Primer Design
Dave Palmer
dpalmer@zdap.com
Why Are Primers Important?
Primers are what gives
PCR its SPECIFICITY!!!
Good primer design: PCR works
great.
Bad primer design: PCR works
terrible.
Very-Brief PCR Reminder
PCR is a method to
amplify large
quantities of a DNA
covering a specific
sequence.
Factors That Affect Priming
Melting / Annealing
Temperature

Of primers to target
Secondary Structure

Within target
Complementarity


Primers to target
Primers to each other
Factor 1:
Melting / Annealing Temperature
aagtcagtcagtactagtgatgta
aagtcagtcag
PRIMER LENGTH

Longer primers stick better = melt at a
higher temperature.
GC CONTENT

More G-C content = more triple bonds =
primers stick better = melt at higher
temperature.
PCR Annealing Temp = Melt T - 5°C
Tm = [4(G + C) + 2(A + T)] °C
Tm = 58.3°C + 0.41°C (%G-C) - 500/length
http://www.alkami.com/primers/refprmr.htm
Factor 2:
Secondary Structure
Primers will have difficulty
annealing:
if they anneal to regions of
secondary structure within the
target that have a higher melting
point than the primer.
http://www.alkami.com/primers/refprmr.htm
Factor 3:
Complementarity
PRIMER-PRIMER (BAD)
atcggactatcga

gctatacttatggcca
Excessive similarity between primers,
especially at the 3’ ends, leads to the
formation of “primer dimers”
PRIMER-TARGET (GOOD)
atcggactatcga

tagcctgatagctatacttatggcca

Ideally should be 100% similar for
maximal specificity.
Primers don’t HAVE to be perfectly similar
to target to work.
http://www.alkami.com/primers/refprmr.htm
What is a Primer-Dimer
An unwanted
extension product
Results from primers
annealing to
themselves, or each
other, at 3’ ends
Extended primers are
no longer available to
prime target for PCR
atcggactatcga
gctatacttatggcca
atcggactatcgatatgaataccgga
tagcctgatagctatacttatggcca
Two Strategies for Primer
Design
Pick a primer pair and
optimize PCR conditions
for it.
If an exact sequence
site needs to be primed
or amplified.

If you’re working with
someone else’s primers.

?
Optimize the primer
design to work in a
specific set of PCR
conditions.
If you’ve got flexibility
around the amplified
site.

Allows more
“standardized” PCR
conditions.

Strategy 1 for Primer Design:
Fixed Primers, Vary Conditions
With a given primer pair, the Tm can be calculated.
Run multiple PCR reactions, each using a different
annealing temperature (= Tm - 5).
“Bracket” Ta: – 10C, -5C, 0C, +5C, +10C
Temp too low: Smearing due to non-specific priming
Temp too high: No amplification due to no priming
Choose conditions which give the best results.
http://www.iscpubs.com/pubs/abl/articles/b9812/b9812pre.pdf
Strategy 2 for Primer Design:
Optimizing Primers for Set Conditions
PCR conditions (esp. annealing
temp) are kept constant.
Select primers for a theoretical
Tm.
Best to select multiple primers,
then experiment to see which
combination works best.
95C – 65C – 72C
F1: atcgatcgatcgatcagtcatcg
F2: gtactgagctagctgcagctc
R1: atgactgagctgctagcttg
R2: atgcatgctcgtgactgtg
R1
R2
F2
F1/R1
F1/R2
F2
F2/R1
F2/R2
Designing Primers
Primer Design on the Web

Example: “Primer3”
http://frodo.wi.mit.edu/
Example gene: GFP5 Green Fluorescent Protein

GFP5, Genebank 1848286
1 ggatccaagg agatataaca atgagtaaag gagaagaact tttcactgga gttgtcccaa 61 ttcttgttga attagatggt
gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg 121 aaggtgatgc aacatacgga aaacttaccc ttaaatttat
ttgcactact ggaaaactac 181 ctgttccatg gccaacactt gtcactactt tctcttatgg tgttcaatgc ttttcaagat 241
acccagatca tatgaagcgg cacgacttct tcaagagcgc catgcctgag ggatacgtgc 301 aggagaggac catcttcttc
aaggacgacg ggaactacaa gacacgtgct gaagtcaagt 361 ttgagggaga caccctcgtc aacaggatcg agcttaaggg
aatcgatttc aaggaggacg 421 gaaacatcct cggccacaag ttggaataca actacaactc ccacaacgta tacatcatgg 481
ccgacaagca aaagaacggc atcaaagcca acttcaagac ccgccacaac atcgaagacg 541 gcggcgtgca actcgctgat
cattatcaac aaaatactcc aattggcgat ggccctgtcc 601 ttttaccaga caaccattac ctgtccacac aatctgccct
ttcgaaagat cccaacgaaa 661 agagagacca catggtcctt cttgagtttg taacagctgc tgggattaca catggcatgg 721
atgaactata caaataagag ctc
Designing Primers
Primer Design on the Web Using Primer3
Enter sequence
Pick Primers
Designing Primers
Primer3 Advanced Controls
Primer Size
Primer Tm
Complementarity
Designing Primers
Primer3 Output
Details:
-Start
-Length
-Tm
-GC
-Sequence
Where they bind:
Designing Primers
Primer 3 Output, continued
Primer Evaluation
Let’s assume we selected the first
primer pair (for + rev)
Website for online primer
evaluation:
Enter Sequence
TCATTGTTTGCCTCCCTGC
TAGAAACCCCAACCCGTGAAA
Primer Evaluation
Website displays potential problems
with primer self-annealing
TCATTGTTTGCCTCCCTGC
TAGAAACCCCAACCCGTGAAA
More advanced software can
examine interactions between
primers
Graphical
Output
Primer Evaluation
Just for fun, let’s assume we
selected a really BAD primer...
GGGCCCCTCACCAACCCGTGCCCGGG
Live Example Primer Design
Primer Design Workflow:
1. Pick a gene.
ie. BRCA1
2. Pull up sequence for the gene.
a. http://www.ncbi.nlm.nih.gov/
b. search Nucleotide Database for brca1
c. scroll through accessions for desired one
3. Copy sequence to text editor.
4. Pull up a primer design website.
a. http://frodo.wi.mit.edu/
b. copy sequence
c. select options and choose Pick Primers
4b. Verify primers find target (optional)
a. http://www.ncbi.nlm.nih.gov/BLAST/
b. select nucleotide blast
c. enter primer sequence, choose blast
4c. Analyse and double-check the primers
a. http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/Default.aspx
b. enter sequence, view
5. Order oligos.
a. http://www.operon.com
End of Primer Design