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Biomedical subjects

Frank Diehl

Publications and source records attributed to Frank Diehl.

10 recordsLinked to original sources
PubMed2007

Digital quantification of mutant DNA in cancer patients.

PURPOSE OF REVIEW: The accumulation of somatic mutations is the major driving force for tumorigenesis. These mutations uniquely differentiate tumor cells from their normal counterparts. Mutations within tumor cells and mutant DNA released by tumor cells into blood, lymph, stool, tissues and other bodily compartments can thereby be used for cancer detection. Here we discuss technologies available for the detection and quantification of mutant DNA in clinical samples and the value of such measurements for patient management. RECENT FINDINGS: Conventional mutation detection technologies are either qualitative or only roughly estimate the abundance of mutant DNA molecules. Recently-developed approaches, however, use single molecule counting to determine the genotype of each individual member of a DNA population, providing a more accurate and precise digital output. SUMMARY: In this review, we discuss the clinical utility of mutant DNA quantification in cancer patients in the context of recent technical advances made in digital mutation detection.

DNA Mutational Analysis↗
PubMed2006

BEAMing up for detection and quantification of rare sequence variants.

BEAMing allows the one-to-one conversion of a population of DNA fragments into a population of beads. We used rolling circle amplification to increase the number of copies bound to such beads by more than 100-fold. This allowed enumeration of mutant and wild-type sequences even when they were present at ratios less than 1:10,000 and was sensitive enough to directly quantify the error rate of DNA polymerases used for PCR.

DNA↗
PubMed2005

Detection and quantification of mutations in the plasma of patients with colorectal tumors.

The early detection of cancers through analysis of circulating DNA could have a substantial impact on morbidity and mortality. To achieve this goal, it is essential to determine the number of mutant molecules present in the circulation of cancer patients and to develop methods that are sufficiently sensitive to detect these mutations. Using a modified version of a recently developed assay for this purpose, we found that patients with advanced colorectal cancers consistently contained mutant adenomatous polyposis coli (APC) DNA molecules in their plasma. The median number of APC DNA fragments in such patients was 47,800 per ml of plasma, of which 8% were mutant. Mutant APC molecules were also detected in >60% of patients with early, presumably curable colorectal cancers, at levels ranging from 0.01% to 1.7% of the total APC molecules. These results have implications for the mechanisms through which tumor DNA is released into the circulation and for diagnostic tests based on this phenomenon.

Adult↗
PubMed2004

Human papillomavirus type 16 E6 promotes retinoblastoma protein phosphorylation and cell cycle progression.

We show that E6 proteins from benign human papillomavirus type 1 (HPV1) and oncogenic HPV16 have the ability to alter the regulation of the G(1)/S transition of the cell cycle in primary human fibroblasts. Overexpression of both viral proteins induces cellular proliferation, retinoblastoma (pRb) phosphorylation, and accumulation of products of genes that are negatively regulated by pRb, such as p16(INK4a), CDC2, E2F-1, and cyclin A. Hyperphosphorylated forms of pRb are present in E6-expressing cells even in the presence of ectopic levels of p16(INK4a). The E6 proteins strongly increased the cyclin A/cyclin-dependent kinase 2 (CDK2) activity, which is involved in pRb phosphorylation. In addition, mRNA and protein levels of the CDK2 inhibitor p21(WAF1/CIP1) were strongly down-regulated in cells expressing E6 proteins. The down-regulation of the p21(WAF1/CIP1) gene appears to be independent of p53 inactivation, since HPV1 E6 and an HPV16 E6 mutant unable to target p53 were fully competent in decreasing p21(WAF1/CIP1) levels. E6 from HPV1 and HPV16 also enabled cells to overcome the G(1) arrest imposed by oncogenic ras. Immunofluorescence staining of cells coexpressing ras and E6 from either HPV16 or HPV1 revealed that antiproliferative (p16(INK4a)) and proliferative (Ki67) markers were coexpressed in the same cells. Together, these data underline a novel activity of E6 that is not mediated by inactivation of p53.

3T3 Cells↗
PubMed2003

Multicolor in vitro translation.

