Probe Coverage Research Articles

Abstract P1-09-18: A clinically validated DNA microarray for high-resolution HER2 testing defines a new genomic subtype in high-risk breast cancer with equivocal results by IHC and FISH

Abstract Background: In all stages of breast cancer, the HER2 status of a patient's tumor is critically important as both a prognostic indicator, and for predicting response to targeted anti-HER2 therapies. CAP/ASCO 2013 guidelines recommend that newly diagnosed, recurrent, and metastatic breast tumors be evaluated for HER2 positivity by protein-based immunohistochemistry (IHC) and/or chromosome-based fluorescence in situ hybridization (FISH). In the majority of cases, these testing modalities provide a clearly actionable “positive” or “negative” answer. However, in an estimated 10- to 20% of breast cancers, both tests are reported as “equivocal” leaving the clinician with a treatment decision dilemma and no definitive alternative testing method. Here we report validation of an IHC-targeted DNA microarray comparative genomic hybridization (array CGH) assay for HER2 equivocal breast cancer, and definition of a new genomic subtype of HER2 status in high-risk breast cancer with equivocal IHC and FISH results. Methods: IHC-targeted HER2 receptor “hot spot” DNA samples extracted from 25 formalin fixed paraffin embedded (FFPE) breast tumor tissue samples previously characterized by IHC and FISH, were analyzed by array CGH. Eight tumors were known to be highly HER2 positive, seven tumors had IHC scores of 0 with negative FISH, and ten tumors had HER2 receptor staining by IHC (1-2+) and equivocal results by FISH (4-6 HER2 gene copies.) Tumor DNA (test) and human genomic DNA (reference) were fluorescently labeled, and competitively hybridized to a custom-designed genomic DNA microarray with high-density probe coverage of the HER2 amplicon on chromosome 17 (Agilent Technologies, Santa Clara CA). The array design includes over 4,600 chromosome 17 probes representing the p arm, q arm, telomeric and centromeric regions with 66 tiling probes over the HER2 (ERBB2) gene. Following hybridization, average HER2 gene copy number was calculated for each tumor sample by converting mean log2 signal intensity ratio value into genomic region copy number adjusted for % clonal fraction and experimentally established log2 ratio compression of the assay. Results: 25/25 (100%) of samples yielded adequate DNA for analysis and all highly HER2 positive and HER2 negative results were confirmed by array CGH. In 10/10 IHC equivocal cases with HER2 gene copy number 4-6 by FISH, CGH results confirmed HER2-Low gene copy number. Results for 25 Validation SamplesNumber of CasesIHC ScoreFISHCGH Copy NumberResult83+Positive> 6HER2-Positive101-2+Equivocal4-6HER2-Low70Negative< 6HER2-Negative Conclusions: High-resolution HER2 testing by IHC-targeted DNA microarray analysis accurately classifies HER2 status in breast cancer and better defines the HER2-Low genomic subtype most often called “equivocal” by standard IHC and FISH testing. This subcategory is characterized at the protein level by IHC evidence of anti-HER2 therapy target receptor expression on the surface of the cells, and at the genomic level by HER2 gene copy number < 6. Results of the NSABP-B47 clinical trial and further studies with larger numbers of HER2-Low patients are needed to determine if these patients benefit from anti-HER2 therapy. Citation Format: Gunn S, Yaziji H, Sims C, Govender S, Moore M, Cotter P, Jones S. A clinically validated DNA microarray for high-resolution HER2 testing defines a new genomic subtype in high-risk breast cancer with equivocal results by IHC and FISH [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-09-18.

