Micrococcal Nuclease Does Not Substantially Bias Nucleosome Mapping

We have mapped sequence-directed nucleosome positioning on genomic DNA molecules using high-throughput sequencing. Chromatins, prepared by reconstitution with either chicken or frog histones, were separately digested to mononucleosomes using either micrococcal nuclease (MNase) or caspase-activated DNase (CAD). Both enzymes preferentially cleave internucleosomal (linker) DNA, although they do so by markedly different mechanisms. MNase has hitherto been very widely used to map nucleosomes, although concerns have been raised over its potential to introduce bias. Having identified the locations and quantified the strength of both the chicken or frog histone octamer binding sites on each DNA, the results obtained with the two enzymes were compared using a variety of criteria. Both enzymes displayed sequence specificity in their preferred cleavage sites, although the nature of this selectivity was distinct for the two enzymes. In addition, nucleosomes produced by CAD nuclease are 8–10 bp longer than those produced with MNase, with the CAD cleavage sites tending to be 4–5 bp further out from the nucleosomal dyad than the corresponding MNase cleavage sites. Despite these notable differences in cleavage behaviour, the two nucleases identified essentially equivalent patterns of nucleosome positioning sites on each of the DNAs tested, an observation that was independent of the histone type. These results indicate that biases in nucleosome positioning data collected using MNase are, under our conditions, not significant

Base pair mismatch identification with DNA nanoswitch and long lifetime acridine fluorophore

Detection of single base pair mutations in an unlabelled nucleic acid target sequence is demonstrated using the time-resolved measurement of the Förster Resonance Energy Transfer (FRET) between a long lifetime acridine-based fluorophore and a non-fluorescent quencher molecule covalently bound to a DNA Holliday junction-based nanoswitch

Bait-and-switch molecular recognition in nucleic acid sensors: Time-resolved fluorescence, single nucleotide polymorphism detection

We investigate the properties of a simple DNA-based nanodevice capable of detecting single nucleotide polymorphisms (SNPs) in unlabelled nucleic acid target sequences. Detection is achieved by a two-stage bait-and-switch process combining complementary-base hybridization and switching as molecular recognition criteria. A probe molecule is constructed from a single DNA strand designed to adopt a partial cruciform structure with a pair of exposed (unhybridized) strands. Upon target binding, a switchable cloverleaf construct (similar to a Holliday junction) is formed where the states are the open and closed junction conformations. Switching between these occurs by junction folding in the presence of divalent ions. A combination of steady-state and time-resolved fluorescence spectroscopy is used to measure Forster resonance energy transfer

Detection of single nucleotide polymorphisms using a DNA Holliday junction nanoswitch?a high-throughput fluorescence lifetime assay

We report a simple DNA sensor device, using a combination of binding and conformational switching, capable of rapid detection of specific single nucleotide polymorphisms in an unlabelled nucleic acid target sequence. The detection is demonstrated using fluorescence lifetime measurements in a high-throughput micro plate reader instrument based on the time-correlated single-photon counting technique. The sensor design and instrumental architecture are capable of detecting perturbations in the molecular structure of the probe–target complex (which is similar to that of a Holliday junction), due to a single base pair mismatch in a synthetic target. Structural information, including fluorophore separations, is obtained using time-resolved Förster resonance energy transfer between two fluorophores covalently bound to the probe molecule. The two probes required are designed to detect a single nucleotide polymorphism from a sequence present on each of the two copies of human chromosome 1