NDLI: Unlabeled-probe high-resolution melting to detect KRAS codon 12 and 13 mutations in pancreatic adenocarcinoma tissues.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Guo, Wei Zhang, Chunyan Wu, Jiong Song, Binbin Wang, Beili Zhou, Yan Zhou, Jiaye Shen, Minna Zhang, Chen Zhang, Xinju Guan, Ming Pan, Baishen
Description	Country affiliation: China Author Affiliation: Guo W ( Department of Laboratory Medicine, Zhongshan Hospital, Fudan University, Shanghai, China.)
Abstract	BACKGROUND: The aim of our study was to establish an unlabeled-probe high-resolution melting (HRM) approach to the detection of Kirsten RAS (KRAS) codon 12 and 13 mutations in pancreatic adenocarcinoma (PA) tissues as a novel and effective diagnostic technique. METHODS: We tested the sensitivity and specificity of this genotyping approach in cell lines with known KRAS mutations using 166 bp amplicons and 37 bp wild-type probe to detect KRAS codon 12 and 13 mutations. We screened 49 PA tissues to be subsequently sequenced to confirm the mutations. Simultaneously, we tested the specimens using Sanger sequencing and then used target-DNA cloning and sequencing for verification. RESULTS: It was found that unlabeled-probe HRM was reliable in detecting 3% of mutant cell lines DNA diluted with that of the wild-type, whereas Sanger sequencing could only discriminate 20% mutant cell ratios. In detecting 49 specimens, the former was capable of detecting 23 mutations (46.9%); and the latter could observe 15 (30.6%). For further verification, T-A DNA cloning and sequencing was applied to the differences, with the results matching those of the unlabeled-probe HRM. CONCLUSIONS: It was concluded that the unlabeled-probe HRM approach can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in diagnosing and treating PA.
File Format	HTM / HTML
ISSN	14346621
Issue Number	6
Volume Number	50
e-ISSN	14374331
Journal	Clinical Chemistry and Laboratory Medicine
Language	English
Publisher	Walter de Gruyter
Publisher Date	2012-01-31
Publisher Place	Germany
Access Restriction	Subscribed
Subject Keyword	Adenocarcinoma Research Support, Non-u.s. Gov't Proto-oncogene Proteins P21(ras) Humans Middle Aged Male Journal Article Codon Genetics Adult Female Pancreatic Neoplasms Nucleic Acid Denaturation Discipline Clinical chemistry Proto-oncogene Proteins Aged, 80 And Over Dna Probes Reproducibility Of Results Pathology Chemistry Discipline Clinical Chemistry Ras Proteins Dna Analysis Aged Transition Temperature Methods Dna Mutational Analysis
Content Type	Text
Resource Type	Article
Subject	Biochemistry (medical) Clinical Biochemistry

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