Major upgrades of the high frequency B-dot probe diagnostic suite on ASDEX Upgrade

The high frequency B-dot (HFB) probe diagnostic on the ASDEX Upgrade tokamak has undergone a considerable upgrade during the 2016 opening of the torus. The probe coverage is now greatly expanded toroidally, as well as radially with the addition of probes on the high field side and the removable manipulator head. A new 2-channel fast digitizer now allows to examine and record radio frequency (RF) wave emissions emanating from the plasma in the ion cyclotron range of frequencies (ICRF). Possible studies that can be achieved now include: a study of core ICRF power absorption efficiency; a study of ion cyclotron emissions from the plasma generated by energetic ions; and study of ICRF wave/plasma turbulence interactions in the scrape-off layer region.

Analytical and Clinical Validity Study of FirstStepDx PLUS: A Chromosomal Microarray Optimized for Patients with Neurodevelopmental Conditions.

Introduction:Chromosomal microarray analysis (CMA) is recognized as the first-tier test in the genetic evaluation of children with developmental delays, intellectual disabilities, congenital anomalies and autism spectrum disorders of unknown etiology.Array Design:To optimize detection of clinically relevant copy number variants associated with these conditions, we designed a whole-genome microarray, FirstStepDx PLUS (FSDX). A set of 88,435 custom probes was added to the Affymetrix CytoScanHD platform targeting genomic regions strongly associated with these conditions. This combination of 2,784,985 total probes results in the highest probe coverage and clinical yield for these disorders.Results and Discussion:Clinical testing of this patient population is validated on DNA from either non-invasive buccal swabs or traditional blood samples. In this report we provide data demonstrating the analytic and clinical validity of FSDX and provide an overview of results from the first 7,570 consecutive patients tested clinically. We further demonstrate that buccal sampling is an effective method of obtaining DNA samples, which may provide improved results compared to traditional blood sampling for patients with neurodevelopmental disorders who exhibit somatic mosaicism.

The dependence of the measured surface energy of graphene on nanosheet size

The surface energy of graphene nanosheets is surprisingly poorly known, probably due to size effects and energetic heterogeneities. Here we use finite-dilution inverse gas chromatography to measure the surface energy of liquid-exfoliated, few-layer graphene nanosheets of different sizes as a function of probe coverage. In all cases, the surface energy falls with probe coverage from a defect-controlled, low-coverage value to a value that approaches the basal plane surface energy at high coverage. We find an intrinsic basal plane dispersive surface energy of 61 ± 4 mJ m−2, close to the value of 63 mJ m−2 found for graphite. By comparison with similar data measured on graphite and using simple models, we can use the length dependence of the low coverage surface energy to differentiate between the effects of edge and basal plane defects, finding these to contribute ∼130 and 180 mJ m−2 to the surface energy respectively. From this data, we estimate a basal plane defect content of ∼6 × 1014 defects m–2 for both graphite and graphene in reasonable agreement with Raman data. This work shows that, in terms of surface energetics, few-layer graphene nanosheets behave exactly like graphite with the only differences associated with platelet dimensions.

The eddy current probe array for Keda Torus eXperiment.

In a reversed field pinch device, the conductive shell is placed as close as possible to the plasma so as to balance the plasma during discharge. Plasma instabilities such as the resistive wall mode and certain tearing modes, which restrain the plasma high parameter operation, respond closely with conditions in the wall, in essence the eddy current present. Also, the effect of eddy currents induced by the external coils cannot be ignored when active control is applied to control instabilities. One diagnostic tool, an eddy current probe array, detects the eddy current in the composite shell. Magnetic probes measuring differences between the inner and outer magnetic fields enable estimates of the amplitude and angle of these eddy currents. Along with measurements of currents through the copper bolts connecting the poloidal shield copper shells, we can obtain the eddy currents over the entire shell. Magnetic field and eddy current resolutions approach 2 G and 6 A, respectively. Additionally, the vortex electric field can be obtained by eddy current probes. As the conductivity of the composite shell is high, the eddy current probe array is very sensitive to the electric field and has a resolution of 0.2 mV/cm. In a bench test experiment using a 1/4 vacuum vessel, measurements of the induced eddy currents are compared with simulation results based on a 3D electromagnetic model. The preliminary data of the eddy currents have been detected during discharges in a Keda Torus eXperiment device. The typical value of toroidal and poloidal eddy currents across the magnetic probe coverage rectangular area could reach 3.0 kA and 1.3 kA, respectively.

