Mapping Protein-DNA Interactions Using ChIP-exo and Illumina-Based Sequencing

Chromatin immunoprecipitation (ChIP) provides a means of enriching DNA associated with transcription factors, histone modifications, and indeed any other proteins for which suitably characterized antibodies are available. Over the years, sequence detection has progressed from quantitative real-time PCR and Southern blotting to microarrays (ChIP-chip) and now high-throughput sequencing (ChIP-seq). This progression has vastly increased the sequence coverage and data volumes generated. This in turn has enabled informaticians to predict the identity of multi-protein complexes on DNA based on the overrepresentation of sequence motifs in DNA enriched by ChIP with a single antibody against a single protein. In the course of the development of high-throughput sequencing, little has changed in the ChIP methodology until recently. In the last three years, a number of modifications have been made to the ChIP protocol with the goal of enhancing the sensitivity of the method and further reducing the levels of nonspecific background sequences in ChIPped samples. In this chapter, we provide a brief commentary on these methodological changes and describe a detailed ChIP-exo method able to generate narrower peaks and greater peak coverage from ChIPped material.

Detection of aberrant behaviour in home environments from video sequence

We introduce an application for the detection of aberrant behaviour within home based environments, with a focus on repetitive actions, which may be present in instance of persons suffering from dementia. Video based analysis has been used to detect the motion of a person within a given scene in addition to tracking them over the time. Detection of repetitive actions has been based on the analysis of a person's trajectory using the principles of signal correlation. Along with the ability to detect repetitive motion the developed approach also has the ability to measure the amount of activity/inactivity within the scene during a given period of time. Our results showed that the developed approach had the ability to detect all patterns in the data set examined with an average accuracy of 96.67%. This work has therefore validated the proposed concept of video based analysis for the detection of repetitive activities.

Detection of single nucleotide polymorphisms within a sequence of a gene associated with prostate cancer using a fluorophore-tagged DNA probe

Single nucleotide polymorphisms within a sequence of a gene associated with prostate cancer were identified using oligodeoxynucleotide probe sequences bearing internal anthracene fluorophores proximal to the SNP site. Depending upon the nature of the synthesised target sequences, probe-target duplex formation could lead to enhanced or attenuated fluorescence emission from the anthracene, enabling detection of a proximal base-pair as either matching or mismatching. © 2011 Elsevier Ltd. All rights reserved.

Detection of a single DNA base-pair mismatch using an anthracene-tagged fluorescent probe

A novel anthracene-tagged oligonucleotide can discriminate between a fully-matched DNA target sequence and one with a single mismatching base-pair through a remarkable difference in fluorescence emission intensity upon duplex formation.

Iterative Multiuser Detection and Decoding for DS-CDMA System With Space-Time Linear Dispersion

This paper considers a Q-ary orthogonal direct-sequence code-division multiple-access (DS-CDMA) system with high-rate space-time linear dispersion codes (LDCs) in time-varying Rayleigh fading multiple-input-multiple-output (MIMO) channels. We propose a joint multiuser detection, LDC decoding, Q-ary demodulation, and channel-decoding algorithm and apply the turbo processing principle to improve system performance in an iterative fashion. The proposed iterative scheme demonstrates faster convergence and superior performance compared with the V-BLAST-based DS-CDMA system and is shown to approach the single-user performance bound. We also show that the CDMA system is able to exploit the time diversity offered by the LDCS in rapid-fading channels.

