PCR (the chemical reaction) isn’t near optimal, but thermocyclers (the device) are hard to improve on.
For PCR, one of the innovations I’m excited about is the development of PCR that preserves chemical properties of the input DNA, like CG methylation. This is a critical epigenetic mark on cytosines (C DNA bases). When cytosine’s followed by guanine (G base), forming the sequence CG, its complement is also CG. There’s an enzyme called a maintenance methyltransferase that copies CG methylation from the template ssDNA strand to the new reverse strand during DNA replication.
Normally this mark gets diluted into invisibility during PCR, because there’s no maintenance methyltransferase to preserve it as the input DNA is copied. A thermostable maintenance methyltransferase can preserve CG methylation throughout PCR. This is brand new technology that’s just making its way into the scientific marketplace now. It’s the kind of PCR innovation my lab’s excited about.
This is about modern PCR, which is already optimized a lot compared to early PCR. And if you're in a "normal" lab, everything around the PCR, all the handling and preparation will be such a large chunk of time that improving the PCR time alone doesn't really matter that much.
In a very automated, high-throughput setting I'd imagine that parallelizing the PCR would be the best way to increase throughput. There probably isn't that much potential in speeding up the time compared to just multiplying the number of reactions. Which is part of the point of the article.
Regarding the cheaper lab instruments, I'm not quite convinced by these ultra cheap examples. Many lab instruments need quite a bit of precision and reliability, and I would be suspicious that the cheap examples here could compete in that regard. Even PCR needs pretty exact temperature control across many individual reaction vessels. Of course the margins on lab instruments are likely enormous, and there should be plenty of potential for cheaper ones. But I don't think the ultra-cheap DIY stuff will convince people, and it'll likely also fail at the purchasing process anyway for larger institutions.
The way to sell new cheap stuff is to give it away for a period of time, with the obligation to buy the reactives from you (you just buy them and re-tag, as if they where specific for your machine). This way the lab can test the reliability of the machine without any risk.
Also, don't dismiss the user end here: people using the thermo are used to other interface(s), and they will complain endlessly about how bad it is if they fail to, for example, program the machine even if it is their fault. They don't want to think or struggle. If they do, they will tell their supervisor that the machine is crap, and you won't sell it even for 10$.
$20 for the glucose meter. $1 for each test strip. And you are expected to be testing yourself around 10 times per day.
CGMs are much cheaper by comparison since it's a one time sensor installment for 15 days worth of monitoring. Though you are still looking at anywhere from $100/$300 per 15 day sensor.
Nuts considering these sensors are literally just a metal spike hooked into a small DSP sensing the voltage and transforming it into a glucose value.
The spike is coated in a thin layer of silver cloride which interacts with the glucose in the blood to make that voltage, pretty neat [1]
But hard to justify that $600/month price tag when you really bust out what these little plastic boxes are actually doing.
Also trust in PCR cyclers ist often low... PCR is often not straight forward, chemicals can go bad, primers don't work reliably, input DNA has inhibitors, etc. So people are quick to blame the thermocycler if things don't work for an unknown reason, or some have their favorite cycler that "always works for me", and don't what to switch to an unknown one. I guess part of the reason is that there is no log where you can check the exact temperature in every well of the block after the run. Failed PCRs cost money and especially time, so I can see why people don't want to try machines some guy built in his garage.
When practitioners say "PCR" they don't (usually) just mean amplifying DNA for use as part of the input to another process.
What they usually mean is PCR with chemistry that selectively amplifies some specific sequence of DNA. This chemistry has dyes in it which fluoresce when illuminated at some specific wavelength. The point of all this is to answer a "yes/no" question for the presence of some DNA sequence in the sample. This is done at scale with multiple chemistries looking for different DNA sequences. This is also known as "real-time PCR".
It's sort of like the biological-assay version of the kid's game "20-questions". If you do it right, it's an enormously powerful detection technique for medical purposes. It gives you your "answer" in a reasonable amount of time on your desk while you wait.
That said, there are biological assays that don't need the thermo cycling anymore. These newer assays use more sophisticated chemistry that amplifies at a constant heating temperature. In the simplest terms, they're just heaters combined with a fluorometer. It's potentially MUCH faster than realtime-PCR.
