Wanna play with fruit DNA? Me too! I really miss the lab – and while I can’t do the experiments I really want/need to do (can’t really work with radioactively-labeled stuff at home…) there *is* an experiment I can (try to) do here – and you can do it in your home too! All you need is some fruit, water, salt, and rubbing alcohol (isopropanol) or ethanol.
EXTRACTION works by separating molecules based on their SOLUBILITY (and insolubility) in different liquids. It’s kinda like having a mixture get a “divorce” and letting the “kids” decide which parent’s house they want to live in after the split – in our case they can choose between an aqueous (water-based) “phase” or an organic, alcohol “phase” which don’t mix with each other, so will separate into layers.
Being soluble means that each copy of a molecule has its own full coat of solvent. Most of the time in biochemistry, the solvent we’re talking about is WATER. Our bodies are mostly water, so if we want to study what goes on in our bodies, we use water-based solutions, and we call such water-based solutions AQUEOUS
It’s not just for the sake of “authenticity” that we study compounds in water. Because most molecules in our body have to work in a watery environment, they’ve evolved to function optimally in that environment, which involves being maximally soluble. If something’s NOT soluble, it means that the solute molecules (things you’re dissolving) would rather bind to each other than to the solvent molecules (thing in which you’re dissolving) &/or the solvent molecules would rather bond to each other than to the solute.
If molecules bind to each other instead of the solute, they can clump up – aggregate – as precipitate. This maximizes their contact w/each other & minimizes their contact w/the solvent, but it also makes them non-functional. You definitely don’t want this occurring in your cells, so you need the molecules to be soluble. But there are solvents other than water, and some molecules like some solvents better than others, largely due to their charge distributions & if you give them options they can choose a different solvent instead of just panicking and clumping up.
There are parts of your body, like your cell membrane that are “fatty,” like the lipid membranes surrounding your cells, so the molecules that work there (at least the membrane-embedded parts) have to be fat-soluble, so they go through special processing steps to ensure they don’t clump.
So, in our bodies, precipitation is a bad thing, but in the lab we can exploit different molecules’ different abilities to clump under different conditions to remove things from solution that we don’t want in there. Or to remove things from solution that we want so we can then remove all the stuff we don’t want in the liquid. Some types of precipitation are “reversible” – remember, you’re not breaking any strong covalent bonds, not “cutting any chains” just unspooling the yarn balls. So if you take something that’s precipitated and put it back in the solvent it likes, it might redissolve and refold.
Problem is, folding complex things like proteins is a complex process that often requires the help of other proteins called chaperones (which is why we still can’t predict exactly how a protein will fold based on its sequence alone). So they often can’t re-fold properly. BUT DNA & RNA have more “simple” structures that can re-fold. So we can take precipitate the nucleic acids, remove the other stuff, and then redissolve the nucleic acids, heat up a bit, cool them off, & they’ll “anneal” back to their original shapes. Or, we can just take them out in their clumpy form and admire them 🙂
Today I tried to admire some cantaloupe DNA. You can theoretically do this with any sort of fruit – strawberry is great cuz it has a ton of DNA due to some chromosome duplication. wheat germ also works really well. Cantaloupe apparently not… but it’s all I had 😩 To get to the fruit’s DNA we have to break down the fruit cells’ starchy walls and the lipid (fatty) cellular membranes underneath those. That will get us into the general cellular interior (the cytoplasm). Then there’s just one barrier between us and the DNA – the nuclear envelope, which partitions of a little spherical “room” inside the cell called the nucleus where the DNA is stored. Thankfully, this nuclear envelope is just another one of those lipid membranes and both this and the outer membrane (the plasma membrane that surrounds the cytoplasm) can be broken down by soap. But we need to make the membranes soap-accessible, which means breaking down the cell walls and with some good ‘ole smooshing (it works well to stick the fruit in a ziploc back and smoosh away).
Once sufficient smooshing is achieved, it’s time to lyse (break open) the cells by adding soap or detergent (artificial soap). Soap is amphiphilic – it has a part that likes water (a hydrophilic polar head) and a long tail part that doesn’t like water (it’s hydrophobic). The hydrophobic tails look a lot like the lipids making up the cells’ membranes, so they sneak in there, disrupting the membrane’s structure and causing the cells’ contents to spill out – including the DNA!
Now we just need to isolate the DNA from all the other stuff. Which is where the extraction part comes in. When you add the soap, instead of just pouring on some Dawn, you mix the soap with water and add salt. If you’re using table salt, this means adding sodium chloride (NaCl). When this dissolves it “dissociates” into a positively-charged Na+ cation and a negatively-charged Cl- anion.
The salt is really important for a couple of reasons. One is that it helps destick any stuck on proteins – basically the salt gives the proteins more options – instead of just having to choose between the solvent (e.g. water molecules) and the protein, there are now a bunch of ions hanging around they can hang out with.
Another is that it helps keeps the DNA strands stuck to each other. DNA has a negatively-charged backbone, and like charges repel each other, so if you tried to get DNA strands to hang out together without salt, they’d say “no way” – but when you add salt, the positively-charged component of the salt (the cation) can coat the negatively-charged backbone, neutralizing it so that the strands can stay together – and so that the DNA is less soluble, causing it to precipitate.
BUT this precipitation doesn’t happen until you add alcohol. Without the alcohol, the salt ions can’t find the DNA because the DNA’s coated in a layer of tightly-bound water molecules. And the salt ions are too. This is because water’s really polar – it has partly-negative and partly positive parts because of unequal electron sharing between the oxygen & the hydrogens. Opposite charges attract, so water is attracted to the charged parts of the DNA & the ions – and to other water molecules, thereby forming “solvation shells” – tight networks of water molecules around the DNA & ions that hide their charges.
But, the alcohol helps break down this shell by lowering the “dielectric constant” of the solvent – it’s less polar so it forms “looser” networks than water, therbey reducing shielding around charges so the + salts can see the negative phosphates and bind to them -> neutralize charge -> nucleic acids become less soluble & they don’t have a better solvent alternative to flee to, so they cling to each other -> precipitates
This precipitate shows itself as some white stringy/gloopy stuff at the boundary between the water & alcohol layers, and you can pull it out using a q-tip or a paper clip, or toothpick or something.
So, the basic overview is
- add soap/water/salt mixture (for this mix you can do ~1/2 cup water + 2 tsp dish soap + 1/2 tsp salt) & wait 10-20 min
- filter it to remove the unbroken-down plant parts – any DNA we have a chance at recovering is now in the liquid part – most protocols recommend using a coffee filter but I didn’t have one of those… but I did have an old sock…
- add alcohol (some sort of alcohol that’s at least 70% alcohol – can be rubbing alcohol (isopropanol) or ethanol) – when you add it you just want to carefully pour it down the side of the glass to make a layer of alcohol on top of the water – you don’t want them to actually mix (most protocols call for the alcohol to be cold either because it helps lower solubility even further and/or it inhibits any potential DNA chewers (DNAses) from eating up your bounty, but the pre-chilling isn’t required
- wait & watch
- pull out the precipitated DNA
I first did this experiment for an elementary-school science fair demo when I started a science club in undergrad. And then I taught a version of this at CSHL’s Dolan DNA Learning Center (DNALC). So it has a fond spot in my heart.
more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0