Thursday, July 31, 2008

Who Makes the Most Unique DNA polymerase

Well the results from our first survey are in. I have to say, I was very surprised. When I was at a large multinational, we subscribed to various industry reports. All of them, as I recall, pointed to Invitrogen Platinum taq as being the leader in the industry when it comes to DNA least Hot Start. In this survey, Invitrogen scored a lowly 10% of the votes. The highest votes went to Kapa Biosystems (31% of the votes), followed by Qiagen (26% of the votes). Other votes went to Roche (15%) TaKaRa (10% tied with Invitrogen), and finally Finnzymes (5%).

So let's look at the top two:

Qiagen has a number of enzymes that have been carefully selected with optimized buffers. These include: Hot Star High Fidelity with a "special additive to the buffer", Hot Star Taq with a special blend of enzymes, Proof Start with a special buffer to prevent primer degradation, and QuantiTect SYBR Green kit which is optimized over a large linear range. When you look at all of these polymerases, they do perform very nicely as the scientists at Qiagen have spent a great deal of time optimizing the buffers. As an example, SYBR green generally inhibits PCR. Qiagen has used a careful balance with the amount of SYBR green used in its formulation to make sure the reaction efficiency is high. While this sacrifices intensity of signal, you can be assured that your reactions are indeed linear.

Kapa Biosystems takes a very different approach. Where Qiagen has focussed on the buffers, Kapa has spent its time engineered the enzyme specifically to perform better than anything else available. Their lead enzymes are Kapa2G FAST Hot Start. This enzyme can amplify at speeds up to 1 kb per second (not per minute, per second!). That means exension times of about 1-5 seconds. This, according Kapa, turns your slow cycler into a fast cycler, and save you a lot of time...and money. I can appreciate this point. Although we all say we can put the PCR reaction on, do something else for 2 hours, and then come back, the question is "why wait?" If I could get the results in 1/2 an hour or 15 minutes, I could get another run on very quickly and move my research on that much quicker....I wish that happened when I was post-doc'ing (I didn't do that much PCR when I was in grad school). We were almost scooped on a big paper. We were scooped on the follow-on study (at least part of it) and the the PCR was a time sink.

The real gems from Kapa, however, are the enzymes that handle inhibitors. Their Kapa Robust enzyme deals with a wide range of different inhibitors. It comes with two different buffers, and two sets of enhancers. A similar technology went into building their Kapa FAST SYBR kit. In comparison to Qiagen which limits the amount of SYBR, Kapa loads up on the SYBR since its enzymes are not inhibited. As a result the flouresence is through the roof, linearity is great, and combined with their FAST enzymes, it can run a slow protocol, or keep up with today's fast real-time systems to finish reactions in less than 40 minutes. The other notable enzyme is the Kapa HiFi. It claims to have the lowest error rate in the industry, runs 3 times faster than other enzymes, and handle high GC templates very well.

I look forward to hearing about how the changes in enzyme technology helps those of us at the bench. I'm sure we'll see new developments from the likes of Invitrogen to keep up. It should prove very interesting.

Tuesday, July 15, 2008

New age of Researchers in Canada

There has been much made about the disparity in Funding between Canadian Universities and their counterparts in other parts of the world. In the 1990's and into the early part of this century, when the Canadian Dollar was at 65 cents, we saw a mass migration to the U.S. In the past few years, we have seen this reverse.

Now, U.S. is stagnant, and Canada has ramped up its funding, particularly in the fields of genomics and applied sciences. The funding situation in Canada has improved, much to the thanks of the Canadian Foundation for Innovation. We have seen large research buildings go up all across this country from Victoria to Halifax, and all the way in between. Every major Research University has been flush with cash spending it on large capital equipment, and undertaking huge projects.

None of this has been as impressive as what we have seen in Vancouver, with the explosive growth of the Genome Sciences Centre. With their bank of ABI sequencers, MJ Research thermal Cyclers, and massive amounts of supercomputing power, no other research group has created as large an impact (open to debate, I'm sure).

In addition, we are now seeing the likes of Canadian Researcher Tom Hudson, who used to head up the Whitehead (now the Broad) Institute in Boston, come back to Canada. He landed first in Montreal, but now heads up an exciting new group at the Ontario Insitute for Cancer Research in Toronto. One of his first major hires was John McPherson, who is another Canadian who made it big in the U.S. John headed the team at Baylor's Genome Sciences Center to take over as Director of the Cancer Genomics.

What is interesting is the complete shift in technology. Both Tom and John "grew-up" using tradition Sanger based sequencing, relying heavily on PCR and cycle sequencing to get through the mounds of DNA they needed to isolate. Now, using their Next Generation Sequencers, they are able to produce as much data in a week than Baylor or the Broad could put out in year. All of this with much less manpower.

