Dam – where is your parent plasmid! DpnI needs to cut it out! On the template plasmid, METHYL marks the spot ❌ but your new PCR products, this methyl group they have not 👍 Dpn-ding on your template & your further plans, you may need to DPNI DIGEST your PCR reaction 👇
In biochemistry and molecular biology we use a lot of protein machinery (enzymes) that come from bacteria that are super useful. But, believe it or not, those bacteria don’t make that stuff just so that we can take it out of them – they make it for themselves, we just steal it.
Take, for example, RESTRICTION ENDONUCLEASES (aka restriction enzymes)(aka DNA scissors). These molecules recognize specific sequences in nucleic acid chains (DNA or RNA) and cut it. They use this as a way to destroy foreign DNA or RNA coming from foreigners like bacteria-invading viruses called bacteriophages (“phages”)
But the bacteria face a dilemma – they have their own DNA too – and it’s even written in the same language – which is super cool that all organisms and even phages share this common “genetic code,” but it also means that the bacteria can’t just cut up all the DNA & RNA inside of itself or it will destroy its own genetic instructions – mutual annihilation
So the bacteria needs a way to distinguish between its own DNA & the phages. One way is to limit the scissors to cutting at really specific sequences (usually 4-8 letter palindromes (think kayak or racecar) & making sure that those sequences aren’t in their own DNA. A lot of the restriction enzymes we use in the lab are this kind – we utilize that precise specificity to cut precisely where we want them to in a piece of DNA.
This comes in super handy in MOLECULAR CLONING – if we want to study a gene and/or the protein it has instructions for, we can use restriction enzymes to cut that gene out of one place and put it somewhere else that we can manipulate better. Often, we transfer genes into circular pieces of DNA called PLASMIDS that we put into bacteria – the bacteria will then copy the plasmid, make protein from it, etc. (another way we take advantage of these guys)
BUT, remember, that’s not “why” the bacteria exist and make the normal things they make. They make restriction enzymes to protect themselves from those invaders. And the invaders can be sneaky – what if they don’t have those specific sequences? The phages have small genomes, so the chances of that sequence just randomly being there are small. Bacteria “know” this, so have evolved to target sequences that many phages need. But the phages can evolve to have different sequences.
So the bacteria needs more “generic,” snip-happy scissors as backup. But, *Dam,* how will the bacteria protect its own DNA? Other enzymes called METHYLTRANSFERASES come to the rescue. These guys add methyl (-CH3) “tags” on the bacteria’s DNA. This methylation acts as a kinda uniform to tell the DpnI hey we’re on the same team!
So methylation “hides” the cut sites of some restriction enzymes, but some bacteria have taken the opposite approach, potentially because some of those phages got tricky and started methylating their DNA. So the bacteria “decided” to swap roles -> Streptococcus pneumoniae has a restriction enzyme, DpnI that ONLY cuts methylated sites. And this endonuclease can come in really handy in the lab as well!
A lot of times when we’re doing molecular cloning, we’re doing something called SITE-DIRECTED MUTAGENESIS – basically we want to change just a small piece of the gene but keep it in its same plasmid home.
We can do this in different ways, but they mostly involve using PCR to make copies of the plasmid with the typo we want. It gives you lots of copies of the new, mutated, plasmid, but these are mixed in with all those “originals” and we need a way to weed out those template plasmids. More in yesterday’s post http://bit.ly/2oiw6wL
When we’re cloning, we usually use a selectable marker like an antibiotic resistance that’s in that plasmid home so that only bacteria with our plasmid can grow if we spike their food with that antibiotic. But antibiotic selection markers won’t help us distinguish between un-mutated “template” plasmid & the mutated plasmid we want because the sequence is in the same plasmid, so they both have the selection marker.
So we need another way to weed out the template plasmid. But they look almost the same right? There’s a key difference we can exploit -> the methylation gets added in bacterial replication, but not during the replication we do in PCR (no methyltransferases in there). So the original templates will be coated in methyl groups but our new copies will be methyl-free.
So, before we put the plasmid we’ve made into bacteria, we add some DpnI and some salts, etc. it likes, get it nice and cozy at 37°C and let it do its thing for a few hours -> the DpnI will cut up the template plasmid but won’t touch your new copies. This way, when you stick the plasmid into the bacteria, only the new copies will be “viable” and only bacteria that take it up will be allowed to live because they have the plasmid selection marker.