Bisulfite-converted Duplexes for the Strand-specific Detection and Quantification of Rare Mutations

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Unmet Need
One of the most important opportunities in the future of the fight against cancer is the detection of cancer-related genetic mutations in the blood or bodily fluids at early stages of cancer, at the early stages of recurrence, or to become aware of changing cancer genetics during a treatment course. Studies have shown that blood, saliva, pap smear, or pancreatic cyst fluid samples can be used to detect circulating tumor DNA (ctDNA) at early-mid stages of cancer progression. However, most approaches to analyzing the DNA samples depend on sequencing instruments with relatively high error rates, which limits the detection sensitivity to around 1 mutant among 100 wild-type molecules.  Unfortunately, in liquid biopsies the incidence of ctDNA in normal DNA is often closer to 1 in thousands of molecules. In these cases, for the earliest possible detection of mutant sequences, a new, more sensitive approach is needed.
Technology overview
Johns Hopkins researchers have developed a method of greatly increasing the sensitivity of mutation detection from fluid patient samples by filtering out DNA mutations made in the process of analyzing the samples. The important new step is initial bisulfite treatment of the DNA, which converts all non-methylated C residues to U residues, allowing researchers to differentiate the two original strands of DNA that are input. Then, a molecular barcode is added to the strands, and they are amplified by PCR. These two steps allow scientists to separate mutations that get introduced in error by sequencing instruments or from PCR: If a mutation is from the original patient sample, it will be found in every sample with the same barcode and in both strands of the template DNA. This method identifies and removes errors from high-throughput sequencing and PCR, increasing sensitivity of detection to better than 0.02%, or 1 in 5000. Additionally, the inventors can barcode all DNA from a specific patient, allowing patient samples to be combined and analyzed more efficiently.
Stage of Development
The inventors have demonstrated the ability to detect and quantify cancer-related mutations at very low frequency in patient samples.
Mattox AK, et al. PNAS 114. 4733-4738, 2017
Patent Information:
Title App Type Country Serial No. Patent No. File Date Issued Date Expire Date Patent Status
Bisulfite-converted Duplexes for the Strand-specific Detection and Quantification of Rare Mutations PCT: Patent Cooperation Treaty PCT PCT/US2018/022664   3/15/2018     Pending
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Jeanine Pennington
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