Differentiating Defect and Basal Plane Contributions to the Surface Energy of Graphite Using Inverse Gas Chromatography

Historically, reported values for the surface energy of graphite have covered a very wide range. Here, we use finite-dilution inverse gas chromatography (FD-IGC) to show that the dispersive component of the surface energy of graphite has contributions from edge and basal plane defects as well as from the hexagonal carbon lattice. The surface energy associated with the defect-free hexagonal lattice is measured at high probe-coverage to be 63 ± 7 mJ/m2, independent of graphite type. However, the surface energy measured at low probe coverage varied from 125 to 175 mJ/m2 depending on the graphite type. Simulation of the FD-IGC output for different binding site distributions allows us to associate this low-coverage surface energy with the binding of probe molecules to high energy defect sites. Importantly, we find the rate of decay of surface energy with probe coverage to carry information about the defect density. By analyzing the dependence of these properties on flake size, it is possible to separate out th...

Signal Amplification of a Universal Four-Way Junction Based Nucleic Acid Sensor through Modification of Electrochemical Technique Parameters

Here, we report the characterization of a four-way junction (4J) universal electrochemical sensor that consists of an electrode-immobilized DNA stem-loop (SL) probe and two adaptor strands (m and f). The “m” adaptor strand was labeled with a methylene blue (MeB) redox marker. The adaptor strands hybridize to the DNA SL probe and target nucleic acid to form a 4J structure. In this orientation the MeB is in close proximity to the electrode’s surface thus enabling its reduction and subsequent electrochemical signal. The experimental timing for immobilization of the DNA SL probe and hybridization with adaptor strands and miRNA-122 was investigated. In addition, the density of the DNA SL probe coverage was evaluated using chronocoulometry to determine the ideal concentration of immobilized DNA SL probe. The sensor performance was investigated using cyclic voltammetry (CV), alternating current voltammetry (ACV), differential pulse voltammetry (DPV) and square wave voltammetry (SWV). Specifically, a single parameter from each voltammetric technique was modified to evaluate the sensor’s response. The DNA SL probe demonstrated an efficient immobilization after 30 minutes and optimal hybridization occurred at 1.5 hours. The 4J sensor was characterized with ACV at a frequency range from 1 to 5000 Hz and the current increased from 1 to 600 Hz where the threshold of the sensor was reached, followed by a drastic decrease in signal at higher frequencies. The 4J sensor was also characterized with SWV at frequencies ranging from 1 to 5000 Hz and exhibited an increase in signal from 1 to 1500 Hz, where the signal stabilized until reaching the threshold frequency of 4000 Hz, where the current then began to decrease. CV was used to evaluate the sensor’s performance at scan rates ranging from 0.1 to 800 V/s. The peak separation increased at faster scan rates (529 mV) and the signal was saturated after 500 V/s. Finally, the sensor performance was monitored using DPV at pulse widths ranging from 1 to 100 ms. The current was highest at short pulse widths (1 ms) and the current decreased rapidly as the pulse width was increased. Amplification of the 4J sensor was achieved by modifying a specific parameter for each electrochemical technique which can provide insight into the sensor performance to facilitate lower limits of detection of nucleic acids.

Molecularly resolved label-free sensing of single nucleobase mismatches by interfacial LNA probes.