Multiplex Solid-Phase PCR for Rapid Detection and Identification of Salmonella spp. at Sub-species

This study presents a solid-phase PCR (SP-PCR) for rapid detection, identification, and sub-typing of various Salmonella species, the major food-borne cause of salmonellosis. The target DNA is firstly amplified with PCR primers (one primer is labeled with fluorophores) in the liquid phase. Simultaneously on the solid phase, the amplified PCR amplicons interact with the nested DNA probes immobilized on the solid substrate as an array. If the immobilized probes match the sequence of the DNA templates they are extended by the polymerase and serve as template for the second strand elongation primed by the liquid phase primer thus generating new templates for the SP-PCR. After the reaction, PCR products labeled with fluorophores remain attached to the substrate and can be visualized directly by fluorescence readout devices. Using this method, S. enteritidis, S. typhimurium and S. dublin can be detected at the same time. The method offers several advantages over conventional multiplex PCR: less competition between different primer pairs thus increasing multiplexing capability, only single wavelength optical readout needed for the multiplexing detection, and less time-consuming owing to reduction of the post-PCR gel electrophoresis. The method will be useful for development of point-of-care devices for rapid detection and identification of Salmonella spp. A solid-phase PCR for rapid detection and identification of S. enteritidis, S. typhimurium and S. dublin is developed. The method offers advantages such as better multiplexing capability, only single wavelength optical readout needed, and less time-consuming.

Molecular characterisation of Alternaria linicola and its detection in linseed

Nucleotide sequences of the ribosomal DNA (rDNA) internal transcribed spacers (ITS) 1 and 2 and a 1068 bp section of the beta-tubulin gene divided seven designated species of Alternaria into five taxa. Stemphylium botryosum formed a sixth closely related taxon. Isolates of A. linicola possessed an identical ITS sequence to one group of A. solani isolates, and two clusters of A. linicola isolates, revealed from beta-tubulin gene data to show minor variation, were as genetically similar to isolates of A. solani as they were to each other. We suggest, therefore, that A. linicola falls within the species A. solani. Similar results suggest that A. lini falls within the species A. alternata. RAPD analysis of the total genomic DNA from the Alternaria spp. concurred with the nucleotide sequence analyses. An oligonucleotide primer (ALP) was selected from the rDNA ITS1 region of A. linicola/A. solani. PCR with primers ALP and ITS4 (from a conserved region of the rDNA) amplified a c. 536 bp fragment from isolates of A. linicola and A. solani but not from other Alternaria spp. nor from other fungi which may be associated with linseed. These primers amplified an identical fragment, confirmed by Southern hybridization, from DNA released from infected linseed seed and leaf tissues. These primers have the potential to be used also for the detection of A. solani in host tissues.

Report of the workshop for life detection in samples from Mars

The question of whether there is or was life on Mars has been one of the most pivotal since Schiaparellis' telescopic observations of the red planet. With the advent of the space age, this question can be addressed directly by exploring the surface of Mars and by bringing samples to Earth for analysis. The latter, however, is not free of problems. Life can be found virtually everywhere on Earth. Hence the potential for contaminating the Mars samples and compromising their scientific integrity is not negligible. Conversely, if life is present in samples from Mars, this may represent a potential source of extraterrestrial biological contamination for Earth. A range of measures and policies, collectively termed ‘planetary protection’, are employed to minimise risks and thereby prevent undesirable consequences for the terrestrial biosphere. This report documents discussions and conclusions from a workshop held in 2012, which followed a public conference focused on current capabilities for performing life-detection studies on Mars samples. The workshop focused on the evaluation of Mars samples that would maximise scientific productivity and inform decision making in the context of planetary protection. Workshop participants developed a strong consensus that the same measurements could be employed to effectively inform both science and planetary protection, when applied in the context of two competing hypotheses: 1) that there is no detectable life in the samples; or 2) that there is martian life in the samples. Participants then outlined a sequence for sample processing and defined analytical methods that would test these hypotheses. They also identified critical developments to enable the analysis of samples from Mars.

