Open Source PCR Thermocycler | Hacker News Reader

Open Source PCR Thermocycler

openpcr.org

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There's also PocketPCR[0] and MiniPCR[1]. $499 for OpenPCR is still pricey imo when you can buy a proper programmable thermocycler with heated top for less than half on eBay, presuming it actually works.

I'm documenting my journey in self-learning genetic engineering[2][3] and in the market for PCR and electrophoresis setup, so I'd be happy to hear what folks recommend.

[0] https://www.minipcr.com/

[1] http://gaudi.ch/PocketPCR/

[2] https://everymanbio.com/

[3] https://www.instagram.com/everymanbio/

Okay... now with my trusty PocketPCR... I shall... what does a PCR do again?

It helps you sequence a genome . On the website they give examples like sequencing beer yeast.

Well, it replicates DNA. There's other uses for it other than just sequencing, but that is a common one.

It doesn't sequence, it amplifies DNA/RNA (through reverse transcription) and lets you detect a particular sequence or produce more of a sequence you want to use for downstream steps

No reverse transcription. Just amplification by extending a primer using DNA polymerase (rtPCR -- reverse transcription PCR -- of RNA does do an initial reverse transcriptase step.) The PCR process is also used in modern DNA sequencing, but that is not what a PCR machine does.

And a non-qPCR machine does not detect the sequence -- it just produces enough of it by amplification so that it can be detected using other methods.

No, you can use a thermocycler for reverse transcription. In fact, you need one to do rtPCR, and I don't see why this cycler wouldn't be able to do that.

And to clarify, I didn't say the device detects a sequence. I said it lets you detect the presence of a sequence. This isn't a biology forum, so I deliberately didn't go into excessive detail and technicalities, just conveying the information that one of the things you use this for is detecting a DNA sequence (and sure, there are post processing steps like running a gel or sending it off for sanger sequencing etc), and the other sort of thing you can do is RT-PCR. You can also do all sorts of cloning techniques like gibsons. And technically, neither device nor a qPCR machine let you detect a particular sequence exactly. It just amplifies a piece of dna enclosed by the relevant primers that you are using to amplify. I'm well aware of all these technicalities with saying that a thermocycler does sequence detection, I left them out on purpose. And as mentioned before, the pocketPCR, like any other thermocycler, is absolutely capable of doing RT etc. I know the parent comment said "what does a PCR do again?," but it's pretty clear from context and use of the article "a" that they are asking about the pocketPCR machine's capabilities, not about polymerase chain reaction in general. If they were, then yes you are technically correct regarding RT.

This is overly pedantic but you did literally say lets you detect the sequence in a context where the average reader would have parsed the sentence as referring to the capabilities of the device.

A thermocycler is simple: it heats up and cools down in series of programmable cycles. The thing being heated and cooled is often a tube of DNA and reagents. The strands of DNA denature, or separate into individual strands, at a specific temperature and the reverse happens (annealing) at cooler temps. Imagine peeling apart a double strand of DNA creating two single strands. Now imagine making each single strand whole again by plugging in free-floating nucleotides. You'd now have two strands of DNA when you started with one. This is a simplified explanation of DNA amplification and is quite useful for taking a small sample of DNA and making lots of copies for analysis like sequencing.

so your pocket PCR is a thermocycler, which is a thing that heats and cools samples of liquid to set temperatures for set times for a programmed number of cycles. Tube gets hot, tube gets cold, makes a few stops along the way, repeat. Now, what heating and cooling the liquid to precise temperatures a bunch of times actually does depends on what's in the liquid. The most common use case is something called Polymerase Chain Reaction (or PCR, hence the name of the product). As another comment explains, this basically lets you take a little bit of DNA and make a lot of DNA. You can run other reactions as well, such as reverse transcription, where you take some RNA and generate a DNA sequence out of it. There are a lot of reasons you might want to do that, but for example that's a common procedure in diagnostics when testing for viruses like SARS-COV-2. You can do molecular cloning work, which is editing and modifying DNA, stitching DNA sequences together, etc. It's a very useful took and a core component of most biological labs.

