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The use of COLD‐PCR,DHPLC and GeneScanning for the highly sensitive detection of c‐KIT somatic mutations in canine mast cell tumours 下载免费PDF全文
The conventional polymerase chain reaction (PCR)/sequencing methods may be poorly suited for the detection of somatic mutations in canine mast cell tumour (MCT) samples owing to limited sensitivity. This study was aimed at establishing novel and more sensitive methods, assessing their limit of detection and comparing their sensitivity with conventional methods.Two different ‘driver’ somatic mutations of c‐KIT, together with the wild‐type counterparts, were cloned in plasmids to prepare standard samples with known concentrations of mutated alleles in a background of wild‐type alleles; the plasmids standards were assayed using either conventional or novel, highly sensitive technique. Conventional PCR/sequencing showed a sensitivity of 50–20%. Conversely, all the novel methods obtained higher sensitivities allowed reaching as low as 2.5–1.2% of the mutated DNA.The study demonstrates that early conventional methods could likely have underestimated the prevalence of KIT mutations of MCTs, therefore affecting the assessment of their relevance in prognosis and tyrosine kinase inhibitor (TKI) treatment effectiveness. 相似文献
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Here, we describe the establishment of mutant‐specific polymerase chain reaction (PCR) for detection of a c‐KIT c.1430G>T mutation in feline mast cell tumours. Several mutations in feline c‐KIT have been identified, with the c.1430G>T mutation accounting for a significant portion of feline mast cell tumour mutations. The c.1430G>T mutation in c‐KIT exon 9 was detected in 15.7% (11 of 70) of samples by mutant‐specific PCR but in only 7.1% (5 of 70) by PCR–restriction fragment length polymorphism (RFLP) in the genomic DNA isolated from 70 formalin‐fixed paraffin‐embedded sections or cells collected by fine needle aspiration. Mutant‐specific PCR showed remarkably higher detection rate than did PCR–RFLP. DNA sequence analysis did not always yield identical results to those of mutant‐specific PCR, suggesting heterogeneity of tumour cells. Mutant‐specific PCR is a valid and efficient screening tool for detection of the c‐KIT c.1430G>T point mutation in feline mast cell tumours compared with PCR–RFLP and sequencing analysis. 相似文献
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QIN Ke HUANG Xiang ZENG Jian-hua WANG Chen SUN Guan-jie SONG De-qing CHEN Yao-sheng LIU Xiao-hong HE Zu-yong 《中国畜牧兽医》2017,44(3):773-781
In order to assess the purity of Luchuan pig populations, four South Chinese local pig breeds including Putian Black pig, Yuedong Black pig, Dahuabai pig, Bama miniature pig and three foreign pig breeds including Duroc pig, Yorkshire pig and Landrace pig were studied as controls by sequencing of PCR products, MC1R and KIT genotypes in 56 Luchuan pigs were analyzed in this study. Sequencing results indicated that a splicing mutation (G>A) was presented in the first base in intron 17 of KIT gene in both Yorkshire pig and Landrace pig, in contrast, the wildtype GG of KIT gene was presented in Luchuan pig, four south Chinese local pig breeds and Duroc pig.Compared with Hainan wild boar, South Chinese local pig breeds had two missense mutations 95Val > Met and 102Leu > Pro in the coding region of MC1R gene;Compared with Yorkshire pig, Landrace pig and Duroc pig, South Chinese local pig breeds had 5 to 6 SNPs in MC1R gene 5'UTR, and in addtion, an A base deletion in MC1R gene 3'UTR. Furthermore, we found one litter of Luchuan pig with abnormal coat color.The results showed that the presentation of two distinct MC1R genotypes ED1 and Ep in both litters and the sow,but only ED1 in the boar. Considering Ep was derived from Pietrain pig, we preliminarily considered that the genome of the sow might be infiltrated with foreign pig breeds. In summary, we detected the genotypes of the coat color genes KIT and MC1R in eight pig breeds, confirmed the molecular differences of coat color between Chinese local pig breeds and foreign pig breeds, which could be useful for the further investigation of the molecular mechanism of pig coat color. 相似文献