In any case, the only real serious money-making business for these instruments is in-vitro diagnostics. That requires FDA approval, and that means a ~10K minimum for the instrument and tens of dollars for the consumables containing the assays, and definitely a pricey service agreement for the instrument (eg Bio-Rad instruments).
A distant second money-making business would be research-use-only instruments, but these are not going to be inexpensive little devices.
> When practitioners say "PCR" they don't (usually) just mean amplifying DNA for use as part of the input to another process.
I definitely do.
> What they usually mean is PCR with chemistry that selectively amplifies some specific sequence of DNA. This chemistry has dyes in it which fluoresce when illuminated at some specific wavelengt
This is called qPCR (and qRT-PCR, and RT-PCR and ‘Taqman assay’... But it's not called PCR because it's not just PCR).. It has uses outside of diagnostics (which is what it seems you're most familiar with).
Although his details are likely correct, this is not an analysis from physical laws (and other hard requirements, eg usability requirements) which is what you need to show optimality, it's an exploration of the known options.
He makes the point that labs wouldn't adopt a cheaper machine - yes, much cheaper processes are often adopted first by outsiders, who couldn't afford the current ones. Not clear if there's a huge market for home PCR or similar (many DNA-active chemicals need to be used under controlled conditions because mucking with your DNA causes cancer -not sure about PCR specifically)
From a physical point of view, I wonder if energy- transfer thermal cyclers could be replaced by adiabatic compression, which is likely much faster. Depends how well these enzymes work at high pressure. Could be problematic at a process level though.
one of the coolest parts about the original inception was the application of taq polymerase. veritasium did a video of about it not too long ago. a friend of mine and a man whom i very much respect was mullis's lab manager at the time.
PCR (the chemical reaction) isn’t near optimal, but thermocyclers (the device) are hard to improve on.
For PCR, one of the innovations I’m excited about is the development of PCR that preserves chemical properties of the input DNA, like CG methylation. This is a critical epigenetic mark on cytosines (C DNA bases). When cytosine’s followed by guanine (G base), forming the sequence CG, its complement is also CG. There’s an enzyme called a maintenance methyltransferase that copies CG methylation from the template ssDNA strand to the new reverse strand during DNA replication.
Normally this mark gets diluted into invisibility during PCR, because there’s no maintenance methyltransferase to preserve it as the input DNA is copied. A thermostable maintenance methyltransferase can preserve CG methylation throughout PCR. This is brand new technology that’s just making its way into the scientific marketplace now. It’s the kind of PCR innovation my lab’s excited about.
This is about modern PCR, which is already optimized a lot compared to early PCR. And if you're in a "normal" lab, everything around the PCR, all the handling and preparation will be such a large chunk of time that improving the PCR time alone doesn't really matter that much.
In a very automated, high-throughput setting I'd imagine that parallelizing the PCR would be the best way to increase throughput. There probably isn't that much potential in speeding up the time compared to just multiplying the number of reactions. Which is part of the point of the article.
Regarding the cheaper lab instruments, I'm not quite convinced by these ultra cheap examples. Many lab instruments need quite a bit of precision and reliability, and I would be suspicious that the cheap examples here could compete in that regard. Even PCR needs pretty exact temperature control across many individual reaction vessels. Of course the margins on lab instruments are likely enormous, and there should be plenty of potential for cheaper ones. But I don't think the ultra-cheap DIY stuff will convince people, and it'll likely also fail at the purchasing process anyway for larger institutions.
The way to sell new cheap stuff is to give it away for a period of time, with the obligation to buy the reactives from you (you just buy them and re-tag, as if they where specific for your machine). This way the lab can test the reliability of the machine without any risk.
Also, don't dismiss the user end here: people using the thermo are used to other interface(s), and they will complain endlessly about how bad it is if they fail to, for example, program the machine even if it is their fault. They don't want to think or struggle. If they do, they will tell their supervisor that the machine is crap, and you won't sell it even for 10$.
This is how the consumer Glucose meter market works. The machine is free and it only accept their rebranded sticks.
It's really crazy.
$20 for the glucose meter. $1 for each test strip. And you are expected to be testing yourself around 10 times per day.
