A novel cassette method for probe evaluation in the designed biochips

A critical step in biochip design is the selection of probes with identical hybridisation characteristics. In this article we describe a novel method for evaluating DNA hybridisation probes, allowing the fine-tuning of biochips, that uses cassettes with multiple probes. Each cassette contains probes in equimolar proportions so that their hybridisation performance can be assessed in a single reaction. The model used to demonstrate this method was a series of probes developed to detect TORCH pathogens. DNA probes were designed for Toxoplasma gondii, Chlamidia trachomatis, Rubella, Cytomegalovirus, and Herpes virus and these were used to construct the DNA cassettes. Five cassettes were constructed to detect TORCH pathogens using a variety of genes coding for membrane proteins, viral matrix protein, an early expressed viral protein, viral DNA polymerase and the repetitive gene B1 of Toxoplasma gondii. All of these probes, except that for the B1 gene, exhibited similar profiles under the same hybridisation conditions. The failure of the B1 gene probe to hybridise was not due to a position effect, and this indicated that the probe was unsuitable for inclusion in the biochip. The redesigned probe for the B1 gene exhibited identical hybridisation properties to the other probes, suitable for inclusion in a biochip

Genetic diversity of eukaryotic ultraphytoplankton in the Gulf of Naples during an annual cycle

Eukaryotic ultraphytoplankton (<5 μm) are an important component of phytoplankton populations, Dot blot hybridisation analysis using class level 16S rRNA gene probes as well as clone libraries were used to investigate the diversity of these ultraphytoplankton during a 15 mo period (2003 to 2004) in the Gulf of Naples. Hybridisation data showed the presence of 3 main classes, Cryptophyceae, Chrysophyceae and Prymnesiophyceae, along with lower signals from the Pelagophyceae. Clone libraries also contained these 4 classes as well as sequences from the Dictyochophyceae, Bacillariophyceae and Prasinophyceae. However, the Prymnesiophyceae gave the dominant hybridisation signal and constituted the majority of each clone library. Their diversity, with a total of 190 sequences belonging to 114 operational taxonomic units (OTUs), probably allows them to dominate the ultraphytoplankton throughout the whole year under differing environmental conditions. Over 100 of these OTUs were unique to different libraries, suggesting a succession of different taxa during the year. The Cryptophyceae were present most of the year with 1 OTU, corresponding to a Plagioselmis prolonga strain from the Gulf of Naples, being the dominant taxon (28 % of sequences). A striking result was the high hybridisation signal from the Chrysophyceae, which showed a preference for the summer months. The Pelagophyceae were present between December and March. Most (80 %) of the sequences found in the clone libraries were not identical to available 16S rRNA gene sequences, indicating a high amount of hidden diversity for these algal classes. However, sequences from Prasinophyceae Clade II (Mamiellales) were not detected in the clone libraries

Multicolour interphase cytogenetics: 24 chromosome probes, 6 colours, 4 layers

From the late 1980s onwards, the use of DNA probes to visualise sequences on individual chromosomes (fluorescent in-situ hybridisation - FISH) revolutionised the study of cytogenetics. Following single colour experiments, more fluorochromes were added, culminating in a 24 colour assay that could distinguish all human chromosomes. Interphase cytogenetics (the detection of chromosome copy number in interphase nuclei) soon followed, however 24 colour experiments are hampered for this application as mixing fluorochromes to produce secondary colours produces images that are not easily distinguishable from overlapping signals. This study reports the development and use of a novel protocol, new fast hybridising FISH probes, and a bespoke image capture system for the assessment of chromosome copy number in interphase nuclei. The multicolour probe sets can be used individually or in sequential hybridisation layers to assess ploidy of all 24 human chromosomes in the same nucleus. Applications of this technique are in the investigation of chromosome copy number and the assessment of nuclear organisation for a range of different cell types including human sperm, cancer cells and preimplantation embryos

Kondo hybridisation and the origin of metallic states at the (001) surface of SmB6

SmB6, a well-known Kondo insulator, has been proposed to be an ideal topological insulator with states of topological character located in a clean, bulk electronic gap, namely the Kondo hybridisation gap. Seeing as the Kondo gap arises from many body electronic correlations, this would place SmB6 at the head of a new material class: topological Kondo insulators. Here, for the first time, we show that the k-space characteristics of the Kondo hybridisation process is the key to unravelling the origin of the two types of metallic states observed directly by ARPES in the electronic band structure of SmB6(001). One group of these states is essentially of bulk origin, and cuts the Fermi level due to the position of the chemical potential 20 meV above the lowest lying 5d-4f hybridisation zone. The other metallic state is more enigmatic, being weak in intensity, but represents a good candidate for a topological surface state. However, before this claim can be substantiated by an unequivocal measurement of its massless dispersion relation, our data raises the bar in terms of the ARPES resolution required, as we show there to be a strong renormalisation of the hybridisation gaps by a factor 2-3 compared to theory, following from the knowledge of the true position of the chemical potential and a careful comparison with the predictions from recent LDA+Gutzwiler calculations. All in all, these key pieces of evidence act as triangulation markers, providing a detailed description of the electronic landscape in SmB6, pointing the way for future, ultrahigh resolution ARPES experiments to achieve a direct measurement of the Dirac cones in the first topological Kondo insulator.Comment: 9 pages, 4 Figures and supplementary material (including Movies and CORPES13 "best prize" poster

