Monthly Archives: June 2010

PCR Machine Patents are Dead

One of the goals of the OpenPCR project is “4. A basic understanding of PCR patents and how they affect this project“. When Josh and I were first discussing OpenPCR back in March, I looked into the original PCR patents and found that they were expired as of March 2005 (US) and March 2006 (Worldwide). These patents have been expired for a little over 4 years now. This was covered somewhat by the media, in articles from Frost (2005) and PatentBarista (2006). There are other articles, but they are not freely accessible — email us PDFs if you like and we may share them.

If you’re interested in reading the original patents that were fought over for years:

USPTO 4965188
USPTO 4683202
USPTO 4683195

What’s also interesting is that “expired PCR patents” doesn’t seem to be common knowledge — lots and lots of people know *about* the patents on PCR, but not too many know they expired. Staying current is really important for those of you who want to innovate. The landscape is quickly changing. In the early 2000s, the question was “Can the human genome really be sequenced?”, the answer was YES, DEFINITELY — and we must move on to bigger and better questions. Current questions are “Can a community biotech lab be a source of innovation?“, ”Will every home want a DNA barcode reader and biotech engineering tools before 2025″? We’ll need to answer them and move on. The hurdles in front of us will soon be behind us.

One area that we want to shed light on for future projects is patents around the qPCR process (update – 7/18, also add Gradient PCR to the list, I’m not sure what the patents around that technology are). qPCR is an important tool as it allows you to easily quantify (q) the DNA in your sample. In many cases this can save a lot of time and shorten the “test cycle” of biotech. Imagine if every iGEM team and community lab had not just an OpenPCR, but a qPCR machine at their fingertips!

Are these questions interesting to you?

  1. Is there any reason to think the basic PCR patents aren’t expired? Lawyers, scientists want to help clarify?
  2. Looking forwards — What are the existing patents on qPCR and other advanced processes?
  3. Looking back — Did the patent for PCR help to encourage innovation and proliferation of PCR?

Thanks to Adam Braithwaite for inspiring this post.

The heated lid

Now that the heat sink is ordered, on to the heated lid. The lid is a major component of the OpenPCR machine. It eliminates the mineral oil that screws up experiments of newbies and professionals alike. More importantly, it’s the one part of OpenPCR that you’ll interact with *every* time you do PCR. It needs to be done right.

As I’ve shared with a lot of you, our prototype lid is nichrome wire and silicone tape, sandwiched between two plates of aluminum in a wooden lid and it needs a lot of help. A few posts ago, Josh figured out that a peltier device + aluminum plate would fit the bill, and a custom board heater was suggested as something to try out long term.

Getting hot is only half of the story. The hot piece of aluminum needs to make good contact with the top of the tubes (which can have minor variations in size) — which means flat AND compressed. One idea that was suggested was a double hinged lid that would be able to rotate to be flat for a range of tube heights – kind of like the lid on a scanner, which can accommodate paper, thin magazines, and thick books. We also need a good downward force on the tubes, so that good contact is made. After wrangling on it for awhile, I’m going to try a lid on a single springd-hinge to handle compression and a piece of thermal pad (stock image below) to make contact with the tubes. Bought some on Mcmaster (spring hinge) and digikey (thermal pads, various softness, 2.5 – 3 mm, capable of 100-150C+). I think this will be straightforward to use, cheap, and reliable. We will see :)

We’ve got the aluminum parts on hand to test out these components, but we’ll need some better plates to test with. I’ll be working on putting together a design doc for Ponoko to get the flat aluminum parts made, as well as the case (wood or plastic).

If you want detailed specs, sign up as a donor to OpenPCR. For only $32 you get behind the scenes months before anyone else with access to photos, design documents, and software for the OpenPCR project. http://openpcr.org/kickstarter

A heat sink

Hi all,

Just watching James Bond in Casino Royale and looking for a new heat sink for the OpenPCR machine. Thought I would share the process. We need a heat sink for pulling all the heat away from the aluminum block. Our aluminum block is 4 cm square to match our peltier, which is 4 cm square. If our heat sink had a mounting surface of 4 cm x 4 cm that would be perfect to press against the peltier. The current heatsink has a surface smaller than 4 cm square, which makes it necessary to have an 4 cm square 1/8″ aluminum plate between the peltier and the heat sink. This is an extra layer, which means extra cost and extra complexity. If there’s a heat sink out there with a 4 cm x 4 cm surface with a vertical profile and a way to mount the whole heat sink with screws, I want to find it.

