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PCR is a surprisingly near-optimal technology

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Re: PCR is a surprisingly near-optimal technology

#11

Post by bonsai_spool »

crispyambulance wrote: post #8

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 s…

> 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.

Re: PCR is a surprisingly near-optimal technology

#12

Post by directevolve »

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.

Re: PCR is a surprisingly near-optimal technology

#14

Post by shellfishgene »

crispyambulance wrote: post #8

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 s…

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.

Re: PCR is a surprisingly near-optimal technology

#16

Post by shellfishgene »

fabian2k wrote: post #2

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 probabl…

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.
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