Dataset Information

Detection of KRAS and BRAF mutations in colorectal carcinoma roles for high-sensitivity locked nucleic acid-PCR sequencing and broad-spectrum mass spectrometry genotyping.

ABSTRACT: KRAS and BRAF mutations predict the resistance of colorectal carcinomas to therapy targeted to the epidermal growth factor receptor, but their detection can be challenging because of high testing volume, frequently low tumor content, and the spectrum of rarer mutations in these genes. To address these issues, we evaluated a locked nucleic acid (LNA)-PCR sequencing assay to detect low levels of mutant DNA, and we also evaluated a mass spectrometry genotyping assay (Sequenom, San Diego, CA) that is suitable for broad mutation screening. Clinical cases (n = 308) previously tested for KRAS and BRAF by standard sequencing were retested by LNA-PCR sequencing incorporating an LNA oligonucleotide to suppress amplification of nonmutant DNA, and by a Sequenom assay panel targeting common mutations in both genes. Standard sequencing detected 121 KRAS (39%) and 10 BRAF mutations; retesting with the LNA-based method and the Sequenom assay detected 19 (140/308, 45%) and 6 (127/308, 41%) additional KRAS mutants, respectively. One additional BRAF mutant was detected by the Sequenom assay. The analytical sensitivities were 0.3% for both KRAS and BRAF by LNA-PCR and from 1% to 10% for the Sequenom assays, depending on the specific mutation. Given these results, standard sequencing is suboptimal for mutation detection in metastatic and treated lesions even with predissection for tumor enrichment. High-sensitivity LNA-PCR sequencing detects significantly more mutations, whereas the Sequenom platform shows intermediate sensitivity but offers significant advantages for broader mutation screening.

SUBMITTER: Arcila M

PROVIDER: S-EPMC3070595 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Detection of KRAS and BRAF mutations in colorectal carcinoma roles for high-sensitivity locked nucleic acid-PCR sequencing and broad-spectrum mass spectrometry genotyping.

Arcila Maria M Lau Christopher C Nafa Khedoudja K Ladanyi Marc M

The Journal of molecular diagnostics : JMD 20101223 1

KRAS and BRAF mutations predict the resistance of colorectal carcinomas to therapy targeted to the epidermal growth factor receptor, but their detection can be challenging because of high testing volume, frequently low tumor content, and the spectrum of rarer mutations in these genes. To address these issues, we evaluated a locked nucleic acid (LNA)-PCR sequencing assay to detect low levels of mutant DNA, and we also evaluated a mass spectrometry genotyping assay (Sequenom, San Diego, CA) that i ...[more]

PMID: 21227396

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Project description:BackgroundMultiplexing technologies, which allow for simultaneous detection of multiple nucleic acid sequences in a single reaction, can save a lot of time, cost and labor compared to traditional single reaction detection methods. However, the multiplexing method currently used requires precise handiwork and many complicated steps, making a new, simpler technique desirable. Oligonucleotides containing locked nucleic acid residues are an attractive tool because they have strong affinities for their complementary targets, they have been used to avoid dimer formation and mismatch hybridization and to enhance efficient priming. In this study, we aimed to investigate the use of locked nucleic acid pentamers for genomic DNA amplification and multiplex genotyping.ResultsWe designed locked nucleic acid pentamers as universal PCR primers for genomic DNA amplification. The locked nucleic acid pentamers were able to prime amplification of the selected sequences within the investigated genomes, and the resulting products were similar in length to those obtained by restriction digest. In Real Time PCR of genomic DNA from three bacterial species, locked nucleic acid pentamers showed high priming efficiencies. Data from bias tests demonstrated that locked nucleic acid pentamers have equal affinities for each of the six genes tested from the Klebsiella pneumoniae genome. Combined with suspension array genotyping, locked nucleic acid pentamer-based PCR amplification was able to identify a total of 15 strains, including 3 species of bacteria, by gene- and species-specific probes. Among the 32 species used in the assay, 28 species and 50 different genes were clearly identified using this method.ConclusionAs a novel genomic DNA amplification, the use of locked nucleic acid pentamers as universal primer pairs in conjunction with suspension array genotyping, allows for the identification of multiple distinct genes or species with a single amplification procedure. This demonstrates that locked nucleic acid pentamer-based PCR can be utilized extensively in pathogen identification.

Dataset Information

Detection of KRAS and BRAF mutations in colorectal carcinoma roles for high-sensitivity locked nucleic acid-PCR sequencing and broad-spectrum mass spectrometry genotyping.

Publications

Detection of KRAS and BRAF mutations in colorectal carcinoma roles for high-sensitivity locked nucleic acid-PCR sequencing and broad-spectrum mass spectrometry genotyping.

Similar Datasets

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