New England Biolabs has released a new DNA polymerase that it claims provides the highest fidelity amplification currently available while offering robust amplification across the broadest range of amplicons, including those that are GC- and AT-rich.
According to company officials, the new polymerase, called Q5, is expected to help address the growing concern of amplification bias in next-generation sequencing library amplification.
High-fidelity amplification is essential for experiments whose outcome depends upon the correct DNA sequence, such as sequencing, SNP detection, protein expression, and gene function studies, according to NEB.
Results from such amplifications can be compromised by a DNA polymerase incapable of evenly amplifying diverse types of sequences, the company said. For example, high-fidelity PCR of GC-rich regions and other challenging amplicons can lead to lower yields and even non-specific amplification.
In addition, any bias in library amplification for NGS can lead to challenges in sequence assembly and even missing sequence, a problem of growing concern in the sequencing community, NEB said.
"GC rich is certainly the easiest way to think about difficult amplicons, but clearly we know that there is more than meets the eye when it comes to difficult amplicons," Lynne Apone, application and development scientist for New England Biolabs' NEBNext product line, told PCR Insider. "They can't only be explained by high GC content. However, that's the simplest way to think about it."
NEB has for several years been selling an enzyme called Phusion — originally distributed on behalf of Finnzymes but now manufactured by NEB. Phusion is "very robust, with very high fidelity," Fiona Stewart, marketing manager for next-generation sequencing and DNA amplification products, told PCR Insider.
Phusion, however, "doesn't always have the same robustness of amplification with maybe some more difficult types of sequences, including GC-rich regions," Stewart said.
NEB's new Q5 products provide all the attributes of Phusion DNA polymerase, including its ultra-low error rates, alongside a significant improvement in the amplification of very high and very low GC targets, the company said.
More specifically, the polymerase is fused to the processivity-enhancing Sso7d DNA-binding domain, which improves fidelity by more than 50-fold over Taq polymerase. The novel construct also improves speed and performance reliability, amplifying targets up to 20 kb long with extension times as low as 10 seconds per kb.
NEB has yet to publish data supporting its claims, nor has it released the name of any early-access testers of the product. However, internal testing "has been really extensive," Apone said. "We have an entire PCR test panel that essentially interrogates human genomic DNA, as well as other commonly used samples in the lab."
A hot-start version of the Q5 polymerase, which utilizes a synthetic aptamer for room-temperature setup, is also available. Both the Q5 and Q5 Hot Start DNA Polymerases are available as standalone enzymes or in 2X master mix format, especially for NGS applications as part of the company's NEBNet product line.
"For the NGS formulation, we certainly have tested [the enzyme] with the Illumina and Ion Torrent sequencing systems," Apone said.
NEB's Stewart added that in the development of the Q5 formulations, "something that was highlighted to me is the importance of buffer optimization. Sometimes it's easy to focus on the polymerase, but the optimization we've done here has been really extensive and has made major differences in how the product actually works with real-life samples."
Apone and Stewart noted that the new enzyme is "something that people have been asking for" to improve the evenness of coverage for specific types of amplicons in a variety of applications, but particularly NGS.
"Across the board, the major sequencing centers were probably the first to comment about these areas for potential improvement, and they're sequencing so many different types of samples in so many types of applications," Stewart said.
"One of their concerns really across the board is the reduction of bias in amplification steps, and that's really what this enzyme gets you," Apone added. "Because the enzyme is so robust, you can reduce the number of amplification cycles that need to be done, so that also can reduce bias. So, it really has broad application."