Hot Start PCR, heated lids, polymerase options, etc. - basically random PCR stuff...

Hot Start PCR, heated lids, polymerase options, etc. - basically some random PCR stuff to get the best chance at a successful PCR experiment! for more, see blog post: https://bit.ly/hotstartPCR

Topic #1: Hot Start PCR…

PCR is a fundamental technique in biochemistry and molecular biology, and, like most experiments, there are lots of places where things can go wrong… One “bad outcome” is formation of NONSPECIFIC PRODUCTS - if primers bind multiple locations they’ll amplify multiple stretches of DNA, giving you multiple products of different lengths.

The most obvious way to avoid this is to make sure primers have HIGH SPECIFICITY for sequence you want them to bind. You can use free software programs like NCBI BLAST or Primer3 to help you check for specificity & design good primers. More on this in yesterday’s post.

But even if your sequence is super specific to a single region of the template, you can still get problems from binding of primer to primer! You can get “wrong pairing” within primers themselves (e.g. hairpins —u—) or between primers (primer dimers). This leads to less primer available to bind template and lower yield (less copies made). Artifact-wise, when you get primer dimers, DNA Pol can end up using primers as a (really short) template, amplifying primer “artifacts” instead of desired amplicon. And the high primer concentrations needed to prevent template-template zipping make such primer pairing more likely because there are more primer fish & fewer template fish in the sea. 

When you set up a PCR reaction, you have to mix together ingredients - template, primers, nucleotides, buffer (solution of salts & pH-stabilizers), Mg²⁺, & DNA Pol… We buy a pre-mixed “master mix” of Pol, buffer, nucleotides, & Mg² that makes this a lot simpler, but we still have to add template & primers, set up machine, etc. Because Pol’s part of the mix, it can get a “head start” before you start the cycling. Even if you add the DNA Pol last, right before you stick your (tiny) tubes in thermal cycler, there’s a lag time where, if you’re unlucky, Pol can start working & temp’s low, so you’re at risk of primers settling & giving you nonspecific products.

In HOT START PCR, you “hide” Pol until you’re ready to go. The “hider” is often an ANTIBODY that binds to Pol & blocks its active site. Antibodies are like primers in the sense that they recognize & bind to specific parts of specific molecules. BUT antibodies are PROTEINS whereas the primers are DNA. And antibodies can recognize & bind to parts of different things (proteins, small molecules etc.) by “mimicking” their surfaces with a combination of well-placed amino acid building blocks (kinda like making a mold of a keyhole) 🗝 Different antibodies recognize specific parts of different molecules, kinda like having a specific key for a specific molecular lock.

The first melt step you separate the template & also separate the antibody, freeing Pol to go to work & since you’re at higher temps, primers have enough energy to seek out their soulmates, so fewer non-specific matches. So you get less nonspecific priming, less nonspecific products you don’t want, & more of the specific product you do want.

In the really olden days (no offense meant to anyone) there weren’t thermal cyclers so ppl had to manually transfer tubes back & forth between heated water baths. Then thermal cyclers came along, freeing arms & minds of grad students in labs around the world! But you still had the nonspecificness problem. The best way to prevent it was to add Pol at the very last minute, but this could be hard when you had a lot of tubes to add to. Then there were some methods introduced using wax to physically separate components in the tube in “layers” until heated. And now there are antibody-based hot start polymerases which are even easier to use.

Other methods hide DNA Pol with aptamers (short oligonucleotides that can kinda fold up & bind things specifically (but not through base-pairing because we’re talking protein-DNA here) that fall off when heated. Or they sequester the DNA Pol in wax beads that melt with heat.

Or you can hide other critical components - some hide the dNTPs or primers with thermolabile (heat-sensitive) chemical groups on the 3’ OH that prevent them from being used until heated. Others hide magnesium in phosphate-magnesium precipitates that won’t dissolve until heated, etc. etc. etc.

see blog post for more...

Topic #2: Heated Lids
Topic #3: DNA Pol choices