In vitro translation is a widely used tool for both analytical and preparative purposes. For analytical purposes, small amounts of proteins are synthesized and visualized by detection of labeled amino acids incorporated during translation. The original strategy of incorporating radioactively labeled amino acids, such as [35S]methionine or [14C]leucine, has been superseded by the addition of antigenic tags or the incorporation of biotin-labeled or BODIPY-FL-labeled amino acids. Such nonradioactive tags are easier to visualize after translation and do not pose a radiation hazard. Among the nonradioactive tags, BODIPY-FL-lysine offers the advantage that proteins that have incorporated this amino acid can be directly visualized after gel electrophoresis. We show here that multiple fluorophores introduced into proteins can considerably extend their usefulness, particularly for the comparison of in vitro-translated proteins from related sources. This technology can be applied in various situations, including the simplified detection of rare truncating mutations in clinical samples from cancer patients.

Boron Compounds↗
PubMed2003

Antibody microarrays: an evaluation of production parameters.

Antibody microarrays could have an enormous impact on the functional analysis of cellular activity and regulation, especially at the level of protein expression and protein-protein interaction, and might become an invaluable tool in disease diagnostics. The array surface is bound to have a tremendous influence on the findings from such studies. Apart from the basic issue of how to attach antibodies optimally without affecting their function, it is also important that the cognate antigens, applied within a complex protein mixture, all bind to the arrayed antibodies irrespective of their enormous variety in structure. In this study, various factors in the production of antibody microarrays on glass support were analysed: the modification of the glass surface; kind and length of cross-linkers; composition and pH of the spotting buffer; blocking reagents; antibody concentration and storage procedures, in order to evaluate their effect on array performance. Altogether, data from more than 700 individual array experiments were taken into account. In addition to home-made slides, commercially available systems were also included in the analysis.

Absorption↗
PubMed2002

Analysis of stage-specific gene expression in the bloodstream and the procyclic form of Trypanosoma brucei using a genomic DNA-microarray.

A microarray comprising 21,024 different PCR products spotted on glass slides was constructed for gene expression studies on Trypanosoma brucei. The arrayed fragments were generated from a T. brucei shotgun clone library, which had been prepared from randomly sheared and size-fractionated genomic DNA. For the identification of stage-specific gene activity, total RNA from in vitro cultures of the human, long slender form and the insect, procyclic form of the parasite was labelled and hybridised to the microarray. Approximately 75% of the genomic fragments produced a signal and about 2% exhibited significant differences between the transcript levels in the bloodstream and procyclic forms. A few results were confirmed by Northern blot analysis or reverse-transcription and PCR. Three hundred differentially regulated clones have been selected for sequencing. So far, of 33 clones that showed about 2-fold or more over-expression in bloodstream forms, 15 contained sequences similar to those of VSG expression sites and at least six others appeared non-protein-coding. Of 29 procyclic-specific clones, at least eight appeared not to be protein-coding. A surprisingly large proportion of known regulated genes was already identified in this small sample, and some new ones were found, illustrating the utility of genomic arrays.

Animals↗
PubMed2002

Manufacturing DNA microarrays from unpurified PCR products.

For the production of DNA microarrays from PCR products, purification of the the DNA fragments prior to spotting is a major expense in cost and time. Also, a considerable amount of material is lost during this process and contamination might occur. Here, a protocol is presented that permits the manufacture of microarrays from unpurified PCR products on aminated surfaces such as glass slides coated with the widely used poly(L-lysine) or aminosilane. The presence of primer molecules in the PCR sample does not increase the non-specific signal upon hybridisation. Overall, signal intensity on arrays made of unpurified PCR products is 94% of the intensity obtained with the respective purified molecules. This slight loss in signal, however, is offset by a reduced variation in the amount of DNA present at the individual spot positions across an array, apart from the considerable savings in time and cost. In addition, a larger number of arrays can be made from one batch of amplification products.

Animals↗
PubMed2002

The genome structure of Pseudomonas putida: high-resolution mapping and microarray analysis.

As part of a collaborative project aimed at sequencing and functionally analysing the entire genome of Pseudomonas putida strain KT2440, a physical clone map was produced as an initial resource. To this end, a high-coverage cosmid library was arrayed and ordered by clone hybridizations. Restriction fragments generated by rare-cutting enzymes and plasmids containing the rrn operon and 23S rDNA of Pseudomonas aeruginosa were used as probes and, parts of the cosmids were end-sequenced. This provided the information necessary for merging and comparing the macro-restriction map, cosmid clone order and sequence information, thereby assuring co-linearity of the eventual sequence assembly with the actual genome. A tiling path of clones was selected, from the shotgun clones used for sequencing, for the production of DNA microarrays that represent the entire genome including its non-coding portions.

Cloning, Molecular↗