title: Detection of BRAF mutations from solid tumors using Tumorplex™ technology.
abstract: Allele specific multiplex sequencing (Tumorplex™) is a new molecular platform for the detection of single base mutation in tumor biopsies with high sensitivity for clinical testing. Tumorplex™ is a novel modification of Sanger sequencing technology that generates both mutant and wild type nucleotide sequences simultaneously in the same electropherogram. The molecular weight of the two sequencing primers are different such that the two sequences generated are separated, thus eliminating possible suppression of mutant signal by the more abundant wild type signal. Tumorplex™ platform technology was tested using BRAF mutation V600E. These studies were performed with cloned BRAF mutations and genomic DNA extracted from tumor cells carrying 50% mutant allele. The lower limit of detection for BRAF V600E was found to be 20 genome equivalents (GE) using genomic DNA extracted from mutation specific cell lines. Sensitivity of the assay was tested by challenging the mutant allele with wild type allele at 20 GE, and was able to detect BRAF mutant signal at a GE ration of 20:1 × 10(7) (mutant to wild-type). This level of sensitivity can detect low abundance of clonal mutations in tumor biopsies and eliminate the need for cell enrichment. •Tumorplex™ is a single tube assay that permits the recognition of mutant allele without suppression by wildtype signal.•Tumorplex™ provides a high level of sensitivity.•Tumorplex™ can be used with small sample size with mixed population of cells carrying heterogeneous gDNA.
authors.affiliation: MultiGEN Diagnostics LLC, 854 Paragon Way, Rock Hill, SC 29730, United States.
authors.forename: Jacob
authors.initials: J
authors.lastname: Yo
authors.affiliation: MultiGEN Diagnostics LLC, 854 Paragon Way, Rock Hill, SC 29730, United States.
authors.forename: Katie S L
authors.initials: KS
authors.lastname: Hay
authors.affiliation: MultiGEN Diagnostics LLC, 854 Paragon Way, Rock Hill, SC 29730, United States.
authors.forename: Dilanthi
authors.initials: D
authors.lastname: Vinayagamoorthy
authors.affiliation: MultiGEN Diagnostics LLC, 854 Paragon Way, Rock Hill, SC 29730, United States.
authors.forename: Danielle
authors.initials: D
authors.lastname: Maryanski
authors.affiliation: MultiGEN Diagnostics LLC, 854 Paragon Way, Rock Hill, SC 29730, United States.
authors.forename: Mark
authors.initials: M
authors.lastname: Carter
authors.affiliation: MultiGEN Diagnostics LLC, 854 Paragon Way, Rock Hill, SC 29730, United States.
authors.forename: Joseph
authors.initials: J
authors.lastname: Wiegel
authors.affiliation: MultiGEN Diagnostics LLC, 854 Paragon Way, Rock Hill, SC 29730, United States.
authors.forename: Thuraiayah
authors.initials: T
authors.lastname: Vinayagamoorthy
keywords: BRAF V600E; Formalin fixed paraffin embedded; Lowest level of detection; Multiplex Sanger sequencing platform; Tumorplex™; Tumors; Weighted sequencing primers