I’ll start with Newegg, I’ve honestly done this search before where I found the first 2 vertical heat sinks we tried. One had a way to mount it but is a small surface , the other had no way to mount it, but the surface was the right size. Since I have a better idea about what we need, I’m giving newegg a second shot. The current $15 heatsink, and the price for a cut piece of aluminum would probably be $5, and I’ll just put a rough estimate that the reduced complexity of the design is worth about $10, so something that meets the spec for under $30 would be great. Its outer dimensions are nice and small, 83.5 x 68 x 117 mm, which I looked up before ordering and measured myself once I received it.

“Oh of course, just find a part with a 4 cm square surface, easy easy!”. What would be great is if there were CAD files for everything out there, but there really aren’t. People who want a heat sink don’t want to mess around with dimensions. Instead, they can ask a wonderfully short question “I have a Intel Core 2 Extreme processor – does this heat sink fit?”. If I’m looking for heat sinks with a 4 cm x 4 cm surface, I really need to be talking about processor sizes. Which is an interesting turn.

So, the first one I’m looking at says “Intel Celeron D (Socket LGA 775)”. I google “Intel Celeron D”, which brings back nothing useful, and then I google “Socket LGA 775″, and wikipedia provides the “processor size” of 1.47″ square. My assumption here is that the surface of the heatsink must be at least as big as the processor it fits, so it’s a bit smaller than I need. This could be an incorrect assumption!

The first heat sink spec also mentions it fits other processors, but they’re all the same socket size. Maybe it would be better to work backwards, starting with the socket size I need, and then find heatsinks that fit that processor. Wikipedia pulls back the “CPU socket” category, and I’m on my way. Looks like LGA 1366 is a bit bigger than what I need, which is great — and Newegg has this one, this one, this pricey one.

http://www.newegg.com/Product/Product.aspx?Item=N82E16835114089 : Gargantuan, 122 x 108 x 157 mm.
http://www.newegg.com/Product/Product.aspx?Item=N82E16835150113 : Masscool AWA743, OK, kind of short and stocky, 95 x 95 x 104. Interestingly enough, looking at the manufacturer’s page, it looks like they just include a plate as an adapter for the 1366 socket.
http://www.newegg.com/Product/Product.aspx?Item=N82E16835192017 : Titan NK35TZ. Again, gargantuan, the overall specs are unavailable. specs say the fan is 95 x 95  25 cm, and doing simple estimation I’m guessing the overall size is around 100 x 175 x 100 mm, over 2.5x the volume of the fan we’re using now, arg.

I went ahead and ordered the Mascool AWA743 and Titan NK35TZ and we’ll see how they look. One concern is that it was difficult to find these, so a design that used a simple aluminum plate but allowed for use of a much wider range of heatsinks would be good.

Dieter Rams 10 principles for good design

Any designers out there who want to make biotechnology that is welcome and accessible?

http://www.designsojourn.com/dieter-rams-and-his-10-design-commandments/

• Good design is innovative.

• Good design makes a product useful.

• Good design is aesthetic.

• Good design helps us to understand a product.

• Good design is unobtrusive.

• Good design is honest.

• Good design is durable.

• Good design is consequent to the last detail.

• Good design is concerned with the environment.

• Good design is as little design as possible.

Make it hot

A good discussion on the OpenPCR heated lid over at O’Reilly Answers:
http://answers.oreilly.com/topic/1641-silicone-heaters-for-openpcr/page__p__3131&#entry3131

We’ve been thinking a lot about the heated lid, and have had some great ideas suggested.
Windell Oskay from Evil Mad Scientist Labs suggested actually making a custom PCB printed with copper trace to form a heater. What an awesome idea, definitely want to try it out.

I also found this great “wire making” tip on the EMSL site, looks good for fixing the ‘rats nest’ of wires we run into from time to time and thought I would share: link

Tonight Josh tried out a simple $16 peltier to do our heating and it worked, so we’ll go with that for now and focus on getting the mechanical design down. The heated lid must make flat contact with all the tubes, so a double hinged lid and some sort of downward pressure (magnets? latch?) are needed.

Tito

The Importance of Speed in PCR

The job of a thermocycler is to implement a set of temperature changes in a reaction volume. The biological requirement for PCR is to implement a set of temperature changes such as this:

Repeat 30 times:

  1. Denature at 95 C for 30 seconds
  2. Anneal at 64 C for 45 seconds
  3. Extend at 73 C for 45 seconds

If you add up the times required, the biological requirements of this reaction take 50 minutes. However it also takes time for the PCR machine to transition between each set of temperatures, which is effectively wasted time. Here’s where the speed of the PCR machine comes in. If the PCR machine is capable of changing 2 C/s, then starting from 20 C the above reaction would take 66 minutes. However if it was only capable of changing 0.5 C/s, the above would take nearly twice as long: 114 minutes.

