if you are going to perform
sequencing as the first part I read as noise in
sequencing machine
81

82. Adjust general settings as following
and click pick primers
82

83. No.1 choice is the best
and they appear as pair 1 and 2 and so on
83

84. • Product size in different pairs are variable
• The first with repeated base more than 5 so
choose another one or modify general
settings as following
84

85. Change settings again as following
85

86. 86

87. Copy in a word file as following
• Left forward
• F: gtatttcctcgctgtgtgactct
• Reverse right
• R:gtcaggtgtagttaatggcttgg
• You can save also the parameters chosen to
get these primers to avoid them if the primers
are not working.
87

88. To check if primers are ok or not
• Use oligo calc to check for self complementary
• Add sequence click calculate reverse
complement strand will appear then click
check for self complementary
88

89. 89

90. 90

91. 91

92. To check for cross dimer
Primer-dimer estimation**
• Multiple Primer Analyzer | Thermo Fisher
Scientific
• >forward
• Add forward primer sequence
• > reverse
• Add reverse primer sequence
92

93. 93

94. 94

95. Insilico PCR to check specificity of the
primer
• Using Primer blast
•
95

96. Add the sequence, reverse and forward primers
detect Homo sapiens then get primers
96

97. 97

98. In real time PCR
• Quantification of the amount of the RNA
present into the sample by using the
fluorescent dye or probe by synthesizing cDNA
from RNA using the reverse transcriptase
enzyme
98

99. One step RT PCR

100. Two step RT PCR

101. Two types of chemistry are available
for the real-time quantitative PCR:

102. Taqman probe

103. Molecular beacons:

104. Output curve of real time PCR
104

105. Melting curve
105

106. Relative quantification by real time
PCR
106

107. Example for relative quantification of
PCR
107
GOI-HSG ….gene of interest house keeping gene

108. Actual steps to find primers for
your gene

109. Using pubmed or any website to look for paper with
real time PCR for your gene of interest

110. Real time PCR of AKT3
• miR-224-5p inhibits proliferation, migration,
and invasion by targeting PIK3R3/AKT3 in
uveal melanoma.

111. • https://onlinelibrary.wiley.com/doi/full/10.10
02/jcb.28507

113. AKT3 Forward
5′‐GGATCACAGATGCAGCTA
CC‐3′
Reverse
5′‐GTAGAAAGGCAACCTTC
CACAC‐3′

114. First you have to use primer blast to
check specificity of primers

116. Adjust these parameters to be as
following and click pick primers

117. Result

118. • Check this pair with oligo calc and multiple
primer analyzer from ThermoFisher Scientific.

119. Oligo calc result

120. Thermofisher Scientific multiple primer analyzer result
confirm self dimer

121. If ok
• Just change the bases with mispriming to the
right one and use this primer set

122. Not ok
• design a new primer set

123. Designing primers for real time PCR
• RT -PCR amplification of a particular
mRNA sequence requires two PCR
primers that are specific for that mRNA
sequence.
• The primer design should also allow
differentiation between the amplified
product of cDNA and an amplified
product derived from contaminating
genomic DNA. 123

124. Factors that affect the efficiency of a
qPCR
• include the amplicon length and primer
quality.
124

125. In a SYBR® Green‐based qPCR application,
specificity is very important
• SYBR® Green dye will bind to any dsDNA
present in the reaction mix, so amplification of
nonspecific products produces invalid data.
• Primer dimers may increase fluorescence,
resulting in inaccurate quantification of the
amplicon.
125

126. • Design primers that span exonl exon
junctions on the mRNA to avoid
introns sequences. Such primers
should not amplify genomic DNA.
126

127. Primer design for RT-PCR
 Design amplicon to be 75–210 bp.
 An amplicon should be at least 75 bp to easily
distinguish it from any primer-dimers that might form.
 Avoid templates with long (>4) repeats of single bases
 Maintain a GC content of 50–60%
 Place Gs and Cs on ends of primers
 Check sequence of forward and reverse primers to
ensure no 3‘ complementarity (avoid primer-dimer
formation)
 Maintain a melting temperature (Tm) between 60C and
68C.
 Verify specificity using
(http://www.ncbi.nlm.nih.gov/blast/)

