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Adv 4.

The document discusses primers and their design for PCR. It defines primers as short DNA or RNA sequences that provide a starting point for DNA replication. It notes that primers are essential for DNA detection, cloning and sequencing. It discusses primer types, including RNA primers used in vivo and DNA primers synthesized for in vitro use. The document provides guidelines for optimal primer length of 18-24 bases, GC content of 40-60%, and melting temperature of 55-65°C. It also cautions against primer dimers. Bioinformatics software can help design primers adhering to these guidelines.

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harseen rahim
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29 views

Adv 4.

The document discusses primers and their design for PCR. It defines primers as short DNA or RNA sequences that provide a starting point for DNA replication. It notes that primers are essential for DNA detection, cloning and sequencing. It discusses primer types, including RNA primers used in vivo and DNA primers synthesized for in vitro use. The document provides guidelines for optimal primer length of 18-24 bases, GC content of 40-60%, and melting temperature of 55-65°C. It also cautions against primer dimers. Bioinformatics software can help design primers adhering to these guidelines.

Uploaded by

harseen rahim
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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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

SnapGene Free Trial | Software for everyday molecular biology

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