Running a PCR experiment and need to design primers? We all have been there!
Primer design is essential for a successful PCR, but where do you start?
To recap, PCR is an in vitro process that uses thermal cycling to amplify specific regions of DNA.
For more information on PCR, you can find one of our earlier videos in the description below!
In this video, we will go through important
considerations and tips for good primer design. Let's go!
Primer Length
the optimal primer length is between 18 and 30 nucleotides.
Longer primers will promote highly specific annealing but
the downside is that these long oligos will have a higher melting temperature
and tendency to form secondary annealing. Shorter primers on the other hand,
bind more easily to the template but at the sacrifice of lower specificity.
Considerations for melting temperature and annealing temperature
The Primer's Melting Temperature and Annealing Temperature go hand-in-hand. By definition, the melting temperature (Tm)
is the temperature at which 50 percent of the DNA duplex separates and becomes single stranded.
This is highly dependent on the length of the oligo and the composition of the DNA molecule.
The annealing temperature (Ta) is the optimal temperature for your PCR reaction that allows primers to successfully
bind to the DNA. When designing primers, most software calculate the melting temperature for you.
The optimal melting temperature of primers is between 50 to 65 degrees Celsius
It is important to have the forward and reverse primer pairs' melting temperature be within
5 degrees of each other. So both primers can bind simultaneously and efficiently to the target DNA.
Once you have the melting temperature for the primers, you can calculate
the annealing temperature to use in PCR using the formula on the screen.
Generally speaking, the annealing temperature should be set to no more than 5 degrees lower than
the melting temperature of the primers. Annealing temperature that is too low will tolerate partial
annealing with mismatched bases, and give you non-specific amplifications. Conversely, if the
annealing temperature is too high, it will reduce template primer hybridization and PCR yield.
GC Content
Another design parameter that is related to the melting and annealing temperature is the percentage of cytosine
and guanine in the primer, or GC content.
Higher GC content indicates a higher melting temperature. 40 to 60% GC is recommended for primer designs.
Ending the 3' terminal with Gs or 's can also promote specific binding.
This is referred to as the GC clamp. But you should have no more than 3 GCs in the last 5 bases!
Avoid Repeats in Your Primer Sequence
Another consideration is to avoid runs of 4 or more single bases or 4 or more di-nucleotide repeats in the primer design.
Both situations can lead to mispriming resulting in undesired products.
Avoid Complementary Sequences Within the Primer
Any complementary sequences within the primer or between a forward and reverse primers
can lead to poor or no yield of the product.
This is because the intermolecular or intramolecular interactions lead to self-dimers or primer dimers
Both situations will reduce the availability of primers to bind to its intended target sequence.
Avoid Secondary Structures in Target Sequences
Lastly, if your target template contains secondary structures, avoid designing your primers to bind
within those sequences. Your primers may not be able to bind to the template if the
secondary structures are stable during the PCR reaction. This will result in poor yield.
Let's recap what we learned today! Here are some parameters to keep in mind when designing primers:
the primers should be 18 to 30 nucleotides long. The annealing temperature should be
set to no more than 5 degrees lower than the melting temperature of the primers.
The optimal melting temperature for primer should be between 50 and 65 degrees Celsius
The forward and reverse primer should have melting temperature that are within 5 degrees
of each other. The GC content of the primer should be between 40 to 60%
And here is a list of what you should avoid. Make sure there are no more than three GCs in the last
5 bases of the primer. Avoid having more than 4 nucleotide runs or di-nucleotide repeats
Ensure the design primers do not form self-dimers or primer dimers
And lastly, avoid designing primers targeting the secondary structures in the template.
There are some powerful primer design tools available online which can be used
to simplify the primer design process. But if you're in a pinch, check out our
Primer Design and Synthesis Services if you need some expert help with your designs.
For more resources, visit us at abmgood.com or follow us on our social media.
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