Cells and microorganisms are already so small, that I find it hard to imagine how someone can be looking through a microscope with tiny scissors or something cutting DNA or RNA from the nucleus of a cell. Also they are able to cut specific genes from those strands of DNA/RNA. How do they even read the genes. Also then how do they extract it out the cell and then insert it in another, or set it aside etc.
In: 8
I am not qualified to explain this, but I can give a Snapple-cap explanation. We got bacteria and viruses to do it for us.
Bacteria in general have the ability to edit their own genetic sequence, and they will often exchange genetic info with other bacteria they come across, even if they aren’t the same kind of bacteria! Others will straight up steal genetic info from other bacteria/viruses/cells they eat, incorporating that new info into their own genome.
Viruses do the reverse, in that they’re really good at injecting their code into living cells and making that cell create more virus copies. Most of the time, a virus knows how to do literally 2 things: Latch onto a cell, and inject a genetic payload into that cell.
So, we have bacteria that can cut, copy, and paste extremely specific parts of a genetic sequence, and we have viruses that can perfectly inject a genetic payload to edit the genome of a single cell. Scientists harness these abilities to do what no machine can do.
They can use the bacteria in the lab to analyze, edit, and fabricate a perfect genetic sequence, and then use viruses to actually put that new sequence into a living organism.
PLEASE remember that this is extremely oversimplified. I am no scientist, let alone a geneticist.
How we do it has changed a lot over the past 40 years. Exactly what we do depends on what we’re trying to accomplish.
Most of the time, we just want a bunch of copies of a chunk of DNA. In that case, we use something called PCR. If you imagine DNA as a long ribbon, we can copy any part by knowing a tiny bit of sequence at either end of the chunk we want to copy. In PCR, you mix the fragments at the ends with the DNA, and the enzyme from a cell that duplicates DNA and they you just warm and cool it and the enzyme pumps out copies of the DNA segment you wanted.
We can also use enzymes that will cut DNA at specific patterns (restriction enzymes), and even “program” enzymes to cut at very specific places (CRISPR+Cas9) using short DNA pieces to tell it where to cut.
Much of this relies on our ability to make short DNA sequences to order. You can get a shoebox-sized thing that plugs into a computer that can “print” short custom DNA sequences.
It also relies on DNA sequencing (now very cheap, and there’s lots of databases of DNA sequence).
For RNA, we use an enzyme to make DNA copies of it (reverse transcriptase) and just do the DNA stuff.
Biologist here! Cutting up and reassembling DNA and RNA was literally a daily task for me at one point.
In short, we don’t do this directly. We use a lot of little tools, almost all of which we discovered inside the cells of various creatures. There’s 5 steps here: first, we have to find a specific gene. Then we have to remove it without damaging it. Third, we have to fish it out. Fourth, we have to insert in somewhere else. And finally, we have to check that our experiment worked.
Step 1: searching.
You can think of DNA and RNA as charm bracelets. They have 4 different types of charms, broadly, and they use a special code to store information using these charms. You may have seen the genetic code written as a sequence of letters, like ACTGGCA. Each letter is the name of a specific chemical (Adenine, Cytosine, Guanine, and Thymine) that can be attached to the ‘string’ that forms the backbone of DNA/RNA
The special thing about these chemicals is that they stick to each other like magnets, but they’re picky. A and T stick to each other, and G and C stick to each other, and that’s it. (there’s exceptions of course, but we can set those aside right now). When you see a double helix of DNA, you’re seeing two strings, with matching pairs of charms on each side, latched onto each other.
This means that if I have a little charm bracelet that reads ATGC, and I put it a soup of random DNA bracelets, it will selectively latch onto any sequence that reads TACG, and bounce off pretty much anything else.
That is exactly how we target specific genes – we find a sequence of letters that’s only in that gene, and manufacture a ‘mirror’ sequence that will latch on to just that sequence. This is our search tool.
Step 2: removing
The next step involves enzymes. Enzymes are like tiny little biological machines. The vast majority of enzymes we use in research are things we’ve found in the biology of all sorts of creatures. Enzymes that do things to DNA or RNA typically have a built in string of RNA that’s used specifically for searching. This means we can swap that out for our own custom RNA, and redirect that enzyme to do what we want it to do.
For example, you may have heard of CRISPR. The full name is CRISPR-Cas9. This is an enzyme which functions as a pair of molecular scissors. There’s lots and lots of these sorts of enzymes. Our bodies use them for defending against viruses and bacteria (by attacking their DNA), for repairing damage to our own DNA, and a whole ton of other uses. What makes CRISPR-Cas9 so special is that it’s very easy to repurpose it with a new target. Meaning if we know what we want to cut, we can do so very specifically.
Step 3: fishing
Once we’ve chopped up some DNA, how do we retrieve it? We use another small piece of RNA. We make this specific to a sequence in the middle of the segment we want. Then we glue something to the RNA that we can fish out easily. Imagine our chopped up target DNA was like a needle in a haystack. Now imagine we took a neon blue ping pong ball, glued it to a tiny magnet, threw it in the haystack, and shook it up loads. Eventually, the magnet would latch on to the needle, and once it does, the ping pong ball will make it easy for us to find. We do pretty much that. Our magnet + ping pong ball is called a label.
In the lab, we find something that’s easy to glue onto our searching RNA (there’s a looooot of options), and combine them to make a molecular label. To fish out our labels, we typically use antibodies. Antibodies are little molecular flags that our immune systems make whenever something they don’t recognise shows up in our bodies. We can make antibodies that specifically latch onto our labels, and use them like fish hooks to fish out our searching RNA and whatever it’s attached to. Then we use some clever chemical tricks to release our chopped up DNA fragments from the label.
Step 4: inserting
Now, we have to insert our chopped up DNA fragment in somewhere else. Typically, we have a specific spot in mind. So we go back to our CRISPR scissors, and use them to chop up some DNA in a new place. But only one cut this time – we’re making an opening, not removing a chunk. We then mix in loads of the the DNA chunk we hope to insert. Remember when I told you we have enzymes to repair damage to our DNA? Well one type of this damage is a split. Most of the time these enzymes will repair the split caused by CRISPR correctly. Very rarely, they’ll mistakenly insert the chunk of DNA we’ve injected. We rely on this to attach our new DNA.
Step 5: checking
Because this happens rarely, we have to repeat all this loads of times, and then filter out our successes from our failures. How we do this depends greatly on what we’re trying to do. Typically, though, once we extract a bit of DNA, we attach it to a pre- prepared piece of DNA that has something we can use to filter. If we’re studying bacteria, we might add an antibiotic resistance gene to the snippet of DNA we want to insert. That means we can expose the bacteria to that antibiotic, and any cells that survive probably managed to absorb our new chunk of DNA.
Another useful tool we use sometimes is a gene found in many coral called Green Fluorescent Protein, or GFP. GFP is useful because when you shine UV light on it, it glows green. However, it’s extremely small and does very little else, so most cells don’t notice it’s there. If we add this into our DNA chunk, then any cells that have managed to take it in properly will start producing GFP, and thus will glow slightly green under UV light.
The most common/simplest way is to not cut it at all, but to instead just copy the small region you want many, many times using PCR.
In PCR you make two short sequences called primers that will bind specifically to either side of the region you want to amplify. These are typically made based on sequencing data you can find online but in some cases you might need to sequence the DNA yourself to know how to target either side. The actual primers are made by companies you can just order them online. You then add an enzyme which will copy everything between the primers many times (like 2^30 times, massive numbers) and you are left with a solution that is essentially 99.99% the sequence you want.
Interestingly, you can add some chemicals that glow when amplification happens and use a machine to look for their glow and this is how PCR tests (such as COVID tests) work.
The way it’s is shown in presentations and the vocabulary used (“cut”) does make one think the scientist uses litttle siccors. But it is very far from that.
It has been answered here already but I thought I’d clarify a bit. The mechanisms we use come from bacteria and viral mechanism for changing around DNA or breaking it down. Nothing has been specifically developed it’s just been discovered and we use it to our advantage.
Cut: as mentioned before there are things called restriction enzymes and restriction cut-sites. A specific restriction enzyme will sever the DNA at a certain sequence (cut -site). these sequences, when cut, produce mirrored ends that can then be matched back up. So it follows that If you cut two peices of DNA with the same cut sequence, they can be matched together and a recombinent DNA sequence can be formed.
Amplify: use PCR, which is essentially the process every organism uses to copy it’s DNA but at a higher temperature and many many times. This gives us a large quantity of DNA so we can measure it, or extract it, and we’re more statistically likely to get the desired DNA from it.
Transformation: this step is a bit more complicated but like the others it uses the methods already present in organism, just to our advantage.
There are things called plasmids which are circular peices of DNA and are essential used to transfer DNA from bacteria to bacteria , it’s a method they use to evolve. These plasmids can be inserted in a bacteria or a yeast cell and be read by it.
We use the cuting step to create a plasmid with the DNA we want on it. Just by matching cut sites. Amplify it using PCR to make a lot of it. And then insert it in the cells(mix it all together in a vial ). Insertion is yet again done using mechanism already present in the organism and not by anything fancy (usually). You can use a virus that will enter the cell. You can shock the cell and make it think it’s dying (at which point it will suck up neighbouring DNA fragments in an effort to “evolve”).
A “new” method is CRISPR that is more targeted and can be done on cells that can’t read plasmids(human cells). It’s a “programmable ” cut site maker essentially. Again, a mechanism discovered in an organism, not engineered.
Edit: how to read the DNA.
DNA is made of two strands. When it’s copied it seperates and each strand is used as a template to make new DNAs one nucleotide at a time in order.
To read and determine the sequence of DNA. you essentially make it copy (PCR) but with nucleotides that are both radiolabeled and will force the reaction to stop. You end up with a bunch of DNA strands of every possible length that terminates with a radiolabeled nucleotide. So now you now the position and the specific nucleotide.
(You can separate DNA by size using gels and electricity ).
I think there’s a very efficient way to do this that uses the same base concepts but is all automated and fast
Latest Answers