DNA is now DIY: OpenPCR ships worldwide(openpcr.org)
openpcr.org
DNA is now DIY: OpenPCR ships worldwide
http://openpcr.org/2011/07/dna-is-now-diy-openpcr-ships-worldwide/
16 comments
I think it's also useful to note _just_ how versatile a PCR machine is in biotech. If every EE hacker needs an oscilloscope, every bioengineer hacker needs a PCR machine. PCR is good for much more than just amplification -- a huge number of variations on the basic technique have surfaced in the past few years, here are just a few:
- Need to synthesize a new gene or genetic circuit? Take a bunch of DNA fragments, design overlapping fragments which serve as the "glue", and use PCR to sew the whole thing together
- Want to know if your bacteria is the right strain? Microwave a colony and toss it into the PCR machine with some probes to check. Same thing if you want to test yourself for common disease risk factors such as the BRCA1 gene.
- Want to know the expression of protein A compared to protein B? Use Q-PCR to get quantitative data on the difference.
- Made a mistake in your latest gene synthesis order? Use PCR to change base-pairs or even edit out regions of DNA to fix the problem region. Conversely, if you want to _introduce_ mutations / randomize segments of DNA, PCR can do that too!
There are many more uses of the general technique including next-gen sequencing and DNA origami that are just too numerous to list. PCR is like the IDE of biology, containing an editor, a compiler, and a debugger all in one!
Really, my only complaint is that a PCR run takes an hour or more to run depending on the complexity.
- Need to synthesize a new gene or genetic circuit? Take a bunch of DNA fragments, design overlapping fragments which serve as the "glue", and use PCR to sew the whole thing together
- Want to know if your bacteria is the right strain? Microwave a colony and toss it into the PCR machine with some probes to check. Same thing if you want to test yourself for common disease risk factors such as the BRCA1 gene.
- Want to know the expression of protein A compared to protein B? Use Q-PCR to get quantitative data on the difference.
- Made a mistake in your latest gene synthesis order? Use PCR to change base-pairs or even edit out regions of DNA to fix the problem region. Conversely, if you want to _introduce_ mutations / randomize segments of DNA, PCR can do that too!
There are many more uses of the general technique including next-gen sequencing and DNA origami that are just too numerous to list. PCR is like the IDE of biology, containing an editor, a compiler, and a debugger all in one!
Really, my only complaint is that a PCR run takes an hour or more to run depending on the complexity.
That's a good summary.
To put it another way, this is really just a box that does programmed temperature cycling. This is a critical part of PCR, but there's nothing too terribly high-tech here. PCR is a relatively simple process.
PCR is frustrating for the undergrad? Sometimes. Historically dependent on overpriced equipment? Absolutely. Dependent on small temperature variations? You bet. Complex overall? No.
To put it another way, this is really just a box that does programmed temperature cycling. This is a critical part of PCR, but there's nothing too terribly high-tech here. PCR is a relatively simple process.
PCR is frustrating for the undergrad? Sometimes. Historically dependent on overpriced equipment? Absolutely. Dependent on small temperature variations? You bet. Complex overall? No.
Yup. Highschool / Undergraduate labs that teach PCR with two water baths and a stopwatch really kill a lot of the "magic" bestowed on these absurdly expensive machines.
Is it possible to do projects like glowing bacteria and stuff like that with this kit ? (if that sounded like a noob question, that's because I'm one :) )
Not usually. The way that generally works is this:
- You get some competent cells ("competent" means "able to accept external DNA" there are different ways you can make different cells competent) - You place plasmids (circular DNA strands) containing the trait you're interested in imparting (e.g. glowing) unto the bacteria - Under favorable conditions the bacteria will take on the genetic information contained in the plasmids
You can order plasmid vectors much like primers, though iirc it's a bit more complicated--may be wrong about that.
Caveat: As Cixelyn mentions there are actually protocols for constructing DNA using PCR and and thus there are also protocols for constructing plasmids using PCR. So yes, you could technically use an OpenPCR machine to construct a plasmid vector which you then introduce to competent cells.