CGMs are much cheaper by comparison since it's a one time sensor installment for 15 days worth of monitoring. Though you are still looking at anywhere from $100/$300 per 15 day sensor.
Nuts considering these sensors are literally just a metal spike hooked into a small DSP sensing the voltage and transforming it into a glucose value.
The spike is coated in a thin layer of silver cloride which interacts with the glucose in the blood to make that voltage, pretty neat [1]
But hard to justify that $600/month price tag when you really bust out what these little plastic boxes are actually doing.
[1] https://pmc.ncbi.nlm.nih.gov/articles/PMC8764584/
Also trust in PCR cyclers ist often low... PCR is often not straight forward, chemicals can go bad, primers don't work reliably, input DNA has inhibitors, etc. So people are quick to blame the thermocycler if things don't work for an unknown reason, or some have their favorite cycler that "always works for me", and don't what to switch to an unknown one. I guess part of the reason is that there is no log where you can check the exact temperature in every well of the block after the run. Failed PCRs cost money and especially time, so I can see why people don't want to try machines some guy built in his garage.
When practitioners say "PCR" they don't (usually) just mean amplifying DNA for use as part of the input to another process.
What they usually mean is PCR with chemistry that selectively amplifies some specific sequence of DNA. This chemistry has dyes in it which fluoresce when illuminated at some specific wavelength. The point of all this is to answer a "yes/no" question for the presence of some DNA sequence in the sample. This is done at scale with multiple chemistries looking for different DNA sequences. This is also known as "real-time PCR".
It's sort of like the biological-assay version of the kid's game "20-questions". If you do it right, it's an enormously powerful detection technique for medical purposes. It gives you your "answer" in a reasonable amount of time on your desk while you wait.
That said, there are biological assays that don't need the thermo cycling anymore. These newer assays use more sophisticated chemistry that amplifies at a constant heating temperature. In the simplest terms, they're just heaters combined with a fluorometer. It's potentially MUCH faster than realtime-PCR.
In any case, the only real serious money-making business for these instruments is in-vitro diagnostics. That requires FDA approval, and that means a ~10K minimum for the instrument and tens of dollars for the consumables containing the assays, and definitely a pricey service agreement for the instrument (eg Bio-Rad instruments).
A distant second money-making business would be research-use-only instruments, but these are not going to be inexpensive little devices.
> When practitioners say "PCR" they don't (usually) just mean amplifying DNA for use as part of the input to another process.
I definitely do.
> What they usually mean is PCR with chemistry that selectively amplifies some specific sequence of DNA. This chemistry has dyes in it which fluoresce when illuminated at some specific wavelengt
This is called qPCR (and qRT-PCR, and RT-PCR and ‘Taqman assay’... But it's not called PCR because it's not just PCR).. It has uses outside of diagnostics (which is what it seems you're most familiar with).
Either way, the article is not about qPCR.
In normal biology labs real-time PCR is used much less than normal PCR, I'd guess 5% of PCRs across labs are run in real-time machines.
This is well said and good illustration of why optimality a fragile concept. High impact improvements often involve reframing the goal.
Although his details are likely correct, this is not an analysis from physical laws (and other hard requirements, eg usability requirements) which is what you need to show optimality, it's an exploration of the known options.
He makes the point that labs wouldn't adopt a cheaper machine - yes, much cheaper processes are often adopted first by outsiders, who couldn't afford the current ones. Not clear if there's a huge market for home PCR or similar (many DNA-active chemicals need to be used under controlled conditions because mucking with your DNA causes cancer -not sure about PCR specifically)
From a physical point of view, I wonder if energy- transfer thermal cyclers could be replaced by adiabatic compression, which is likely much faster. Depends how well these enzymes work at high pressure. Could be problematic at a process level though.
A PCR machine is a lot like a lathe or a milling machine. The price of the machine is absolutely dwarfed by the tooling and consumables.
one of the coolest parts about the original inception was the application of taq polymerase. veritasium did a video of about it not too long ago. a friend of mine and a man whom i very much respect was mullis's lab manager at the time.
https://youtu.be/zaXKQ70q4KQ
Optimized? Perhaps.
Optimal? Not even close.
Yeah, tell the thousands of PhD students who question their life choices over non-working PCRs every year that it's "optimal". ;)