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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
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
aagtcagtcagtactagtgatgtaaagtcagtcag
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) Excessive similarity between primers,
especially at the 3’ ends, leads to the formation of “primer dimers”
PRIMER-TARGET (GOOD) 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
atcggactatcga
gctatacttatggcca
atcggactatcga
tagcctgatagctatacttatggcca
What is a Primer-Dimer
An unwanted extension productResults from primers annealing to themselves, or each other, at 3’ endsExtended primers are no longer available to prime target for PCR
atcggactatcga
gctatacttatggcca
atcggactatcgatatgaataccgga
tagcctgatagctatacttatggcca
Two Strategies for Primer Design
?
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.
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.
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 different annealing temperature (= Tm - 5).“Bracket” Ta: – 10C, -5C, 0C, +5C, +10CTemp too low: Smearing due to non-specific primingTemp too high: No amplification due to no primingChoose 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
F2 F2R1 F1/R1 F2/R1 R2 F1/R2 F2/R2
Designing Primers
Primer Design on the Web Example: “Primer3”
Example gene: GFP5 Green Fluorescent Protein GFP5, Genebank 18482861 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
http://frodo.wi.mit.edu/
Designing Primers
Primer Design on the Web Using Primer3
Enter sequence
Pick Primers
Designing PrimersPrimer3 Advanced Controls
Primer Size
Primer Tm
Complementarity
Primer3 Output
Details:-Start-Length-Tm-GC-Sequence
Designing Primers
Where they bind:
Designing Primers
Primer 3 Output, continued
Primer EvaluationLet’s assume we selected the first primer pair (for + rev)Website for online primer evaluation:
TCATTGTTTGCCTCCCTGCTAGAAACCCCAACCCGTGAAA
Enter Sequence
Primer EvaluationWebsite displays potential problems with primer self-annealingMore advanced software can examine interactions between primers
TCATTGTTTGCCTCCCTGCTAGAAACCCCAACCCGTGAAA
GraphicalOutput
Primer EvaluationJust 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 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
4b. Verify primers find target (optional)a. http://www.ncbi.nlm.nih.gov/BLAST/b. select nucleotide blastc. enter primer sequence, choose blast
4c. Analyse and double-check the primersa.
http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/Default.aspxb. enter sequence, view
5. Order oligos.a. http://www.operon.com
End of Primer Design
