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.
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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.
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