So far, there has been no report on molecularly resolved discrimination of single nucleobase mismatches using surface-confined single stranded locked nucleic acid (ssLNA) probes. Herein, it is exemplified using a label-independent force-sensing approach that an optimal coverage of 12-mer ssLNA sensor probes formed onto gold(111) surface allows recognition of ssDNA targets with twice stronger force sensitivity than 12-mer ssDNA sensor probes. The force distributions are reproducible and the molecule-by-molecule force measurements are largely in agreement with ensemble on-surface melting temperature data. Importantly, the molecularly resolved detection is responsive to the presence of single nucleobase mismatches in target sequences. Since the labelling steps can be eliminated from protocol, and each force-based detection event occurs within milliseconds' time scale, the force-sensing assay is potentially capable of rapid detection. The LNA probe performance is indicative of versatility in terms of substrate choice - be it gold (for basic research and array-based applications) or silicon (for ‘lab-on-a-chip’ type devices). The nucleic acid microarray technologies could therefore be generally benefited by adopting the LNA films, in place of DNA. Since LNA is nuclease-resistant, unlike DNA, and the LNA-based assay is sensitive to single nucleobase mismatches, the possibilities for label-free in vitro rapid diagnostics based on the LNA probes may be explored.

Membrane gene ontology bias in sequencing and microarray obtained by housekeeping-gene analysis

Microarray (MA) and high-throughput sequencing are two commonly used detection systems for global gene expression profiling. Although these two systems are frequently used in parallel, the differences in their final results have not been examined thoroughly. Transcriptomic analysis of housekeeping (HK) genes provides a unique opportunity to reliably examine the technical difference between these two systems. We investigated here the structure, genome location, expression quantity, microarray probe coverage, as well as biological functions of differentially identified human HK genes by 9 MA and 6 sequencing studies. These in-depth analyses allowed us to discover, for the first time, a subset of transcripts encoding membrane, cell surface and nuclear proteins that were prone to differential identification by the two platforms. We hope that the discovery can aid the future development of these technologies for comprehensive transcriptomic studies.

Intragenic deletions of ALDH7A1 in pyridoxine-dependent epilepsy caused by Alu-Alu recombination.

To investigate the role of intragenic deletions of ALDH7A1 in patients with clinical and biochemical evidence of pyridoxine-dependent epilepsy but only a single identifiable mutation in ALDH7A1. We designed a custom oligonucleotide array with high-density probe coverage across the ALDH7A1 gene. We performed array comparative genomic hybridization in 6 patients with clinical and biochemical evidence of pyridoxine-dependent epilepsy but only a single detectable mutation in ALDH7A1 by sequence analysis. We found partial deletions of ALDH7A1 in 5 of 6 patients. Breakpoint analysis reveals that the deletions are likely a result of Alu-Alu recombination in all cases. The density of Alu elements within introns of ALDH7A1 suggests susceptibility to recurrent rearrangement. Patients with clinical pyridoxine-dependent epilepsy and a single identifiable mutation in ALDH7A1 warrant further investigation for copy number changes involving the ALHD7A1 gene.

Abstract P5-10-18: The chromosomal genomic landscape and targetable co-amplified genes in HER2 positive breast cancer patients who relapsed on an adjuvant trastuzumab chemotherapy trial

Abstract Background Early stage HER2 amplified breast cancer has a generally favorable prognosis with over 95% of patients showing 2-year disease free survival (DFS) when treated with adjuvant trastuzumab. However, a subset of these tumors are refractory to treatment and present a challenge for the oncologist, particularly when clinical and histologic parameters, including the patient’s nodal and hormonal status, are indicative of lower-risk HER2 positive disease. In this study we describe the genomic landscape of three clinically lower-risk patients with HER2 amplified tumors who relapsed on adjuvant docetaxel and cyclophosphamide plus 1 year of trastuzumab in a phase 2 study (Jones et al, Lancet Oncology 14: 1121, 2013). All patients’ tumors showed high-level HER2 amplification ratios by FISH (8.51-14.46) and were analyzed in parallel with a fourth clinically matched 2-year DFS patient’s tumor from the same trial with high HER2 gene amplification (FISH ratio 11.38). Methods Primary tumor genomic DNA analysis was performed from archival tissue by next generation sequencing (NGS) on the Illumina HiSeq 2500 platform in a CLIA certified laboratory. Tumors were screened for point mutations and copy number alterations (CNAs) by NGS using a targeted-whole exome 613 gene panel. CNAs detected by NGS were confirmed on a DNA microarray featuring high-density probe coverage of the same 613 genes on the targeted panel. CGH chromosome ratio plots were overlaid with algorithmically derived NGS copy number data to generate a map of the chromosomal genomic landscape for each patient’s tumor. Results High-level HER2 gene amplification status was confirmed, and co-amplified chromosome 17 genomic regions were detected in all three relapsed patients’ tumors. High-level HER2 amplification was also confirmed in the non-relapsed patient’s tumor but co-amplified regions were not detected on chromosome 17 or elsewhere in this patient’s tumor genome. Two of the relapsed ER negative tumors shared focal high-level CCNE1 gene amplifications on chromosome 19. High-level MAP3K3 gene amplification on chromosome 17 was detected in the one ER positive tumor from a relapsed patient. Focal PIK3CA gene amplifications were not identified in any of these tumors, but two tumors (one from the relapse group and one non-relapse) were positive for activating H1047R mutations. Conclusions Combined NGS and CGH analysis of HER2 positive early stage breast cancer can be performed in the clinical laboratory to reveal the tumor’s chromosomal genomic landscape. Combined with other test results, this tumor map can help identify patients at high-risk for relapse and reveal alternative predictive biomarkers of therapeutic response. Citation Format: Shelly Gunn, Chris McCaskill, Linda Daley, Agnes Puskas, Lina Asmar, Yunfei Wang, Stephen Jones. The chromosomal genomic landscape and targetable co-amplified genes in HER2 positive breast cancer patients who relapsed on an adjuvant trastuzumab chemotherapy trial [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-10-18.

Off to a Flying Start!

Off to a Flying Start!

Abstract 2361: Identification and analysis of gene fusion and splice variant events in the NCI sarcoma cell line panel

Abstract Sarcomas represent approximately 7% of newly diagnosed childhood cancers in the United States per year. A rare form of cancer but one that is highly variable and predominantly characterized by specific chromosomal translocations in pediatric occurrences. Sarcoma tumors can progress through several cellular pathways and are highly dependent on the function of the genes involved in the fusion event. Therefore, the identification of novel fusion candidates or altered genomic functions from splice variation is essential for understanding genomic heterogeneity among sarcomas and for the development of sarcoma specific therapeutic protocols. Within the Division of Cancer Treatment and Diagnosis (NCI) we developed a cell line panel of 67 human sarcoma and measured exon expression using the Affymetrix Exon 1.0 ST arrays. These data have been used to generate gene expression profiles for all the cell lines while the probe coverage across all exons of these genes facilitates identification of putative splice variants and fusion gene candidates. In order to predict gene fusions in these cell lines, we implemented computational prediction algorithms such as generalized linear models and separately random forest procedures training on features determined only from the EWSR1-FLI1 exon expression profiles. These resulted in 83% accuracy and 90% coverage of potential EWSR1-FLI1 fusions and the ability to identify 60% of the known individual sarcoma fusion gene partners, as annotated by the COSMIC database, within all 67 sarcoma cell lines. Specifically, the ASPSCR1-TFE3 fusion gene in alveolar soft part sarcoma is captured in the ASPS-1 cell line; currently the only ASPS cell line known. We also find the alveolar rhabdomyosarcoma PAX3-FOXO1, PAX7-FOXO1 fusion genes and the Ewing's sarcoma EWSR1's C-terminal fusion partners DDIT3, ERG, ETV1 and ETV4, in addition to FLI1. Next, splice variant analysis in all sarcoma cell lines based on exon arrays was performed using the FIRMA algorithm and the computational package AltAnalyze. Using individual gene-exon expression profiles we successfully segregated the sarcoma cell lines based on sarcoma type. Among theses clustering profiles, differential inclusion exon expression was exclusively observed in the gene DMKN (dermokine) primarily in the adult sarcoma types such as chondrosarcoma, fibrosarcoma, leiomyosarcoma and uterine sarcoma and may suggest tissue and sarcoma type alternate splice variations. Additionally, the enzyme phosphatidylserine synthase 1 (PTDSS1) showed exon specific variation in 6 of 8 rhabdomyosarcoma (both ARMS and ERMS) examined but not in other types of sarcoma. Therefore, our computational analysis and discovery of putative gene fusion and splice variant events is 67 sarcoma cell lines has further identified points of heterogeneity within sarcoma subtypes and can potentially be exploited to better identify and treat sarcoma patients based on their tumor profiles. Citation Format: Kazimierz O. Wrzeszczynski, Eric C. Polley, Curtis Hose, Anne Monks, Beverly A. Teicher. Identification and analysis of gene fusion and splice variant events in the NCI sarcoma cell line panel. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2361. doi:10.1158/1538-7445.AM2014-2361

