AGAROSE GEL ELECTROPHORESIS provides a way to separate differently-sized pieces of DNA! Last Friday we looked at how we can use a technique called SDS-PAGE to separate proteins by size by sending them through a gel mesh that acts like a molecular sieve, slowing down bigger molecules more. Well, we can do something similar with DNA too! We use a gel made up of agarose, cast horizontally, instead. But, thanks to DNA’s natural negative charge, we still run to red!
If you buy synthesized DNA, they might charge you by base. But DNA is naturally charged! Does that mean they charge us twice? At least phosphate charges at an even rate! DNA’s generic backbone has a negative charge included thanks to the phosphate (more below) & we can use this natural ➖ charge to our advantage to send pieces of DNA (like we might get from using PCR to copy specific sequences or using restriction endonuclease to cut DNA up) through an agarose gel mesh towards a ➕ charged electrode. The DNA wants to go there bc opposites attract ➕➖ 😍 BUT longer pieces have a harder time getting there, so they travel more slowly. The agarose gel thus acts as a “molecular sieve” separating different sized DNA pieces so we can 👀 what’s in there &, if we want to, use gel extraction to take them out & work with them.
DNA (DeoxyriboNucleic Acid) (& its biochemical sibling RNA (RiboNucleic Acid) are POLYMERS (long chains of similar repeating units 🔗) made up of linked NUCLEOTIDES. NUCLEOTIDES contain a sugar (RIBOSE in RNA or DEOXYRIBOSE in DNA (has 1 fewer oxygen)) linked to 1 or more PHOSPHATE (PO43-) groups on its 5’ “leg” & a NITROGENOUS BASE (aka NUCLEOBASE or just BASE) more on nucleic acids here: http://bit.ly/2FqasfN
It’s the base part that’s different between different nucleotide “letters” & the different bases (A, C, G, & T (in DNA) or U (in RNA)) offer different HYDROGEN BONDING (H-bonding) opportunities that allow for specific “base pairing.” The chemical makeup & spatial orientation of the bases makes it so C wants to bind to G (w/3 H-bonds) & A to T (in DNA) or U (in RNA)(w/2 H-bonds). This 1:1 matching is why DNA is double-stranded & we can use 1 strand as a template to recreate the other – either to copy all the DNA in our cells before they divide (replication) or in a test tube in a thermal cycler (PCR) to copy specific parts of DNA we’re interested in working with. more here: http://bit.ly/31GrYoJ
These H-bonds form because of unequal charge *distribution* (the O’s an electron hog) BUT the NUCLEOBASES are NEUTRAL overall (have an equal # of protons (+) & electrons (-). It’s the BACKBONE that’s NEGATIVELY CHARGED (has whole extra electrons). And that part’s generic, so the sequence “doesn’t” matter in terms of the overall charge, just the length
For each nucleotide you add you get another phosphate group that adds another ➖ charge, so DNA comes with a “built-in” constant length/charge ratio. When you add more bases, you add more charge so you might think that it will travel faster, but it turns out this added charge is less significant than the added bulk & since the charge/mass ratio is constant, it “normalizes” itself
🔑 in short 👉 Different pieces of DNA have different orders of bases that “spell out” instructions for doing things your cells need to do like make proteins. So 2 pieces of DNA of the same length can be really different, but they’ll still have the same net charge because the only fully charged part of DNA is the phosphate in the backbone, which is the same regardless of what base is attached. The more nucleotides you add, the more negatively charged the molecule but the bulkier it is, and that’s what matters in these gels.
