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Development and Analytical Validation of a DNA Dual-Strand Approach for the US Food and Drug Administration-Approved Next-Generation Sequencing-Based Praxis Extended RAS Panel for Metastatic Colorectal Cancer Samples.

J Mol Diagn. 2020; 
Udar N, Iyer A, Porter M, Haigis R, Smith S, Dhillon S, Meier K, Ward D, Lu J, Wenz P, Buchner L, Dunn T, Wise A, Mueller A, Gutekunst K.
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Molecular Biology Reagents … Plasmids with panel mutations were purchased from GenScript (Piscataway, NJ, USA) … Two of the five sample mixes contained a RAS panel MNV in a plasmid from GenScript (Piscataway, NJ, USA) spiked into a RAS mutant CRC FFPE sample … Get A Quote

摘要

A next-generation sequencing method was developed that can distinguish single-stranded modifications from low-frequency somatic mutations present on both strands of DNA in formalin-fixed paraffin-embedded colorectal cancer samples. We applied this method for analytical validation of the Praxis Extended RAS Panel, a US Food and Drug Administration-approved companion diagnostic for panitumumab, on the Illumina MiSeqDx platform. With the use of the TruSeq amplicon workflow, both strands of DNA from the starting material were interrogated independently. Mutations were reported only if found on both strands; artifacts usually present on only one strand would not be reported. A total of 56 mutations were targeted wit... More

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