Primer Design

DESIGN OF OLIGONUCLEOTIDE PRIMERS FOR BASIC PCR

JEREMY G. VICENCIO
D E PA R T M E N T O F B I O L O G Y COLLEGE OF ARTS AND SCIENCES UNIVERSITY OF THE PHILIPPINES MANILA

Before you start designing primers find and use the right resources!
o What are the primers for? o What do you have to begin o General purpose with?
o o o o o o amplification? SNPs detection/validation Methylation study? Real-time PCR? Microarray probes? Degenerate PCR? Multiplex PCR? o Single DNA/protein sequence?

o Multiple DNA/protein sequence files?

o GenBank ID/Gene ID/Gene Symbol/rsSNP ID?

After you have designed your primers Consider a second opinion!

o Several different software are available for designing primers o Various software may differ significantly in:
o o o o Concrete and overall approaches Designing criteria and default settings Comprehensiveness Usability, accessibility, and speed

o Consider a second opinion when

o You are new to such a design task/application o You dont have a lot of confidence in the initial result

Question 1
What is the chief goal of primer design?

A. Specificity B. Sensitivity

Selecting PCR Primers

o Analysis of the target gene for potential priming sites
o free of homopolymeric tracts o have no obvious tendency to form secondary structures o not self-complementary o have no significant homology with other sequences on either strand of the target genome

o Creation of lists of possible forward and reverse primers

Selecting PCR Primers

o Selection of well-matched pairs of forward and reverse primers
o similar in G+C content o must generate an amplified product of the appropriate size and base composition

o Refining the length and/or placement of the oligonucleotides

Properties of Oligonucleotides That Influence the Efficiency of Amplification

Base Composition
o G+C content should be somewhere between 40% and 60%, with an even distribution of all four bases along the length of the primer
o avoid polypurine tracts or polypyrimidine tracts

o avoid long runs of a single base (max 4 bases)

o avoid dinucleotide repeats (max 4 repeats)

Length
o Primer length determines the specificity and significantly affects its annealing to the template
o Too short low specificity, resulting in non-specific amplification o Too long decrease the template-binding efficiency at normal annealing temperature due to the higher probability of forming secondary structures such as hairpins

o Optimal primer length

o 18-25 nucleotides long for general applications o 30-35 nucleotides for multiplex PCR o Members of a primer pair should not differ in length by >3 bp.

Length
o Optimal amplicon size
o 300-1000 bp for general applications, avoid >3 kb
o 50-150 bp for real-time PCR, avoid >400 bp

Repeated and SelfComplementary Sequences

o No inverted repeat sequences or selfcomplementary sequences >3 bp in length should be present. o If primers can anneal to themselves, or anneal to each other than anneal to the template, the PCR efficiency will be decreased dramatically. They shall be avoided.

Secondary structures

Hairpin
o Formed via intra-molecular interactions o Negatively affect primertemplate binding, lead to poor or no amplification o Acceptable G (free energy required to break the structure): >-2 kcal/mol for 3end hairpin; >-3 kcal/mol for internal hairpin

Secondary structures

Self-dimer (homodimer)
o Formed by intermolecular interactions between the two same primers

o Acceptable G: >-5 kcal/mol for 3 end selfdimer; >-6 kcal/mol for internal self-dimer

Complementarity between members of a primer pair

o The 3 terminal sequences of one primer should not be able to bind to any other site on the other primer. o Formation of primer dimers can be reduced by
o careful primer design o use of hot start or touchdown PCR o use of specially formulated DNA polymerases

Secondary structures

Cross-dimer (heterodimer)
o Formed by intermolecular interactions between the sense and antisense primers

o Acceptable G: >-5 kcal/mol for 3 end cross-dimer; >-6 kcal/mol for internal cross-dimer

What is a primer dimer?

o An unwanted extension product o Results from primers annealing to themselves, or each other, at 3 ends o Extended primers are no longer available to prime target for PCR
atcggactatcga gctatacttatggcca

atcggactatcgatatgaataccgga tagcctgatagctatacttatggcca

Melting Temperatures (Tm)

o Tm is the temperature at which 50% of the DNA duplex dissociates to become single-stranded
o Determined by primer length, base composition, and concentration

o Also affected by the salt concentration of the PCR reaction mix

Melting Temperatures (Tm)

o Wallace rule: Tm (in C) = 2(A+T)+4(G+C)
o can be used to calculate the melting temperature for perfect duplexes 15-20 nucleotides in length in solvents of high ionic strength (e.g., 1 M NaCl):

o Baldino algorithm
Tm (in C) = 81.5C + 16.6 (log10[K+]) + 0.41(%[G+C]) (675/n) where n is the number of bases in the oligonucleotide

o predicts reasonably well the melting temperature of oligonucleotides, 14-70 nucleotides in length, in cation concentrations of 0.4 M or less:

Melting Temperatures (Tm)

o Should not differ by >5C between members of a primer pair o The Tm of the amplified product should not differ from the Tm values of the primer pairs by >10C

Melting Temperatures (Tm)

o Optimal Tm
o 52C to 60C
o Tm above 65C should be avoided because of the potential for secondary annealing

o Higher Tm is recommended for amplifying high GC content targets

3 Termini
o If possible, the 3 base of each primer should be G or C.
o However, primers with a .NNCG or .NNGC sequence at their 3 termini are not recommended since this promotes the formation of hairpin structures and may generate primer dimers.

o GC Clamp refers to the presence of G or C within the last 5 bases from the 3 end of primers
o Essential for preventing mis-priming and enhancing specific primer-template binding

Adding restriction sites, bacteriophage promoters, and other sequences to the 5 termini of primers
o In general, the presence of additional sequences does not significantly affect annealing of the oligonucleotide to its target DNA. o Restriction sites: the primer should be extended by at least three additional nucleotides beyond the recognition sequence of the restriction enzyme.

Placement of priming sites

o Various constraints on the placement of priming sites:
o o o o o mutations restriction sites coding sequences microsatellites cis-acting elements

o Use forward and reverse primers that bind to different exons when designing primers for use on cDNA templates.

Cross-homology
o Cross homology may become a problem when PCR template is genomic DNA or consists of mixed gene fragments. o BLASTing PCR primers against the NCBI non-redundant sequence database is a common way to avoid designing primers that may amplify non-targeted homologous regions. o Use primers spanning intron-exon boundaries to avoid nonspecific amplification of gDNA due to cDNA contamination. o Use primers spanning exon-exon boundaries to avoid nonspecific amplification cDNA due to gDNA contamination.

Annealing temperature (Ta)

o Ta vs. Tm
o Ta is determined by the Tm of both primers and amplicons:
optimal Ta = 0.3 x Tm (primer) + 0.7 x Tm (amplicon) 25 o General rule: Ta is 5C lower than Tm

o Higher Ta enhances specific amplification but may reduce yield

Test your understanding

EVALUATE WHETHER THE FOLLOWING PRIMERS MEET THE GUIDELINES FOR EACH SPECIFIED CRITERION

Question 2
o Length

5 ACT GCC TCG ACT ACT TAC GC 3

Question 3
o Length

Forward: 5 GCT TCA ACG GAC CAT TGC 3 Reverse: 5 CTT ACG ACT TCC ACT TCC GCA C 3

Question 4
o Base composition

5 GCCACGTCGCGCATGCGC 3

Question 5
o Base Composition

5 ACACACACTTTTGCC 3

Question 6
o Self-complementary sequences

5 GCTTCACGGATCTGAAGC 3

Question 7
o Hairpins 5 ACGCTCTCCACGAGTCACGC 3

Question 8
o Homodimers 5 GCGTTAGGCACGAATCACGC 3

Question 9
o Heterodimers
Forward: 5 GCGTTAGGCACGAATCACGC 3 Reverse: 5 GCGTCAGAACCGTACGAGCG 3

Question 10
o Melting Temperature

5 ATTGAATCTACTACTTACGC 3 Tm: 50.7C

Question 11
o Melting Temperature Forward: 5 GATTTAAGCACGAATCACGC 3 Tm: 54.7C Reverse: 5 TAGTCAGCATCGTATGAGCG 3 Tm: 58.0C

Amplified Product: 851 bp, Tm: 64.0C

Question 12
o 3 Terminus

5 GATTTAAGCACGAATCACGC 3

Computer-Assisted Design of Oligonucleotide Primers

Primer3
o Web-based tool for primer design http://frodo.wi.mit.edu/ o Example gene: GFP5 Green Fluorescent Protein
o GenBank: U87973.1

Primer3
Enter sequence

Pick Primers

Primer Size Primer Tm

Complementarity

Primer3 Advanced Controls

Details: -Start -Length -Tm -% GC -Sequence

Where they bind:

Primer3 Output

Primer3 Output

Primer Evaluation
o OligoAnalyzer 3.1 (web-based)
http://sg.idtdna.com/analyzer/Applications/OligoAnalyzer/

OligoAnalyzer 3.1: Analyze

OligoAnalyzer 3.1: Hairpin

OligoAnalyzer 3.1: Homodimer

OligoAnalyzer 3.1: Heterodimer

AmplifX
o Standalone desktop software

AmplifX

AmplifX

OligoAnalyzer 1.5
o Standalone desktop software

OligoAnalyzer 1.5

OligoExplorer 1.5
o Standalone desktop software

OligoExplorer 1.5

Primer-BLAST
o Web-based tool
o Provides primers specific to the PCR template sequence.
o Includes primer pair specificity checking against a selected database.

Primer-BLAST