TY  - JOUR
A1  - Klamp, Tobias
A1  - Camps, Marta
A1  - Nieto, Benjamin
A1  - Guasch, Francesc
A1  - Ranasinghe, Rohan T.
A1  - Wiedemann, Jens
A1  - Petrášek, Zdeněk
A1  - Schwille, Petra
A1  - Klenerman, David
A1  - Sauer, Markus
T1  - Highly Rapid Amplification-Free and Quantitative DNA Imaging Assay
JF  - Scientific Reports
N2  - There is an urgent need for rapid and highly sensitive detection of pathogen-derivedDNAin a point-of-care (POC) device for diagnostics in hospitals and clinics. This device needs to work in a ‘sample-in-result-out’ mode with minimum number of steps so that it can be completely integrated into a cheap and simple instrument. We have developed a method that directly detects unamplified DNA, and demonstrate its sensitivity on realistically sized 5 kbp targetDNA fragments of Micrococcus luteus in small sample volumes of 20 mL. The assay consists of capturing and accumulating of target DNA on magnetic beads with specific capture oligonucleotides, hybridization of complementary fluorescently labeled detection oligonucleotides, and fluorescence imaging on a miniaturized wide-field fluorescence microscope. Our simple method delivers results in less than 20 minutes with a limit of detection (LOD) of,5 pMand a linear detection range spanning three orders of magnitude.
KW  - laboratory techniques and procedures
KW  - diseases
KW  - infectious diseases
KW  - assay systems
Y1  - 2013
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130500
VL  - 3
IS  - 1852
ER  - 
TY  - JOUR
A1  - Lando, David
A1  - Endesfelder, Ulrike
A1  - Berger, Harald
A1  - Subramanian, Lakxmi
A1  - Dunne, Paul D.
A1  - McColl, James
A1  - Klenerman, David
A1  - Carr, Antony M.
A1  - Sauer, Markus
A1  - Allshire, Robin C.
A1  - Heilemann, Mike
A1  - Laue, Ernest D.
T1  - Quantitative single-molecule microscopy reveals that CENP-A\(^{Cnp1}\) deposition occurs during G2 in fission yeast
JF  - Open Biology
N2  - The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and 'counts' individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-A\(^{Cnp1}\) with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-A\(^{Cnp1}\) levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-A(Cnp1) is deposited solely during the G2 phase of the cell cycle.
KW  - nucleosome
KW  - fission yeast
KW  - identification
KW  - propagation
KW  - CSE4, CENP-A
KW  - CENP-A
KW  - schizosaccaromyces-pombe
KW  - fluorescent protein
KW  - centomeres
KW  - superresolution
KW  - chromatin
KW  - centromere
KW  - ingle-molecule microscopy
Y1  - 2012
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134682
VL  - 2
IS  - 120078
ER  -