A commentary on the promise of whole-exome sequencing in medical genetics

The dawn of next-generation sequencers (NGSs) and innovative sequencing technologies have brought a paradigm shift in medical research and clinical practice. Furthermore, the cost reduction of NGSs enables personalized medicine to come to fruition. However, whole-genome sequencing (WGS) remains expensive when applied to personal genome analysis. WGS generates a large amount of data that requires highperformance computer processing. Targeted whole-exon capture and sequencing [wholeexome sequencing (WES)] is more costeffective when compared with WGS because exons represent only B1–2% of the genome and also higher sequence coverage can be achieved by NGSs. In addition, most Mendelian disorders are caused by exonic mutations or splice-junction mutations, and protein-coding genes harbor B85% of the mutations that have large effects on diseaserelated traits.1 Thus, WES will provide many advantages and lower costs than WGS when analyzing personal genomes. WES was first successfully used in 2010 to discover the gene responsible for Miller syndrome, a Mendelian disorder.2 Since then, WES has been increasingly used as a fast and accurate genomic discovery approach to investigate both rare genetic disorders and common diseases. WES is widely applied across different areas of medicine, because it has the added advantage of reduced cost and requires analysis of a much smaller but essential dataset when compared with WGS. In addition, recent clinical molecular diagnostics have used WES to detect heterogeneous Mendelian diseases.3,4 A recent review of WES approaches in medical genetics describes the usefulness of WES in medicine and medical research and the impact of WES on clinical diagnoses.5 WES approaches have greatly facilitated the discovery of candidate genes or gene variants in Mendelian disorders and rare variants in common diseases and genomic characterization in cancer. Currently, WES is increasingly being applied to disease gene discovery, cancer typing and molecular diagnosis.5 Presently, WES is an essential tool in medical genetics, especially in the research of Mendelian disorders. WES or multigene tests using NGSs are widely applied to heterogeneous disorders including deafness or ciliopathy.5,6 WES is also being increasingly applied to genetic testing for undiagnosed patients.4,5 Yang et al.4 performed WES in undiagnosed patients whose phenotypes were suggestive of potential genetic disorders and achieved a molecular diagnosis for 62 of 250 (25%) patients. Because WES detects individual genetic variation, it can be used to construct a variation database of anthropic and ethnic populations. At the same time, because WES can detect groups of genetic variations that are unrelated to the indication for the first diagnostic purpose but are of medical value for individual patient care, such ‘incidental findings’ pose potential ethical problems that should be strongly considered and discussed in clinical practice.5,7 WES is a widely applied technique in medical genetics that is capable of detecting variations in whole exons. However, in practical use, understanding WES methodology and limitations are important. Current WES techniques are not capable of detecting all of the variations surrounding exons. Detecting variation by WES is limited by the experimental methods, probe coverage and/or platforms used.8–10 Hence, WES may not always detect pathogenic or causative variations in a genetic disease. In addition, because WES is a method to detect genomic sequence variations, when a candidate of causative variation in the disease is detected, it requires verification or support by secondary analyses. In particular, further functional analyses are important to confirm whether the variant is pathogenic or benign. Nevertheless, WES enables the unprecedented low cost and highly efficient analysis of whole exons. WES can be easily used to comprehensively detect individual variations in exons. It is without doubt that WES is a powerful tool in genome analysis, and it greatly progresses medical genetics. Although WESs’ limitations need to be overcome, we anticipate that WES will be used not only in medical research but also in clinical practice for example, molecular diagnosis (whole-gene test) and personal genomics before WGS becomes a common place in medical genetics. Thus, a paradigm shift in medicine by advancement in both WES and WGS is expected to continue.

Optimization of the Probe Coverage in DNA Biosensors by a One‐Step Coadsorption Procedure

AbstractAnalytical performance (selectivity, sensitivity, limit of detection) of a sensor is ultimately connected to the interfacial architecture of the sensing surface that needs to be optimized. Self‐assembly of a mixed monolayer of thiolated probes and diluent onto gold surfaces used in the design of biosensing platforms is nowadays commonly performed by a two‐step immobilization procedure. In this work, the merits of a one‐step coadsorption procedure are emphasized, and it is shown that the ionic strength is a very efficient tool to fine control the surface concentration of the immobilized probe. Probe densities were obtained by alternating current voltammetry or fluorescence measurements depending on the type of labeling of the probes. With unlabeled DNA sequences, chronocoulometric measurements were performed in the presence of dissolved hexaammineruthenium(III) cations. The maximum probe density achievable at high ionic strength by the one‐step coadsorption procedure is limited by steric constraints and depends on the structure of the sequence. Various thiolated DNA sequences (single‐stranded DNA, double‐stranded DNA, linear, hairpin, quadruplex) coadsorbed with a common diluent, 4‐mercaptobutan‐1‐ol, were used to discuss the structural factors affecting the maximum surface densities obtained for the different types of probe. Application of the one‐step coadsorption procedure to the optimization of the analytical performance of a protein aptasensor is illustrated by building a sensor for the detection of thrombin on the basis of the thrombin binding aptamer sequence.

Detection limit of intragenic deletions with targeted array comparative genomic hybridization

BackgroundPathogenic mutations range from single nucleotide changes to deletions or duplications that encompass a single exon to several genes. The use of gene-centric high-density array comparative genomic hybridization (aCGH) has revolutionized the detection of intragenic copy number variations. We implemented an exon-centric design of high-resolution aCGH to detect single- and multi-exon deletions and duplications in a large set of genes using the OGT 60 K and 180 K arrays. Here we describe the molecular characterization and breakpoint mapping of deletions at the smaller end of the detectable range in several genes using aCGH.ResultsThe method initially implemented to detect single to multiple exon deletions, was able to detect deletions much smaller than anticipated. The selected deletions we describe vary in size, ranging from over 2 kb to as small as 12 base pairs. The smallest of these deletions are only detectable after careful manual review during data analysis. Suspected deletions smaller than the detection size for which the method was optimized, were rigorously followed up and confirmed with PCR-based investigations to uncover the true detection size limit of intragenic deletions with this technology. False-positive deletion calls often demonstrated single nucleotide changes or an insertion causing lower hybridization of probes demonstrating the sensitivity of aCGH.ConclusionsWith optimizing aCGH design and careful review process, aCGH can uncover intragenic deletions as small as dozen bases. These data provide insight that will help optimize probe coverage in array design and illustrate the true assay sensitivity. Mapping of the breakpoints confirms smaller deletions and contributes to the understanding of the mechanism behind these events. Our knowledge of the mutation spectra of several genes can be expected to change as previously unrecognized intragenic deletions are uncovered.

Solid phase microextraction fills the gap in tissue sampling protocols

Metabolomics and biomarkers discovery are an integral part of bioanalysis. However, untargeted tissue analysis remains as the bottleneck of such studies due to the invasiveness of sample collection, as well as the laborious and time-consuming sample preparation protocols. In the current study, technology integrating in vivo sampling, sample preparation and global extraction of metabolites – solid phase microextraction was presented and evaluated during liver and lung transplantation in pig model. Sampling approaches, including selection of the probe, transportation, storage conditions and analyte coverage were discussed. The applicability of the method for metabolomics studies was demonstrated during lung transplantation experiments.

Technology-Driven and Evidence-Based Genomic Analysis for Integrated Pediatric and Prenatal Genetics Evaluation

The first decade since the completion of the Human Genome Project has been marked with rapid development of genomic technologies and their immediate clinical applications. Genomic analysis using oligonucleotide array comparative genomic hybridization (aCGH) or single nucleotide polymorphism (SNP) chips has been applied to pediatric patients with developmental and intellectual disabilities (DD/ID), multiple congenital anomalies (MCA) and autistic spectrum disorders (ASD). Evaluation of analytical and clinical validities of aCGH showed >99% sensitivity and specificity and increased analytical resolution by higher density probe coverage. Reviews of case series, multi-center comparison and large patient-control studies demonstrated a diagnostic yield of 12%-20%; approximately 60% of these abnormalities were recurrent genomic disorders. This pediatric experience has been extended toward prenatal diagnosis. A series of reports indicated approximately 10% of pregnancies with ultrasound-detected structural anomalies and normal cytogenetic findings had genomic abnormalities, and 30% of these abnormalities were syndromic genomic disorders. Evidence-based practice guidelines and standards for implementing genomic analysis and web-delivered knowledge resources for interpreting genomic findings have been established. The progress from this technology-driven and evidence-based genomic analysis provides not only opportunities to dissect disease-causing mechanisms and develop rational therapeutic interventions but also important lessons for integrating genomic sequencing into pediatric and prenatal genetic evaluation.

Development of Assisted Software for TOFD Testing

The detection of flaws is essential as the defects of pipes and containers affect the safe operation of many kinds of equipment. During the industrial production process, it is usually use the ray method to detect the flaws which has radiant effect on the testing personnel. The ultrasonic time-of-flight diffraction (TOFD) is a new type acoustic detection method and it is relatively safe for users. When using the TOFD technique, the selection of the probe separation is the important parameter which related to the effect of the test and the coverage range of the probe identifies the effective range of the detection. In this paper, the probe separation distance and the probe coverage range procedures which can help the testing personnel set the parameters quickly is devised. The experimental results indicate that this algorit

Label-free DNA microarray bioassays using a near-field scanning microwavemicroscope

A near-field scanning microwave microscope (NSMM) is used to readout and visualize homemade 10-mer oligonucleotide microarrays and an Agilent 60-mer DNA microarray as a realistic test of NSMM applicability to multiplexed sequence analysis. Sensitive characterization of DNA coverage and high resolution mapping of DNA spots in the microarray were realized by measuring the change of microwave reflection coefficient (S11) at about 4GHz operating frequency. Hybridization between target (free) and capture (immobilized) sequences leads to changes in the microwave reflection coefficient, which were measured by the NSMM. These changes are caused by hybridization-induced modification of the dielectric permittivity profile of the DNA film. The dynamic range based on analysis of the 10-mer microarrays is over 3orders of magnitude with the detection limit estimated below 0.01strands/μm2. The NSMM method should be readily capable of detecting target coverages down to 98% of probe coverage. We also directly image the patterned DNA microarray by NSMM with a 2μm resolution. The complementary optical image of the DNA microarray visualized by using a relative fluorescent intensity metric agrees well with the NSMM results.