- You get some competent cells ("competent" means "able to accept external DNA" there are different ways you can make different cells competent) - You place plasmids (circular DNA strands) containing the trait you're interested in imparting (e.g. glowing) unto the bacteria - Under favorable conditions the bacteria will take on the genetic information contained in the plasmids
You can order plasmid vectors much like primers, though iirc it's a bit more complicated--may be wrong about that.
Caveat: As Cixelyn mentions there are actually protocols for constructing DNA using PCR and and thus there are also protocols for constructing plasmids using PCR. So yes, you could technically use an OpenPCR machine to construct a plasmid vector which you then introduce to competent cells.
Whenever I come across this sort of DIY (or at least low-end) biotech I wonder, "Is this what computers looked like before the PC revolution?" I'm sure that's a terrible comparison, but it's easy to make, and there's just enough substance to it that I wonder if I'm missing out on the next revolution by not being involved in biotech.
What can I exactly do with it? Can I find any gene and how does that really work? The video introduced some gels which reveal the DNA bits, but without further info it's not clear how does it find a particular gene. Really awesome stuff anyway.
PCR requires short (typically synthesized) DNA primers to initiate replication. That is, you must know the DNA sequences at the beginning and end of the region you want to amplify. Ideally you would know the entire sequence so you can synthesize primers that have one and only one binding site.
Amplifying DNA is useful/necessary for all sorts of biological procedures. High concentrations of DNA are needed for efficiently transfecting microbes, for example.
Amplifying DNA is useful/necessary for all sorts of biological procedures. High concentrations of DNA are needed for efficiently transfecting microbes, for example.
Fortunately, primers are relatively cheap, typically under $0.30/base, so for two 20bp primers, you may end up paying as much for shipping as for the synthesis itself.
These days, it's also pretty rare that you won't know the complete genome of the species you're working with, and there are free web tools that can help you design unique primers.
These days, it's also pretty rare that you won't know the complete genome of the species you're working with, and there are free web tools that can help you design unique primers.
there are free web tools that can help you design unique primers.
Wow, you all know a surprising amount about PCR! Are any of you regular PCR users?
Tito OpenPCR
Tito OpenPCR
Hey Tito, congrats on shipping! This is Patrick from Baltimore-- we talked for a bit at iGEM and the FBI DIY-BIO thing last year.
In the dry lab, I'm really more of a regular PCR consumer than a regular PCR user, but yeah, my attitude towards PCR is more or less my attitude towards snakes: try to keep a respectful distance :-)
In the dry lab, I'm really more of a regular PCR consumer than a regular PCR user, but yeah, my attitude towards PCR is more or less my attitude towards snakes: try to keep a respectful distance :-)
Are there any good guides or tutorials for DIY bioinformatics? Targeted for people who are interested to learn about bioinformatics in practice in addition to theory.
The "virtual lab" animation on bottom this page actually does a great job of showing how PCR works: http://openpcr.org/what-is-openpcr/
From the wikipedia article [1]
"PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR methods typically amplify DNA fragments of up to ~10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.[5]"
What that means is this: you have a sample of some DNA or protein. Typically it might be only one copy or only a handful of copies. Nothing in bioscience works on tiny copies; you typically need thousands or millions, particularly for operations like dna sequencing. PCR allows you to amplify -- ie copy -- your sample by causing the DNA helix to split from heat, including a polymerase or enzyme that does the replication work, including spare dna bases, then cooling to cause the helixes to reform. It's essentially a doubling operation; you run k heating/splitting/replication/cooling cycles and at the end hopefully have (#{ starting copies }) * 2^k
Doing the previous operation requires a pcr device which very accurately and evenly heats and cools a microtitre plate; the polymerase; something to dump all this into like very clean water; and raw dna base pairs.
[1] http://en.wikipedia.org/wiki/Polymerase_chain_reaction
What that means is this: you have a sample of some DNA or protein. Typically it might be only one copy or only a handful of copies. Nothing in bioscience works on tiny copies; you typically need thousands or millions, particularly for operations like dna sequencing. PCR allows you to amplify -- ie copy -- your sample by causing the DNA helix to split from heat, including a polymerase or enzyme that does the replication work, including spare dna bases, then cooling to cause the helixes to reform. It's essentially a doubling operation; you run k heating/splitting/replication/cooling cycles and at the end hopefully have (#{ starting copies }) * 2^k
Doing the previous operation requires a pcr device which very accurately and evenly heats and cools a microtitre plate; the polymerase; something to dump all this into like very clean water; and raw dna base pairs.
