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Primer Design Dalia 1
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Primer Design
ByDr/ Dalia Shaalan
Lecturer of Medical Biochemistry
and Molecular BiologyMansoura University
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Why Are Primers Important?
Primers are what gives PCR itsSPECIFICITY!!!
Good primer design: PCR work is great.
Bad primer design: PCR work is terrible.
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Very-Brief PCR Reminder
PCR is a method to
amplify largequantities of a DNAcovering a specificsequence.
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PCR
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General notes on primer design in PCR
Perhaps the most critical parameter for
successful PCR is the design of primers
Primer selection
Critical variables that affect priming
are:- primer length
- specificity
- melting temperature (Tm)- G/C content
- 3-end sequence
- complementary primer sequences
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Primer length
Specificity and the temperature of annealing are dependenton primer lengthOligonucleotides between 20 and 30 bases are highlysequence specific
Primer length is inversly proportional to annealing efficiency:in general, the longer the primer, the more inefficient theannealingThe primers should not be too short as specificity decreases
Primer Size:
Too small, may bind to more than one site in the genome
Too Large (Long primers), takes a longer time to hybridize
and would slow down the PCR cycle.
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Factors affecting Tm: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 highertemperature.
http://www.alkami.com/primers/refprmr.htm
Melting temperature (Tm)Not all primers will work the same; primers perform differently at
different temperatures. Melting temperature(Tm) is temperature at which the primer will
dissociate.
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Annealing temperature (Ta):
Optimal temperature for primers to attachto the template DNA
If you use too high Ta:
Bonds dont work
Primer doesnt anneal If you use too low Ta:
Primer may attach anywhere
Non-specific amplification
Depends on strength of bonds
Remember:
G-Cthree hydrogen bonds
A-Ttwo hydrogen bonds
Annealing temperature depends on GC content
Melting temperature (Tm)
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Melting temperature (Tm)the goal should be to design a primer with anannealing temperature of at least 50C.the relationship between annealingtemperature and melting temperature is one
of the Black Boxes of PCR.a general rule is to use an annealingtemperature that is 5C lower than themelting temperature.
PCR Annealing Temp = Melt T - 5C
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Melting temperature (Tm)both of the primers should be designed suchthat they have nearly similar meltingtemperatures.If primers are mismatched in terms of Tm,
amplification will be less efficient or may noteven work:
One primer with the higher Tm will mis-
prime at lower temperatures; Other primer with the lower Tm may not
work at higher temperatures.
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-A good working approximation of this value can be calculatedusing the Wallaceformula:
Tm= 4x (C+G) + 2x (A+T) C
-The melting temperatures of oligos are most accuratelycalculated using nearest neighbor thermodynamiccalculations with the formula:
Tm= H [S+ R (c/4)] 273.15 C + 16.6 log 10[K+]
(H is the enthalpy, S is the entropy for helix formation, R isthe molar gas constant and c is the concentration of primer)
Melting temperature (Tm)
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G/C content
Ideally a primer should have a randommix of nucleotides and about 50% GCcontent.
There should be no PolyG or PolyCstretches that can promote non-specific
annealing.
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3-end sequence
The 3' terminal position in PCR primers isessential for the control of mis-priming.
Inclusion of a G or C residue at the 3'end of primers helps to ensure correctbinding (stronger hydrogen bonding = 3bonds of G/C residues).
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Complementary primer sequences
Primers need to be designed with absolutelyno intra-primer homologybeyond 3 basepairs. If a primer has such a region of self-homology, snap back can occur.
Another related danger is inter-primerhomology:partial homology in the middle
regions of two primers can interfere withhybridization. If the homology should occurat the 3' end of either primer, primer dimerformation will occur
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Complementarity
PRIMER-PRIMER (BAD) Excessive similarity between
primers, especially at the 3 ends,
leads to the formation of :
primer dimers
PRIMER-TARGET (GOOD)
Ideally should be 100% for
maximal specificity.
atcggactatcga
gctatacttatggcca
atcggactatcga
tagcctgatagctatacttatggcca
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What is a Primer-Dimer
An unwantedextension product
Results from primersannealing tothemselves, or eachother, at 3 ends
Extended primers areno longer available toprime target for PCR
atcggactatcga
gctatacttatggcca
atcggactatcgatatgaataccgga
tagcctgatagctatacttatggcca
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Two Strategies for Primer Design
?
