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

A Calorimetric Characterization of Cr(VI)-Reducing Arthrobacter oxydans

This is the first of a series of calorimetric studies designed to characterize and understand survival mechanisms of metal-reducing bacteria isolated from metal-polluted environments. In this paper we introduce a new concept of thermal spectrum of the endothermic melting of complex biological systems (e.g., proteins, nucleic acids, ribosomes, membrane structures) in intact cells. All thermal spectra measured are thermograms that describe the temperature dependence of heat capacity change of the complex systems of biologically active substances in bacterial cells. This new concept of thermal spectrum was applied to investigate spectral features from intact cells of Cr(VI)-reducer Arthrobacter oxydans at different points of their growth conditions and stages. Over the temperature range of 40–105°C, we observed that spectral changes are particularly significant in the 40–90°C interval. This may correspond to the orderly changes in subcellular structural elements: proteins, ribosomes and RNA, membranes, and various structural elements of the cell wall during different points of the growth cycle and growth conditions. Spectral changes in the 90–105°C region are less pronounced, implicating that the structural composition of DNA-Protein (DNP) complexes may change little

Linker histone subtypes are not generalized gene repressors

Antibodies to the six chicken histone H1 subtypes and the variant histone H5 have been used in immunoprecipitations of crosslinked chromatin fragments (xChIPs) to map linker histones across the β-globin locus and the widely expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and carbonic anhydrase (CA) genes in three cell types: 15-day embryo chicken erythrocytes, 15-day embryo chicken brain and the early erythroid cell line HD24. In erythrocytes, where the β-adult and β-hatching genes are active, the H1.01, H1.11L and H1.11R subtypes are substantially depleted throughout the β-globin locus and the neighboring heterochromatin, in contrast to the other four subtypes, in particular the more abundant H5. Active genes therefore carry high levels of some but not all linker histone subtypes. The situation is similar in HD24 cells, except that substantial depletions are found at the promoters of the adult β(A) and embryonic β(ρ) and β(ε) genes, despite these genes not yet being active in HD24 cells. The distributions in the brain tissue are characterised by the absence of H1.02, H1.03 and H5 from the hypersensitive site HS3 and from the β-adult 3' enhancer for the H1.11L and H1.11R subtypes. The data show that although linker histone subtypes play distinct cell-type specific roles in gene regulation, their widespread distribution indicates they are not intrinsically inhibitory to basic chromatin transactions

Characteristics of the probes used in this study.

<p>Characteristics of the probes used in this study.</p

Schematic diagrams and quantitative analysis of the designed cassettes hybridization with biochip.

<p>The arrangement of the probes on the biochips (panels A, B, C, and D) was as follows: a1, a2 – CT1; a3, a4 – HSV; a5 – RV; b1, b2 – CT2; b3, b4 – TOXO1; b5 – RV; c1, c2 – CT3; c3, c4 – TOXO2; d1, d2 – CMV1; d3, d4 – TOXO3; e1, e2 – CMV2; e3, e4 –TOXO4; e5 – bacterial DNA (negative control); c5, d5 were left empty to give background signals. White circles represent gene probes that hybridized with the respective targets on the cassette; grey circles represent gene probes that showed no hybridization and background. Error bars in the histograms are from three replicates and present the results of the Student's <i>t</i>-Tests procedure (*p<0.05). Panel (A) Cassette N1 shows the hybridization signals for three <i>Chlamydia trachomatis</i> probes (probes CT1 and CT2), and for a cryptic plasmid sequence (probe CT3). Panel (B) shows the hybridization signals for the probes CMV1 and CMV2 for <i>Cytomegalovirus</i>, the probe HSV for <i>Herpes simplex virus</i> (HSV), the TOXO1 probe for <i>Toxoplasma gondii</i> (TOXO1), and the RV probe for <i>Rubela virus</i> (RV) with cassettes N2 (shaded grey in the histogram) and cassette N3 (shaded black in the histogram). Panel (C) shows the hybridization patterns for the probes TOXO1, TOXO2, TOXO3 and TOXO4 for <i>Toxoplasma gondii</i> with cassette N4, revealing that the TOXO1 probe was unsuitable for inclusion in the TORCH biochip. Panel (D) shows the hybridization signals for probes CT1, CMV2, HSV, RV, and TOXO2 with cassette N5.</p