[1] http://en.wikipedia.org/wiki/Polymerase_chain_reaction
Really cool stuff.
Of course, you need to get the sequence after you've got your amplified DNA. That involves performing electrophoresis or using a more modern DNA sequencer machine.
Questions:
Do people still work with gels and electrophoresis anymore? That seems like a barrier for a DIY shop. Not only does it involve a lot of equipment, but the interpretation of results seems like it would be hard.
Is it possible to send out the amplified DNA and get it sequenced by a service? I know that sequencing costs have gone down drastically with new technology, perhaps the DIYer can benefit from that?
Of course, you need to get the sequence after you've got your amplified DNA. That involves performing electrophoresis or using a more modern DNA sequencer machine.
Questions:
Do people still work with gels and electrophoresis anymore? That seems like a barrier for a DIY shop. Not only does it involve a lot of equipment, but the interpretation of results seems like it would be hard.
Is it possible to send out the amplified DNA and get it sequenced by a service? I know that sequencing costs have gone down drastically with new technology, perhaps the DIYer can benefit from that?
Yes, DNA sequencing is about $5 to $10 a sample. Just send in your DNA!
Electrophoresis is nice and fast relative to sending in your sample, but currently much more complicated than dropping a tube of DNA in the mail.
Tito
Electrophoresis is nice and fast relative to sending in your sample, but currently much more complicated than dropping a tube of DNA in the mail.
Tito
What's your typical use case for one of these? I've read the wiki page on PCR but I still don't see it.
Many of the "next-gen" high-throughput whole-genome sequencing technologies are actually PCR using fancy substrates. This isn't that, but maybe it will turn you on a bit to PCR.
In general, PCR is useful anytime you want to isolate, amplify, or manipulate relatively short stretches of DNA.
Do you want to isolate a gene and knock-in a mutation that is known to cause a genetic defect so you can study it? Do you want to tag that gene with a fluorescent tail? Do you need to stitch together several shorter DNA components into a single unit (perhaps joining a zinc finger to a nuclease and creating your very own targeted DNA-cutting enzyme)?
If so, PCR might be for you.
In general, PCR is useful anytime you want to isolate, amplify, or manipulate relatively short stretches of DNA.
Do you want to isolate a gene and knock-in a mutation that is known to cause a genetic defect so you can study it? Do you want to tag that gene with a fluorescent tail? Do you need to stitch together several shorter DNA components into a single unit (perhaps joining a zinc finger to a nuclease and creating your very own targeted DNA-cutting enzyme)?
If so, PCR might be for you.
Hi,
Tito here from OpenPCR. That's a fantastic question! The typical use case for PCR is "doing stuff with DNA" along the lines of what you read on the wiki page about PCR.
We hope that OpenpCR will enable new uses of PCR, where perhaps the user doesn't need to know much about DNA. What if PCR could "tell you whether you have the flu or a cold", or "is this sushi really yellowfin tuna", or "is this genetically modified corn?".
What do you think of those apps?
Tito
Tito
The first two definitely seem like things that one could easily imagine a kit for: include primers for the species of interest, a bit of positive control, and perhaps even the Taq and other reagents you'd need. Then, the user could just collect their sample and run the PCR and a gel.
The third might be a bit harder to make a commercial kit for while steering clear of patents. It's also not clear to me what kinds of samples you'd be able to test. An ear of fresh corn would probably work, but what about (un-)popped popcorn, or cornflakes, or corn starch? It would definitely take some experimentation.
To make these kits really user friendly, ideally you could figure out some quick, easy way to determine the result of the PCR without running a gel, which require a relatively high voltage DC power supply and usually use Ethidium Bromide (a chemical that, depending on who you believe, is either nasty or really, really nasty) and UV light. I'm thinking maybe some kind of spectrophotometer-like setup, which are used in things like a NanoDrop, but I don't know how easy/economical it would be to build a DIY-level work-alike.
