Fast, Accurate and Green PCR

Image: Bill Liao

About the author

Suzanne Kennedy

Suzanne is Director of R&D at Mo Bio Laboratories in California, and the author of their blog, The Culture Dish. She has a PhD in Microbiology and Immunology from Virginia Commonwealth University.

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Attending the 2009 American Society of Microbiology meeting this week in Philadelphia, I decided to take my own advice and visit the exhibits to see whether any of the exhibiting companies had any really innovative and unique products on display.

I focused mainly on products I might use in my own lab, such as PCR reagents and basic lab products. And there was one company that had something that really made me stop and say “wow!”.

That company was Finnzymes, and the “wow” came from three products that are individually impressive but together make a superb PCR ensemble that is compact, versatile and offers excellent speed and accuracy.

A Compact Thermal Cycler…

piko_size_comparison2First up was the Piko Thermal Cycler, which is the smallest thermal cycler I have ever seen. Amazingly, this tiny instrument was around the size of a vortex mixer; with a footprint of only 16cm wide by 17 cm deep and 23 cm high and a weight of only 4 kg. Check out the picture on the right for a comparison of footprint sizes for the Piko and a conventional thermal cycler.

Even better, the Piko uses a 1/4 of the power of a normal thermal cycler, so this is one way for our experiments to be a bit greener (for more ways, take a look at this article). And it offers signficantly reduced reaction times. More on that later.

The instrument ranges in price from $4500-$6000 depending on the blocks you purchase (there are several options), so it also costs significantly less than traditional thermal cyclers.

A thermal cycler that won’t hog your bench space ticks one box. But your PCR is only as good as your polymerase, and Finnzymes have thought of that too..

A Speedy, Accurate Polymerase…

phusionFinnzymes’ Phusion polymerase is well known to us at Bitesize Bio. In fact it scooped the awards for “Highest Fidelity” and “Highest Processivity” at our 2008 Thermostable Polymerase awards.

At ASM I had the pleasure of hearing about this great enzyme from Managing Director of Finnzymes USA, David Unger.

David explained that the power behind Phusion comes from the unique dsDNA binding domain fused to a Pyrococcus-like proofreading polymerase, which results in a very tight association of the enzyme with the template DNA.

“Taq polymerases work by moving on and off the template. This slows down the enzyme and leads to difficulty if inhibitors are present.

With Phusion, the enzyme stays linked to the template and so has a faster processivity along with the ability to work in inhibiting conditions such as 20% whole blood in the PCR reaction.”

The enzyme is twice as fast as Taq (<15 second/kb extension time) and can amplify long templates, up to 20 kb with less enzyme. Just as importantly, the error rate is 50 fold lower than Taq and 6 fold lower than standard Pyrococcus furiosus DNA Polymerases.

Sounds great (and it is). Together, Piko and Phusion offer fast, accurate and economical PCR.

But throw in Finnzymes’ patent pending UTW (Ultra Thin-wall) tubes and plates and the whole thing gets a lot more powerful.

96 x 5ul Reactions in 10 minutes…

The UTW PCR plates are, as the name suggests, ultra thin, which dramatically cuts down on the time taken to heat and cool the samples.

Piko is available with with a 24-well block for UTW 0.2 ml tubes, but for maximum output the instrument can be equipped to take Finnzyme’s 96-well UTW Piko PCR Plates.

These plates are about the size of a microscope slide and Piko can take up to 4 of them to make the equivalent of a 384-well plate for easy up and downstream processing with other lab equipment.

Using Phusion, your 384 x 5 ul reactions can be completed in just 10 minutes flat. Now that’s bench-scale high throughput.

At that speed and size, even the smallest lab can forget about PCR backlogs or downtime.

Of all the new products on display at ASM from the 250 vendors exhibiting, this little machine and the accompanying enzyme look like stand outs. Next time my lab is looking to purchase a new thermal cycler or needs to increase our capacity, I will be buying a Piko.

If you have tried any or all of these products, we’d love to hear your views…



9 comments on this article already!

  1. J??rgen

    1 year ago

    We use Phusion Polymerase in our lab …and it is indeed a great enzyme, very reliable and fast. Before we used KOD polymerase but we always got problems due to unspecificity of that enzyme when too long extension times were used. This we never observed with the Phusion, since you can use a much higher annealing temperature compared with conventional enzymes you will get, most of the time only the product of interest …and no additional bands as observed frequently with KOD.

  2. Kurt

    1 year ago

    When you talk about “Green PCR”, I thought about Fermentas DreamTaqâ„? Green DNA Polymerase, which comes with ready applied tracking dyes, for direct loading in the gel.

    http://www.fermentas.com/catalog/modifyingenzymes/dreamtaqgreenpol.htm

  3. Johannes

    1 year ago

    Phusion is excellent BUT some long Primers simply will not work with Phusion. Same reaction with Taq gives excellent amplification, but with Phusion…NADA. And yes I have tried the whole portfolio of PCR Tricks and Tweaks.

  4. Suzanne

    1 year ago

    Hi Jurgen,
    Thanks for your comments!

    Hi Kurt,
    I looked up the Fermentas Taq but besides the dye, what makes it “enhanced”, do you know? The website doesn’t give much detail. A lot of companies are adding dye to their taq now for easy direct loading onto gels.

    Hi Johannes,
    When you say long primers do not work, how long is long? What applications are you using the longer primers for? Like for adding in restriction sites for cloning?

    Thanks,
    Suzanne

  5. Johannes

    1 year ago

    @Suzanne
    Long means 80-120 Bp. We use these for adding promotors, Tags or recombination sequences.
    As mentioned before, normally that works quite well, but for some it just will not work with Phusion.
    If anyone else is encountering this problem, please comment here. I am trying to figure out if it is a sequence or secondaray structure specific problem.

  6. Ivan

    1 year ago

    I’ve owned a Piko for 2 years now and find it to be highly reliable and robust. When you first look at the Piko it is hard to believe that it is a full fledged 96-well PCR instrument. A few thousand PCR reactions later I must say that the Piko gets the job done as well as any PCR instrument out there.

  7. Max

    1 year ago

    The Piko cannot do gradients (but I at least don’t use them anymore).

    I’d be very careful with UTW plates as we don’t know how successful they will be and how Finnzymes will change their price. If they don’t sell to many Pikos and they need money one day, they might very well increase the price of the UTW plates (which is already high) I think we all learnt a lesson with the “free” Vector NTI licence from Invitrogen. So I’d rather buy a PIKO for normal tubes…

    I’d be interested to hear more about this story of Phusion and long primers…

  8. Suzanne

    1 year ago

    Hi Max,
    Those are important concerns you bring up so I checked with Finnzymes and they tell me that the plastics have been the same price for the last 3 years and because they own the molds, production cost will not change. Also, the plates can be used in many different cyclers, not just the Piko, so there will never be a reason to increase the price based on sales of the instrument. So no need to worry about the UTW prices spiraling out of control.
    Thanks for commenting!

    Suzanne

  9. Nancy

    1 year ago

    I love using Phusion either when you want a quicker PCR reaction, or when you need a polymerase that is high fidelity. Using Phusion I was able to get large yields of my product, where PWO had either failed completely or given poor yields. In addition, the reaction worked using just the standard HF buffer – I didn’t even need to mess around with mixing buffers!

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