Unfortunately, if you buy a PocketPCR, you end up with a device that doesn't have a heated lid. This means that you have to add mineral oil to all of your samples before cycling them since otherwise you could screw up the reaction/lose your sample. As someone who has dealt with that before, take my word for it that it's a royal pain. Splurge a bit on the heated lid.

Sounds like it heats up and cool down your vials in certain way, that allow enzymes sold separately to replicate DNA chunks of your specification, through Polymerase Chain Reaction reaction, so that DNA in question can be optically or electro-mechanically detected/observed by sheer volume of it

Can't really give you any recommendations in terms of PCR or gel hardware, other than just be really really careful about opening post-reaction tubes. Looks like you've done a bunch of mycology work, so you're part of the way there. Unfortunately, you need like the opposite of a laminar flow setup - you need to be pulling all the air over your workspace away from you and getting it as far away from where you're prepping your PCR reactions. PCR amplicon cleaning is a bitch (basically you're stuck with bleach or maybe blasting with UV).

Amplicon will get onto anything (including the sleeves of your lab coat) and show up as annoying random products for weeks down the line.

Good luck! Bookmarked your site.

I don't believe that is entirely accurate. Positive pressure systems are designed to blow 99.999% sterile air over the work area to prevent contamination. Commercial PCR workstations will typically take dirty outside air, run it through a prefilter, through a HEPA and then over the workstation either horizontally or vertically - blowing away any contamination that might be brought towards the sample. I'm not sure I've run into anyone who do this kind of sterile work who has necessitated the need for the kind of negative pressure system you're suggesting.

Also, I'm learning how to do DNA barcoding and typically, the PCR product isn't opened but rather sent off for sequencing or analysis. Anyway, thank you! Maybe I'm wrong so I'm happy to hear your thoughts. And thanks for the support.

Ah, I can see how what I wrote could be confusing.

For the workspace where you're prepping your reactions, yes the work bench you described is what you'd want. I was referring to a workspace for working with post PCR product (which is what you'd need to do to run a gel), for which you'd want the opposite - since effectively what you're doing is handling a super dirty sample, and you want to contain that as much as possible.

Even if you're pretty confident of your PCR workspace, dealing with random PCR amplicon contamination in your overall lab space can just lead to random frustrations. It's not like it'll for sure ruin everything forever, but it can also just... be very annoying at exactly the most inopportune times.

But right, if all of your stuff is going to be sent off for sequencing or analysis, then my warnings is totally moot - just be care to not accidentally open your post run tubes =P. I just got hooked by you asking about gels.

Who's the market for these? It seems too cheap (and the sites frankly.. not 'professional-looking' enough/not enough 'call for a sales talk') for professional labs. How many hobbyists such as yourself are there? I know next to nothing about it, what can you actually 'do' with a home 'PCR and electrophoresis setup'?

(edit: Maybe I should just read your blog!)

A lot more than I would have expected. Turns out that biology degrees are not as lucrative as computer science degrees (at least amongst my anecdata), so people with biology degrees often fall into the CS career track. But they know how to engineer life!

There are so many fun (for various definitions of fun) things you can do with a home lab. I know a guy who collects fungi, just to sequence them - because he can. You can also design your own experiments to produce materials, engineer plants, and play with organs.

It's like modern alchemy, and it's super cool.

Obligatory HN "co-creator of OpenPCR here, glad to see all the comments".

Appreciate your work, Joshua, and others like you!

I helped launch OpenPCR nearly a decade ago on the belief that cutting edge technology needed to be in the hands of people, specifically in biotech through equipment like OpenPCR, and community labs like BioCurious [0], both of which have flourished over the last decade.

One proof of successful "open sourcyness" is if the code/designs gets replicated by others. To that end, I've been inspired by NinjaPCR [1] by Shingo Hisakawa in Tokyo is one such effort that used OpenPCR design files directly and is still under development. There's also Wild OpenPCR [2] created as an experiment, and I believe Bethan Wolfenden from Bento Bio [3] said OpenPCR was some of the inspiration tho perhaps not at the source level.

