I can’t stand that extra PCR band! Perhaps my PRIMER found too many places to land! Maybe next Tm it’ll work better… Polymerase Chain Reaction (PCR) is a molecular biology technique used to amplify (make lots of copies of) specific regions of DNA from bigger pieces of DNA. But, sometimes instead of producing perfect DNA products, PCR produces problems!

More on how PCR *should *work here 👉 http://bit.ly/31KkGAt But today (after a quick review) I want to talk about when PCR *doesn’t* work and what to do so it doesn’t happen to you too!

in PCR you take some double-stranded DNA containing a stretch you want to amplify, separate the strands by heating it up (melting) ♨️ & use a molecule called DNA Polymerase (DNA Pol) to copy it by traveling along the strands between “train stations” 🚉 using the strand it’s on as a template to lay down track ahead of it as it goes (extension) so it has double-stranded track to travel on 👍 It knows what rack it needs to add because it has to complement the 1/2 track on the other side (e.g. see an A, lay down a T; see a C, gotta put in a G). Like the tracks it’s laying down are puzzle pieces with 1 unique side sticking out. As long as there are track pieces (nucleotides) available, it can build the track and keep moving.

But it can only move in a single direction (from its perspective (like how your left is on the right in the you in the mirror) – Instead of “left” and “right” DNA has “5’” & “3’” ends, which is like whether the end you’re looking at has a puzzle piece with a bulge or a cut-out. Biochemically, this corresponds to whether the nucleotide has a free phosphate group (5’ phosphate) or a free 3’ hydroxyl (-OH) – you need 1 of each to make a bond and the energetic cost of linking them is paid by the one with the 5’ phosphates, so this is the form that gets added – and it needs an OH to add to. More on this here: http://bit.ly/2TFdQN9 so track gets laid from 5’ to 3’ (From the layer’s perspective)

The DNA template like a cross-continental railroad but you only want to amplify a shorter stretch of it (e.g. Kansas City, Kansas to New York, New York ) 👉 You specify the “start” 🏁 & “stop” 🛑 points of this region by designing short pieces of DNA called PRIMERS that bind to those locations (anneal), creating double-stranded platform “stations” 🚉 for DNA Pol train to start from on each strand (just like the 🚂 can only travel on double-stranded track, it can only start on double-stranded track)

The “start” and “stop” stations are really 2 start stations, because instead of stopping the polymerase actually just falls off the end of the tracks (or runs out of track or energy). So the “stop” station really just defines where the tracks end for the other train. 

PCR’s performed in multiple rounds 🔁 so that the track laid down in 1 round becomes the “template” track for the next round ⏩ the start station for 1 round becomes stop station for next round ⏩ so you end up w/lots of copies of the defined region (see pics)

🔑 so: MELT (heat to separate strands (train tracks)) ⏩ ANNEAL (cool slightly to allow primers to bind (create train stations)) ❄️ ⏩EXTEND ⏩ give DNA Pol (train) time to travel between stations, laying down track (nucleotides) ahead of it as it goes ⏩ REPEAT cycle lots of times 👉 each time you have more & more tracks to act as templates so you can make more & more new tracks to act as templates to make more & more new tracks to act as templates to…

It’s really important ⚠️ to design the stations (primers) carefully. Like many things in biochemistry, it’s largely a matter of AFFINITY & SPECIFICITY👇

AFFINITY 👉 We want the primers to have high affinity (attractiveness & stickiness 😍) for the site we want them to bind so that they’ll bind there stably & not fall off randomly during the annealing or extension steps ❄️ 😬 BUT we don’t want the affinity to be too high or it won’t come off during the melt steps ♨️ 😬

Affinity is largely dependent on the primer length (longer primers have more interstrand bonds working together to keep the strands glued shut) & “base composition” 👉 DNA is the biochemical language genetic info is written in ✍️ & its alphabet only has 4 letters (A, T, C, & G) 👉 They have a generic backbone, so they can link in any order WITHIN a strand, BUT bonding ACROSS strands uses each letter’s unique part (the nitrogenous BASE) so a letter can only bind its “soulmate” (A to T and C to G) 😍

When G’s & C’s are across from each other they can form 3 H-bonds 💪🏻 but when A’s & T’s are across from each other they can only form 2 H-bonds, so G-C pairs are stronger than A-T pairs 👉 So a higher “G-C” content (typically given as a % of bases) means stronger binding. Ideal is usually ~40-60% 👍

💡👆is why origins of replication (ORIs) (where DNA strands come apart to be copied before cells divide) tend to be “A-T rich” because it makes it easier to melt them apart 👍

Just like it’s easier to pull off a piece of tape from the end than the middle, it’s easier to pull of primers from the end, so you might want to put a“G-C” clamp at the 3’ end (1 C or G) to help latch it on tight 🗜

A common measure used to calculate/report 👆 affinity is the Tm (melting temperature). It’s the temperature at which 1/2 the primer is bound 👉 the higher the temp, the more energy the molecules have & the harder it is to get the DNA to “stay still” & bind 👉 a high Tm means the affinity’s high enough to hold down the wriggling DNA 🗜 👉 higher Tm, higher affinity 💪🏻 typically want ~60°C & you want the Tm of the 2 primers to be similar to one another (within ~5°C)

If you use too low of a temperature, you can get nonspecific products. You can think of the primersusing some of 👆 energy to seek out ideal binding partners 👉 At ⬇️ temps, primers are more likely to “settle” for non-ideal matches 👉 they just don’t have the energy to seek out their perfectly complementary “soulmates” 😴These non-ideal matches can be on the template dsDNA (just in wrong spot) leading to nonspecific products 😬 And there’s a good chance of such mispairing bc you need to use a big excess of primers so when strands melt apart, the DNA doesn’t just zip back up 👉 but, from primers’ perspective 👀 there’s lots of competition for DNA so primers will settle for something suboptimal rather than fight it out to get the best spots 🥊

SPECIFICITY 👉 We want the primers to bind ONLY where we want them to bind. Say you ask some friends to buy you a train ticket 🎫for a trip from Kansas City to NYC 🚂 Is that Kansas City, Kansas? Or Kansas City, Missouri? 🤷‍♀️ Some friends might think Kansas, others might think Missouri 👉 you end up w/2 types of train tickets 😬

Similarly, if your primer can bind at multiple sites on the DNA, you end up copying different stretches of train track giving you a mix of “nonspecific products” 😬 which show up as multiple bands on an agarose gel you use to separate the DNA pieces by size & visualize them: http://bit.ly/2SDKE8I

When you design primers, you want to make sure they match your site of interest & ONLY that site. Like a computer password 🖥 the longer the sequence, the more likely it is to be “unique” 🤩 👉 if there are multiple occurrences of sequence you initially choose you might have to lengthen it to include more of the surrounding sequence (like saying “find a blue house w/a red house on the left & a green house on the right” instead of just saying “find a blue house”) You can use free software programs like NCBI BLAST or Primer3 to help you check for specificity & design good primers 👍

BUT too long a primer and you can face other problems that lead to a “PCR TLDR” (too long didn’t read)… 👇
🔹 PRIMER DIMERS 👉 this is where the primer binds to itself instead of to your template 👉 can be self-dimers (where 2 “start stations” or “stop stations” bind to themselves or cross dimers (a start & a stop)
🔹SECONDARY STRUCTURE 👉 a single primer can fold up into “hairpins” & ↩️ bind itself 

This leads to less primer available to bind template ⏩ lower yield (less copies made) ☹️ & DNA Pol can end up using primers as a (really short) template, amplifying primer “artifacts” instead of desired amplicon 😬 & high primer concentrations needed to prevent template-template zipping, make such primer pairing more likely bc there are more primer fish & fewer template fish in the sea 🐟

Secondary structure in the *template* can also be a problem 👉some regions of DNA are tightly wound up, making it hard to get to the site to bind (it’s hard to build a train station in the middle of a mountain pass) 🗻

You also want to avoid repetitive stretches (things like “AAAAAA”) because it makes it easier to “slip” & misprime

Typical primers are usually ~20 nucleotides (nt) long, but it depends on experiment type, etc. There’s free software available (like NCBI Primer-BLAST, Primer3, & AmplifiX) to help you design primers to fit your needs. 👍

Even with good design you still have a race against time! 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 ⬆️ temps, primers have enough energy to seek out their soulmates 😍 👉 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 🤗

apologies for formatting and technical-ness but the bumbling biochemist train has run out of steam for the day! Hope someone finds this helpful anyway!

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0

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