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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?
Question 1
What is the chief goal of primer design?
A. Specificity B. Sensitivity
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
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
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
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
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
atcggactatcgatatgaataccgga tagcctgatagctatacttatggcca
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:
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.
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.
Question 2
o Length
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
Question 11
o Melting Temperature Forward: 5 GATTTAAGCACGAATCACGC 3 Tm: 54.7C Reverse: 5 TAGTCAGCATCGTATGAGCG 3 Tm: 58.0C
Question 12
o 3 Terminus
5 GATTTAAGCACGAATCACGC 3
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
Complementarity
Primer3 Output
Primer3 Output
Primer Evaluation
o OligoAnalyzer 3.1 (web-based)
http://sg.idtdna.com/analyzer/Applications/OligoAnalyzer/
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