Target inhibitors are used for melanoma treatment, and their effectiveness depends on the tumor genotype. We developed a diagnostic biochip for the detection of 39 clinically relevant somatic mutations in the BRAF, NRAS, KIT, GNAQ, GNA11, MAP2K1 and MAP2K2 genes.We used multiplex locked nucleic acid (LNA) PCR clamp for the preferable amplification of mutated over wild type DNA. The amplified fragments were labeled via the incorporation of fluorescently labeled dUTP during PCR and were hybridized with specific oligonucleotides immobilized on a biochip. This approach could detect 0.5% of mutated DNA in the sample analyzed. The method was validated on 253 clinical samples and six melanoma cell lines.Among 253 melanomas, 129 (51.0%) BRAF, 45 (17.8%) NRAS, 6 (2.4%) KIT, 4 (1.6%) GNAQ, 2 (0.8%) GNA11, 2 (0.8%) MAP2K1 and no MAP2K2 gene mutations were detected by the biochip assay. The results were compared with Sanger sequencing, next generation sequencing and ARMS/Scorpion real-time PCR. The specimens with discordant results were subjected to LNA PCR clamp followed by sequencing. The results of this analysis were predominantly identical to the results obtained by the biochip assay. Infrequently, we identified rare somatic mutations.In the present study we demonstrate that the biochip-based assay can effectively detect somatic mutations in approximately 70% of melanoma patients, who may require specific targeted therapy.
The genus Deschampsia P. Beauv. (Poaceae) involves a group of widespread polymorphic species, and many of them are highly tolerant to stressful environmental conditions. Genome diversity and chromosomal phylogeny within the genus are still insufficiently studied. Satellite DNAs, including CON/COM families, are the main components of the plant repeatome, which contribute to chromosome organization. For the first time, using PCR-based (Polymerase Chain Reaction) techniques and sequential BLAST (Basic Local Alignment Search Tool) and MSA (Multiple Sequence Alignment) analyses, we identified and classified CON/COM repeats in genomes of eleven Deschampsia accessions and three accessions from related genera. High homology of CON/COM sequences were revealed in the studied species though differences in single-nucleotide alteration profiles detected in homologous CON/COM regions indicated that they tended to diverge independently. The performed chromosome mapping of 45S rDNA, 5S rDNA, and CON/COM repeats in six Deschampsia species demonstrated interspecific variability in localization of these cytogenetic markers and facilitated the identification of different chromosomal rearrangements. Based on the obtained data, the studied Deschampsia species were distinguished into karyological groups, and MSA-based schematic trees were built, which could clarify the relationships within the genus. Our findings can be useful for further genetic and phylogenetic studies.
BackgroundGrasslands in the Arctic tundra undergo irreversible degradation due to climatic changes and also over-exploitation and depletion of scarce resources. Comprehensive investigations of cytogenomic structures of valuable Arctic and sub-Arctic grassland species is essential for clarifying their genetic peculiarities and phylogenetic relationships, and also successful developing new forage grass cultivars with high levels of adaptation, stable productivity and longevity. We performed molecular cytogenetic characterization of insufficiently studied pasture grass species (Poaceae) from related genera representing two neighboring clades: 1) Deschampsia and Holcus; 2) Alopecurus, Arctagrostis and Beckmannia, which are the primary fodder resources in the Arctic tundra.ResultsWe constructed the integrated schematic maps of distribution of these species in the northern, central and eastern parts of Eurasia based on the currently available data as only scattered data on their occurrence is currently available. The species karyotypes were examined with the use of DAPI-banding, multicolour FISH with 35S rDNA, 5S rDNA and the (GTT)9 microsatellite motif and also sequential rapid multocolour GISH with genomic DNAs of Deschampsia sukatschewii, Deschampsia flexuosa and Holcus lanatus belonging to one of the studied clades. Cytogenomic structures of the species were specified; peculiarities and common features of their genomes were revealed. Different chromosomal rearrangements were detected in Beckmannia syzigachne, Deschampsia cespitosa and D. flexuosa; B chromosomes with distinct DAPI-bands were observed in karyotypes of D. cespitosa and H. lanatus.ConclusionsThe peculiarities of distribution patterns of the examined chromosomal markers and also presence of common homologous DNA repeats in karyotypes of the studies species allowed us to verify their relationships. The obtained unique data on distribution areas and cytogenomic structures of the valuable Arctic and sub-Arctic pasture species are important for further genetic and biotechnological studies and also plant breeding progress.
Background Grasslands in the Arctic tundra undergo irreversible degradation due to climatic changes and also over-exploitation and depletion of scarce resources. Comprehensive investigations of cytogenomic structures of valuable Arctic and sub-Arctic grassland species is essential for clarifying their genetic peculiarities and phylogenetic relationships and also successful developing new forage grass cultivars with high levels of adaptation, stable productivity and longevity. We performed molecular cytogenetic characterization of seven insufficiently studied pasture grass species from related genera Alopecurus, Arctagrostis, Beckmannia, Deschampsia and Holcus (Poaceae) which are the primary fodder resources in the Arctic tundra. Results For these species, integrated schematic habitat maps were constructed based on the available data on their distribution in Eurasia. The species karyotypes were examined with the use of DAPI-banding, fluorescence in situ hybridization with 35S rDNA, 5S rDNA and the (GTT)9 microsatellite motif and also sequential rapid genomic in situ hybridization with genomic DNAs of Deschampsia sukatschewii, Holcus lanatus and Deschampsia flexuosa. Cytogenomic structures of the studied species were specified; peculiarities and common features of their genomes were revealed. Different chromosomal rearrangements were detected in Beckmannia syzigachne, Deschampsia cespitosa and D. flexuosa; B chromosomes with distinct DAPI-bands were observed in karyotypes of D. cespitosa and H. lanatus. Conclusions The peculiarities of distribution patterns of the examined chromosomal markers and also presence of common homologous DNA repeats in karyotypes of the studies species allowed us to verify their relationships. The obtained unique data on distribution areas and cytogenomic structures of the valuable Arctic and sub-Arctic pasture species are important for further genetic and biotechnological studies and also plant breeding progress.
Grasslands in the Arctic tundra undergo irreversible degradation due to climatic changes and also over-exploitation and depletion of scarce resources. Comprehensive investigations of cytogenomic structures of valuable Arctic and sub-Arctic grassland species is essential for clarifying their genetic peculiarities and phylogenetic relationships, and also successful developing new forage grass cultivars with high levels of adaptation, stable productivity and longevity. We performed molecular cytogenetic characterization of insufficiently studied pasture grass species (Poaceae) from related genera representing two neighboring clades: 1) Deschampsia and Holcus; 2) Alopecurus, Arctagrostis and Beckmannia, which are the primary fodder resources in the Arctic tundra.
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