53 research outputs found
An ELISA-based high throughput protein truncation test for inherited breast cancer
Abstract
Introduction
Breast cancer is the most diagnosed and second leading cause of cancer deaths in the U.S. female population. An estimated 5 to 10 percent of all breast cancers are inherited, caused by mutations in the breast cancer susceptibility genes (BRCA1/2). As many as 90% of all mutations are nonsense mutations, causing a truncated polypeptide product. A popular and low cost method of mutation detection has been the protein truncation test (PTT), where target regions of BRCA1/2 are PCR amplified, transcribed/translated in a cell-free protein synthesis system and analyzed for truncated polypeptides by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. We previously reported a novel High Throughput Solid-Phase PTT (HTS-PTT) based on an enzyme-linked immunosorbent assay (ELISA) format that eliminates the need for radioactivity, SDS-PAGE and subjective interpretation of the results. Here, we report the next generation HTS-PTT using triple-epitope-tagged proteins and demonstrate, for the first time, its efficacy on clinical genomic DNA samples for BRCA1/2 analysis.
Methods
Segments of exons 11 of BRCA1/2 open reading frames were PCR amplified from either blood derived genomic DNA or cell line mRNA. PCR primers incorporate elements for cell-free transcription/translation and epitope tagging. Cell-free expressed nascent proteins are then antibody-captured onto the wells of a microtiter plate and the relative amount of truncated polypeptide measured using antibodies against the N- and C-terminal epitope tags in an ELISA format.
Results
100% diagnostic sensitivity and 96% specificity for truncating mutations in exons 11 of BRCA1/2 were achieved on one hundred blood-derived clinical genomic DNA samples which were previously assayed using the conventional gel based PTT. Feasibility of full gene coverage for BRCA1/2 using mRNA source material is also demonstrated.
Conclusions
Overall, the HTS-PTT provides a simple, quantitative, objective, low cost and high throughput method for analysis of truncating mutations as an alternative to gel based PTT for BRCA analysis. The technology is readily accessible to virtually any laboratory, with the only major instrumentation required being a PCR thermocycler and a basic micro-well plate reader. When compared to conventional gel based PTT, the HTS-PTT provides excellent concordance
Mismatch between physicians and family members views on communications about patients with chronic incurable diseases receiving care in critical and intensive care settings in Georgia: a quantitative observational survey
Nothing Lasts Forever: Environmental Discourses on the Collapse of Past Societies
The study of the collapse of past societies raises many questions for the theory and practice of archaeology. Interest in collapse extends as well into the natural sciences and environmental and sustainability policy. Despite a range of approaches to collapse, the predominant paradigm is environmental collapse, which I argue obscures recognition of the dynamic role of social processes that lie at the heart of human communities. These environmental discourses, together with confusion over terminology and the concepts of collapse, have created widespread aporia about collapse and resulted in the creation of mixed messages about complex historical and social processes
To be objective in Experimental Phenomenology: a Psychophysics application
Background Several scientific psychologists consider the approach for the study of perceptive problems of the Experimental Phenomenology is problematic, namely that the phenomenological demonstrations are subjectively based and they do not produce quantifiable results. Aim The aim of this study is to show that Experimental Phenomenology can lead to conclusions objective and quantifiable and propose a procedure allowing to obtain objective measuring using the Rasch mathematical model able to describe the experimental data gathered in Experimental Phenomenology procedures. Method In order to demonstrate this, a Psychophysics simulated study is proposed. Results/conclusions It is possible to carry out a fundamental measurement starting from Experimental Phenomenology by way of the Theory of Conjoint Measurement
Family history of cancer, personal history of medical conditions and risk of oral cavity cancer in France: the ICARE study
Development and evaluation of the feasibility and effects on staff, patients, and families of a new tool, the Psychosocial Assessment and Communication Evaluation (PACE), to improve communication and palliative care in intensive care and during clinical uncertainty
Antibiotic prophylaxis in a patient with penicillin allergy and recurrent bacterial endocarditis: A case report
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