Primer design is like art. There is more than one design to cover the region of interest. Are you an “Artist”?
Primers are crucial to the success of target amplification and subsequent sequencing in PCR and Sanger sequencing workflows.
Let’s take a look at our lab book:
In the typical Sanger sequencing workflow from genomic DNA, one needs to first amplify the target by PCR, and then subsequently run the Sanger sequencing reaction. If you start from purified plasmid DNA, one only needs to run the Sanger sequencing reaction. PCR amplification requires 2 primers from opposite strands that determine the region of sequence amplified in the forward and reverse direction.
Sanger sequencing differs from PCR in that only a single primer is used in the reaction. Typically, for a given PCR fragment, two Sanger sequencing reactions are set up, one for sequencing the forward strand, the other one for sequencing the reverse strand. Primer design is an important aspect relating to many forms of PCR including basic PCR, fragment analysis, quantitative analysis and Sanger sequencing.
Here are a few things to keep in mind when designing your own primers.
- Primer length should be in the range of 18 to 22 bases.
- The primer should have GC content of 50% to 55%.
- Primers should have a GC-lock on the 3’ end.
- The melting temperature of any good primer should be in the range of 50OC to 55OC.
- The primer should not include poly base regions.
- Four or more bases that compliment either direction of the primer should be avoided.
In addition, there are some PCR specific guidelines to help you design good PCR primers. These guidelines can be found on our website.
Since primer design is not easy. There is risk of designing the wrong primers which could be costly in your experiments. You may be asking: Is there an easier way? The answer is Yes, and Thermo Fisher Scientific has a free tool to help you out. Primer Designer tool is a free PCR/Sanger primer online search tool that includes over 600,000 primer pairs covering the human exome and human mitochondrial genome. You can choose the range of amplicon length for your sample and your research interest to optimize it for your experiment.
If you are doing NGS confirmation with your capillary electrophoresis genetic analyzer, this tool can really simplify your workflow. You can use this online by uploading your .vcf file, or if you have an Ion Torrent sequencer, even better, as there is a seamless integration with your ion reporter software to upload your data into this Primer Designer tool. Now you can find the right primers with just a few simple clicks.
I hope this video was helpful on primer design, and I am sure you’ll have more questions.
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And remember, when in doubt, just Seq It Out