Detection of BRAF mutations from solid tumors using Tumorplex™ technology

Abstract

Graphical abstract

Can detection of Braf p.V600E mutation be improved? Comparison of allele specific multiplex sequencing to present tests

“…To further confirm the specificity of the ASMS assay, we tested DNA extracted from a Braf p.V600E negative cell line and blood samples obtained from a normal population; all were negative for Braf p.V600E/K. These results, together with our earlier publication, [14] confirms the specificity of ASMS assay. The ultra-specificity of the ASMS assay is attributed to the combination of the following features.…”

“…Further, each of these primers has a modification at the 5' end so that the cohort of truncated molecules generated from each sequencing primer do not overlap. [14] The mutant sequencing primer (which is a part of the antisense strand) binds to the sense strand of the Braf p.V600E target, and generates a sequence (CTGTAGCTAGA) where 'C' is the first nucleotide. Similarly, the wild-type sequencing primer (which is a part of the antisense strand) binds to the sense strand of the wild-type human DNA and generates a sequence (CTGTAGCTAGA) where 'C' is also the first nucleotide (see Figure 2).…”

“…In contrast, with ASMS, allele-specific primers are used in the identification step (sequencing step) after amplification, generating a Braf p.V600E specific nucleotide sequence that can be verified. [14] Preliminary analytical validation of ASMS shows a lower limit of detection (LLOD) of 20 GE for Braf p.V600E, and a sensitivity ratio of mutant to wild-type of 1: 100,000. , and wild-type human gDNA was also obtained (Promega). Human genomic DNA was extracted from wild-type human cell line NCI-PBCF-CCL247-HCT 116 (Horizon Discovery, UK.…”