The third might be a bit harder to make a commercial kit for while steering clear of patents. It's also not clear to me what kinds of samples you'd be able to test. An ear of fresh corn would probably work, but what about (un-)popped popcorn, or cornflakes, or corn starch? It would definitely take some experimentation.
To make these kits really user friendly, ideally you could figure out some quick, easy way to determine the result of the PCR without running a gel, which require a relatively high voltage DC power supply and usually use Ethidium Bromide (a chemical that, depending on who you believe, is either nasty or really, really nasty) and UV light. I'm thinking maybe some kind of spectrophotometer-like setup, which are used in things like a NanoDrop, but I don't know how easy/economical it would be to build a DIY-level work-alike.
GMO testing: there's a kit available for high schoolers (and we've used it too)
http://www.carolina.com/category/teacher+resources/classroom...
Pretty sweet!
Tito
Pretty sweet!
Tito
Congrats on shipping Tito!
(This is Mike Katsevman btw :>)
(This is Mike Katsevman btw :>)
[deleted]
Looks like the site might be down--here's a repost of most of the content on Make Magazine's blog:
http://blog.makezine.com/archive/2011/07/dna-is-now-diy-open...
http://blog.makezine.com/archive/2011/07/dna-is-now-diy-open...
I just built our KickStarter ordered OpenPCR today. The directions were easy to follow. And the assembled version is strong. Waiting on our order for a dremelfuge (a centrifuge attachment for a dremel) and our electrophoresis box. Can't wait to try it out!
You rock, Brett!
Tito
I have been out of the lab for a while, but as I recall the consumables were an expensive part of PCR. Taq polymerase used to be pretty costly. Perhaps scale has caused that to come down recently? I see a lot of comments from hackers here questioning why this would be useful. In general, you can't get anything done in molecular biology without PCR. It's a critical tool for manipulating DNA. If you're going to do even the most basic biological manipulation (inserting genes into E. coli, comparing dan samples, amplifying small quantities of DNA for further work, etc...) you need PCR.
Yes, the cost of the reagents (taq++) is now cheap, about ~$1 per run, and DNA sequencing is cheap ($5 to $10 a run).
Tito
Tito
That's disruptive tech. Very impressive guys.
Could you talk about the most difficult technical challenge you came across? The temperature regulation (i.e. the heart of the pcr machine) seems to me like the toughest part to get right.
I had the luck of working in a couple of well funded research labs, and I wonder if some of the tall prices we see for this type of lab equipment is due to "using someone else's" money for research.
In any case, I'm sure there'll be plenty of cash strapped labs around the world who'll make some science with it.
Could you talk about the most difficult technical challenge you came across? The temperature regulation (i.e. the heart of the pcr machine) seems to me like the toughest part to get right.
I had the luck of working in a couple of well funded research labs, and I wonder if some of the tall prices we see for this type of lab equipment is due to "using someone else's" money for research.
In any case, I'm sure there'll be plenty of cash strapped labs around the world who'll make some science with it.
Thanks, we hope to get the word out to a lot of cash strapped labs. Suggestions where find them?
Re: Challenges -- Sounds like a good title for a future blog post: "The Toughest Challenge of OpenPCR". Off the top of my head, 2 challenging areas were the mechanical design of the heated lid and super-hacking Arduino for our USB control. For instance with the heated lid, we knew we had to have a flat hot surface come down on top of the PCR tubes. We went through several mechanical prototypes, and learned that the high tolerance of the heated lid meant that the tolerance for the rest of the device needed to match. Not giving you enough details at this point, but look out for that "challenges" blog post in the future :)
Tito
Re: Challenges -- Sounds like a good title for a future blog post: "The Toughest Challenge of OpenPCR". Off the top of my head, 2 challenging areas were the mechanical design of the heated lid and super-hacking Arduino for our USB control. For instance with the heated lid, we knew we had to have a flat hot surface come down on top of the PCR tubes. We went through several mechanical prototypes, and learned that the high tolerance of the heated lid meant that the tolerance for the rest of the device needed to match. Not giving you enough details at this point, but look out for that "challenges" blog post in the future :)
Tito
I'll keep an eye out for that, it'll be an interesting read.
