Why your plasmid runs weird on an agarose gel: DNA topology meets agarose gel electrophoresis

Agarose gel electrophoresis separates DNA fragments by size by using electricity to send them through gel meshes. It “expects” 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 - biased reptation as in REPtilian. bit.ly/DNAtopology
The premise 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. 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
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. This is where we engineer plasmids to act as vectors (vehicles) for a gene/protein we want to study.
In the process of designing these vectors & making sure we made them correctly, we generate short linear pieces of DNA. 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 topoisomerase 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)
Biochemists use “topology” to describe DNA’s “landscape.” We call different 3D-structured versions of the same DNA sequence topological isomers or topoisomers. The other day 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 double-stranded DNA plasmids exist in 1️⃣ supercoiled (aka covalently closed circular DNA, ccc) 2️⃣ nicked (aka relaxed, aka open-circular, oc) & 3️⃣ linear⠀
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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.⠀
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And linear? You get this if you cut both strands & it runs like you’d expect it to (finally!)⠀
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So, from top to bottom (what you think should be biggest to smallest) you’ll see⠀
- nicked⠀
- linear⠀
- supercoiled⠀
      
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com

[@31-canaan44]

great video for learning about electrophoresis!! it's simple, fun, and yet full of knowledge.

[@thebumblingbiochemist]

Thanks so much - so glad you found it helpful!

[@arkanshsharma850]

Best explanation on youtube

[@arkanshsharma850]

nice work really appreciate your efforts thanks for the awesome explanation

[@thebumblingbiochemist]

Thank you so much!

[@sophiebaldwin5380]

this is super interesting! I’ve been learning something similar at university lately. Great video! :)

[@thebumblingbiochemist]

Thank you so much! And best of luck with school :)