I probably already have a pretty strange reputation so why not discuss the strange but cool REPTATION? This cool word does have a relation to our talk of gel electrophoretic separation! So allow me to talk “somer” about DNA TOPOISOMERS!
AGAROSE GEL ELECTROPHORESIS & SDS-PAGE (Sodium Dodecyl Sulfate – PolyAcrylamide Gel Electrophoresis) separate biological macromolecules by size (DNA for agarose gels and proteins for SDS-PAGE) by using electricity to send them through gel meshes. More here: http://bit.ly/2Ik1g0s
Both these methods “expect” your sample to be LINEAR, so it can wind its way snakelike through the gel’s mesh. Longer things get tangled up more along the way, so they travel more slowly &, when you pull the plug on the experiment they will have traveled less far down the gel. This snakelike motion has an awesome name – REPTATION as in REPtilian
The premise of these gels is – things of the SAME LENGTH will run at the SAME RATE so will travel the SAME DISTANCE in the same amount of time. This means if you run a mix of LINEAR pieces of known size – what we call a standard “ladder” – alongside your samples you can use it as a sort of “ruler” to see how big your samples are
⚠️ BUT this ONLY works if your samples are LINEAR⚠️
Imagine a snake traveling through a wadded up mesh net. Or, if you’re afraid of snakes – which, has another cool name, by the way 👉 OPHIDIOPHOBIA (though phobia part’s not cool) imagine a jumprope or something. A COILED up snake would travel differently through a mesh then an uncoiled snake 👉 you could have 2 snakes of the same length, 1 coiled, the other stretched out & they’d travel very differently
If the COILED snake were coiled so TIGHTLY that it was smaller than the holes in the mesh, it could slide through easily & since it doesn’t have any loose ends to get tangled up, it would travel MORE QUICKLY than the stretched snake that would get tangled lots & experience a lot of friction (drag) that slows it down. BUT if the coiled snake were coiled more LOOSELY & /or the mesh were tighter, it would have a hard time pushing its bulk through, encountering more friction & moving more slowly
PROTEINS are like coiled-up snakes made up of AMINO ACID building blocks. You get them to uncoil & stay uncoiled by heating them up & coating them in a detergent called SDS so you don’t have to worry about their shape getting in the way. If you don’t, but instead leave the proteins in their “native” state, their shapes *will* affect how they run, which can be taken advantage of in NATIVE-PAGE, BUT we’re usually using SDS-PAGE, which is DENATURING (we’ve removed the NATURal shape so we don’t have to worry about it
DNA “snakes” are made up of NUCLEOTIDE building blocks. DNA holds the genetic “recipes” for making proteins & other things needed to make you & keep you working. This requires a LOT of recipes & your cells house them in CHROMOSOMES (like volumes of your genetic cookbook). Your chromosomes *are* linear BUT they are SOOOOO long that, in order to fit in your cells, they have to be VERY coiled up 👉 SUPERCOILED!
Bacteria have circular genomes that are smaller than ours & bacterial cells can also host even smaller circular pieces of DNA called PLASMIDS. We use this to our advantage in MOLECULAR CLONING 👉 we engineer plasmids to act as VECTORS for a gene/protein we want to study 👉 we put a gene into bacteria & have the bacteria make copies of the gene (& possibly the protein it has the recipe for) for us
In the process of designing these vectors & making sure we made them correctly, we generate short linear pieces of DNA (either by copying a specific stretch of DNA using PCR or by cutting it up using restriction enzymes ✂️) http://bit.ly/2sQaiYw Because these pieces are short they don’t get too twisted up w/themself & they can run snakelike
BUT what if you tried to run the WHOLE PLASMID? It’s a circle so it’s, by definition, NOT linear. How would it run?
Even though these plasmids are a lot smaller than your chromosomes, they still SUPERCOIL in bacterial cells so they don’t take up too much room (the cells have lots to do & they need space to do it!) SUPERCOILING is like what happens if you take a rubber band or a phone cord & “overtwist” it, giving you coils on coils on coils
Plasmids, like your chromosomes, are DOUBLE-STRANDED 👉 If only 1 of these strands is cut we call it “NICKED.” Nicking is done in cells by enzymes called TOPOISOMERASES that nick 1 strand to relieve some of the tension that comes from supercoiling so that cellular machinery can access the genes it contains & do whatever they need to do (don’t worry they also have ligase activity to seal them back)
Where does this cool word come from? 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. Yesterday we looked at stereoisomers – when the same atoms are connected differently in space – that involves the actual connections between the atoms so you can’t just “mold it differently”. But with topoisomers, you *can* change forms (although you might have to cut it to make some forms possible)
There are 3 major ones that 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 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 👀
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 👀 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) ^ 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 🔑