Strong 3p -T1u Hybridization in Ar@C60

Multilayers of fullerenes with and without endohedral Ar units, C60 and Ar@C60, were investigated by photoemission and density functional theory. The stoichiometry and the endohedral nature of Ar is checked by x-ray photoelectron spectroscopy and x-ray photoelectron diffraction. Valence band ultraviolet photoemission spectra show a strong hybridisation of the Ar 3p valence shell with the 6T1u molecular orbital of C60. A hybridisation gap of 1.6 +/- 0.2 eV is found. This is in agreement with density functional theory (DFT) that predicts 1.47 eV, and indicates Ar@C60 to be a noble gas compound with a strong coupling between Ar and the C60 cage. No giant Ar photoemission cross section as predicted for the gas phase in [Phys. Rev. Lett. 99, 243003 (2007)] was found

Flow equation approach to heavy fermion systems

We use Wegner's flow equation method to investigate the infinite-$U$ periodic Anderson model. We show that this method poses a new approach to the description of heavy fermion behaviour. Within this scheme we derive an effective Hamiltonian where the $c$ and $f$ degrees of freedom are decoupled. By evaluating one-particle energies as well as correlation functions we find an electronic structure which comprises two gapped quasiparticle bands. We also address the lattice Kondo temperature, which shows a typical exponential dependence on the hybridisation energy. This energy scale exhibits a significant decrease compared to that of the single impurity Anderson model

T-DNA promoter tagging in Nicotiana tabacum : a thesis presented in fulfilment of the requirements for the degree of Master of Philosophy in Genetics at Massey University, Palmerston North, New Zealand

Plant development is primarily controlled at the level of gene expression. In order to analyse this regulation it is necessary to isolate genes which are involved in organ development through cellular and tissue determination or which respond to environmental signals. Promoter tagging was chosen in order to identify genes potentially associated with plant development by their spatial and temporal pattern of expression. The introduction of a promoterless reporter gene tag allows the expression patterns of plant genes to be readily characterised. A new series of promoter tagging vectors were constructed from the plasmid pPCV604 (Koncz, 1989). The selectable kanamycin resistance marker gene from pBin6 (Bevan, 1984) was cloned into pPCV604 to create pGT. The hygromycin phosphotransferase gene in pGT was then replaced with a promoterless (β-glucuronidase (gus) gene coupled with octopine synthase termination sequence subcloned from pKiwi101a (Janssen and Gardner, 1989) creating pGTG. This binary transformation vector required the helper pRK replication functions of Agrobacterium tumefaciens strain GV3101. In order to bypass this restriction, the vector sequence of pBin19 was combined with the T-DNA of pGTG to create pBin19-GTG. The latter plasmid was found to have a higher Agrobacterium tumefaciens-mediated Nicotiana tabacum transformation efficiency in strain LBA4404 than pGTG in strain GV3101. In both the pGTG and pBin19-GTG promoter tagging vectors the promoterless gus gene has an initiation codon 62 base pairs inside the T-DNA. This sequence includes translation termination codons in all three reading frames. Therefore, insertion of the T-DNA into a plant gene could lead to activation of the gus gene, under the control of the plant gene promoter, via transcriptional fusion. Nicotiana tabacum leaf segments were transformed with pGTG or pBin19-GTG and transgenic plants selected on kanamycin. A population of 87 transgenic tobacco plants were fluorometrically screened for GUS activity in leaf and root material; 37% were found to contain GUS activity, indicating a high frequency of promoter tagging. Two transgenic plants with root specific gus expression were analysed histochemically. Progeny after self-fertilisation lacked GUS activity, though this was restored in progeny of one plant with 5-azacytidine treatment, suggesting involvement of methylation in the gene silencing. Southern hybridisation, inverse PCR cloning of T-DNA flanking sequences and segregation on kanamycin indicated the presence of multiple T-DNA copies within the primary transformants. Furthermore, inverse PCR sequence from one plant indicated multiple and truncated T-DNA insertions at one or more loci. A further population of transformed plants was generated with pBin19-GTG and histochemically screened for GUS activity in roots (14 positive from 147 tested), shoots (27 positive from 147) and floral organs (14 positive from 56). Overall, combining results from all plant organs tested, an average of 33% of plants were found with GUS activity in one or more organs. A diverse range of patterns of gus expression were observed and described including patterns involving root branching. Forty four plants from this population were analysed for T-DNA copy number via Southern hybridisation with a gus probe (right border junction T-DNA) and nptII probe (central T-DNA). Multiple copies were frequently found with an average of 3.3 T-DNA copies per transgenic plant. Overall, an average of 11% of T-DNA insertions were found to be involved in gus activation. Comparison of the fluorometric (37% positive, 87 plants tested) and histochemical (22% positive, 147 plants tested) screens for GUS activity in root and shoot material was discussed and it is suggested that further care is needed in assigning promoter tagging hits from fluorometric screening. Variable expression was observed with promoter tagged genes. It is suggested that further research is required to determine whether this variation was due to silencing, perhaps by methylation, or was a result of the tagged promoters' normal expression patterns