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The Art of PCR Primer Design

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Primer design can sometimes feel like more of an art than a science, and designing the best primer can significantly affect the success or failure of your experiments.  Here are a few tips on optimizing primer design for several different applications:

PCR amplification/cloning

One of the most common primer-based applications is cloning.  The desired amplicon and the vector restriction sites will largely dictate the composition of the primers.  A few tweaks, however, can increase the chance of cloning your gene on the first try:

  • Use a “GC clamp” at the 3’ end of each primer.  The G-C bond is stronger than the A-T bond, so a 3’ end enriched in G and C residues will bind with higher affinity.
  • Match the melting temperatures of paired primers.  If both primers have a melting temperature within 1-2 degrees of each other, it will be much simpler to optimize your PCR conditions.
  • Check for primer dimers or hairpins.  Any annealing between your primer pair or of one primer to itself will drastically affect the efficiency of your PCR reaction.
  • Add a few extra nucleotides upstream of restriction sites.  This will increase the efficiency of the restriction reaction (by giving the enzyme something to “hold on to”).  Since the extra bases do not anneal to the template, they do not need to be included in the melting temperature calculation.

Mutagenesis

Primer-based mutagenesis is a quick and easy way to mutate a gene of interest.  Mutagenesis kits contain extensive information on how to design optimal primers for mutagenesis, which can vary significantly from cloning primers in length, composition, and melting temperature.  Here are a few additional things to keep in mind:

  • Use the company’s Tm formula to calculate melting temperature.  The formula provided in the kit information will often yield a different melting temperature than that calculated by standard methods; be sure to use the formula suggested by the kit to get the right primer length and composition.
  • Try incorporating a restriction site.  If you can introduce a novel restriction site through a silent mutation, you can screen the mutated plasmids by digest.  This can save time and money while waiting for sequencing info on the putative mutated clones.

Websites and programs

Most sequence analysis programs include primer design features.  In addition, there a several fantastic primer design programs available on the web.  Here are a few of my favorites:

  • IDT OligoAnalyzer. This free online tool will analyze single primers or primer pairs for melting temperature, dimer formation, and hairpins.
  • Invitrogen OligoPerfect Designer. This online tool, which requires an Invitrogen account, will design primers for you based on target sequence.
  • Primer3.  A popular online tool which will design primers based on sequence or analyze your pre-designed primers by every conceivable parameter.
  • UCSC Genome Browser InSilico PCR. This website contains genome data for an extensive collection of research organisms, and will perform an in silico PCR reaction to confirm that your primers only amplify one product.

What are your tips for primer design?

13 Comments

  1. Lamaksha Egamag on September 14, 2010 at 8:32 am

    “Add a few extra nucleotides upstream of restriction sites. This will increase the efficiency of the restriction reaction (by giving the enzyme something to “hold on to”). Since the extra bases do not anneal to the template, they do not need to be included in the melting temperature calculation.”

    Adding extra bases upstream of the restriction site will, like the restriction site sequence itself, not anneal to the initial template. However, from the second round of PCR onwards, the template sequence would have this ‘foreign’ sequence incorporated, and hence will contribute to the Tm.

  2. Catherine Kibirige on July 28, 2010 at 8:56 pm

    Does anyone have any experience with nucleotide/nucleoside analogues. I am working with HIV and trying to design some real time PCR primers that will cross react with as many different subtypes of HIV as possible. I can’t seem to find any good reviews or booklets on the matter that give some simple pointers on what to do for positions where I have variability and want to increase primer cross reactivity… help.

  3. Alejandro Montenegro-Montero on July 26, 2010 at 2:13 pm

    For qPCR primers I use primer3plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi).
    Works perfectly.

  4. Åsa Hagström on July 23, 2010 at 7:03 am

    When designing degenerate primers, I strongly recommend CODEHOP (http://bioinformatics.weizmann.ac.il/blocks/codehop.html). All my primers made with this algorithm have worked nicely.

    Sometimes when designing a specific primer it may be hard to find a region with a good GC-content. I then just add 5-10 extra nt (and control for hairpin and dimer formations!)

    I almost never design short primers (only for qPCR). Mine are usually 25-35 nt (I need a Tm over 60°C since I usually work with Phire DNA polymerase) and they always work great.

  5. Emily Crow on July 22, 2010 at 1:44 pm

    I actually use OligoCalc all the time – I didn’t realize it was available to users outside of Northwestern! It’s a great tool.

    Dan, I do very little qPCR, but PrimerBank and AutoPrime look like useful sites for designing qPCR primers.

  6. Fiona Mumoki on July 22, 2010 at 12:46 pm

    Thanks for the article Emily.

    I love oligoCalc too! Really user friendly when it comes to Primer parameters!

    As a tip, I love to keep my primers short, about 20 bp. I find that this reduces chances of obtaining non-specific amplifications, and in some cases, hairpin formations too!

    Also, I avoid long runs of (single type) nucleotides e.g., GGG, for the aforementioned reasons!

  7. Pranay Dogra on July 22, 2010 at 10:20 am

    Does anyone have experience using scprimer. It is supposed to automate the choice of the forward and reverse primer… I tried it once, but is was too confusing for me to comprehend

    Help anyone.
    Thanks and regards.

  8. Dan Rhoads on July 22, 2010 at 7:13 am

    Great post. Do you also have some tips for qPCR, especially for probe design for both multiplex and melting curve analysis?

  9. Suzanne Kennedy on July 22, 2010 at 2:25 am

    Hi Mike,
    I love OligoCalc. I used it a lot when I worked at Qiagen to help customers with PCR and siRNA.
    Suzanne

  10. Yakko yak on July 21, 2010 at 11:27 pm

    I was also expecting a link to the NCBI Primer-BLAST website.
    http://www.ncbi.nlm.nih.gov/tools/primer-blast/

    “Primer-BLAST was developed at NCBI to help users make primers that are specific to the input PCR template. It uses Primer3 to design PCR primers and then submits them to BLAST search against user-selected database. The blast results are then automatically analyzed to avoid primer pairs (all combinations including forward-reverse primer pair, forward-forward as well as reverse-reverse pairs) that can cause amplification of targets other than the input template. “

  11. Mike Morgan on July 21, 2010 at 7:23 pm

    Not a primer design program per se, rather one for checking primer parameters: Oligocalc – http://www.basic.northwestern.edu/biotools/oligocalc.html

  12. Jan Terhag on July 21, 2010 at 1:01 pm

    Another great online tool is WatCut (http://watcut.uwaterloo.ca/watcut/watcut/template.php)
    There you can check your oligo sequence for possible silent mutations introducing restriction sites (assuming you haven’t ordered your primer yet), which is absolutely great when it comes to point mutations.

  13. Jola Kopec on July 21, 2010 at 12:18 pm

    When designing primers for different constructs of a protein,
    ccd is very handy. http://xtal.nki.nl/ccd/Welcome.html

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