Optimizing Primersfor Set Conditions:
Optimize the primerdesign to work in a
specific set of PCRconditions.
If youve got flexibilityaround the amplifiedsite.
Allows morestandardized PCRconditions.
Fixed Primers, VaryConditions:
Pick a primer pair andoptimize PCRconditions for it.
If an exact sequencesite needs to beamplified.
If youre workingwith someone elsesprimers.
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Strategy 1 for Primer Design:Fixed Primers, Vary ConditionsWith a given primer pair, the Tm can be calculated.
Run multiple PCR reactions, each using a differentannealing temperature (= Tm - 5).
Ta: 3C, -2C, -1C, 0C, +1C, +2C, +3C
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.
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Strategy 2 for Primer Design:Optimizing Primers for Set Conditions
PCR conditions (esp. annealingtemp) are kept constant.
Select primers for a theoreticalTm.
Best to select multiple primers,then experiment to see which
combination works best.
95C 65C72C
F1: atcgatcgatcgatcagtcatcg
F2: gtactgagctagctgcagctc
R1: atgactgagctgctagcttg
R2: atgcatgctcgtgactgtg
F1 F2
R1 F1/R1 F2/R1
R2 F1/R2 F2/R2
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Where do we get primer sequences from?
Somebody may have isolated them
Check databases
Freely available on internet (GenBank)
Results not publishable without primer
information Heterologous primers (from related species)
Primer design from published sequences.
Primer isolationVery lengthy and expensive procedure
several months work
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Primer design
Sequence should be reverse compliments
(i.e. 3 ends point toward one another).
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CATACGATGCTAGCTAGCTGCTAGTGCTG
ATCGTAGTCGTAGCTAGTCGTACGTACGT
CGATGTAATTCGCATCGATATGCGCTAGC
GATATGCGCATCGATCGATATCGATATGC
5
3
Primer 1
5-CATACGATGCTAGCT-3
Primer 2
3-GCTATAGCTATACG-5
Primer design
Primer 1: 5-CATACGATGCTAGCT-3 Primer 2: 5-GCATATCGATATCG-3
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CATACGATGCTAGCTAGCTGCTAGTGCTG
ATCGTAGTCGTAGCTAGTCGTACGTACGT
CGATGTAATTCGCATCGATATGCGCTAGC
GATATGCGCATCGATCGATATCGATATGC
5
3
Primer 1: 5-CTGCTAGTGC-3 Primer 2: 5-ATCGATGCGCAT-3
Primer design
Primer 1
5-CTGCTAGTGC-3
Primer 2
3-TACGCGTAGCTA-5
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PCR
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Designing Primers
Primer Design on the Web
Example: Primer3
Example gene: GFP5 Green Fluorescent Protein
GFP5, Genebank 1848286301 aggagaggac catcttcttc aaggacgacg ggaactacaa gacacgtgct gaagtcaagt 361 ttgagggaga caccctcgtc
aacaggatcg agcttaaggg aatcgatttc 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 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
http://frodo.wi.mit.edu/
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Designing Primers
Primer Design on the Web Using Primer3
Enter sequence
Pick Primers
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Primer3 Output
Details:-Start-Length
-Tm-GC-Sequence
Designing Primers
Where they bind:
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Designing Primers
Primer 3 Output (continued)
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Primer Evaluation
Lets assume we selected the firstprimer pair (for + rev)
Website for online primer
evaluation:
TCATTGTTTGCCTCCCTGC
TAGAAACCCCAACCCGTGAAA
Enter Sequence
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Primer Evaluation
Website displays potential problemswith primer self-annealing
More advanced software can
examine interactions betweenprimers
TCATTGTTTGCCTCCCTGC
TAGAAACCCCAACCCGTGAAA
Graphical
Output
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Primer Evaluation
Just for fun, lets assume weselected a really BAD primer...
GGGCCCCTCACCAACCCGTGCCCGGG
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Live Example Primer DesignPrimer 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 brca1c. 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 sequencec. select options and choose Pick Primers
5. Analyse and double-check the primersa.http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/Default.a
spxb. enter sequence, view
6. Order oligos.a. http://www.operon.com
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Conclusion on Primer design
Primer pairs should have similar annealing temp
length, %GC content
Tm = 4(G + C) + 2(A + T) oC.
Minimal (
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