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.


DNAcowboy said...

Interesting post. Congratulation for starting such a blog.

I'm surprised to see that (you think) Eppendorf being the leader in fast cycling!!!!!

Idaho has been the 1st to launch such instruments, then Roche came up with massive distribution of the Lightcycler. To my opinion, Roche IS the leader in fast cycling. At least they have the biggest experience in this field.

But, sure, now there's competition, and like you mentioned, I am waiting to see Qiagen integrating Corbbett's into their module3 of the QiaSymphony (something to happen presumably in the 2010's).

To stay in this field, I should be curious to have news about Beckman Coulter who is actually building the DxN which is supposed to integrate a thermocycler. Where the hell have they been shopping for such a thermocycler???

PCR Guru said...

I think Roche is the leader for Q-PCR FAST cycling. For traditional PCR, however, the Idaho format is a little difficult to use (or that's my overall impression). I think Eppendorf has promoted their fast cyclers better than most, but I've seen huge variations across their block (ie. don't use the outer two rows all around).

It's too bad MJ was dismantled before they could produce a fast cycler. I think those that stayed at Bio-Rad weren't the cream of the crop....they went to Finnzymes.

I think ABI will have the most dominant position will be interesting to see how Invitrogen handles it.

More info on these topics