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Transgenomic Improves Mutation Detection Limit for Sanger Sequencing


By Julia Karow

Transgenomic has developed a new method, called BLOCker sequencing, that increases the sensitivity of Sanger sequencing for specific mutations, for example in tumors or viral populations.

Earlier this month at the American Association for Cancer Research meeting in Orlando, Fla., the company presented results from a study in which it used BLOCker sequencing to analyze mutations in the K-RAS gene with up to 8-fold better sensitivity than standard Sanger sequencing. Combined with a mutation enrichment technique called Ice COLD-PCR, detection limits can be further lowered, according to the firm.

BLOCker sequencing — short for BLocking Oligonucleotides in Cycle sequencing — uses an oligo that includes locked nucleic acid to block wild-type DNA from being sequenced at a certain position while allowing DNA containing a mutation at that position to be sequenced. The blocking oligo is added during Sanger cycle sequencing and anneals to the template. The temperature is then raised to the point where the oligo peels off the mutant DNA but remains attached to the wild-type DNA, thus preventing it from being sequenced.

According to Transgenomic CEO Craig Tuttle, BLOCker sequencing lowers the detection limit to 1 percent or better, from about 10 percent in standard Sanger sequencing. Surprisingly, the researchers found they could use the method to detect several mutations in the K-RAS gene, even with an assay that only targeted one mutation. They are currently testing whether this effect is generally applicable. This would allow them, for example, to design one assay kit for all mutations in K-RAS.

BLOCker's sensitivity matches that of current next-generation sequencing platforms, Tuttle claimed. "You are able to gain at least second-gen or more than second-gen sensitivity levels using Sanger sequencing equipment," he said.

Transgenomic also holds licenses to COamplification at Lower Denaturation temperatures, or COLD-PCR, a mutation enrichment method developed by researchers at the Dana Farber Cancer Institute. A version of it, called Ice COLD-PCR, lowers the detection limit to 0.01 percent, and combining it with BLOCker sequencing provides even greater sensitivity.

Since December, the company's pharmacogenomic services laboratory has been offering to pharmaceutical partners research assays that combine the two techniques for detecting mutations in a number of cancer genes.

Pharmas like the assay, Tuttle said, because the output is Sanger data, which the Food and Drug Administration requires them to submit as part of studies. "The FDA prefers Sanger data over pyrosequencing, allele-specific assay data, and second-gen [sequencing] data, because they are familiar with it," he said.

The high sensitivity afforded by Ice COLD-PCR and BLOCker sequencing enables researchers to look for cancer mutations in blood, he said, which could be helpful for resurrecting studies that have run out of tumor material. Transgenomic is currently involved in a study of pancreatic cancer with an academic partner that uses the techniques to look for prognostic markers in blood.

BLOCker sequencing also works well in formalin-fixed paraffin-embedded tumor samples, he added, though most of the firm's service work is currently conducted on blood samples.

"We believe that we have one of the most sensitive techniques, and also one that can find any mutation, rather than being allele-specific," he said. "It's much more cost-effective [than other methods], and easy to multiplex and then profile these tumors with small amounts of material."

Another application, besides cancer, is mutation detection in viral populations, which Transgenomic is offering as a service to partners as well. According to Tuttle, it is possible to enrich at least 90 percent for a specific viral strain with the method.

Next quarter, Transgenomic plans to launch research assay kits that combine Ice COLD-PCR and BLOCker sequencing to detect mutations in important cancer genes, such as K-RAS, NRAS, BRAF, TP53, EGFR, and PIC3CA. "It's very clear that we need to continue to build out that menu to allow both pharma partners as well as clinicians and other researchers to look at other important and emerging biomarkers," Tuttle said.

Have topics you'd like to see covered in Clinical Sequencing News? Contact the editor at jkarow [at] genomeweb [.] com.