So, now that we understand where the charge does (& doesn’t) come from, how can we take advantage of it? The usefulness of the charge is that we use it as a driving force to get the DNA to “get a move on!” & travel through a gel mesh made up of the polysaccharide (sugar chain) AGAROSE. AGAROSE is great for these gels because it’s neutral itself so it doesn’t interact with the DNA – our DNA doesn’t get “sidetracked” on its journey. Agarose is related to AGAR which we use for making bacterial food beds in Petri dishes, but agar is a less pure form that also has other polysaccharides that are charged so *would* interfere. more on agarose here: http://bit.ly/2CWPRil
Unlike in PAGE, when we prepare AGAROSE gels, we are NOT doing the polymerization (chain-making) – that’s already done for us (thanks red algae!) Instead of adding strong, covalent bonds between individual subunits to form long chains called POLYMERS, we simply need to free existing polymer chains from their clumped-up-ness to form a more spread out matrix. The chains stay chains, we just need to change the ways they interact w/one another/
💭 You can think of it like yarn. You have long strands of yarn that can be all balled up. This would not act as a good molecular sieve. BUT you can unspool the ball & knit a more mesh-like product. When preparing an agarose gel, its like you have lots of identical balls of yarn & you’re knitting them together👇
1st you have to unspool them (& untangle strands that may be tangled up), which you can do by heating them up this breaks the existing *non-covalent* interactions (the yarn unballs & untangles BUT you don’t “cut” any yarn). Then when you cool it down, new attractions can form. BUT, before they do, the yarn gets hydrated (puts on a water coat) so you end up with a gel. more on this gelation here: http://bit.ly/2Rztc4L
We put the gel in a pool of buffer (stable-pH salt water) in a gel box “swimming pool” hooked up to a power source & set up the gel so that electricity flows through it (from a ➖ electrode (cathode) at one end to a ➕ electrode (anode) at the other end), generating a ➖ charged end where your gel starts & a ➕ charged end where your gel ends. There’s no charge gradient until you turn on the power and the electricity starts splitting water (electrolysis) like w/SDS page except that here our gel’s horizontal. More here: http://bit.ly/2XMLMbG
We pipet our DNA samples into wells in the gel at the ➖ end & because they’re ➖ charged, they’ll get repelled from this end & pulled toward the ➕ end, BUT they’ll be slowed down as they go because they’re basically having to swim through jello. And they get differentially slowed depending on how big they are compared to how tight the gel’s mesh is.
You want a mesh tightness that differentially separates the size of the fragments you expect. Imagine you were trying to shoot hoops with different kinds of sports balls. And once you get through one hoop there’s another, & then another. If your hoops were the size of basketball, they’d be able to separate basketballs that were slightly too big to easily fit but can push their way through eventually from softballs (straight on through!) BUT not softballs from from golf balls (because both are significantly smaller than the hole). And things that are too big, like yoga balls, couldn’t get through at all and would get stuck.
It’s easier to intuit this in terms of balls & the end result is the same, BUT what’s actually happening isn’t just that the DNA is traveling like a ball – it’s traveling more like a jump rope – the linear DNA travels snake-like through the gel, sometimes getting coiled around some of the agarose strands as it goes (like getting wrapped around the basketball hoop rim)
This snakelike, or REPTilan motion has a cool name – BIASED REPTATION & the longer the DNA fragment, the more tangled up around the gel matrix it will get -> experiences more friction -> gets slowed down more.
And the tighter the mesh (smaller the basketball hoop radius & thus more hoops in the same amount of space) the more opportunities for tangling and the more the friction. You can change the meshiness by changing the agarose concentration. I usually use a 1% agarose gel (1g agarose in 100mL TAE buffer -> microwave to bring to boil -> pour into casting well -> let set). 1% is good for separating pieces that are 400-8,000 nucleotides long.
You can decrease the concentration to make bigger pores better for separating bigger pieces) or increase the concentration to get smaller pores better for separating smaller pieces
You can’t 👀 the DNA fragments until you dye them with fluorescent dye & look at them with UV light (more on this here: http://bit.ly/2U4na9r ) But when you do, they’ll appear as “bands” with bands towards the end (furthest away from the wells) corresponding to smaller, thus faster-migrating, DNA fragments and bigger fragments closer to the wells. The bands that you see while the gel’s running are just tracking dye that helps you know how far the gel’s run. I got it to make a smiley face by loading a couple wells, letting it run a little, loading other wells …
Assuming that you aren’t making smiley faces and have loaded all the samples at the same time, you can use the tracking dye to see where different size things “should be.” Some tracking dyes have multiple dyes. Different dyes run at different rates and they tend to run “as if” they were DNA fragments of certain sizes, so you can look to a migration chart, look back at your gel and say something like “this cyan band is xylene cyanol FF, and in a 1% gel like this it should run alongside (though not attached to or anything) pieces of double-stranded DNA that are ~4000bp. I’m expecting a 1000bp product so, it should be somewhere between this band and the blue-ier band below it – FF bromophenol blue – which runs alongside ~300bp DNA.”
Then, when we’re at the UV light part, we can get a “real” comparison by comparing the sizes of the bands to the sizes of a “ladder” containing DNA pieces of known sizes that we run alongside it – it’s important to note that these sizes are for LINEAR, double-stranded, DNA – if you’re DNA’s not linear it will travel differently. More here: http://bit.ly/2SZbasf
Because DNA’s double-stranded, we often speak of lengths of DNA pieces in “basepairs” or “bp” And when dealing with pieces that are thousands of bases long, it’s easier to work with “kilobasepairs” or “kbp” (1 kilobase is 1000 bases)
In addition to the tracking dye, the loading buffer has glycerol in it which is heavier than water so it helps keep your samples from floating out.
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.
more on SDS-PAGE: http://bit.ly/2GZc3tG