Browsing by Subject "LCR (Ligase Chain Reaction)"

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    Beiträge zur Verbesserung der Analytik von Mutationen im Protoonkogen Kirsten-ras und epigenetische Untersuchungen zur Eignung von DUSP9/MKP4 als CIMP-Marker
    (2012) Jenner, Stefan; Preiss, Anette
    The present study extends over two different fields of applications, which contribute to improvements for the analysis of colorectal cancers. First, a ligase-based method was developed which allows a quick, inexpensive, specific and highly sensitive detection of point mutations in the mutation hot spot codon12 and codon13 of the K-ras gene. The establishment and validation of the technique was performed on clinical samples, which were present as FFPE tissues and gone through routine diagnostics using conventional molecular biological techniques (microarray analysis, Sanger Sequencing) to determine the K-ras mutation status. In addition, a comparison between the new developed technology and the conventional technologies should be performed. The evaluation of the gLCR approach was done by ABI310er capillary electrophoresis. Among the tested LCR variants the gLCR-monoplex had been the most robust, specific and sensitive technique. The presence of very weak mutations in the samples had been successfully confirmed by gLCR-monoplex, whereas several conventional techniques had to be applied together to detect the mutations unambiguously. The signal strengths for all tested samples were high, coming along with a low standard deviation. The superiority of gLCR-monoplex over the conventional techniques had been further highlighted impressively by performing a comparison of methods on a dilution series. While the mutation detection using Sanger Sequencing or microarray analysis had been successful only up to the 1:10-dilution, or 1:100-dilution, it was possible to detect the K-ras mutation by gLCR-technique up to the 1:1 million-dilution. In routine diagnostics a single monoplex reaction for each possible mutation has to be performed. Therefore the monoplex technique is only suitable as a confirmatory test of weak or doubtful mutation screening results, which had been previously indicated by conventional techniques. A multiplex approach would be desirable to use the gLCR technology as a main detection technique in routine diagnostics. Therefore a single discriminating base at the mutation-specific and color-labeled oligo probe appears to be insufficient. In future studies an additional mismatch should be integrated at position (-3), in relation to the 3'-end of the dye-labeled and mutation-specific oligo probe. In this way it could come to a significant reduction of false-positive signals, thereby gLCR technology could possibly be used as a multiplex approach. In the second part of the work the methylation status of the DUSP9/MKP4 promoter region had been evaluated. By methylation, the accessibility of the promoter, and thus the transcriptional activation of a gene can be reduced. The promoter regions of tumor suppressors are frequently strong methylated in colorectal cancer (CRC). The methylation is resulting in an increased tumorigenesis and cannot be observed in the corresponding normal tissues. This alteration is called CIMP (CpG-island-methylator-phenotype) and is an independent tumor phenotype, which is linked to other clinical aspects. Involved genes are classified as CIMP markers. The DUSP9/MKP4 gene product is a potent tumor suppressor, but it´s suitability as a marker for CIMP in CRC has not been studied so far. In this study, the degree of methylation of 79 colorectal cancer FFPE tissue samples and 22 corresponding normal FFPE tissues was determined quantitatively. A broad variation of methylation strength has been observed in the examined tumor tissues, ranging from nearly unmethylated to nearly completely methylated. Only minor differences between tumor and normal tissues had been detected for the 11 DNA samples with the lowest methylation strength. On the other side, there was a significant correlation with CRC for the 11 strongest methylated DNA samples. About 80% of the DNA from normal tissue showed clearly weaker methylation, leading to the conclusion, that an aberrant DNA methylation status is present in these tumor tissues. Additionally, 9 out of 11 strong methylated DNA samples showed CIMP criteria, which were completely missing at the weakly methylated DNA samples. This work represents, as far as published, the first study that reveals a difference in the methylation pattern of the DUSP9/MKP4 promoter from human colorectal carcinoma and their corresponding normal tissues. Strong methylation could be associated with all tested CIMP criteria. This relationship needs to be confirmed in further studies on a larger sample collective. In addition, the investigations should include the detection of microsatellite instabilities, as an additional CIMP-criterion, and a functional protein detection method should be established.