PCR
SnapGene
Dr. Sirwan Sleman PhD in Molecular Virology
Content
• What is Primer?
• Primer Types
• Primer Designing Guidelines
• Primer Designing Resource and Bioinformatics
Software's
What is primer?
• A primer is a short, single strand of oligonucleotides (RNA
or DNA) that act as a starting point for DNA synthesis
• If there is no primer there will be no DNA replication
because the DNA polymerases, can only add new
nucleotides to an existing strand of DNA.
• Application:
Primers are essential for the purpose of Detection,
Cloning and Sequencing
Types of Primers
RNA Primers: In vivo : DNA replication by Primase
DNA Primers: Invitro : synthesized chemically (PCR,
and DNA sequencing)
RNA Primers: In vivo DNA replication
Video
RNA Primers: In vivo DNA replication
https://www.youtube.com/watch?v=TNKWgcFPHqw
DNA Primers: In vitro DNA replication
DNA amplification process
Genomic DNA OR cDNA
Primer Design
It is important to choose the right primer for a successful DNA
amplification of your target amplicon because your designed
primer affects the entire DNA amplification process
Primer Design Guidelines
I. Primer Length
II. Melting and Annealing Temperature
III. Primer Base Compositions
IV. Secondary Structure
Primer Length
The primer length should not be too short or too long.
The primer should ONLY bind to a unique target site in
the template DNA
Too short - low specificity, resulting in non-specific
Amp.
Too long – slowdown the primer-template
hybridization process and increase probability of
primer dimer.
Primer Length
For example
Too short
Primer candidate 1 5’-TGCTAAGTTG-3’ NOT UNIQUE!
Primer candidate 2 5’-CAGTCAACTGCTACAGTAC-3’ UNIQUE!
Long enough
Template DNA
5’...TCAACTTAGCATGATCGGGTA.GTACTGTAGCAGTTGACTGTACAACTCAGCAA...3’
CAGTCAACTGCTACAGTAC
Primer 1 Primer 2
Primer 1
Optimal primer length: 18-24 bp. Avoid >3 bases difference
Forward primer Length = 19 bp
Reverse primer Length = 22 bp
Optimal Amplicon size: 150-1000 bp, avoid > 3 kb length
Melting and Annealing Temperature ( Tm/Ta)
Melting Temperature (Tm) –the temperature at which the DNA strands
are dissociated. Whilst Annealing Temperature (Ta) -the temperature at
which primers anneal to the template DNA
Higher G+C content or primer length would require a higher Tm due to
more hydrogen bonds.
Optimal Tm = (55°C - 65°C) e.g. Tm = 60°C
Optimal Ta is approximately 5°C below the Tm Ta = 55°C
Ta can be calculated from Tm Ta Opt = 0.3 x(Tm of primer) + 0.7 x(Tm of
product) - 14.9
Base Composition
Primer G/C content (G/C %):
o Optimal range is 40 %- 60%.
o Try to put G or C, or CG or GC at the last 5 bases of the 3’ prime end
to promote stability. This is known GC Clamp. BUT more than 3 G's or
C's should be avoided as it may result in non-specific amplification of
the target genes
o Try to avoid runs of one base or dinucleotide repeats ≥ 4 (e.g.
AGCGGGGGATGGGG, or ACTATATATATCAGGTCA)
For example
Template DNA
5’...TCAACTTAGCATGATCGGGGCA...AAGATGCACGGGGCCTGTACACAA...
3’
Caution for designing PCR Primers
Primer dimer
Primers can anneal to themselves, or each other rather than anneal
to the template.
Self-Dimer (homodimer)
An interactions between the nucleotides of the primer itself (the
same sense). The primer is homologous to itself e.g. Hairpins
Cross-Dimer (heterodimer)
An interactions between the nucleotides of the sense and
antisense primers (forward and reverse primers)
Hairpins
C A A A T C C T A T G G G G
G T T T A G G A T A C C C C
Caution for designing PCR Primers
e.g. Cross Primer Dimer
F Primer 5’-ACGGATACGTTACGCTGAT-3’
R Primer 5’-TCCAGATGTACCTTATCAG-3’
Complementarity of primer 3’ ends
F Primer 5’-ACGGATACGTTACGCTGAT-3’
R Primer 3’GACTATTCCATGTAGACCT-5’
Results in Primer dimer formation
F Primer
5’-ACGGATACGTTACGCTGATAAGGTACATCTGGA-3’
3’-TGCCTATGCAATGCGACTATTCCATGTAGACCT-5’
R Primer
Summary
Acceptable primer length is 18-24 bases
(G+C) base composition should be 40-60%
a G or C, or CG or GC in the last five bases (3') end of
primer : this reduce false priming;
Tm between 55-65oC are preferred;
Primer dimer should be avoided
Primer Designing Resource and Bioinformatics
Software's
Primer Design
Retrieve from Retrieve from Retrieve from
Genome Browser Gateways NCBI Gene banks, Published
primers..
Bioinformatics Software Primer BLAST
SnapGene
Vector NTI
Primer3
PrimerZ
.... ect.
SnapGene Software
A bioinformatics software enables DNA sequence visualization, sequence
annotation, sequence editing, cloning, protein visualization, and simulating
common cloning methods, primer designing, and PCR.
The software also enables documentation and sharing of data
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