Biomedical subjects

Ina Schwope

Publications and source records attributed to Ina Schwope.

3 recordsLinked to original sources

PubMed2004

A real-time PCR assay for DNA-methylation using methylation-specific blockers.

DNA methylation-based biomarkers have been discovered that could potentially be used for the diagnosis of cancer by detection of circulating, tumor-derived DNA in bodily fluids. Any methylation detection assay that would be applied to these samples must be capable of detecting small amounts of tumor DNA in the presence of background normal DNA. We have developed a real-time PCR assay, called HeavyMethyl, that is well suited for this application. HeavyMethyl uses methylation-specific oligonucleotide blockers and a methylation-specific probe to achieve methylation-specific amplification and detection. We tested the assays on unmethylated and artificially methylated DNA in order to determine the limit of detection. After careful optimization, our glutathione-S-transferase pi1 and Calcitonin assays can amplify as little as 30 and 60 pg of methylated DNA, respectively, and neither assay amplifies unmethylated DNA. The Calcitonin assay showed a highly significant methylation difference between normal colon and colon adenocarcinomas, and methylation was also detected in serum DNA from colon cancer patients. These assays show that HeavyMethyl technology can be successfully employed for the analysis of very low concentrations of methylated DNA, e.g. in serum of patients with tumors.

Adenocarcinoma↗

PubMed2002

Tumour class prediction and discovery by microarray-based DNA methylation analysis.

Aberrant DNA methylation of CpG sites is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation occurs in a tumour type-specific manner. However, large-scale analysis of candidate genes has so far been hampered by the lack of high throughput assays for methylation detection. We have developed the first microarray-based technique which allows genome-wide assessment of selected CpG dinucleotides as well as quantification of methylation at each site. Several hundred CpG sites were screened in 76 samples from four different human tumour types and corresponding healthy controls. Discriminative CpG dinucleotides were identified for different tissue type distinctions and used to predict the tumour class of as yet unknown samples with high accuracy using machine learning techniques. Some CpG dinucleotides correlate with progression to malignancy, whereas others are methylated in a tissue-specific manner independent of malignancy. Our results demonstrate that genome-wide analysis of methylation patterns combined with supervised and unsupervised machine learning techniques constitute a powerful novel tool to classify human cancers.

Algorithms↗

PubMed1999

Synthesis of 3',5'-Dipeptidyl Oligonucleotides.

Peptide-DNA hybrids are richly functionalized analogues of biomolecules that may have applications as hybridization probes and antisense agents with tunable binding and targeting properties. So far, synthetic efforts have mainly focused on hybrids bearing a single peptide chain, either at the 5'- or the 3'-terminus. Such singly modified analogues are vulnerable to nuclease attack at the unmodified terminus. Here we report a convenient and high-yielding solid-phase synthesis of 3'- and 5'-modified analogues of DNA with aminoacyl and peptidyl appendages at both termini. Using MALDI-TOF mass spectra of crude products as the criterion, serine, glycolic acid, hydroxylauric acid, and dimethyl hydroxypropionic acid were tested as 3'-linker residues. The latter, together with a direct amide link at the 5'-terminus, gave the highest yields of hybrids. The optimized procedure assembles hybrids on a controlled pore glass support bearing three consecutive omega-hydroxy lauric acid linkers. This support greatly reduces side reactions observed with conventional supports, probably due to its ability to increase steric accessibility during coupling ("swelling") and its rapid hydrolysis during deprotection with ammonium hydroxide. Dihybrids with aromatic, basic, and acidic amino acid residues were prepared, including H-Phe-Gly-TGCGCA-DP-Phe-OH, where DP denotes the dimethyl hydroxypropionic acid linker, whose structure was confirmed via mass spectrometry and one- and two-dimensional NMR. Further, a mixed coupling with seven Fmoc-protected amino acids was shown to produce a combinatorial library of dipeptidyl DNA hybrids.

Journal Article↗