The SALSA MLPA Probemix P298 BRAF-HRAS-KRAS-NRAS is a research use only (RUO)
assay for the detection of deletions or amplifications in the RAS
) and the BRAF
gene, all core components of the Ras/Raf/MEK/ERK pathway. RAS
) code for small guanine-nucleotide-binding proteins which are essential for signalling networks controlling cellular proliferation, differentiation and survival. In the Ras/Raf/MEK/ERK pathway, RAS proteins are regulated by receptor tyrosine kinases (RTKs) which are activated upon growth factor binding. These RTKs mediate addition of an active GTP by GTPase activating proteins (GAPs) or an inactive GDP by GTP exchange factors (GEFs) (Simanshu et al., 2017). Downstream proteins will only be activated by the active RAS-GTP, but not by the inactive RAS-GDP. Oncogenic mutations in RAS
occur in about 15% of human cancers, predominantly affecting the G12 residue of the protein. These oncogenic mutations result in the constitutive activation of RAS by keeping the protein in its GTP-bound state, thereby bypassing the need for growth factors to activate the pathway (Simanshu et al., 2017; Braicu et al. 2019).
BRAF is activated by RAS-GTP as a result of ligand-induced RTK activation in healthy cells. Mutations in the activating kinase domain of BRAF cause overactive downstream signalling via MEK and ERK, leading to excessive cell proliferation and survival independent of growth factors. The BRAF p.V600E (c.1799T>A) mutation is the most frequent mutation which confers oncogenic BRAF function (Davies et al., 2002; Braicu et al. 2019).
This SALSA MLPA probemix is not CE/FDA registered for use in diagnostic procedures. Purchase of this product includes a limited license for research purposes.
The SALSA MLPA Probemix P298 BRAF-HRAS-KRAS-NRAS contains 57 MLPA probes with amplification products between 115 and 504 nucleotides (nt). This includes 19, seven, nine and seven probes for the BRAF, HRAS, KRAS
These 42 probes include one probe specific for the BRAF
p.V600E (c.1799T>A) mutation which will only generate a signal when the mutation is present, and two probes for KRAS c.34G and c.35G, both located in codon 12, which will only generate a signal when the wildtype allele is present. In addition, 15 reference probes are included that detect autosomal chromosomal locations and target relatively copy number stable regions in various cancer types. Complete probe sequences and the identity of the genes detected by the reference probes are available online (www.mrcholland.com
) and in Table 3.
This probemix contains nine quality control fragments generating amplification products between 64 and 105 nt: four DNA Quantity fragments (Q-fragments), two DNA Denaturation fragments (D-fragments), one Benchmark fragment, and one chromosome X and one chromosome Y-specific fragment. More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol and online at www.mrcholland.com
SALSA Binning DNA SD029
The SD029 Binning DNA provided with this probemix can be used for binning of all probes including the mutation-specific probe (BRAF probe 08780-SP0039-L21281 for the p.V600E (c.1799T>A) mutation). SD029 Binning DNA is a mixture of genomic DNA from healthy individuals and plasmid DNA that contains the target sequence detected by the above mentioned probe. Inclusion of one reaction with 5 μl SD029 Binning DNA in initial MLPA experiments is essential as it can be used to aid in data binning of the peak pattern using Coffalyser.Net software. Furthermore, Binning DNA should be included in the experiment whenever changes have been applied to the set-up of the capillary electrophoresis device (e.g. when capillaries have been renewed). Binning DNA should never be used as a reference sample in the MLPA data analysis, neither should it be used in quantification of mutation signal(s). It is strongly advised that all samples tested are extracted with the same method and derived from the same source of tissue. For further details, please consult the SD029 Binning DNA product description, available online: www.mrcholland.com
. This product is for research use only (RUO).
Sample DNA developed for this product: