The SALSA MLPA
Probemix P298 BRAF-HRAS-KRAS-NRAS is a research use only (RUO)
assay for the detection of deletions or duplications in the RAS
) and the BRAF
) code for small guanine-nucleotide-binding proteins which are essential for signalling networks controlling cellular proliferation, differentiation and survival. In the normal Ras/Raf/MEK/ERK pathway these RAS proteins are regulated by growth factors through tyrosine kinase receptors, which mediate addition of an active GTP by GTPase activating proteins (GAPs) or an inactive GDP by GTP exchange factors (GEFs). Downstream proteins will only be activated by the active RAS-GTP, but not by the inactive RAS-GDP. However, when RAS
is mutated to an oncogenic form (in about 15% of human cancers) this regulation is eliminated and, as a consequence, bypass the regulation of cell growth factors. BRAF plays an important role as intermediary in the Ras/Raf/MEK/ERK pathway. In a normal situation, BRAF is activated by RAS-GTP. When mutated, BRAF causes 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 allows BRAF to signal independently.
The SALSA MLPA Probemix P298-A1 BRAF-HRAS-KRAS-NRAS contains 57 MLPA probes with amplification products between 115 and 504 nucleotides (nt). This includes 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 which detect 15 different autosomal chromosomal locations. These target relatively copy number stable regions in various human tumours. Complete probe sequences and the identity of the genes detected by the reference probes is available online (www.mlpa.com
This probemix contains nine quality control fragments generating amplification products between 64 and 121 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.mlpa.com
SALSA Binning DNA SD029:
The SD029 Binning DNA provided with this probemix can be used as Binning DNA sample for binning of one 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), as for this purpose true mutation positive patient samples or cell lines should be used. It is strongly advised to use test DNA sample and reference DNA samples extracted with the same method and derived from the same source of tissue. For further details, please consult the SD029 Binning DNA product description provided. This product is for research use only (RUO).
Sample DNA developed for this product: