Probe Spacing Research Articles

A three-point-probe method for measuring resistivity and the Hall coefficient using Hall devices with minimal design complexity

A novel empirical method for express measurement of resistivity and the Hall coefficient, i.e. the sign, the concentration and the bulk mobility of the majority carriers in semiconductor (silicon) wafers is presented and tested. This approach is based on the parallel-field Hall devices with minimal design complexity containing only three contacts–two input and one (Hall) output. The unique simplicity of this three-point-probe method, the lack of numerical coefficients’ calculation and particular requirements for the geometrical dimensions, probe spacing, wafer form, etc. allows to obtain the necessary information following experimentally clear and technically reproducible steps. The data obtained from non-structured n- and p-type silicon wafers at carrier’s concentration of 10 15 ⩽ n; p ⩽ 10 16 cm −3 by appropriate probe arrangements corroborate very well with the results from wafers’ certificates and the results obtained using common measurement techniques. The error does not exceed 5–6% which is about the same as the accuracy of other approaches used for this purpose. The results are especially promising in IC fabrication and complementing the classical Van der Pauw method.

Direct detection of grain boundary scattering in damascene Cu wires by nanoscale four-point probe resistance measurements

Four-terminal conductivity measurements of damascene copper (Cu) wires with various widths have been performed using platinum-coated carbon nanotube (CNT) tips in a four-tip scanning tunneling microscope. Using CNT tips enabled the probe spacing to be reduced to 70 nm, which is the shortest probe spacing in interconnect wire measurements achieved so far. The measured resistivity of Cu increased as the line width decreased and direct evidence of individual grain boundary scattering was observed when the probe spacing was varied on a scale comparable to the grain size of the Cu wires (∼200 nm).

Identification of small gains and losses in single cells after whole genome amplification on tiling oligo arrays

Clinical DNA is often available in limited quantities requiring whole-genome amplification for subsequent genome-wide assessment of copy-number variation (CNV) by array-CGH. In pre-implantation diagnosis and analysis of micrometastases, even merely single cells are available for analysis. However, procedures allowing high-resolution analyses of CNVs from single cells well below resolution limits of conventional cytogenetics are lacking. Here, we applied amplification products of single cells and of cell pools (5 or 10 cells) from patients with developmental delay, cancer cell lines and polar bodies to various oligo tiling array platforms with a median probe spacing as high as 65 bp. Our high-resolution analyses reveal that the low amounts of template DNA do not result in a completely unbiased whole genome amplification but that stochastic amplification artifacts, which become more obvious on array platforms with tiling path resolution, cause significant noise. We implemented a new evaluation algorithm specifically for the identification of small gains and losses in such very noisy ratio profiles. Our data suggest that when assessed with sufficiently sensitive methods high-resolution oligo-arrays allow a reliable identification of CNVs as small as 500 kb in cell pools (5 or 10 cells), and of 2.6–3.0 Mb in single cells.

Array analysis and karyotyping: Workflow consequences based on a retrospective study of 36,325 patients with idiopathic developmental delay in the Netherlands

Anomalies of chromosome number and structure are considered to be the most frequent cause of unexplained, non-syndromic developmental delay and mental retardation (DD/MR). High-resolution, genome-wide, array-based segmental aneusomy profiling has emerged as a highly sensitive technique for detecting pathogenic genomic imbalances. A review of 29 array-based studies of DD/MR patients showed that a yield of at least ∼19% pathogenic aberrations is attainable in unselected, consecutive DD/MR referrals if array platforms with 30–70 kb median probe spacing are used as an initial genetic testing method. This corresponds to roughly twice the rate of classical cytogenetics. This raises the question whether chromosome banding studies, combined with targeted approaches, such as fluorescence in situ hybridisation for the detection of microdeletions, still hold substantial relevance for the clinical investigation of these patients. To address this question, we reviewed the outcome of cytogenetic studies in all 36,325 DD/MR referrals in the Netherlands during the period 1996–2005, a period before the advent of array-based genome investigation. We estimate that in a minimum of 0.78% of all referrals a balanced chromosomal rearrangement would have remained undetected by array-based investigation. These include familial rearrangements (0.48% of all referrals), de novo reciprocal translocations and inversions (0.23% of all referrals), de novo Robertsonian translocations (0.04% of all referrals), and 69,XXX triploidy (0.03% of all referrals). We conclude that karyotyping, following an initial array-based investigation, would give only a limited increase in the number of pathogenic abnormalities, i.e. 0.23% of all referrals with a de novo, apparently balanced, reciprocal translocation or inversion (assuming that all of these are pathogenic), and 0.03% of all referrals with 69,XXX triploidy. We propose that, because of its high diagnostic yield, high-resolution array-based genome investigation should be the first investigation performed in cases of DD/MR, detecting >99% of all pathogenic abnormalities. Performing both array investigation and karyotyping may not be a feasible option when laboratories are faced with a need to limit the number of genetic tests available for each patient. However, laboratories that supplant karyotyping by array-based investigation should be aware that, as shown here, a chromosomal abnormality, with possible pathogenic consequences for the patient or the family, will escape detection in about 0.78% of all DD/MR referrals.

Neurobeachin ( NBEA) is a target of recurrent interstitial deletions at 13q13 in patients with MGUS and multiple myeloma

Chromosome 13 deletions (del[13]), detected by metaphase cytogenetics, predict poor outcomes in multiple myeloma (MM), but the gene(s) responsible have not been conclusively identified. We sought to identify tumor-suppressor genes on chromosome 13 using a novel array comparative genomic hybridization (aCGH) strategy. We identified DNA copy number losses on chromosome 13 using genomic DNA isolated from CD138-enriched bone marrow cells (tumor) from 20 patients with MM, monoclonal gammopathy of undetermined significance, or amyloidosis. We used matched skin biopsy (germline) genomic DNA to control for copy number polymorphisms and a novel aCGH array dedicated to chromosome 13 to map somatic DNA gains and losses at ultra-high resolution (>385,000 probes; median probe spacing 60 bp). We analyzed microarray expression data from an additional 262 patient samples both with and without del[13]. Two distinct minimally deleted regions at 13q14 and 13q13 were defined that affected the RB1 and NBEA genes, respectively. RB1 is a canonical tumor suppressor previously implicated in MM. NBEA is implicated in membrane trafficking in neurons, protein kinase A binding, and has no known role in cancer. Noncoding RNAs on chromosome 13 were not affected by interstitial deletions. Both the RB1 and NBEA genes were deleted in 40% of cases (8 of 20; 5 patients with del[13] detected by traditional methods and 3 patients with interstitial deletions detected by aCGH). Forty-one additional MM patient samples were used for complete exonic sequencing of RB1, but no somatic mutations were found. Along with RB1, NBEA gene expression was significantly reduced in cases with del[13]. The NBEA gene at 13q13, and its expression are frequently disrupted in MM. Additional studies are warranted to evaluate the role of NBEA as a novel candidate tumor-suppressor gene.

Assessment of tissue oxygen saturation during a vascular occlusion test using near-infrared spectroscopy: the role of probe spacing and measurement site studied in healthy volunteers

IntroductionTo assess potential metabolic and microcirculatory alterations in critically ill patients, near-infrared spectroscopy (NIRS) has been used, in combination with a vascular occlusion test (VOT), for the non-invasive measurement of tissue oxygen saturation (StO2), oxygen consumption, and microvascular reperfusion and reactivity. The methodologies for assessing StO2 during a VOT, however, are very inconsistent in the literature and, consequently, results vary from study to study, making data comparison difficult and potentially inadequate. Two major aspects concerning the inconsistent methodology are measurement site and probe spacing. To address these issues, we investigated the effects of probe spacing and measurement site using 15 mm and 25 mm probe spacings on the thenar and the forearm in healthy volunteers and quantified baseline, ischemic, reperfusion, and hyperemic VOT-derived StO2 variables.MethodsStO2 was non-invasively measured in the forearm and thenar in eight healthy volunteers during 3-minute VOTs using two InSpectra tissue spectrometers equipped with a 15 mm probe or a 25 mm probe. VOT-derived StO2 traces were analyzed for base-line, ischemic, reperfusion, and hyperemic parameters. Data were categorized into four groups: 15 mm probe on the forearm (F15 mm), 25 mm probe on the forearm (F25 mm), 15 mm probe on the thenar (T15 mm), and 25 mm probe on the thenar (T25 mm).ResultsAlthough not apparent at baseline, probe spacing and measurement site significantly influenced VOT-derived StO2 variables. For F15 mm, F25 mm, T15 mm, and T25 mm, StO2 ownslope was -6.4 ± 1.7%/minute, -10.0 ± 3.2%/minute, -12.5 ± 3.0%/minute, and -36.7 ± 4.6%/minute, respectively. StO2 upslope was 105 ± 34%/minute, 158 ± 55%/minute, 226 ± 41%/minute, and 713 ± 101%/minute, and the area under the hyperemic curve was 7.4 ± 3.8%·minute, 10.1 ± 4.9%·minute, 12.6 ± 4.4%·minute, and 21.2 ± 2.7%·minute in these groups, respectively. Furthermore, the StO2 parameters of the hyperemic phase of the VOT, such as the area under the curve, significantly correlated to the minimum StO2 during ischemia.ConclusionsNIRS measurements in combination with a VOT are measurement site-dependent and probe-dependent. Whether this dependence is anatomy-, physiology-, or perhaps technology-related remains to be elucidated. Our study also indicated that reactive hyperemia depends on the extent of ischemic insult.

Synthetic Aperture Imaging of Multiple Point Targets in Rician Fading Media

This paper presents a study of stability, resolution, and detection for broad-band synthetic aperture (SA) imaging in Rician fading media. The stability condition $BNK^2/(K+1)\gg M$ is derived, where $K$ is the Rician factor, $B$ is the effective number of coherence bands, $N$ is the effective number of array elements, and $M$ is the number of (widely separated) targets. The imaging method is tested numerically with randomly distributed discrete scatterers, and comparisons with the imaging with the full response matrix (RM) are made. The resolution study reveals several interesting effects: First, given the same measurement resources, SA imaging has better resolution performance, although less stable, than RM imaging; second, for both imaging methods, the cross-range resolution measure (i.e., “full width at half maximum”) decreases with the aperture ($N$ fixed) and the probe spacing (the total aperture fixed) while the range resolution increases with both parameters. A statistical scheme is introduced to reduce the uncertainty when the stability regime is not realized.

Assessment of tissue oxygen saturation during a vascular occlusion test using near-infrared spectroscopy: role of the probe spacing and measurement site studied in healthy volunteers

To assess potential metabolic and microcirculatory alterations in critically ill patients, near-infrared spectroscopy (NIRS) has been used, in combination with a vascular occlusion test (VOT), for the noninvasive measurement of tissue oxygen saturation (StO2), oxygen consumption, and microvascular reperfusion and reactivity. However, the methodologies for assessing StO2 are very inconsistent in the literature and results vary from study to study. In this study we investigated the effects of the probe spacing and measurement site, using both a 15 mm and a 25 mm probe spacing on the thenar and the forearm, in healthy volunteers.

NOTCH2 Is Neither Rearranged nor Mutated in t(1;19) Positive Oligodendrogliomas

The combined deletion of 1p and 19q chromosomal arms is frequent in oligodendrogliomas (OD) and has recently been shown to be mediated by an unbalanced t(1;19) translocation. Recent studies of 1p/19q co-deleted OD suggest that the NOTCH2 gene is implicated in oligodendrocyte differentiation and may be involved in this rearrangement. The objective of the present study was to analyze the NOTCH2 locus either as a chromosomal translocation locus that may be altered by the 1p/19q recurrent rearrangement or as a gene that may be inactivated by a two hit process. We performed an array-CGH analysis of 15 ODs presenting 1p/19q co-deletion using a high-density oligonucleotide microarray spanning 1p and 19q pericentromeric regions with 377 bp average probe spacing. We showed that the 1p deletion extends to the centromere of chromosome 1 and includes the entire NOTCH2 gene. No internal rearrangement of this gene was observed. This strongly suggests that the t(1;19) translocation does not lead to an abnormal NOTCH2 structure. The analysis of the entire NOTCH2 coding sequence was performed in four cases and did not reveal any mutation therefore indicating that NOTCH2 does not harbor genetic characteristics of a tumor suppressor gene. Finally, the detailed analysis of chromosome 19 pericentromeric region led to the identification of two breakpoint clusters at 19p12 and 19q11–12. Interestingly, these two regions share a large stretch of homology. Together with previous observations of similarities between chromosome 1 and 19 alphoid sequences, this suggests that the t(1;19) translocation arises from complex intra and interchromosomal rearrangements.This is the first comprehensive deletion mapping by high density oligo-array of the 1p/19q co-deletion in oligodendroglioma tumors using a methodological approach superior to others previously applied. As such this paper provides clear evidence that the NOTCH2 gene is not physically rearranged by t(1;19) translocation of oligodendroglioma tumors.

Efficient Biosensor Interfaces Based on Space-Controlled Self-Assembled Monolayers

In this paper we demonstrate control over the spacing of surface-modifying probe molecules through the use of labile dendron spacers. During this process, anchor molecules are first adsorbed to a surface, with dendron modifiers attached. Steric interactions of the bulky dendrons control the density of anchor molecules bound to the surface. The dendron branches are subsequently detached from the anchor molecules, and the anchors are chemically modified with probe molecules, resulting in a surface with controlled spacing between probe molecules. Control over this spacing is important when the probe size is small in comparison with the target molecule. This importance is demonstrated for the binding of protein (streptavidin) targets to the probe (biotin) surface. The effect of probe space control on the efficiency of target capture is evaluated by examining the binding of streptavidin to thiolated biotin for a series of mixed monolayers. Surface modification is monitored by Fourier transform infrared reflection absorption spectroscopy (FTIR-RAS). The relative concentration of probe molecules at the surface is measured using X-ray photoelectron spectroscopy (XPS) measurements. Thiolated-biotin surfaces with optimized spacing show an increased capture efficiency for streptavidin relative to surfaces with nonoptimal or no control over probe spacing, as measured by surface plasmon resonance (SPR) spectroscopy. These results are of potential significance for the optimization and fabrication of micro- and nanoarrays used in chemical and biochemical measurements.

A Snapshot of CNVs in the Pig Genome

Recent studies of mammalian genomes have uncovered the extent of copy number variation (CNV) that contributes to phenotypic diversity, including health and disease status. Here we report a first account of CNVs in the pig genome covering part of the chromosomes 4, 7, 14, and 17 already sequenced and assembled. A custom tiling oligonucleotide array was used with a median probe spacing of 409 bp for screening 12 unrelated Duroc boars that are founders of a large family material. After a strict CNV calling pipeline, 37 copy number variable regions (CNVRs) across all four chromosomes were identified, with five CNVRs overlapping segmental duplications, three overlapping pig unigenes and one overlapping a RefSeq pig mRNA. This CNV snapshot analysis is the first of its kind in the porcine genome and constitutes the basis for a better understanding of porcine phenotypes and genotypes with the prospect of identifying important economic traits.

Ultra-High Resolution Analysis of Genomic Alterations in High-Risk Acute Lymphoblastic Leukemia.

Previous genomic analyses identified a high frequency of genetic alterations targeting B-lymphoid development (>40%) and cell cycle/tumor suppression (CDKN2A/B, 35%) in pediatric B-progenitor acute lymphoblastic leukemia (B-ALL). The frequency and nature of copy number alterations (CNAs) differs significantly across ALL subtype with less than one CNA/case in MLL ALL and over 8 lesions/case in BCR-ABL1 and ETV6-RUNX1 ALL; and the near obligate presence of IKZF1 (Ikaros) deletion in BCR-ABL1 ALL, but never in ETV6-RUNX1 ALL. Although important insights have been obtained from these genome-wide CNAs, it is important to realize that the SNP array platforms that have been used are limited by uneven probe spacing and poor coverage of some genes (including IKZF1 and CDKN2A/B). Moreover, additional CNAs are detected as array resolution increases (e.g. 6.5 lesions per BCR-ABL1 case using 315,000 markers, and 8.8 for 615,000 marker data). Thus, the true frequency of CNAs in ALL remains unknown. To address this question, we now report ultra-high resolution CNA analysis using 2.17 million feature oligonucleotide arrays (Roche Nimblegen) of 20 MLL, 20 BCR-ABL1, and 4 miscellaneous karyotype B-ALL cases. All had previously been examined using Affymetrix 500K SNP arrays. We identified a mean of 6.2 deletions (range 0–38) and 1.4 gains (0–13) per case. There were more lesions in BCR-ABL1 than MLL ALL cases (mean 11.2 v 1.15 deletions, P<0.0001; 2.3 v 0.45 amplifications, P=0.01). Notably, more lesions were detected by this 2.17 million feature array in comparison to previous Affymetrix SNP array data in BCRABL1 ALL (13.5 v 8.8 lesions/case, P=0.001) but not MLL ALL (1.6 v 1.3 lesions/case, P=NS). Moreover, the Nimblegen platform robustly identified focal deletions in genes poorly covered by SNP arrays (IKZF1, CDKN2A/B) and detected additional deletions not previously identified (e.g. deletion of HBS1L immediately adjacent to MYB). The most common lesions in ALL were deletions of IKZF1 (80% BCR-ABL1, none in MLL), CDKN2A/B (60% BCR-ABL1, 20% MLL) and CNAs of PAX5 (50% BCR-ABL1, 20% MLL). High array resolution enabled PCR-based mapping and sequencing of the genomic breakpoints of multiple recurring deletions (e.g. ADD3, BTLA, CDKN2A/B, C20orf94, HBS1L, IKZF1 and PAX5), each of which bore hallmarks of aberrant RAG-mediated recombination.Due to the small size of the CDKN2A/B genomic locus (41.5 Kb), previous genomic analyses have been unable to accurately determine the frequency of deletion of each of the three tumor suppressors (INK4A, ARF, and INK4B) encoded by this locus. We performed genomic quantitative PCR (gqPCR) across the locus (CDKN2A exons 1b, 1a and 2; CDKN2B exons 1 and 2), which revealed complete concordance between deletions identified by the Nimblegen array and confirmatory gqPCR. However, gqPCR was required to precisely delineate the extent of deletion in each case. All deletions involved both CDKN2A and CDKN2B loci except one MLL case (CDKN2A only), and the same degree of deletion (mono- or bi-allelic) was usually uniform across the locus for each case. Exceptions included two cases harboring homozygous deletions of INK4B and ARF but hemizygous deletion of INK4A, and a case with deletion of ARF-INK4A but not INK4B. To extend this analysis to epigenetic silencing of CDKN2A/B, we performed quantitative methylation analysis of six CDKN2A and two CDKN2B CpG islands by MALDI-TOF mass spectrometry for all BCR-ABL1 cases and 18 CDKN2A/B non-deleted MLL, ETV6-RUNX, and T-ALL cases. Strikingly, whereas high level CpG methylation of CDKN2B and CDKN2A exons 2 and 3 was observed in half the MLL and T-ALL cases, no high level methylation was observed in BCR-ABL1 ALL.These data demonstrate that identification of all CNAs in BCR-ABL1 ALL is critically dependent on array resolution, whereas MLL -rearranged leukemias harbor very few CNAs. Importantly, the use of high density arrays with even probe distribution improves the ability to robustly identify focal CNAs in genes poorly covered by SNP arrays, and to further characterize the mechanism of deletion by sequencing. Furthermore, even with the highest resolution arrays, complementary quantitative PCR across CDKN2A/B is required to precisely define the extent of deletion of this compact gene cluster. Thus, oligonucleotide array or sequencing-based platforms allowing true tiling across the genome will be required to determine the full complement of CNAs in ALL.

Integrative Analysis of Genomic Aberration and Gene Expression Suggests That MPD with Loss of Heterozygosity or Gain of Chromosome 9 Represents a Distinct Entity

Polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF) can all be associated with the JAK2V617F gain of function mutation. MPDs are also associated with gross cytogenetic anomalies including genomic gain/loss or loss of heterozygosity (LOH) due to mitotic homologous recombination. To determine whether genomic anomalies may be associated with JAK mutation status/disease phenotype we subjected genomic DNA from the granulocytes of 87 patients with PV, ET or IMF to analysis using high resolution Affymetrix 250K Nsp SNP arrays with an average probe spacing of 12Kb. Firstly this analysis precisely mapped previously identified anomalies in MPD patients such as gain of chromosome 1q chromosome 9 (Chr9), chromosome 8, and deletion of 20q and 13q indicating the robustness of the technique. By removing genomic Copy Number Polymorphisms (CNPs) ascertained from 90 normal individuals from the HapMap dataset, we further detected a number of novel alterations such as frequent gain of Chr9p33–34 harboring among other genes, Notch1. MPD patients heterozygous for the JAK V617F mutation exhibited higher genomic instability than JAK2 V617 homozygous or wild type patients. IMF patients had overall more aberrations than PV or ET patients but unsupervised hierarchical clustering of chromosomal anomalies could not distinguish patients with PV, ET or IMF. Whole chromosomal gain or loss was equally frequent among the three diseases, however IMF patients had more frequent alteration of 10Mb or greater when compared to ET or PV (p<0.01) and ET and IMF patients both more frequently exhibited changes ranging from 1–10Mb in size than PV patients (p<0.01). Correlation of gene expression profiles from the granulocytes of 38 MPD patients with copy number profiles from DNA of the patients indicated that gene expression in regions of copy number gain or loss correlated with gene dosages in general. From 2493 genes residing in regions of copy number change region, 259 genes exhibited a positive correlation between copy number and gene expression. By calculating the t–statistics of expression values between samples with and without chromosomal aberrations we further identified 28 genes that showed a change of at least 1.5 fold (p<0.05), 27 of which were located on chromosome 9. Among these were Smarca2, NOTCH1 JAK2 itself. JAK2V617F expression was highly elevated by gain of Chr9 or Chr9p LOH. LOH on chr9p was detected in 16 patients, the majority of whom were confirmed as homozygous for JAK2V617F. To investigate how the over-expression of JAK2V617F might affect the expression of other genes and pathways, gene expression profiles of patients with Chr9 abnormality including Chr9 gain or Chr9p LOH were compared to samples with a normal copy number for Chr9. We identified 493 genes including 210 up-regulated and 283 down-regulated genes by at least 1.5 fold at a p value of 0.05 after multiple-testing correction. As a control gene expression patterns in JAK2-wildtype MPD granulocytes were compared to expression in normal granulocytes. This analysis identified an almost completely different set of genes; 441/493 (89%) of genes differentially expressed in Chr9 abnormal cases versus Chr9 normal cases were not found in the Chr9 normal versus normal control dataset. Up-regulated genes in Chr9 abnormal cases included CD177, CDK5RAP2, BIRC1, STAT5, TLR4 and down-regulated genes include KLRB1, Myc, RUNX3, P53CSV, Pathway analysis of dysregulated genes in Chr9 gain/LOH cases showed that genes involved in p38/MAP kinase, Toll-like receptor and Fc epsilon RI signaling were up-regulated and the genes involved in pathways of protein synthesis/translation and T cell receptor signaling were down-regulated. Collectively these findings suggest that MPD associated with Chr9 gain or 9pLOH and increased expression of JAK2V617F represents a distinct subset of MPD, characterized by less genomic instability than other cases. Furthermore, increased JAK2 and STAT5 expression and activity in these cases may induce distinct downstream pathways and alterations in gene expression.

On the analysis of the activation mechanisms of sub-melt laser anneals

In order to fabricate carrier profiles with a junction depth (∼15 nm) and sheet resistance value suited for sub-32 nm Si-CMOS technology, the usage of sub-melt laser anneal is a promising route to explore. As laser annealed junctions seem to outperform standard anneal approaches, a detailed assessment of the basics of laser induced activation seem appropriate. In this work the electrical activation is studied from a comparison between the dopant profiles as measured by Secondary Ion Mass Spectrometry, and the electrically active fraction as extracted from sheet resistance and mobility measurements. The latter is based on a large variety of techniques. For the sheet resistance we use conventional Four-Point Probe (FPP), Variable Probe Spacing (VPS), contactless junction photo voltage (JPV), Micro Four-Point Probe (M4PP) and an optical technique, namely Model Based Infra-red spectroscopic Reflectrometry (MBIR). For the sheet carrier density and sheet mobility extraction we use conventional Hall (without cloverleaf van der Pauw patterning, to reflect the need for fast turn-round sheet measurements), MBIR, and a recently developed new Hall-like capability using M4PP. By recognizing the interaction between the various parameters as they are not completely independent, it is possible to test the consistency of the various methods and to identify potential short comings. This concept is applied to the activation behavior of low and high implanted Boron doses and indicates that the obtained electrically active concentration level as well as the concurrent mobility is dependent on the dopant concentration level. This implies that the activation of B through the laser anneal process in the explored temperature–time space is governed by kinetic processes (i.e. the dissolution of B–I pairs) and not by the (temperature related) solid solubility.

A complementary metal-oxide-semiconductor compatible monocantilever 12-point probe for conductivity measurements on the nanoscale

We present a complementary metal-oxide-semiconductor compatible, nanoscale 12-point-probe based on TiW electrodes placed on a SiO2 monocantilever. Probes are mass fabricated on Si wafers by a combination of electron beam and UV lithography, realizing TiW electrode tips with a width down to 250nm and a probe pitch of 500nm. In-air four-point measurements have been performed on indium tin oxide, ruthenium, and titanium-tungsten, showing good agreement with values obtained by other four-point probes. In-vacuum four-point resistance measurements have been performed on clean Bi(111) using different probe spacings. The results show the expected behavior for bulk Bi, indicating that the contribution of electronic surface states to the transport properties is very small.

Analytical Solution of Heat Pulse Method in a Parallelepiped Sample Space with Inclined Needles

The heat pulse method enables estimation of soil thermal diffusivity (k), volumetric heat capacity (C), thermal conductivity, and water content. The heater needle and temperature‐sensing needle may deflect during probe insertion into soils. The impact of needle deflection on estimates of C and k has not been fully studied theoretically or experimentally. We defined θ to be the polar angle of needle deviation from the z axis and φ to be the azimuthal angle in the x–y plane. Transient‐state analytical solutions were derived for an inclined and pulsed finite line source in a parallelepiped sample with zero surface temperature and adiabatic boundary conditions. For a heat pulse sensor with 6‐mm needle spacing and a heater needle of 4‐cm length in a given parallelepiped (5 by 5 by 5 cm, assumed to be filled with air‐dry sand), model errors in C and k were about −11.3 and 12.1%, respectively, for an inclined heater needle with θ = 1° and φ = 0°. Model errors in C and k were about −11.2 and 12.1%, respectively, for an inclined sensor needle with θ = 1° and φ = 180°. When −6 ≤ θ ≤ 6° for either the heater or the sensor needle, the temperature curves could be approximated rather well by a pulsed infinite line source model with a modified probe spacing that accounted for the inclination. For various heating durations and strengths, the errors in both k and C were relatively constant when all other parameters were fixed; however, the errors in both C and k decreased monotonically and slowly as k increased. The model errors in C and k were similar for four soil conditions with different thermal properties in the range −6 ≤ θ ≤ 2°.

Electrical Resistivity Measurements for Quality Control During Concrete Construction

In severe environments, new concrete structure production increasingly uses chloride diffusivity requirements as a concrete durability performance-based specification. Chloride diffusivity testing is both elaborate and time-consuming, however, as a concrete quality control basis during concrete construction. Using the Nernst-Einstein equation as a basis, the given concrete's electrical resistivity and chloride diffusivity relationship should therefore first be established. Then routine-based electrical resistivity measurement during concrete construction can indirectly control chloride diffusivity. There are several test methods for concrete electrical resistivity measurement. Such measurements may also be affected by several factors. An experimental program was carried out in order to provide more information about some of those factors, since, in regard to electrical resistivity during concrete construction, they may affect results and allow simple routine-based quality control procedures to be established. Primarily based on four-electrode (Wenner) electrical resistivity testing, the test program included different test specimen geometry and probe spacing. Some two-electrode measurements were also performed for comparison. Test results are primarily presented as relative resistivities that two different test methods obtained in order to describe various factor effects that resistivity may be affected by. That, for given testing conditions, Wenner-method obtained electrical resistivity differs from two-electrode method obtained resistivity is shown in the results. The authors conclude that, however, for a given concrete specimen type with given temperature and moisture conditions, a suitable and reliable test method for performance-based electrical resistivity quality control, and hence, concrete durability, during concrete construction appears to be the Wenner method.

Model-based deconvolution of genome-wide DNA binding

Chromatin immunoprecipitation followed by hybridization to a genomic tiling microarray (ChIP-chip) is a routinely used protocol for localizing the genomic targets of DNA-binding proteins. The resolution to which binding sites in this assay can be identified is commonly considered to be limited by two factors: (1) the resolution at which the genomic targets are tiled in the microarray and (2) the large and variable lengths of the immunoprecipitated DNA fragments. We have developed a generative model of binding sites in ChIP-chip data and an approach, MeDiChI, for efficiently and robustly learning that model from diverse data sets. We have evaluated MeDiChI's performance using simulated data, as well as on several diverse ChIP-chip data sets collected on widely different tiling array platforms for two different organisms (Saccharomyces cerevisiae and Halobacterium salinarium NRC-1). We find that MeDiChI accurately predicts binding locations to a resolution greater than that of the probe spacing, even for overlapping peaks, and can increase the effective resolution of tiling array data by a factor of 5x or better. Moreover, the method's performance on simulated data provides insights into effectively optimizing the experimental design for increased binding site localization accuracy and efficacy. MeDiChI is available as an open-source R package, including all data, from http://baliga.systemsbiology.net/medichi.

Comprehensive Genomic Copy Number and Sequence Analysis of 28 Chromosome 5q31.2 Candidate Genes in De Novo MDS.

Deletions involving the long arm of chromosome 5 are among the most common recurring cytogenetic abnormalities detectable in human myelodysplastic syndromes (MDS). The minimally deleted segment has been mapped by several groups to a 2.3 megabase interval at 5q31.2. This region contains 21 annotated genes, one pseudogene, 4 small RNAs, and 2 predicted genes (SPOCK1, KLHL3, HNRPA0, NPY6R, MYOT, PKD2L2, C5orf5, WNT8A, NME5, BRD8, KIF20A, CDC23, GFRA3, CDC25C, FAM53C, JMJD1B, REEP2, EGR1, ETF1, HSPA9B, SNORD63, LOC391836, LOC729429, CTNNA1, LRRTM2, SIL1, MATR3, SNORA74A). Extensive study of many of these genes by other investigators has raised the possibility that gene(s) in this interval contribute to MDS pathogenesis by haploinsufficiency. To address this hypothesis, we selected samples from 46 de novo MDS patients that had adequate amounts of paired tumor (bone marrow) and germline (skin) DNA available. The 46 patients include all FAB subtypes, with a range of IPSS scores from 0–3 (median = 1), and a median blast count of 5%. The majority of these samples (n=37) do not have cytogenetically apparent 5q31.2 deletions. We first asked whether these samples might contain deletions in this interval below the limit of detection by cytogenetics. We used a custom oligomer based array comparative genomic hybridization (aCGH) platform developed by NimbleGen Systems, Inc. that has ∼385,000 probes spanning human chromosome 5, providing an average probe spacing of 500 base pairs. Analysis of aCGH data for 20 patients demonstrated that cytogenetically apparent 5q31.2 deletions could be detected by this platform, but no cytogenetically “silent” interstitial deletions were seen. We next asked whether genes in this interval might instead be targeted by point mutations. To define the sensitivity of our sequence-based studies, we used standard agarose gel electrophoresis and the Agilent LabChip to detect FLT3 internal tandem duplications (ITD) in 3 of 90 MDS samples. We chose one sample with an ITD and performed 6 serial dilutions of MDS DNA with wild-type genomic DNA and performed PCR amplification followed by DNA sequencing of the PCR products. We could detect the FLT3 ITD when 12% of the alleles harbored the mutation, or when ∼20–25% of cells contain a heterozygous mutation. From these results, we are confident that our strategy can detect mutant alleles in unpurified MDS bone marrow samples, but may miss mutations occurring in a rare cell. We then designed and validated primers for 415 amplicons covering the coding region and proximal introns of all 28 genes in the 5q31.2 interval and produced 7.2 megabases of sequence for these genes in the 46 patient samples. No somatic changes were identified. Twelve novel non-synonymous SNPs were discovered. The allele and genotype frequencies of 49 known SNPs in these genes were similar to the frequencies in race-matched HapMap individuals, apart from an enrichment in a non-synonymous CDC25C SNP in the MDS cohort (rs3734166, Odds Ratio=1.87, 95% confidence interval=1.06–3.32, p=0.036). The over-representation of this SNP and the other rare novel SNPs in the MDS population suggest that they may mark susceptibility alleles for MDS. Taken together, these data suggest that small deletions and/or point mutations of genes in 5q31.2 are not common events in the pathogenesis of MDS, and that larger deletions leading to haploinsufficiency of several genes in the interval appear to be the dominant mechanism.

Coordinate Interstitial Deletion of Retinoblastoma (RB1) and Neurobeachin (NBEA) Is a Recurring Event in Multiple Myeloma.

Monoallelic chromosome 13 deletion detected by cytogenetics predicts poor patient survival in multiple myeloma (MM), but the genes responsible have not been conclusively identified. To this end, we performed array comparative genomic hybridization (aCGH) using a novel, human chromosome 13 oligonucleotide array (Nimblegen) with 385,272 probes and median probe spacing of 60 base pairs. The dense coverage, and the use of germline DNA collected from each patient as internal controls for DNA copy number polymorphisms, enabled unprecedented map resolution of somatic DNA gains and losses on chromosome 13. Array CGH was performed on genomic DNA isolated from CD138+ bone marrow plasma cells purified from 20 patients with MM, monoclonal gammopathy of undetermined significance (MGUS), or amyloidosis. Visual analysis of the aCGH data identified 4 patients with chromosome 13 interstitial deletions that were confirmed using a circular binary segment algorithm (Nimblegen). Monosomy chromosome 13 was detected in 5 patients by cytogenetics, and as expected, appeared normal by aCGH due to data normalization. We also performed an unsupervised analysis of the data, which identified 49 genes with DNA copy number decreases. Both methods identified copy number decreases at 13q14 commonly affected in MM and MGUS patients and thought to harbor a relevant tumor suppressor. Three of the 4 patients with interstitial deletions at 13q14 had striking regions of DNA copy loss whose minimally deleted region was defined by a patient with a small deletion spanning exon 20 of RB1, encoding part of the functionally important ‘pocket domain' responsible for binding E2F transcription factors. We found RB1 protein levels in MM cell lines correlated with RB1 genomic copy number, and therefore considered the model that RB1 haploinsufficiency contributes to MM. However, we found Rb1 heterozygous (HET) and wild type (WT) mice had indistinguishable steady-state B, T and myeloid compartments in addition to plasma cell induction in response to sheep red blood cell stimulation. Disease burden was similar in HET vs. WT Rb1 mice in a model of NRAS induced tumorigenesis. These results suggest other genomic events cooperate with RB1 copy number loss in MM. Unexpectedly, we found the 3 patients that had an interstitial deletion of RB1 at 13q14 concomitantly harbored a separate interstitial loss at 13q13. Every patient with DNA copy number loss of RB1 also had DNA copy loss within 13q13 (5 patients who lost the entire chromosome and 3 patients with interstitial deletions). The minimally deleted region at 13q13 mapped to the 5′ end of Neurobeachin (NBEA), which encodes a Protein Kinase A (PKA) anchoring protein. We detected NBEA transcripts at low levels in normal human plasma cells. NBEA transcripts and protein were robustly expressed in 3/5 MM cell lines. This is the first report of coordinate copy number loss of RB1 and NBEA on chromosome 13 in MM. Taken together, our data suggest that chromosome 13 deletions in MM may target protein dose level of RB1 and at least one other gene, likely NBEA. Our data provide a novel rationale for future studies to examine the biological consequences of coordinate loss of NBEA and RB1.