Are you looking to design a primer for your PCR reaction but you're not sure where to start?
I'm Jennifer, Science Communications and Marketing Coordinator at Addgene
and I'm here to help.
As you know PCR Reactions require two complementary oligonucleotide primers.
Primers are short, single stranded DNA fragments that anneal to the regions
upstream and downstream of the DNA segment to be amplified.
Designing a working primer can be a bit tricky, with so many ways to design them
and a number of small details that can derail your reaction.
So we're here with a few useful tips to guide you through the process.
There are several properties you'll need to consider to design succesful primers.
The length of the primer.
The anealing and melting temperature of the primers.
the GC content of the primer.
and the secondary structure of the primer.
We'll start with the primer length.
Generally you'll want your primers to be
between 18 and 24 base pairs.
This is the optimal range to get the best yields.
If your primers are too short they could
produce more non-specific DNA amplification products.
meaning that some of your DNA products aren't what you were trying to amplify.
But on the other hand if they're too long they can result in slower hybridization rates
Next let's talk about annealing and melting temperatures.
Your annealing temperature is what
allows the primers to base pair with the DNA.
The melting temperature is a temperature where half of the primers
will dissociate from the DNA.
You want the kneeling temperature to be five degrees below the melting temperature,
so that most of the primers bind to the template.
lowering the temperature any further
however could result in more non-specific binding.
The two primers should ideally have similar melting temperatures.
There are many tools that can help you calculate this online
but there's an easy way you can quickly do the rough math for yourself.
Simply add four degrees celsius for every G or C,
and two degrees celsius for every A or T in your primer sequence.
Great so we have the length of the primer and the annealing and melting temperatures set.
But what about the makeup of the
nucleotides in your primer?
The ideal makeup of a primer is between
forty and sixty percent GC content.
The GC content is the percentage of Gs and Cs in the primer.
So for example, if a primer contains twenty base pairs total,
and has six Gs and four Cs,
It would have a 50 percent GC content.
You'll also want to include a GC clamp at the three prime end of the primer.
This means that you include two to three Gs and Cs to the three
prime end of the primer if possible,
given the constraints of your DNA sequence.
GC base pairs are useful because they
because have three hydrogen bonds, one more than the AT pairs
that have two hydrogen bonds.
Therefore GC pairs help promote stronger
stronger and more specific binding to the template DNA,
helping to ensure your reactions will work properly.
Now there's a secondary structure to consider.
Depending on the sequence of the primer there's a chance it could form hairpins
or dimerize with itself or the other primer.
This means it could base pair with itself or base pair with the other primer instead of the template.
To prevent this use online tools to warn
you of potential hair pins self-dimers or cross-dimers.
Check out Addgene's blog post for some programs and tools you can use.
Also be aware of repeats in your nucleotide sequence.
Too many repeats could cause mispriming, and the primer can anneal to unintended locations.
Avoid runs of identical nucleotides in
the three prime portion of your primer too, for the same reason.
Congratulations! You just successfully designed a PCR primer!
If your primers do not give you the PCR product you're looking for
You could try changing the conditions of your PCR reaction.
like the annealing temperature.
If still not having any luck, it might be time
to go back to the drawing board and redesign your primers.
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