When we sat down to design a low cost thermocycler, we had options that were cheap but slow, such as relying on convection and radiation to cool the heat block. We opted instead to go with a more complex peltier cooling solution so the machine would be faster: our prototype currently ramps at about 2 C/s.

Why did we feel speed was so important? There’s two reasons.

My formal background is in computer science. When writing new software code, I do not write it correctly the first time. I may have a design flaw, or simply have made some mistakes. However I’m able to get rapid feedback, correct the issue, and test it again, so making mistakes is not a problem at all. In software, it’s typically possible to get feedback in a time ranging from seconds for small changes to at most about 10 minutes to run a well designed test suite on a modestly sized application. That rapid feedback cycle not only allows engineering projects to proceed rapidly, but it is also a large part of how the novice developer learns the art of software development.

When I work in biology, my experience is anything but that. Experiments (or design-build-test cycles) run anywhere from hours for simple operations, to days if cells must be grown, to weeks if DNA must be synthesized, to years for long-running clinical trials. Faster PCR cannot solve all of that, but it can do its part for the experiments where it is critical.

For example, I’ve been working to create a SNP genotyping protocol which would allow people to read any SNP of interest from their own DNA. It’s complex and involves DNA extraction, PCR, and a gel run. Currently it takes the better part of a day to complete; reducing it so it could be run twice a day would be a big win, especially considering someone new to biology is likely to make mistakes the first couple times. We should all be thinking about how we can make design-build-test cycles faster when we create tools for biology.

The second reason is a little more technical. The PCR reaction makes use of a “thermostable” polymerase enzyme, which really means that the polymerase doesn’t denature and cease to function right away at the denaturing temperatures of PCR. In actuality the polymerase has a half-life which is reduced as temperature increases. At the end of a 25 cycle PCR run, the majority of the polymerase may in fact be destroyed, which is a limiting factor in the amount of amplification possible.

That can be a problem if you’re starting from a very small amount of DNA, as may be the case in a forensics application. It is also important if your upstream DNA preparation protocol is sub-par. For example, suppose a DIYbio enthusiast was doing sushi DNA barcoding. While they have access to an enormous amount of DNA to begin with, they may use a lower-cost DNA extraction method which doesn’t remove as many PCR inhibitors as a Qiagen extraction kit would. That’s not a problem so long as you’re still able to get enough amplification for sequencing, which you can do by extending the number of PCR cycles so long as you have enough working polymerase left.

So faster PCR can introduce resilience to overcome poorer upstream processing, and if that enables you to get the same results with cheaper reagents, that’s another win for everyone.

Why we built OpenPCR

First off, let me extend a warm welcome to everyone that’s taken an interest in this project. Over the past two months I’ve been extremely busy with the design and testing of the OpenPCR machine and getting a prototype ready to demonstrate at the Maker Faire, so haven’t had the time to blog about it until now.

I wanted to talk a little about why we undertook this project. Building a PCR machine is something that Tito and I first talked about nearly a year and a half ago, but the project only got started in earnest this February, after Tito and I discussed it driving back from the Outlaw Biology symposium in LA. What really made me jump on it was that I had heard from one too many people that this needed to be done, and I certainly thought it was doable, but I hadn’t seen much progress in actually building a thermocycler to date.

There are really two core benefits I see to a machine like OpenPCR. The first is a drastically lower price point. The cheapest commercial unit I’m aware of costs $4000, with other units easily running up to $10,000. There is no reason these machines have to cost so much. While they have to be accurate, the basic technology is quite mature. The high price point has more to do with the traditional biotech market than it does with the complexity of the machine. Academic and industrial labs can afford these high prices, but there is increasing demand from garage biotech companies, labs in developing nations, the DIYbio community, and even high schools for PCR machines, and OpenPCR was designed to serve these new users.

A common objection to this argument is that “thermocyclers can be found on eBay for $100″, so why re-invent the wheel? eBay can be the solution for some people, but it’s far from a solution for everyone. The $100 eBay thermocycler is actually quite elusive, $300 machines are much more common. It can take quite some time to find the better deals. Even then, these machines are commonly sold “as-is”, which frequently means broken. I stopped buying lab equipment from eBay three years ago for this reason. And even if you do get a working machine for $300, it’s an antique unit without modern features such as a heated lid, and the temperature ramp times are often pathetic.

The second core benefit to building OpenPCR was to create a substrate for further hacking. We’d eventually like to add quantitative functionality to OpenPCR, and I know others who would like to develop more automation around it. You have to walk before you can run, so building a working thermocycler gives us all a starting ground for the more ambitious projects to come.