128. Common Parameters of Primer
Design
• Primer Length
• The optimal length of primers 18–24 bp
Primer Melting Temperature (Tm)
• Primers of minimal length which have melting
temperatures (Tm) that are between 59 and 68
°C, with an optimal Tm of 63–64 °C.
• Tm of the primer pair should be within 1 °C of
each other.
128

129. • Annealing Temperature
• Optimal real‐time PCR annealing
temperatures are 59 °C or 60 °C.
• Product Size
• An ideal amplicon should be between 80 and
150 bp
• Mg++ Concentration
• The default is set to zero on most primer
design software. SYBR® Green buffer mixes
contain 3 to 6 mM of MgCl2.
129

130. • Repeats
• A repeat is a nucleotide sequence (a dinucleotide) that
is repeated (e.g. TCTCTCTCTC) should be avoided
because they promote mispriming.
• maximum number should be 4 di‐nucleotides.
• Runs
• Runs are repeated nucleotides (e.g. TAAAAAGC has a 5
bp run of Adenine). Runs should also be avoided
because they are prone to mispriming. The maximum
run should be no more than 3–4 bp.
130

131. • 3′ Stability
• the maximum ΔG of the 5 bases from the 3′
end of the primers.
• (ΔG is the Gibbs Free Energy, the energy
required to break the bonds present at the 3′
end).
• A higher 3′ stability will improve the efficiency
of the primer.
131

132. • GC Clamp
• the maximum ΔG of the 5 bases from the 5′
end of the primers.
• the 5′ stability refers to how stable the 5′ end
is due to the amount of Gs or Cs present at
the 5′ end of the primer. Having 1 to 2 GC
clamps are ideal, as it allows the primer to
bind strongly to the template strand, making it
more specific,
• avoid more than 2 GC clamps.
132

133. NCBI nucleotide database for gene of
interest
• In our case it is AKT3
• Filters as species :animals
• Ref seq and mRNA
• Organism Homo sapiens

134. Search results

135. Choose the small sized variant
• Homo sapiens AKT serine/threonine kinase 3
(AKT3), transcript variant 3, mRNA

136. • Idt ----tools ---------primer quest tool

138. Copy and paste sequence
• QPCR primer design +probe
• Customize assays
• Keep all task settings in default format except
Add exon exon junctions in overlap junction list
41,199,325,437,582,780,849,972,1101,1316,140
4,1507,1575
Click get assays ----validating , running ----
download assays

139. Downloaded file

140. Check by
• Oligo calc
• Multiple primer analyzer from Thermofisher
scientific
• Primer blast

141. AssaySet Type Sequence Start Length Tm GC Percent Amplicon
Assay Set 1
(Sequence
1) Forward Primer GCACCAGAGGTGTTAGAAGATAA 1093 23 61.946 43.478
Assay Set 1
(Sequence
1) Probe ACTATGGCCGAGCAGTAGACTGGT 1118 24 68.381 54.167
Assay Set 1
(Sequence
1) Reverse Primer TCATGGTCCTGGTTGTAGAAAG 1208 22 61.903 45.455
Assay Set 1
(Sequence
1) Product 116

142. Primer blast
• Sequence is specific
• Bind to different variant of the same gene is
ok

143. Multiple primer analyzer----ok

144. Oligo calc ----potential hair pin

145. Try another set using the same steps
• Set 4 would be the best

146. Analysis of gel using image J for
semiquantitative analysis of DNA
• https://www.youtube.com/watch?v=eazdPbE
HpDw
• You can follow what is illustrated in this link
146

147. steps you can follow as screen shot
• 1- open image J software from its setuped
icon
147

148. • Open the image of gel you want to analyze
• By clicking file menu then open
148

149. Edit-invert and bands will appear in
black
149

150. Select the lane using rectangular icon
150

151. Analyze-gel –select first lane
151

152. 152

153. Analyze-gel-plot lanes
153

154. 154

155. Use straight tool and shift to detect
lanes
155

156. 156

157. Use wand tool to detect areas of
bands
157

158. Click analyze gel ----label peaks
158

159. 159

160. 160