I don’t mean to be blunt, but things might get sticky -so make sure your RESTRICTION ENZYMES are specifically picky! Bacteria have DNA-specific “scissors” called RESTRICTION ENZYMES (aka restriction endonucleases, or REases) that recognize & cut specific “code words” (RESTRICTION SITES aka recognition sequences) written in DNA that as “dotted lines.” Bacteria use them as a defense against invaders like bacteria-infecting viruses (phages) and biochemists use them to cut and paste pieces of DNA together in RESTRICTION CLONING
In MOLECULAR CLONING, we stick a gene we want to study into a PLASMID VECTOR (circular piece of DNA) that’s easier to work with. We often stick this plasmid in bacteria cells to have them “host” it for us. One way, the “classic” way is RESTRICTION CLONING. In this method, we use DNA “scissors” -RESTRICTION ENZYMES – to cut an “insert piece” with the gene we want to stick in and a “vector piece” with the vector we want to stick it in with the same pairs of scissors so they have complementary cuts. And then we purify the matching pieces and mix them together, adding a “stitcher” called DNA ligase to seal them up tight.
A quick overview and then the deets: Take the DNA where it currently is (such as in a plasmid or from a PCR reaction) ⏩ add restriction enzyme(s) (and a buffer containing salts, pH stabilizers, Mg2+, etc. to keep the enzyme happy) ⏩ heat it up to give the enzymes energy to work & give it time to cut ⏩ purify the pieces ⏩ mix them together ⏩ add ligase to stitch them up ⏩ stick them in bacteria ⏩ bacteria host it and make protein from it
Quick note: The enzymes are “numbered” not “lettered” (e.g. EcoRV isn’t an all-electric RV model, it’s EcoR FIVE (learned this the embarrassing way) 👉 tells you it was the 5th restriction enzyme found in the “RY13” strain of E. coli)
The restriction enzymes we use for cloning are usually of the IIP subtype (more on this at the end) – the sequences they recognize are usually fairly short (4-6 bp long) PALINDROMES (think kayak, racecar…) Since DNA’s 2 strands complement each other (A across from T and G across from C), this “palindromnicity?” means that both strands of the DNA have the cut site 👉 usually working in pairs (homodimers) the enzyme cleaves all the way through the DNA (both strand) instead of just “nicking” it (cutting a single strand).
When they cut, they can make “staggered cuts” that result in STICKY ENDS (2-4 unpaired nucleotides “overhanging” on each end (useful if you want to then stick it to something else…) or BLUNT ENDS (cut straight across – no overhangs)
If you cut 2 things with the same 2 enzymes (and they have cut sites in the same orientation) you can remix and match them. So, for example, you can cut a plasmid vector and your gene of interest with the same enzymes -> generates matching sticky ends -> purify the pieces and mix them together. Some REases have different recognition sequences but make the same cut so they’re “complementary” (kinda like one recognizes ace and one recognizes racecar but both cut after the c leaving you with the same overhand. You want to make sure you’re using “unique cutters” so you only get the right pieces. If a cutter cuts multiple places you’ll get multiple matching pieces
If you’ve cut with a blunt end cutter, any 2 pieces can match, but the orientation might “flip” so if possible we use sticky ends. I say “if possible” because this only works if the “dotted lines” are there – for vectors designed for this sort of thing, this is less of a problem because they’re often defined with “multiple cloning sites” (MCSes) that have several options to choose from.
I used such restriction cloning in undergrad but I’ve now switched the PCR-based method SLIC (more here: http://bit.ly/2oiw6wL ) because it’s a lot more versatile – and the piece-purifying is a lot easier. With PCR you get lots of copies of your insert and no copies of “TheRest” – so you just have to purify out the primers and cut up the parent plasmid.
But with restriction cloning you’re not making copies – so you have to start with a lot of parent plasmid – and each time you make an insert piece you make a “TheRest” piece. So you have to separate the insert from the rest so they don’t just bind back together.
One thing you have going for you is that the pieces might “stick” together on their own because of basepair complementarity, but they can’t sew themselves together – they need DNA ligase for that. http://bit.ly/2kGHfpR
We often use T4 DNA ligase (note of caution – this is DIFFERENT from T4 DNA Polymerase which we use for SLIC – so check the tube labels carefully!)
Speaking of “binding back” – to prevent self-circularization (especially important if you’re using the same enzyme to cut on both sides) you can add a phosphatase. In order to do the strand-stitching, the ligase needs the 5’ ends to be phosphorylated. If you take that phosphate away, the ligase cannot play!
A lot of the times when we’re cloning we’re doing “subcloning” where we’re moving a gene from one plasmid to another (like moving it from the generic plasmid it came in when we ordered it) and putting it into a plasmid that’s ideal for protein-making. And we’re often swapping out a gene that was in the new one so our “TheRest” pieces are big – too big to use PCR purification kits which can remove small things like primers. more on those here: http://bit.ly/2yO5BBt
So usually you purify them gel purification – you start by running an agarose gel (you should do this in any case just to check that the pieces look ok and it all got cut). When you’re going to purify it out of the gel you add it all (not just a little bit to look) and you use a wide comb so you have plenty of room to cut around.
