The team here at Modern Vector have decades of experience with molecular biology.  Over all of that time we have picked up a bunch of rules of thumb and protocols to make doing the science easier, faster and cheaper.  We welcome the opportunity to share these with you in a new series, Tips and Tricks Tuesday!

The polymerase chain reaction (PCR) has been around for decades and is easily one of the most widely adopted technologies in biotech.  The power of PCR makes it extremely versatile and quite helpful.  However there are a bunch of things that you can do with PCR that may not be obvious.  Time for a bit of “insider info”.  Enjoy!

Own your next PCR

Tm is the temperature where 50% of the oligo is annealed to the template assuming complimentary sequence.  There are calculators to determine Tm of any sequence, but the rough approximation of 2 degrees for each A or T base and 3 degrees for each C or G base works well enough.  This only breaks down when you have mismatches and the “bubble” (or non-annealed double stranded sequence) affects the affinity of surrounding bases.  A few mismatches clustered in the 5’ half of the oligo have less impact on Tm than mismatches in the 3’ half of the oligo.  Design carefully.

Hot start polymerases are popular since they theoretically do not have any activity until exposure to high temperature removes the inhibitor (commonly an antibody or aptamer).  Without activity at lower temperatures, the enzyme is less likely to produce off target effects, but they tend to be more expensive than non-hot start enzymes. 

You can replicate this by adding a hold step at 95 - 98C (whatever the recommended denaturing temp for your enzyme) to your cycling parameters so the block is at max denaturing temperature.  Then keep your reaction very cold while setting it up (an aluminum block stored in the fridge works really well – also acts as a rack - don't put the block in the freezer, it will be too cold and could freeze your reaction).  By transferring the cold reaction to the preheated block, the temperature of the reaction will rise so quickly that the polymerase will not have a chance to do anything before the temp rises above the annealing temp of any off-target products. (And you will save some budget too.)

I know there is a great deal of opinion about there about primer design, optimal primer length, GC content, etc.  But, long primers can make PCR runs really fast.  Most companies have a set price for oligos up to certain lengths.  By maximizing the length (or getting close to it) you raise the Tm of that oligo.  If you get the Tm close to the extension temperature of the polymerase you are using (typically between 65 and 72 C – depending on the polymerase – or blend of polymerases – you are using) you can run the PCR as a 2 step instead of a 3 step (since you no longer need an “annealing step”). 

Cutting that extra step out of your cycling greatly reduces the time you need for the run.  2 step PCRs can also have higher specificity by reducing the chance that non-optimal interactions between oligos or oligos and template have a chance to form. (Unexpected amplifications are typically the result of weak polymerase activity at lower temperatures.  2 step PCR eliminates those lower temps.)

Unless you have no other choice, do not end your primers (the 3’ end of the oligo) with 3 Guanine and/or Cytosine in a row.  As the polymerase binds to the oligo/template hybrid, the 3’ end is critical for extension.  A mismatch in the last 3 bases will greatly reduce the chance of extension (you can use this to create SNP detection oligos where the 3’ most base will only match one variant).  But conversely, having 3 (or more) Gs and Cs at the 3’ end (due to their higher affinity) can stabilize the annealing of an oligo to an off-target template allowing for polymerase extension even if the anneal isn’t perfect.  It is best to generate the oligo with the 3’ end having 1 or 2 Gs and Cs in the last three to minimize the GC-clamp effect.

Everyone knows that PCR is a great way to amplify DNA, but with careful design of the oligos you can add mutations of all kinds.  For example, point mutations are best added in the middle of the oligo (if possible), but larger mutations, such as sequence to add an expression tag (like the 6x His tag) should be part of the 5’ end of the oligo.  In fact the 5’ end of the oligo can have a great deal of flexibility in its sequence, as long as the 3’ end has perfect matching sequence.

Remember though, when introducing mutations, the Tm of the oligo will change. At first, the oligo will anneal only to the 3’ end with the perfect match, but, after a few rounds, there will be newly synthesized template that has incorporated the mutation oligo, so the annealing will now be the entire sequence of the oligo, not just the 3’ end. 

You can optimize your PCR conditions by running a 2 phase run.  For a few rounds (5-10) assume that only the matching 3’ end of the oligo will anneal and calculate the Tm accordingly.  After that, switch the annealing temp (or switch to a 2 step PCR if the oligos allow it) to take into account the higher Tm of the entire oligo annealing.