Detection of Lateglacial distal tephra layers in the Netherlands

Three distal tephra layers or cryptotephras have been detected within a sedimentary sequence from the Netherlands that spans the last glacial-interglacial transition. Geochemical analyses identify one as the Vedde Ash, which represents the southernmost discovery of this mid-Younger Dryas tephra so far. This tephra was found as a distinct horizon in three different cores sampled within the basin. The remaining two tephras have not been geochemically ‘fingerprinted’, partly due to low concentrations and uneven distributions of shards within the sequences sampled. Nevertheless, there is the potential for tracing these tephra layers throughout the Netherlands and into other parts of continental Europe. Accordingly, the possibilities for precise correlation of Dutch palaeoenvironmental records with other continental, marine and ice-core records from the North Atlantic region are highlighted.

Detection of sensor decalibration using the asymptotic local approach

A novel method for the detection of linear decalibration of sensors is proposed. The presence of a fault is indicated as a change in the mean of a white noise sequence. A simulation example is described which shows the success of the technique.

Pre-Main-Sequence Accretion in the Low Metallicity Galactic Star-forming Region Sh 2-284

We present optical spectra of pre-main-sequence (PMS) candidates around the Ha region taken with the Southern African Large Telescope in the low metallicity (Z) Galactic region Sh 2-284, which includes the open cluster Dolidze 25 with an atypical low metallicity of Z similar to 1/5 Z(circle dot). It has been suggested on the basis of both theory and observations that PMS mass-accretion rates, (M) over dot(acc), are a function of Z. We present the first sample of spectroscopic estimates of mass-accretion rates for PMS stars in any low-Z star-forming region. Our data set was enlarged with literature data of H alpha emission in intermediate-resolution R-band spectroscopy. Our total sample includes 24 objects spanning a mass range between 1 and 2 M-circle dot and with a median age of approximately 3.5 Myr. The vast majority (21 out of 24) show evidence for a circumstellar disk on the basis of Two Micron All Sky Survey and Spitzer infrared photometry. We find (M) over dot(acc) in the 1-2 M-circle dot interval to depend quasi-quadratically on stellarmass, with (M) over dot(acc) proportional to M-*(2.4 +/- 0.35), and inversely with stellar age, with (M) over dot(acc) proportional to t(*)(-0.7 +/- 0.4). Furthermore, we compare our spectroscopic (M) over dot(acc) measurements with solar Z Galactic PMS stars in the same mass range, but, surprisingly find no evidence for a systematic change in (M) over dot(acc) with Z. We show that literature accretion-rate studies are influenced by detection limits, and we suggest that (M) over dot(acc) may be controlled by factors other than Z(*), M-*, and age.

PTEN mRNA detection by chromogenic, RNA in situ technologies: a reliable alternative to PTEN immunohistochemistry

Immunohistochemical staining for phosphatase and tensin homolog (PTEN) does not have either an acceptable standard protocol or concordance of scoring between pathologists. Evaluation of PTEN mRNA with a unique and verified sequence probe may offer a realistic alternative providing a robust and reproducible protocol. In this study, we have evaluated an in situ hybridization (ISH) protocol for PTEN mRNA using RNAScope technology and compared it with a standard protocol for PTEN immunohistochemistry (IHC). PTEN mRNA expression by ISH was consistently more sensitive than PTEN IHC, with 56% of samples on a mixed-tumor tissue microarray (TMA) showing high expression by ISH compared with 42% by IHC. On a prostate TMA, 49% of cases showed high expression by ISH compared with 43% by IHC. Variations in PTEN mRNA expression within malignant epithelium were quantifiable using image analysis on the prostate TMAs. Within tumors, clear overexpression of PTEN mRNA on malignant epithelium compared with benign epithelium was frequently observed and quantified. The use of SpotStudio software in the mixed-tumor TMA allowed for clear demonstration of varying levels of PTEN mRNA between tumor samples by the mRNA methodology. This was evident by the quantifiable differences between distinct oropharyngeal tumors (up to 3-fold increase in average number of spots per cell between 2 cases). mRNA detection of PTEN or other biomarkers, for which optimal or standardized immunohistochemical techniques are not available, represents a means by which heterogeneity of expression within focal regions of tumor can be explored with more confidence.