Agarose gel electrophoresis separates DNA pieces by their length (DNA’s naturally negative, so you can use positive charge to motivate it through a gel mesh made of the sugar agarose – longer DNA pieces will get slowed down more because they’ll get tangled up in the mesh more (think of trying to drag a jumprope through a net) so they’ll travel slower. more here: http://bit.ly/2lPCUR8
You use a DNA-binding stain that absorbs UV light, so you use UV to tell where your band of interest is – cut out that chunk of gel – extract out the DNA (basically chemically melt away the surrounding mesh) and purify it. So you purify the vector and the insert & mix them (usually at ~1:2-3 ratio of vector to insert but it might take some trial-and-error-ing) and add ligase.
You give it time to work and then you stick into into bacteria (we call this transformation and a common way we do this is “heat shock” where we mix the DNA we want to put in with chemically-weakened bacteria in a tube on ice and then briefly dunk it in a warm water bath then stick it back on ice. The heat shock opens up pores in the bacteria so the DNA can rush in. more here: http://bit.ly/2Jj7L47
After you let it recover a bit you plate it on an agar plate in your classic Petri dish. Agar is related to but different from agarose and it makes a nice solid gel matrix to hold bacteria food. We only want it to be a nice “B&B” for bacteria that have our plasmid (not all the cells will have taken it in and other bacteria could have snuck in at various times) so we need a way to prevent them from staying here. Usually we do this using antibiotic selection. We use a plasmid that has an antibiotic resistance gene and then we spike the food with that antibiotic so that only bacteria with the plasmid can survive). more here: http://bit.ly/2tcW4ky
Since the vector has the antibiotic resistance gene you’re using for selection even if it doesn’t have your gene, it’s important to make sure that there’s none of the original or the cut-but-re-self-sealed. So you can run a couple of controls.
In 1 control you just transform the cut vector alone – if you get colonies on this plate it indicates that not all of the vector got cut (only the circularized vector can survive & replicate inside of bacteria)
If you had one of those cases where you had to dephosphorylate, you do a second control. in control 2 you transform the cut vector + ligase (but NO insert) -> if you get colonies on this plate but not the first it indicates that you got self-circularization, suggesting you didn’t dephosphorylate it sufficiently so the ligase *could* play
If you get colonies on both of those it could be a mix of both or just the non-cuttedness or it could be contamination or something.
You’ll probably have a few colonies on the control plates but you should have way more on the “real plate” (the one with vector + insert + ligase). You then take a few of those colonies and grow them in some liquid broth, isolate their plasmid DNA and check to make sure it actually has your insert – you can do a quick check with with colony PCR or analytical digest then verify with sequencing. more here: http://bit.ly/2TGAvo5
Where do these super useful tools come from? As I mentioned briefly before, restriction enzymes come from bacteria themselves! (like many molecular biology tools). Restriction enzymes are an important natural protection mechanism for bacteria. if a virus infects them, the restriction enzymes will recognize specific sequences in the foreign DNA & cut it so that DNA gets chewed up & does no harm.
To make sure that the bacteria doesn’t cut its own DNA, sites where that recognition site occurs in the bacterial DNA are “hidden” by a modification called methylation (which adds a methyl (-CH3) group to the DNA) Yesterday we saw how methylation can be used to tell apart original “parent” plasmid from copies of it & selectively chew up the methylated parent with an enzyme called DpnI http://bit.ly/2pvfti7
DpnI is a restriction enzyme too, but a different type than the ones we use for cut-and-paste or cut-and-look-ing. There are several different “types” of restriction enzymes, some more well-suited than others for what we need. TYPE II restriction enzymes are the useful ones for these purposes. TYPE I & TYPE III are bigger & have multiple pieces (in part because they also carry out methylation to protect their host DNA) They also don’t cut where you think they “should” 👉 TYPE I cuts at random sites that can be over 1000 base pairs (bp) away from the recognition site & TYPE III cuts ~25 bp away. And, if that weren’t bad enough, they both require energy (in the form of ATP not just heat) to cut
So TYPE II’s the one we use! But within this type we still have hundreds of enzymes to choose from (a market for which New England Biolabs (NEB) has gladly cornered)! Some of the type II can be complicated too but we typically use the less complicated ones! (the IIP subtype).
Some restriction enzymes are more promiscuous than others… Evolution-wise this makes sense – too short a sequence & the viral DNA will definitely get cut up 👍 BUT the bacterial DNA likely will as well because it’ll be hard for the methylators to keep up! 👎 And it will also require a lot of unnecessary energy since you don’t need to make tons cuts 👉 just cut it once & EXOnucleases will chew from the ends, a much “cheaper” process
Longer sequences are less likely to occur by chance,👍 BUT too long & they’d have very limited usefulness for the bacteria since it’s really unlikely that sequence would occur in the DNA of the viral invader 👎 So bacteria found that 4-8’s a good balance
⚠️Don’t confuse restriction enzymes (which are endoNUCLEASES & cut DNA) with endoPROTEASES which cut PROTEINS. Both have their uses in molecular biology & biochemistry but at different points in the process! (for example, we can use endoproteases to cut a protein tag off of a protein)