Get happy – it’s DAPI! 😃 Let me tell you the biochemist’s version of the princess and the pea. This story has a happy ending, DNA you can see! 🤩
Once upon a time there was a fluorescent dye named 4′,6-diamidino-2-phenylindole – it’s friends called it DAPI. It went looking for a home in an agarose gel someone was using to separate DNA fragments by size (more here: http://bit.ly/2SDKE8I ). And it found a nice cozy nook to settle down in in the minor groove of those DNA fragments (when double-stranded DNA forms a double helix it has a more open “major groove” and a narrower “minor groove” http://bit.ly/2CgjVW3). It didn’t find *all* of the minor groove groovy – just the AT rich regions – DNA has 4 nucleotide “letters” A, T, C, & G, and the G has an -NH2 group sticking out into the minor groove, acting as chemical “pea” making that bed uncomfortable
But DAPI found a nice AT rich nook, settled down, and traveled with the DNA on its journey through the gel. The journey was slow because the DNA it was bound to kept getting tangled up in the gel’s agarose mesh but DAPI didn’t mind. It was sleeping peacefully until some biochemist had the gall to wake it up by shining UV light on it. The biochemist couldn’t see this kind of light but DAPI could. It got momentarily excited, but then fell back asleep. And when it did so it let off light that we *can* see thus revealing its location. And the location of its DNA home!
It’s not magic, it’s science – and here’s how it works 👉 Light is made up of pockets of energy called PHOTONS traveling in waves. Different colors of light have photons with different energies and that energy is inversely related to the wavelength (λ) so different colors of light have different wavelengths and those are inversely related to their frequencies (f) – what? 🤯 let’s break this down 👇
consider red light -> it has photons with relatively low-energy (the lowest our eyes can detect) -> so it has long wavelengths. Frequency is just how many waves pass a point in a given period of time. All light travels at the same speed (the speed of light) so if you have longer wavelengths, you can’t fit as many waves into the same time period as if you had shorter wavelengths so with longer waves you have lower frequency.
🔑 so -> low energy -> long wavelengths -> low frequency 👍 more on all this stuff here: http://bit.ly/2ROteC8
As you increase the energy, you go through the OYGBIV of the ROYGBIV spectrum. Past violet, the photons have more energy than we can see we call it ultraviolet or UV light – the light’s still there it’s just not visible to us. Fluorescent dyes like DAPI help us convert that invisible light into visible light by lowering its energy into the visible range.
Regular old visible dyes like the food coloring you add to your frosting to get pretty cakes or the tracking dyes we use to monitor the run’s progress absorb light from the visible spectrum. They steal some of the rainbow. R+O+Y+G+B+I+V = white but O+Y+G+B+I+V doesn’t – if you want to know what it does equal, you can turn to a color wheel -> you see the color across from the absorbed color. So take away the red and you get green.
So when light hits something, most of it will bounce back off (reflect) or go through (get transmitted) but dyes will absorb some of it and only give back or let through the rest. So we detect the absence of the parts it stole.
Colorless things still absorb light, just not *visible* light. So we can’t see them with our naked eyes. But we can make invisible things visible by binding them to something that *is* visible – for example, we can stain proteins with coomassie blue (more here http://bit.ly/2QJNwLy ) which lets us see them.
DNA’s also invisible to us, but we can make it visible with something that’s also invisible to us until called upon. It’s kinda like a game of go-fish -> you have to ask if it’s there -> you do this by shining light on it that has a specific wavelength it can absorb. This light we shine on it’s invisible because it’s in the UV range. So we wouldn’t be able to tell that the molecule absorbed it except that, after it absorbs it, it spits it back out as light with less energy, enough less that it’s now in the visible range and we can see it.
There are lots of fluorophores, each with characteristic wavelengths they absorb and transmit and you can learn more about them here: http://bit.ly/2Or1J3q
What makes DAPI special & especially useful is that it can bind DNA. So we can use it to visualize DNA in agarose gels. Cell biologists often use DAPI to stain nuclei (the part of the cell where DNA lives)
Our lab uses EZ-Vision sample-loading buffer when we load DNA onto agarose gels. It’s sold by VWR and this isn’t a paid endorsement or anything – it’s just what we use – because it makes it EZ to see the DNA – we mix our sample DNA with it so the fluorescent DNA-binding dye gets bound to the DNA before we even run it through the gel. Other methods involve putting the stain in the gel itself or staining the gel afterwards.
If you hit it with UV light, it will absorb some of it, but it really just wants to relax. So it gives that energy back off, but looses some of the energy in the process so the emitted light has lower energy, so lower frequency and longer wavelength & we can see it.
UV light can harm DNA – why you should wear sunscreen on a regular basis & why we limit our exposure to it in the lab while still taking advantage of its usefulness by doing the imaging in a shielded compartment. Don’t worry – we’ll still be able to see it because it’s hooked up to a machine that takes a picture for us. On a related note, if you want to actually take the DNA out of the gel & use it, limit its UV exposure as much as possible.
It may be EZ to see where the DNA is (with UV light of course) but they don’t make it EZ to figure out what’s in it. You won’t find it in it’s brochure, but if you look to it’s safety data sheet it tells you it’s DAPI (more on data-sheet-detectiving here: http://bit.ly/2yDSAKg )
Another fluorescent DNA stain is ethidium bromide (EtBr). It’s not used as much as it used to be because of concerns that it’s dangerous – but it may not be more dangerous than other stains and you can learn more about it and the controversy here: http://bit.ly/2EwcZ82
If you want to play around with the DAPI-DNA structure in 3D, I generated the figure using a cool new tool from the NCI called iCn3D. Dickerson et. al. ( PubMed 2627296 ) did all the hard work crystallizing it and solving the structure, then they deposited that information into a repository called the Protein DataBase (PDB) so anyone can use it. Each thing that’s deposited gets a unique PDB ID, and this one is 1YJ5. lots more on how crystallography works starting here: http://bit.ly/2qlzlRS
But anyways, a cool thing about iCn3D is that it’s web-based and you can save URL links that allow you (or anyone else) to “pick up where you left off” – so go ahead and play around! https://icn3d.page.link/eQ4K
I’m excited to introduce girls to it next week when I teach a “Proteins in 3D” lesson as part of the CHSL WiSE/ Dolan DNA Learning Center summer camp!
This post is part of my weekly “Bri-fings from the Bench” for The International Union of Biochemistry and Molecular Biology (@theIUBMB). Be sure to follow the IUBMB if you’re interested in biochemistry! They’re a really great international organization for biochemistry.