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Dana-Farber Team Developing High-Throughput Cold-PCR for NGS-Based Mutation Analysis

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By Ben Butkus

A research group at the Dana-Farber Cancer Institute that invented the Cold-PCR method has been awarded a $131,000 grant from the National Cancer Institute to develop a massively parallelized version of the technique to enrich mutant sequences from colon cancer samples prior to next-generation sequencing analysis.

The researchers are hoping that the work will serve as a stepping stone toward using next-generation sequencing to screen individual patients' tumors for multiple cancer-associated mutations and help guide therapy, according to a recently published grant abstract.

Mike Makrigiorgos, an associate professor and director of medical physics and biophysics at Dana-Farber, will serve as principal investigator on the project, which began receiving funding earlier this month.

Makrigiorgos' lab first described "co-amplification at lower denaturation temperature," or Cold-PCR, in 2008. The technique selectively amplifies mutant DNA by recognizing that mutant DNA strands denature at lower temperatures in a PCR reaction than normal DNA strands.

As such, the method can be used to preferentially amplify minority alleles from mixtures of wild-type and mutation-containing sequences, irrespective of where the mutation lies, allowing for strong enrichment of mutant alleles prior to downstream analysis.

Omaha, Neb.-based Transgenomic has an exclusive license from Dana-Farber to use the Cold-PCR method with pyrosequencing, Sanger sequencing, and mitochondrial DNA analysis, and as such is attempting to work with pharmaceutical partners to screen for various mutations in blood that could serve as early indicators of cancer or help guide therapy (PCR Insider, 10/22/09).

Cold-PCR would also be powerful in front of next-generation sequencing methods in order to sequence mutant alleles from cancer samples, potentially from individual patients. However, the relatively low throughput of Cold-PCR could be a bottleneck in such a scenario.

"We know it works well for single reactions," Makrigiorgos told PCR Insider this week. "However, if one is to combine it with next-generation sequencing, which is what we are trying to do here, it would be very helpful to parallelize and have a high-throughput version of Cold-PCR."

"Up to now we have been talking about enrichment of a specific amplicon," Makrigiorgos added. "Now because of the highly parallel nature of the approach, we are talking about enriching the mutated genome from a clinical sample."

The grant abstract identifies RainDance Technologies' nanodroplet technology as a possible solution for this problem, and Makrigiorgos' group is investigating the use of that platform under the new grant.

However, RainDance's technology "is not the only way," Makrigiorgos noted. "Another [possibility] is Fluidigm. And a third is emulsion-based PCR. We are working on all fronts at the same time."

The group hopes to work with RainDance and Fluidigm to test their platforms prior to purchase to see if they work for the proposed problem. The emulsion PCR approach, Makrigiorgos added, can be performed in the group's lab at Dana-Farber without purchasing a specific technology platform.

The grant abstract also identified Illumina as a potential provider of next-generation sequencing technology, but again Makrigiorgos noted that his group will go with whatever platform works the best at the lowest cost.

"We proposed the Illumina platform, but NCI understands that the technology is developing very fast," Makrigiorgos said. While the group is considering Illumina's upcoming benchtop sequencer, the MiSeq, "it could be that by the time we're ready to apply [the technology], if there is a different company like [Life Technologies'] Ion Torrent that is offering the same thing 10 times cheaper, then we have no ties to Illumina. Being an academic institution, of course cost is a major [concern] for us."

Once Makrigiorgos and colleagues have combined Cold-PCR with the various high-throughput PCR and sequencing technologies, they plan to evaluate each platform by identifying mutational fingerprints, including low-level mutations, in tumors from 20 colon cancer patients, and then follow these fingerprints in plasma during the course of radio-chemotherapy to provide a molecular surrogate to therapy response, the grant's abstract states.

Makrigiorgos' lab also recently developed a new version of Cold-PCR called "improved and complete enrichment," or Ice Cold-PCR, which further enhances the sensitivity of the original technique through the use of locked nucleic acid base pairs and specially engineered reference strands. Earlier this year, Transgenomic also licensed that technology from Dana-Farber for use with Sanger sequencing and pyrosequencing (PCR Insider, 3/17/11).

"At the time our grant was submitted, Ice Cold-PCR had not yet been invented, so this [grant] is for the standard Cold-PCR method," Makrigiorgos said. "It may be that as the work develops we end up using Ice-Cold-PCR."


Have topics you'd like to see covered in PCR Insider? Contact the editor at bbutkus [at] genomeweb [.] com.

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