As to where to find cash strapped labs, definitely not the elite institutions that we always hear of, but perhaps more local or state universities.
This might be a tool that international labs in less well funded countries could appreciate, have you reached out internationally?
As to where to find cash strapped labs, definitely not the elite institutions that we always hear of, but perhaps more local or state universities.
This might be a tool that international labs in less well funded countries could appreciate, have you reached out internationally?
Awesome! Are you guys going to make an open dna microarray next? ;)
http://en.wikipedia.org/wiki/DNA_microarray
http://en.wikipedia.org/wiki/DNA_microarray
This is fantastic!
Imagine you want to do something with DNA, perhaps you'd like to sequence something, or stick some in on orange, or beer yeast.
So you have this bit of DNA, but it's so incredibly tiny and fragile, it's almost not real, it's more like the idea of a thing then a thing.
So you use one of these PCR machines to make your idea real. You put your tiny, fragile sequence in and out comes a robust bucket full of DNA!
Imagine you want to do something with DNA, perhaps you'd like to sequence something, or stick some in on orange, or beer yeast.
So you have this bit of DNA, but it's so incredibly tiny and fragile, it's almost not real, it's more like the idea of a thing then a thing.
So you use one of these PCR machines to make your idea real. You put your tiny, fragile sequence in and out comes a robust bucket full of DNA!
As a former biotech grad now starting to finding my feet at contributing on github, this just put a massive smile on my face.
Congrats on shipping, Tito. Glad to see you carrying the iGEM(-inspired) torch onward.
[deleted]
so if I'm new to this, how can I use this machine to test out for diseases? That seems like a good first practical use.
[deleted]
Here's one way of doing it with a PCR machine:
1. First find a risk gene of interest. You can find a gene name via flipping through scientific articles about the disease, and then finding the exact sequence of the gene on NCBI (http://www.ncbi.nlm.nih.gov/projects/genome/guide/human/inde...).
2. Design primers that amplify around the region of interest. ($15 maybe tops?)
3. Toss everything into a PCR machine and amplify it up. ($3 or so for per-run reagents?)
4. Take your amplified product and send it off for sequencing (another like $10 or so?). When you get the sequence back, compare your sequence to the published sequence and see how it looks!
There are a few caveats in primer design, and there are some other ways of detecting disease markers, but this is one easy relatively low-cost approach.
1. First find a risk gene of interest. You can find a gene name via flipping through scientific articles about the disease, and then finding the exact sequence of the gene on NCBI (http://www.ncbi.nlm.nih.gov/projects/genome/guide/human/inde...).
2. Design primers that amplify around the region of interest. ($15 maybe tops?)
3. Toss everything into a PCR machine and amplify it up. ($3 or so for per-run reagents?)
4. Take your amplified product and send it off for sequencing (another like $10 or so?). When you get the sequence back, compare your sequence to the published sequence and see how it looks!
There are a few caveats in primer design, and there are some other ways of detecting disease markers, but this is one easy relatively low-cost approach.
Comment [dead] for some reason (false positive):
http://news.ycombinator.com/item?id=2738007
http://news.ycombinator.com/item?id=2738007
- You buy some primers which are basically chunks of nucleic acid that match whatever gene you want to look at.
- You put DNA you've extracted together with these primers into the PCR machine. This makes lots of copies of the gene you're interested in.
- Then you put the resultant amplified DNA into an electrophoresis gel box (such as the one sold by http://www.pearlbiotech.com/ who also provide free plans. This is also run by Tito Jankowski of the OpenPCR project) which lets you look at the actual genotype (you compare the striations from your sample against other samples or a reference).
Primers are cheap and easy to get hold of and as other comments mentioned there are various software tools for letting you design them (you then place an order with a company like Invitrogen).
Edited to add:
The reason you need to amplify stuff is because biology is annoying like that. Let's use a simile:
If particle physics is figuring out how a watch works by smashing it against a wall and studying the debris
Then biotech is throwing gears and springs into a test tube and carefully stirring at certain temperatures until you can hear ticking.