When it comes to PCR, the thing I typically care most about is specificity. I want my sequence of interest to be copied (amplified) and NOT anything else. So when I go to the imager and see multiple bands in my gel lane, indicative of the presence of other, “non-specific” products I know there’s optimization ahead! Here are some things I turn to…
blog: bit.ly/pcrspecificity
There are some things you can test out that include changes to the primer, but first I want to talk about what I usually try first - just changing the PCR reaction conditions without having to design and buy new primers…
First and foremost, try raising the anneal temperature. This is the temperature during the step when the primers bind to the template after each cycle (after the melt step that unzips the double strands of the previous cycle’s products (amplicons)). The higher the temperature, the more energy molecules have to squirm. And the more they squirm, the harder it is for them to stay stuck to anything. So it’s harder for the primers to stick to the template at higher temperatures. The more attracted a molecule is to something, the more energy (heat) is required to unstick. So, by using an anneal temperature that is too high to stay stick to those nonspecific sites, but low enough that the molecules will stay stuck to the target site, you can increase your specificity.
*key point: the higher the anneal temperature the greater the specificity (assuming your target sequence is “the best site” on the template which we will come back to when discussing primer design).
*increase the anneal temperature to decrease production of specific bands
How do you know how high to set the anneal temperature? You might need to test out a range (temperature gradient settings on the PCR machine are great for this) but a good place to start is the Tm. This is the “melting temperature”, the temperature at which, on average, half of the primer copies are predicted to be bound. Typically you set the anneal temp a couple degrees below this as a starting point (but try using out Tm prediction tools that take into account the polymerase type and reaction conditions to get a more realistic Tm).
*The higher the Tm of the primers, the higher you can take your anneal temp.
And note that I said primerS. You have two of them. And you will be limited by the lower one. So it’s best to design your primers to be close to one another in Tm (within a couple degrees) so you can optimize them together and not risk going too high for one and too low for another.
If your Tms are really high, you can skip the anneal step altogether and go straight from melt to extend (this is referred to as 2-step PCR).
What makes a Tm high? Things that make it stickier and/or increase the number of binding interactions between the primer and the template. So,
*the longer the primer (assuming it binds the whole length) - if you need to raise the Tm of one primer to match the other, simply lengthening it a few bases typically works
*the higher the Tm & the higher the GC content, the higher the Tm
So why not just chock your primer full of Gs & Cs? This runs the risk of causing weird secondary structures (folded shapes), primers binding to themselves or the other primer (primer diners), etc. And those things will take primer out of the search for template (reduce your effective concentration) and can even cause short, primer-amplified-primer products to form.
I recommend using a primer checking program (I use AmplifiX) to check your primers for predicted problems like that when you design them. If you can’t avoid them by simply shortening or lengthening your primer, try moving your primer binding site if possible.
While we’re on the topic of primer design… be sure to check that your primer isn’t predicted to have strong partial-complementarity (part of it matches) to other potential sites. The larger the template is, the more other sites there are which you need to worry about. So start with as pure a sample as possible (eg make sure your plasmid isn’t contaminated with E. coli genomic DNA from the cells you grew it in).
If you’re doing something where you want to amplify from that big thing, the chances of those off-target “targets” occurring by chance are higher. But thankfully you can make lower the chance by increasing the primer length (similarly to why you increase your password length to make it harder to guess).
Now let’s get back to the anneal temperature…
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