Mario Menschikowski1, Carsten Jandeck2, Markus Friedemann2, Susan Richter2, Dana Thiem3, Björn Sönke Lange3, Meinolf Suttorp3. 1. Institute of Clinical Chemistry and Laboratory Medicine, University Hospital "Carl Gustav Carus", Technical University of Dresden, Dresden, Germany Mario.Menschikowski@uniklinikum-dresden.de. 2. Institute of Clinical Chemistry and Laboratory Medicine, University Hospital "Carl Gustav Carus", Technical University of Dresden, Dresden, Germany. 3. Department of Pediatrics, University Hospital "Carl Gustav Carus", Technical University of Dresden, Dresden, Germany.
Abstract
BACKGROUND/AIM: DNA methylation plays an important role in the initiation and propagation of carcinogenesis; however, the role of heterogeneously methylated epialleles is currently not well studied, also due to the lack of sensitive, unbiased and high throughput methods. Here, a newly developed droplet digital PCR (ddPCR)-based method was evaluated regarding its ability to quantify such heterogeneously methylated epialleles with sufficient analytical sensitivity and specificity. MATERIALS AND METHODS: Genomic DNA from blood leukocytes and bone marrow aspirate of an 8-year old male with B-cell acute lymphoblastic leukemia (B-ALL) and from normal and malignant prostate cell lines were analysed using ddPCR. RESULTS: By using these DNA samples, the specificity of an applied set of fluorescence-labeled probes was demonstrated as a proof of concept. CONCLUSION: All individual heterogeneously-methylated epialleles were quantifiable by a set of fluorescence-labeled probes with complementary sequences to epialleles in a closed-tube and high-throughput manner. The new method named epiallele-sensitive droplet digital PCR (EAST-ddPCR) may give new insights in the generation and regulation of epialleles and may help in finding new biomarkers for the diagnosis of benign und malignant diseases. Copyright
BACKGROUND/AIM: DNA methylation plays an important role in the initiation and propagation of carcinogenesis; however, the role of heterogeneously methylated epialleles is currently not well studied, also due to the lack of sensitive, unbiased and high throughput methods. Here, a newly developed droplet digital PCR (ddPCR)-based method was evaluated regarding its ability to quantify such heterogeneously methylated epialleles with sufficient analytical sensitivity and specificity. MATERIALS AND METHODS: Genomic DNA from blood leukocytes and bone marrow aspirate of an 8-year old male with B-cell acute lymphoblastic leukemia (B-ALL) and from normal and malignant prostate cell lines were analysed using ddPCR. RESULTS: By using these DNA samples, the specificity of an applied set of fluorescence-labeled probes was demonstrated as a proof of concept. CONCLUSION: All individual heterogeneously-methylated epialleles were quantifiable by a set of fluorescence-labeled probes with complementary sequences to epialleles in a closed-tube and high-throughput manner. The new method named epiallele-sensitive droplet digital PCR (EAST-ddPCR) may give new insights in the generation and regulation of epialleles and may help in finding new biomarkers for the diagnosis of benign und malignant diseases. Copyright
Authors: Mathias Ehrich; Matthew R Nelson; Patrick Stanssens; Marc Zabeau; Triantafillos Liloglou; George Xinarianos; Charles R Cantor; John K Field; Dirk van den Boom Journal: Proc Natl Acad Sci U S A Date: 2005-10-21 Impact factor: 11.205
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Authors: Mario Menschikowski; Carsten Jandeck; Markus Friedemann; Brit Nacke; Saskia Hantsche; Oliver Tiebel; Olga Sukocheva; Albert Hagelgans Journal: Oncotarget Date: 2018-11-16