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MessagePosté le: Ven 10 Oct - 10:57 (2014) Sujet du message: The most common web pages of PNET onset will be the cerebru Répondre en citant

 BRAF mutations were largely discovered in codon 600, codon 469 and codon 594 of non little cell lung cancer samples. Moreover, therapies focusing ARN-509 ic50 on BRAF mutant tumors have recently been recognized in NSCLC. Tumor material and pigmentation was assessed by an experienced pathologist. The proportion of tumor cells ranged from 15 100% and pigmentation was scored as no, very low and large pigmentation. Higher resolution melting examination and Sanger sequencing Applying the higher resolution melting strategy and Sanger sequencing, 81 of 82 samples could possibly be amplified and analyzed employing precisely the same PCR items. Situations have been viewed as as mutated applying HRM if a significant variation with the fluorescence level was detected that was outside the variety of variation from the wildtype management.

Samples in in between wildtype manage plus a mutant melting habits were viewed as as bor derline results. All mutated likewise as borderline samples were subjected to Sanger sequencing to determine the spe cific mutation kind. The assay was AUY922 価格 setup with an amplicon of 163 base pairs and it is as a result in a position to detect all hotspot mutations also as uncommon mutations inside the entire exon 15 of BRAF. That is in concordance using the studies of Colomba et al. and Tol et al. Figure 1 displays representative variation plots for BRAF mutations. p. V600E mutation could be clearly distinguished from p. V600K mutation and p. V600R. Furthermore, electro pherograms with widespread mutations in codon 600 in the BRAF gene analyzed by Sanger sequencing are shown, Only one sample with p.

V600E mutation could neither be analyzed by Sanger sequencing nor by HRM mainly because of amplification Alvocidib CDK 阻害剤 failure. Other folks have proven, that melanin binds to and interferes with DNA polymerases resulting in invalid check final results. But this situation had a tumor material of 80% and showed no pigmentation. For that reason, the failure of amplification on the 163 bp frag ment for Sanger sequencing and HRM is rather as a result of high degradation of FFPE utilised material than to pigmentation. This high degradation of FFPE used ma terial could also describe the larger Sanger sequencing failure rate described in other research utilizing a bigger PCR merchandise for analysis.

The sensitivity of Sanger sequencing is described during the literature as 20% mutated alleles within a background of wildtype alleles, but within the existing examine, we had been in a position to detect six. 6% mutated alleles. Figure two exhibits six electropherograms of samples analyzed in this research with unique allele frequencies ac cording to following generation sequencing. B displays that a sample with six. 6% allele frequency could be distinguished from a wildtype sample and that an allele frequency of 15% is usually obviously detected as p. V600E mutation applying Sanger sequencing. HRM examination has an even reduce detection limit of 6. 3% mutated alleles as reported by our group pre viously. Carbonell et al. showed an even reduce detec tion restrict ranging from one 5%. This was also supported by Balic et al. who showed that analyzing DNA methylation 1% methylated DNA inside the background of unmethylated DNA may very well be reproducibly detected in fresh frozen at the same time as in FFPE samples. 99% of all mutations can be detected by HRM as well as by Sanger sequencing.


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MessagePosté le: Ven 10 Oct - 10:57 (2014) Sujet du message: Publicité

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