Spin column nucleic acid purification - how it works

If you’re looking for a spin class, have you tried out spin column DNA/RNA 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 (or RNA) - just make sure you get the right kit! These columns are a form of “solid-phase extraction” where you bind the nucleic acid to a silica gel membrane, wash off the other stuff, then “un-bind” pure DNA or RNA. 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

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. 

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. 

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.

finished in comments

[thebumblingbiochemist]

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 (below 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. 


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!

 
                 

[doviende]

thanks! as someone new to genetics, i really appreciate the detail you've put into this. a lot of intro resources just say sort of "here are some steps", but i really like that you've included a bunch of the background reasoning.

[novoscientist]

Hello, my name is Benjamin, I am a geneticist and biotechnologist. I love your videos, you inspire me to continue researching. Many blessings!

[thmhlckdio.]

Thanks to your video, it's a good source for me to do my presentation!!!

[sekayitigere]

Thank you for this. This is really the best explanation I’ve had on this 🙌🏾🙌🏾🙌🏾

[mn-mwpc]

What the purpose of adding proteinase enzyme together with lysis buffer in DNA extraction?
Thanks.

[vibriofischeri]

So do you use guanidinium thiocyanate or guanidinum chloride? I didn`t get it because on picture it`s guanidinium thiocyanate, but later you talk about guanidinum chloride. And thank you for a great video! You explain biochemistry really cool!

[basilbaby]

Could you explain the purpose of using preheated water (70°C) for nucleic acid extraction ?

[teguhsantoso]

Thank you. Its complete my understading of purification DNA methode.

[chuguavuitinhchannel]

guys this is the org chem tutor in practice