AGAROSE GEL ELECTROPHORESIS provides a way to separate differently-sized pieces of DNA! With the start of the new year, many people are likely starting biochemistry and molecular biology classes soon. So I thought I’d do a series of posts on some of the core techniques and experiments you’re likely to encounter. Thus, as I exhaust myself in the lab working on research for my thesis, let’s review AGAROSE GEL ELECTROPHORESIS!
A lot of molecular biology involves working with pieces of DNA, such as those we might get from using PCR to copy specific sequences or using restriction endonuclease to cut DNA up. Agarose gel electrophoresis gives us a way to see what’s in there (for example, is there a band of the size you’d expect if a particular mutation was present) and, if we want to, use gel extraction to take them those pieces out & work with them.
The basic idea of this technique is that you separate pieces of DNA by size by means of using electricity to send them swimming through a gel mesh made out of the sugar agarose. DNA has a negatively-charged backbone, so it’s motivated to swim towards the positively-charged end of the gel, but it won’t have an easy journey! The mesh acts like a molecular sieve, slowing down bigger molecules more. So, when you turn off the electricity, the bigger pieces won’t have traveled as far and, when you visualize the DNA (typically by using a fluorescent DNA-binding stain and a UV light) the bigger pieces will show up as bands closer to the “top” of the gel and the smaller pieces will show up as bands closer to the “bottom” of the gel.
note: “Top” and “bottom” can be a bit weird if you’re used to running SDS-PAGE gels. SDS-PAGE stands for for Sodium DodecylSulfate PolyAcrylamide Gel Electrophoresis, and it’s a related method we use to separate proteins by size (length of their amino acid chains). SDS-PAGE gels are thin and run vertically, sandwiched between two glass plates, so “top” and “bottom” make more sense. Agarose gels, on the other hand are thicker slabs that are run horizontally without the glass plates. Since you’re running horizontally, “top” just means “the end nearest the wells where you put in your samples” and “bottom” refers to “the end furthest from the wells.” In both cases, smaller things travel further and thus will be closer to the bottom. Assuming you set up the electrodes correctly!
The whole “electro-“ part of electrophoresis relies on electricity, the movement of charged particles. When you set up an electrophoresis gel, you use a power box to create an electric gradient running through the gel, with the positive charge at the bottom of the gel and the negative charge at the top of the gel. Opposite charges repel, so negatively-charged things (like DNA) will move through the gel towards the positive end. The positive end has the red electrode, so you can use the mnemonic Run to Red! to remember how to set up the gel (be careful not to set the electrodes up backwards or your DNA will run the wrong way, out of the top of the gel instead of through it!
DNA has a natural negative charge so we don’t even have to modify it to get it to run. This is unlike the case with protein gels, where, since proteins have different charges, you you have to coat your samples with the negatively-charged (anionic) detergent SDS in order to give them a uniform negative charge to motivate their swim.
To understand why DNA is already “ready to go” let’s take a look at the molecular level…
DNA (DeoxyriboNucleic Acid) (& its biochemical sibling RNA (RiboNucleic Acid) are POLYMERS (long chains of similar repeating units ) made up of linked NUCLEOTIDES, which are like DNA and RNA letters. 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/nucleicacids2
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.” You don’t need to worry about the details of these bonds yet, just know that 1) they’re reversible, unlike the bonds linking the nucleotides into chains and 2) the H-bonds the different bases make to one another are “specific.” 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/pcrtrain
As I mentioned, we call these “bonds” but they’re really just partial-charge:partial-charge based attractions. Atoms (like all the individual carbons, hydrogens, oxygens, nitrogens, and phosphoruses making up nucleotides) are themselves made up of smaller parts – subatomic particles called protons (+), electrons (-), and neutrons (neutral). Atoms link together to form molecules (like nucleotides) by sharing pairs of electrons in strong “covalent bonds.” Some atoms don’t share fairly – instead, “electronegative” atoms like oxygen and nitrogen tend to hog electrons, and this can lead to unequal charge distribution even though the molecule may be neutral overall.
This happens in the nucleotide bases, which is how you can get those H-bonds, which 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, let’s see how we can 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/agaroses
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/agaroses
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 see 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 for that pic by loading a couple wells, letting it run a little, loading other wells .
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.
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.”
jargon watch: Because DNA’s usually 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)
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.
Much more here: http://bit.ly/DNAtopologybut when you have circular pieces of DNA like plasmids, they snake through the gel weirdly. And how fast they move depends in part on how “compact” they are. There’s this phenomenon called “supercoiling” which is like what happens when you take a rubber band or a phone cord & “overtwist” it, giving you coils on coils on coils. Plasmids are often supercoiled, which is great for space-saving, but it makes them run weirdly… So you might see multiple bands from the same plasmid but with different “topologies”
Just like geologists use “topology” to describe a location’s landscape (mountains, valleys, etc. ) biochemists use “topology” to describe DNA’s “landscape.” We call different 3D-structured versions of the same DNA sequence TOPOLOGICAL ISOMERS or TOPOISOMERS. There are 3 major ones that double-stranded DNA plasmids exist in 1️⃣ SUPERCOILED (aka covalently closed circular DNA, ccc) 2️⃣ NICKED (aka relaxed, aka open-circular, oc) & 3️⃣ LINEAR
The cellular benefit of SUPERCOILING is that it makes DNA compact & this compactness makes it run QUICKLY through the gel as if it were *shorter* than it really is. And the cellular benefit of NICKING (cutting one strand) is to “uncompact” it just enough so that regions that need to be accessed become accessible. In terms of movement this is like the worst case possible bc it CANNOT easily move snakelike but it still has “ends” that can get tangled & has a lot of bulk – kinda like a really fat snake. So nicked DNA runs more SLOWLY & will look like it’s LONGER than it really is.
And LINEAR? You get this if you cut BOTH strands & it runs like you’d expect it to (finally!)
So, from top to bottom (what you think should be biggest to smallest) you’ll see
In the pics you can see – in the first lane there’s a ladder of linear DNA pieces of known and conveniently spaced-out length (ladders often have more of one of the fragments int the mix so it’s thicker and stands out more because some of those bands can be pretty smooshed together & hard to tell apart if you don’t run the gel for a long time. In lane 2 is a plasmid that I ran unmodified – and lane 3 has a plasmid that I cut with a restriction enzyme that only has a single cut site on the plasmid so it linearizes it but doesn’t chop it into multiple pieces.
You can see 2 bands in lane 2 – the top faint one is probably nicked plasmid (fat snake) and the lower one is supercoiled. You can also get different amounts of supercoiling, so it’s possible to see multiple bands representing supercoiled products.
In lane 3 is the linearized plasmid – and if I compare where it ends up to the ladder I should get a reasonable estimate of the length &/or confirm that my plasmid’s the right size. The plasmid I cut has about 6.6 thousand DNA letters on each strand (so ~6.6 kilobases (kb)). So it should run between the 6 & 8 ladder bands. And it does – yay! But if you look in lane 3 you see that 1 band (the nicked) runs even higher than the biggest ladder band (10kb) and the lower band runs like it were 5ish. But ALL of these (supercoiled, super-supercoiled, nicked, & linear) are the SAME DNA SEQUENCE, just DIFFERENT TOPOLOGIES