So here we come back to the question about what is happening to the Canadian Biotech community. Newly minted Canadian BSc's, MSc's and PhD's no longer find it as attractive to go to the U.S. CFI has funded large amounts for instrumentation that no longer needs people to run them. It seems that real biology is less important these days, but an understanding of bioinformatics and data analysis are skills that are in demand. That is, if you are in the field of high throughput genomics. The whole lab bench has changed. When you walk into a lab now, you are more likely to see the researchers on their computers, than actually using a pipette.

Is this really biology? What are the essential skills needed by our newest generation of scientists, and how will Canada excel in the Global community? What options do new graduates have with their degrees?

Thursday, July 10, 2008

Will Next Gen Replace Microarrays?

I'm not a user of Microarrays, however, several of my buddies who use this technology tell me they see a growing chance that large applications used by this technology will be replaced by Next Gen sequencers. I'm wondering which platform among the new sequencing companies (Roche, Illumina, ABI, PacBio, Helicos), will slide into the turf of Affymetrix? How will Affymetrix respond? We have already seen the sales for Affy fall off, and so have their corresponding gross margins. How are other players like Agilent doing?

Monday, July 7, 2008

Next Generation in Sequencing, Thermal Cyclers, and Polymerases

For my first post, I would like to stay close to my heart, and talk about what appears to be a new way of doing old science (if you consider PCR old science) least to say there are some new tools for people in the lab using these techniques. It is my hope that as this blog grows, we will have separate strings dedicated to each type of instrument or consumable out there, so that users can show how good, or in some cases how to improve what we are all doing with them. So let's start with the Next Generation Sequencers:

Next Gen, as they are called are promising to replace the traditional Sanger Based sequencers that were dominated by ABI (now part of!). ABI has its own Next Generation platform (which it bought from Agencourt..the SOLiD platform). Unlike Sanger Sequencing, Next Gen will give us choices, and ABI has some good competition from the likes of Roche (purhcased 454 Life Sciences) and Illumina (Solexa). In addition, there are some very interesting newcomers as well, including Helicos and Pacific Biosciences. If you are currently using any of these systems in Canada, or are interested in learning more, post here and we will try to find answers. The more content we can put here, the better off we will all be.

Next Gen is also moving into DNA polymerases. We have all used different Taqs isolated from various organisms. Some gave good fidelity, some great processivity, and the advances were made different buffer blends. That all changed when the scientists at MJ Research (or more correctly, MJ Bioworks) made an entirely new Polymerase by splicing two different proteins together to make "Phusion." A few years later, MJ was sold to Bio-Rad, and this enzyme was re-named iProof, while the company actually making the product was Finnzymes. Finnzymes retained the rights to this technology, and have kept the name Phusion, and have since come out with Phire.

After Beckmann and ABI bought various pieces of Agencourt Biosciences, the scientists there ended up starting a new company making new polymerases. They took the idea of MJ Bioworks (ie. genetically modifying polymerases), but were able to create a full line of polymerases in a fraction of the time MJ did. They now have the Kapa HiFi, which essentially provides the functionality of Phusion (High Fidelity/High Processivty), with better results, particularly with high CG content and short fragments. Has anyone used this?

The Kapa Robust appears to be everyone's dream who has every had to struggle with inhibitors. From Plant to feces, this seems to do the trick. Kapa has a speciatly enzyme for q-PCR that allows you to withstand high levels of SYBR Green. Very interesting.

In addition, the Kapa2G FAST appears too good to be true. A Polymerase that can amplify up to speeds of 1 SECOND per KB? No way. The Kapa folks claim that you don't need to buy a fast cycler, you just need this enzyme. Who's used this one?

That brings me to the new Fast Cyclers. Eppendorf, I think, is the leader in this, but you can never count out ABI of course. And everyone seems to forget Bio-Rad, who purchased MJ. Bio-Rad just came out with their own fast cycler. I have to say, though, it surprises me that the MJ engineers left Bio-Rad, to start their own manufacturing under the Finnzymes brand. These engineers under Finnzymes was able to write patents, and deliver a faster, more accurate cycler to the market in about half the time it took Bio-Rad to release their newest cyclers. I would like to start a string for each instrument so that we can talk about issues for each type of cycler. Post your results here, so that we can all make good decisions.

We can start a second string about Q-PCR. Since Qiagen just purchased Corbett, things are going to get very interesting in clincal world as well as the research lab bench. With Fast Cyclers, FAST kits, and new polymerases, this group will see some changes as well.

More info on these topics