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Murakami A Mori T Sakai H Murakami M Yanai T Hoshino Y Maruo K 《Veterinary and comparative oncology》2011,9(3):219-224
KIT, a transmembrane receptor tyrosine kinase, is one of the specific targets for anti-cancer therapy. In humans, its expression and mutations have been identified in malignant melanomas and therapies using molecular-targeted agents have been promising in these tumours. As human malignant melanoma, canine malignant melanoma is a fatal disease with metastases and the poor response has been observed with all standard protocols. In our study, KIT expression and exon 11 mutations in dogs with histologically confirmed malignant oral melanomas were evaluated. Although 20 of 39 cases were positive for KIT protein, there was no significant difference between KIT expression and overall survival. Moreover, polymerase chain reaction amplification and sequencing of KIT exon 11 in 17 samples did not detect any mutations and proved disappointing. For several reasons, however, KIT expression and mutations of various exons including exon 11 should be investigated in more cases. 相似文献
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Velarde R Mentaberre G Sánchez J Marco I Lavín S 《Veterinary journal (London, England : 1997)》2008,177(3):445-447
Gastrointestinal mesenchymal tumours from two Spanish ibex (Capra pyrenaica hispanica) were examined grossly, histologically and immunohistochemically. One neoplasm was a 1.5 kg tan multinodular cavitated mass in the forestomach. The other tumour was a firm mural mass 1.2 cm in diameter in the colon. Microscopically, both tumours were formed mainly by spindle shaped cells arranged in closely packed interlacing fascicles. Neoplastic cells in both tumours labelled positively for KIT (CD117), vimentin and alpha-smooth muscle actin. These findings suggest that both neoplasms were gastrointestinal stromal tumours and most likely to be derived from the interstitial cells of Cajal or their progenitor cells. 相似文献
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The roan coat color in horses is characterized by dispersed white hair and dark points. This phenotype segregates in a broad range of horse breeds, while the underlying genetic background is still unknown. Previous studies mapped the roan locus to the KIT gene on equine chromosome 3 (ECA3). However, this association could not be validated across different horse breeds. Performing a genome-wide association analysis (GWAS) in Noriker horses, we identified a single nucleotide polymorphism (SNP) (ECA3:g.79,543.439 A > G) in the intron 17 of the KIT gene. The G -allele of the top associated SNP was present in other roan horses, namely Quarter Horse, Murgese, Slovenian, and Belgian draught horse, while it was absent in a panel of 15 breeds, including 657 non-roan horses. In further 379 gray Lipizzan horses, eight animals exhibited a heterozygous genotype (A/G). Comparative whole-genome sequence analysis of the KIT region revealed two deletions in the downstream region (ECA3:79,533,217_79,533,224delTCGTCTTC; ECA3:79,533,282_79,533,285delTTCT) and a 3 bp deletion combined with 17 bp insertion in intron 20 of KIT (ECA3:79,588,128_79,588,130delinsTTATCTCTATAGTAGTT). Within the Noriker sample, these loci were in complete linkage disequilibrium (LD) with the identified top SNP. Based upon pedigree information and historical records, we were able to trace back the genetic origin of roan coat color to a baroque gene pool. Furthermore, our data suggest allelic heterogeneity and the existence of additional roan alleles in ponies and breeds related to the English Thoroughbred. In order to study the roan phenotype segregating in those breeds, further association and verification studies are required. 相似文献
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马毛色是品种鉴定和个体识别的重要依据。马 KIT 基因位于3号染色体,KIT 基因突变影响马毛色及毛色的分布。对德保矮马和哈萨克马69个个体的 KIT 基因21个外显子及部分内含子直接测序,共发现了5个SNPs,其中1个位于5'-UTR区(g.91214T>G),1个位于内含子20 (g.171356C>G),另外3个分别位于外显子15、20和21(g.164297C>T;g.170189C>T;g.171471G>A,p.Ala960Thr)。用PCR-RFLP方法对69个个体进行分型,发现外显子15有3种基因型TT、CT、CC;外显子20有3种基因型TT、CT、CC;外显子21有2种基因型GG与GA,且均为野生型占优势。德保矮马 KIT 基因多态性比哈萨克马更丰富。 相似文献