Gyms might be closed, but if you’re looking for a spin class, have you tried out PCR purification? I hope you don’t mind if my PCR products bind? Biochemists win when we take them for a spin! “Spin Columns” are a quick & easy way to purify pieces of DNA! These columns are a form of “solid-phase extraction” where you bind DNA to silica gel membrane, wash off the other stuff, then “un-bind” pure DNA. There are lots of different versions & kits & they work really similar so, although I’m going to be talking about PCR PURIFICATION (aka “clean-up”) the basic principles apply to other situations
If you want to study DNA from cells, things are more complicated, because you have to break open (lyse) those cells, separate the DNA from all the other stuff (cell membrane, proteins, etc.) & make sure to get it really pure. More on how we use alkaline lysis (minipreps) to isolate whole plasmids in yesterday’s post http://bit.ly/minipreps
For PCR PURIFICATION, you’re starting with a much purer sample, but you still have stuff you need to remove. What kind of stuff?
PCR is a way to amplify (make lots of copies of) specific parts of DNA from larger parts of DNA. You specify the region you want copied using PRIMERS which are short fragments of DNA that bind to where you want the copier (DNA Polymerase) to start & stop.
It’s kinda like the original, bigger, DNA template is like a transcontinental railroad & you only want to copy the stretch from Utah to Colorado – the primers act as “train stations” that the DNA Pol “train” travels between, laying down “tracks” (DNA nucleotides) ahead of it as it goes based on the sequence of the other strand. Lots more about it here: http://bit.ly/pcrtrain
For now, let’s just think about what that train needs – it needs the primers (stations), nucleotides (train tracks), & it also needs salts to keep it happy, & it especially needs magnesium (Mg²⁺), which helps it hold & coordinate the addition of more nucleotides
So you do the reaction & it makes lots & lots of copies, but now you need to separate those copies from that other stuff. We commonly do this using spin columns which have a silica-based membrane. The premise behind these is -> mix reaction mix with stuff that will denature (unfold) the DNA Pol & make big pieces of DNA bind the column, but allow everything else to flow through. Wash it to make sure you really do get everything else to flow through. Then change DNA’s environment so that it unbinds & goes through
The key is to find conditions in which the DNA would rather bind the solid membrane than the liquid & conditions in which the situation’s flipped – the DNA would rather bind the liquid than the solid.
Let’s look at the players.
The membrane in silica-based. silica (amorphous silicon dioxide, SiO₂) can have a lot of modifications & the composition of these membranes are proprietary, so I don’t know exactly what their surfaces actually look like at the molecular level, but silica has hydroxyl (-OH) groups that at low pH (acidic, where there are lots of free protons (H⁺) floating around) will be protonated (-OH) but at higher pHs (where there are less free protons) will be deprotonated & thus negative (-O⁻). In addition to this group, silica also offers up some additional opportunities for hydrogen bonding & even hydrophobic interactions.
& the DNA? DNA’s a polymer (chain of similar repeating units) of nucleotides (DNA letters). These have a generic sugar-phosphate backbone they link through (same-strand bonds) & unique nitrogenous bases (A, T, G, or C) that stick out & form the basis of between-strand base pairing.
The phosphate in that sugar phosphate backbone is negatively charged, & it’s really happy being negatively charged, so even at pretty low pH it’s gonna stay that way. “Normally” the nitrogenous bases are neutral. BUT, as the name suggests, they are (weak) bases, meaning that if the pH is low enough (meaning there are lots of H+ around) they will pick one up & this makes them + charged. So, at lower pH, the overall charge of DNA decreases, so it becomes less water-soluble.
This helps us separate DNA from RNA in liquid-phase RNA extraction. At a low pH, DNA prefers the organic phenol-chloroform phase, whereas RNA’s extra -OH keeps it in the aqueous phase. http://bit.ly/rnaextraction
Here, at a low pH, DNA doesn’t have an “organic phase” – instead, it binds (adsorbs to) the silica membrane. Note: I’m not sure if the pH is low enough here to change those bases, but there’s another important function of the pH.
At a low pH, the sometimes -OH, sometimes O- groups of the silica are more likely to be in the -OH form, so there’s less negative charge that could repel the negatively charged DNA backbone.
It’s not just the low pH that’s important. You also need salts. CHAOTROPIC SALTS like guanidinium hydrochloride bring “chaos” to water -> they disrupt water’s bonding networks, “loosening up” the water coat surrounding the DNA so the DNA can seek out & bind to the silica instead. When it does so, you get an entropic benefit because it frees up water molecules that were stuck so they can move around more, thus increasing entropy (disorder)
There are different ways it can bind, & the actual situations probably a combination of lots of them. The exposed bases could form direct H-bonds or hydrophobic interactions with the silica; Cations from the salts could form “bridges” between the backbone & the silica; etc.
The more interactions, the stronger the binding. The smaller DNA pieces (primers) don’t offer enough binding opportunities to stick well, so they flow right on through with the other stuff
So we start by mixing our (completed) PCR reaction with a solution containing guanidinium hydrochloride (the chaotropic salt to loosen the water shells) &, to help get the DNA to precipitate (come out of solution), ISOPROPANOL (aka isopropyl alcohol, aka propan-2-ol, etc.). This lowers the dielectric constant -> reduces electrostatic shielding so that the salts, DNA, & silica can find each other. & it “dilutes” the water, so there’s less water available to bind the DNA, so the DNA “dehydrates” & really sticks on tight to that silica so it doesn’t come loose when you do the washes.
If you’re using a QIAquick PCR purification kit, this is buffer PB. (This isn’t a paid ad for Qiagen or anything, it’s just what our (and many) labs use).
The kit also includes a pH indicator you can (& should) put in! (unless you’re gonna be using the DNA for a super-sensitive microarray) – you want to make sure that the pH is low enough (<- 7.5). If the solution is orangey-purpley, it’s too high & the DNA won’t bind well. Don’t worry, just add a little acid (sodium acetate, pH 5 does the trick) -> should turn yellow
Now you need to do the actual binding part.
The silica gel membrane is held in a microspin column you can transfer between tubes to collect the flow-through & you pipet your sample into the “cuppy” part above the membrane. Then you centrifuge it (spin it really fast) to pull the liquid through. (alternatively, if you have a lot of samples to do at once, it can be quicker to use a vacuum manifold which sucks it through (though you’ll still have to switch to spin for the elution because it sucks it through into the waste…)).
The bigger pieces of DNA (>100bp) will (hopefully) bind the membrane, but all the other stuff (primers, denatured DNA Pol, salts, etc.) will flow on through & you can toss it (the liquid, not the column!)
Now comes the wash. Now that you’ve gotten DNA stuck on there tight, you want to wash off any lingering extra salts. You do this using a buffer containing ethanol (buffer PE in Qiagen’s kit). just add the liquid the same way you added your sample, spin it through & toss it out.
DNA’s not soluble in ethanol, but salts are, so the extra salts are removed. It’s really important that you remove that guanidinium chloride because, while you wanted to denature the DNA Pol, you don’t want it to stick around & interfere with any reactions you do with the DNA later on.
After the wash & toss, spin it again to give any residual ethanol another chance to get pulled through. It’s really important to remove the ethanol or the DNA will have problems dissolving &/or when you go to load samples into an agarose gel (more here:http://bit.ly/2Z6vmeR) they’ll float up out of the well cuz ethanol’s less dense than the buffer.
Now that you’ve gotten the DNA clean, you need to get it to unstick from the silica. To do this, you need to reverse the conditions that got it to stick. We get it to stick with high salt, low pH -> we get it to unstick with low salt, high pH.
The Qiagen kit comes with an elution buffer (buffer EB – which is 10 mM Tris·Cl, pH 8.5) (Tris is a buffering agent) you can use or you can use plain old (but really pure, nuclease-free) water. If you use water, give it a minute to fully dissolve the DNA before you spin it through.
Speaking of which -> once you redissolve it, you spin it through into a NEW TUBE – this one you want to keep!
Then, take your spun-through, pure DNA over to the spectrometer like a NanoDrop & check out its absorbance spectrum (it should have a 260/280 ratio of ~1.8). More on that here: http://bit.ly/whatabsorbswhere