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German Researchers Make Mutant Polymerase for Mismatch Discrimination, Set to Launch Company

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NEW YORK (GenomeWeb) — Biochemists at the University of Konstanz in Germany have created a mutant polymerase with high activity and high mismatch extension selectivity. The new enzyme is a variant of the large Klenow fragment of Thermus aquaticus DNA polymerase, or KlenTaq.

In a study published this month in PLoS One, the enzyme was used for SNP detection, methylation-specific amplification, and human leukocyte antigen analysis. The new polymerase ─ called HiDi for high discrimination ─ was also shown to be suitable for allele-specific amplification directly from whole blood without the need for sample purification. The researchers plan to launch a company in June to sell HiDi.

In an email interview with PCR Insider, author Andreas Marx explained that "HiDi" is meant to be pronounced like the name Heidi.

"The pronunciation is an allusion to the famous children's TV series 'Heidi, Girl of the Alps.' … Konstanz is [located] at the northern foot of the Alps, close to 'Heidi-land'", he said. This name is an alternative to "KlenTaq R660V."

Marx's group initially did saturation mutagenesis of the five amino acids of KlenTaq that contact the primer strand when the polymerase is in a closed conformation. They selected for mutants that had increased extension selectivity, or, in other words, ones that had a lower cycle threshold with matched DNA than with mismatched substrates. They also selected for mutants that were the most highly active.

The top three mutants were screened for their ability to detect the Factor II prothrombin SNP. Mutant R660V had a mismatch extension discrimination of 18 cycles, compared to 8.5 for wild type KlenTaq, and also had wild type-like high activity.

HiDi was then compared to Life Tech's Platinum Taq DNA polymerase to see how well it could perform SNP genotyping in human genomic DNA. It was shown to double Ct values for mismatches.

The polymerase was then tested in multiplex allele-specific assays, using purified sample as well as sample mixed with whole blood. HiDi performed well, and the researchers wrote in the paper that these types of experiments could be done on "any real-time PCR machine able to perform melting point analysis with a suitable dye such as SYBR Green I."

"We have already started to further investigate this, by using [whole] blood samples and samples from other body fluids in which the presence of PCR inhibitors usually causes significant problems," Marx said.

"Our studies are still ongoing, but [initial] results are extremely promising. In my opinion, direct sample analysis from a patient, without time-consuming and costly sample purification and preparation, will be an important factor of the diagnostics of the future: Lowering costs of diagnostics without compromising high-quality diagnosis of important disease markers," he said.

In the PLoS One study, Marx's group further tested HiDi on a human leukocyte antigen, or HLA, assay. The HLA super-locus contains a number of genes related to immune function, and determining HLA genotype is particularly important in organ transplant.

"HLA is a really interesting application for the new polymerase, as it is a field of high scientific as well as commercial interest," Marx said. The current research showed HiDi performed better than the ‘gold standard’ commonly used for HLA typing, Marx said.

The group also tested HiDi's ability to discriminate between methylated and unmethylated DNA using synthetic Septin 9 gene DNA that contained dU, the product of bisulfite conversion. They compared HiDi to Qiagen's EpiTect kit, and showed HiDi to have higher activity and discrimination ability. The group has since developed a direct method of methylation detection, requiring no bisulfite conversion, which they will publish in the next few weeks, Marx said.

Marx and his colleagues will launch a commercial enterprise in June to "offer several DNA polymerase-based solutions [and] innovations," he said.

In terms of other work, Marx's group is quite active in the polymerase space. "Besides optimizing HiDi DNA polymerase to get it ready for commercial applications, we are always interested in finding new mutation sites that influence the selectivity and substrate scope of DNA polymerases," Marx said. "This is done in order to gain insights into the mechanisms of these complex 'machines,' [but also] often results in properties that are interesting for advanced applications."

Additionally, Marx said his group is working on a DNA polymerase variant able to use RNA as a template. This functions as "a thermostable reverse transcriptase as well as a DNA polymerase," he said. The recently published polymerase will also be commercialized, Marx said, under the name Volcano Reverse Transcriptase.

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