Competitive allele-specific TaqMan PCR (Cast-PCR) is a sensitive, specific and fast method for BRAF V600 mutation detection in Melanoma patients

Abstract

BRAF codon 600 mutation testing of melanoma patients is mandatory for the choice of the most appropriate therapy in the clinical setting. Competitive allele specific TaqMan PCR (Cast-PCR) technology allows not only the selective amplification of minor alleles, but it also blocks the amplification of non-mutant allele. We genotyped codon 600 of the BRAF gene in 54 patients’ samples by Cast-PCR and bidirectional direct sequence analysis. All the mutations detected by sequencing were also identified by Cast-PCR. In addition, Cast-PCR assay detected four samples carrying mutations and was able to clearly identify two mutations of uncertain interpretation by Sanger sequencing. The limit of detection of Cast-PCR was evaluated by constructing dilution curves of BRAFV600E and BRAFV600K mutated clinical samples mixed with a not-mutated specimens. Both mutations could be detected until a 1:100 mutated/not mutated ratio. Cloning and sequencing of the clones was used to confirm mutations on representative discrepant cases. Cast PCR performances were not affected by intratumour heterogeneity, and less affected by melanin content. Our results indicate that Cast-PCR is a reliable diagnostic tool for the identification of melanoma patients as eligible to be treated with TKIs and might be implemented in the clinical setting as elective screening method.

BRAF p.V600E status in epithelial areas of ameloblastoma with different histological aspects: Implications to the clinical practice

“…The latter result should be seen with caution, as long as direct sequencing is a less sensitive technique that might not identify mutations in a low percentage frequency. 28,29 However, our findings support that BRAF p.V600E expression in ameloblastoma is homogeneous in the different areas from the same tumour.…”

Using plasma cell‐free DNA to monitor the chemoradiotherapy course of cervical cancer

“…In recent years, great strides have been made to identify mutations in either whole exome or specifically selected clinically relevant genes . Assays such as allele‐specific qPCR (AS‐qPCR), Scorpion/ARMS‐PCR, PNAClamp PCR, droplet or bead‐based digital PCR, are currently being used or developed to detect genetic alterations in the plasma sample. These techniques can reach the high sensitivity of 0.01–1%, however they are only able to detect known mutations in known genes, and their throughput are usually low .…”

The role of molecular genetic methods in the diagnosis of McCune—Albright syndrome

“…Метод высокопроизводительного параллельного секвенирования (NGS), позволяющий анализировать последовательность ДНК в образцах с малым ее количеством, активно используется при детекции соматических мутаций [6,7]. Метод ПЦР в режиме реального времени с использованием техно-логии детекции соматических мутаций TaqMan (competitive allele-specifi c TaqMan PCR) также оказался успешным при исследовании соматических мутаций по образцам с низким содержанием мутантных аллелей [8,9]. Исходя из этого, представлялось возможным использование данных методов для поиска соматических мутаций в образцах ДНК периферической крови и пациентов с синдромом МОБ или с подозрением на него.…”