Oh and for anyone curious about how the discussion has changed 9 years later, here's OpenPCR's launch on HN on July 7, 2011 [4]

[0] https://biocurious.org

[1] https://www.makery.info/en/2020/04/09/covid-19-au-japon-le-n...

[2] https://hackteria.org/wiki/Wild_OpenPCR

[3] https://www.bento.bio

[4] https://news.ycombinator.com/item?id=2736776

I remember finding OpenPCR too expensive for a student project, and I ended up designing and building a cruder/cheaper alternative for that project: http://2013.igem.org/Team:Paris_Saclay/PS-PCR/detailed_descr...

Nice work! Your BOM looks similar to OpenPCR's. One fundamental advantage of these smaller PCR machines over traditional designs was running fewer samples (i.e. 16 vs 32+), which translates to less fabrication, less peltier devices, and less sensing/cooling, among other things.

Kind of too bad, because I guess it was mildly successful at least because they discontinued the "open" part of, see here: https://www.chaibio.com/openpcr

Their newer product that they've replaced this with is:

1) Way more expensive 2) Not open hardware 3) Remove GPLv3 license (that requires you keep things open) and replaced it with ALv2 (which allows you to take it proprietary)

All good things end up failing because of greed. We'd all be so much better off if we cared about bigger goals than just short term profit.

It is way more expensive because it is way more functional. A PCR machine simply changes the temperature of an aluminum block quickly (and programmably). Their qPCR machine includes a spectrofluorimeter with digital output for measuring the amounts of material present at each PCR cycle. Commercial qPCR instruments cost 5 - 10X (or more) a PCR instrument.

That's a qPCR machine, which is an entirely different beast in terms of capability and cost than your usual thermocycler. $5000 is an amazing deal on a qPCR machine. A piece of new qPCR kit is gonna run you 30-50 thousand dollars minimum, and I've had trouble finding good functional ones from the big players for less than 20k used.

Initiatives like OpenPCR went a long way towards opening up DIY biology to the responsible amateur and making it easier to start a DIY bio hackerspace for a region. Glad to see them featured on HN.

Here here!

They're barking up the right tree here. For want of widely spread PCR testing in February, we let COVID overrun the United States.

If you have the machines and reagents widely distributed, all you need are pathogen specific primers to test for the pathogen du-jour. Then you can do widespread testing and contact tracing like you're supposed to. I really hope we get that in place before the next plague hits.

The reagents have been the limiting ... reagent.

For those of you who are interested in this space (open source/affordable scientific equipment) I recommend the lab on the cheap blog[0]. They have a lot of links to interesting websites/papers in this space.

[0] https://www.labonthecheap.com/

See https://news.ycombinator.com/item?id=24048775 for many diagnostic tests LAMP is superior to PCR on all metrics

I remember when I was doing PCR in the 1980s we needed some really scary centrifuges. The professor I was working for said if we didn't balance them correctly, they could fly out of the building and knock it down.

I am glad that this is on the frontpage. I distinctly recall that Hacker News does not believe in the existence of open source biomedical engineering.

https://news.ycombinator.com/item?id=22578216

Can it be used to detect a virus from a sample of say salvia?

You'll need to run RT-qPCR (Reverse Transcriptase Quantitative PCR) to detect Covid-19 viral sequences.

Simple thermocyclers allow us to amplify sequences but it can't do measurement like a Quantitative PCR machine.

Is this beyond the relm of what is possible with open source parts today?

No you just need more of those parts, and depending on how much manual labor you want to be doing a different number and configuration.

process is:

-take sample -amplify via thermocycler -validate presence

That last step can be done a few different ways depending on what you are looking for and how much time/money you have. (I'm just a hobbyist so not getting into specifics to avoid misspeaking but I'm confident it's possible via open tech for the 'crude' methods -- more advanced are more proprietary i'm sure.)

I know that qPCR is preferred for diagnostics, but is it actually necessary? As in, will the false positive/false negative rates suffer enough for standard RT-PCR to be useless?

No. qPCR is, well, quantitative, so it gives more information. Additionally, it requires less pipetting, and the data is easier to report. All of these things make it more desirable for diagnostics.

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