PrimeSTAR™ HS DNA Polymerase

PrimeSTAR™ HS DNA Polymerase
Takara PrimeStar™ HS DNA Polymerase is touted as having a mutation frequency less than that of enzymes derived from Thermus aquaticus, Pyrocococcus sp and Thermococcus kodakaraenis. The reduced mutation frequency of PrimeStar™ is attributed to the enzyme’s strong exonuclease activity, resulting in 12 errors/250,000 bp. Takara also claims that PrimeStar™ has a higher efficiency than Taq Polymerase resulting in extended product lengths: 8.5 kb for human genomic DNA and 22 kb for λ DNA.

My interest in trying Takara PrimeStar™ HS DNA Polymerase was for the extended product length. Other concerns, such as fidelity, efficiency, and yield, were met with other polymerases, however, I was not able to successfully amplify targets over 5 kb. Previously, when projects were over 2.5 kb, the preferred method was to clone the target in pieces of 2 kb or less. This was because PCR using cDNA for targets over 2 kb could be problematic. Once the pieces were cloned, we would then assemble them for the full-length final clone. The assembly method of cloning proved to be quite laborious. The assembly process often required time-consuming sequence analysis, subcloning, and multiple fragment ligation.

The labor and time-saving answer seemed to be to amplify as much of the target in one attempt as possible. Altering protocols for other polymerases I was using gave mixed results, and one project in particular continued to elude me. It was a 7kb gene, affectionately nick-named "The Anaconda". I tried sample polymerases from various companies that advertised extended product lengths, none of which worked. I expressed this frustration to several of my colleagues and even suggested that I might place a bounty on this gene for anybody who could amplify it in one piece.

The next day, a colleague left a little square of paper on my bench. It was an unpretentious advertisement by Takara Mirus Bio for a "PCR Enzyme". The description seemed to say some of the same things as advertisements from other companies, but the claims were more matter-of-fact rather than marketing hoopla. I figured I could try one more polymerase before throwing in the towel, although I wasn't very optimistic about it. I visited Takara Mirus Bio's website and requested a sample of their PrimeStar™ HS DNA Polymerase. I was immediately contacted by a representatives who said a sample would be sent out the next day.

I set up my PCR reaction exactly as specified in their pamphlet using pooled cDNA as my template for my primary PCR. I gel purified the area I believed the band to be in and used that as template for my secondary PCR. The gel I ran of the secondary PCR gave a bright single band at 7kb. I still have a copy of the gel photo of "The Anaconda" on my wall. Since then I have successfully used PrimeStar™ for 4 other long and difficult targets in the 2.5 to 5kb range, all sequences that are not commonly expressed. As with "The Anaconda" project, other polymerases and protocols failed to amplify these targets. In one case, I was not even able to amplify a 200bp portion of the gene, however with PrimeStar™, I was able to amplify the entire 2.5kb gene in one attempt. I have recommended it to other colleagues that have had difficulties with their PCR and they have had similar successes. In conclusion, PrimeStar™ HS DNA Polymerase delivered on their claims of high fidelity, robust yield, specificity, and its ability to successfully amplify long templates.

Erik Lazarich
Research Associate III
R&D Systems, Inc.
Molecular Biology

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PrimeSTAR™ HS DNA Polymerase
The Good

High fidelity, very efficient, robust, very specific, excellent for long templates

The Bad

May have difficulty amplifying extremely repetitive sequences

The Bottom Line

Excellent choice when a PCR product needs to be generated at all costs

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