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1.  Health Risk Assessment for Air Pollutants: Alterations in Lung and Cardiac Gene Expression in Mice Exposed to Milano Winter Fine Particulate Matter (PM2.5) 
PLoS ONE  2014;9(10):e109685.
Oxidative stress, pulmonary and systemic inflammation, endothelial cell dysfunction, atherosclerosis and cardiac autonomic dysfunction have been linked to urban particulate matter exposure. The chemical composition of airborne pollutants in Milano is similar to those of other European cities though with a higher PM2.5 fraction. Milano winter fine particles (PM2.5win) are characterized by the presence of nitrate, organic carbon fraction, with high amount of polycyclic aromatic hydrocarbons and elements such as Pb, Al, Zn, V, Fe, Cr and others, with a negligible endotoxin presence. In BALB/c mice, we examined, at biochemical and transcriptomic levels, the adverse effects of repeated Milano PM2.5win exposure in lung and heart. We found that ET-1, Hsp70, Cyp1A1, Cyp1B1 and Hsp-70, HO-1, MPO respectively increased within lung and heart of PM2.5win-treated mice. The PM2.5win exposure had a strong impact on global gene expression of heart tissue (181 up-regulated and 178 down-regulated genes) but a lesser impact on lung tissue (14 up-regulated genes and 43 down-regulated genes). Focusing on modulated genes, in lung we found two- to three-fold changes of those genes related to polycyclic aromatic hydrocarbons exposure and calcium signalling. Within heart the most striking aspect is the twofold to threefold increase in collagen and laminin related genes as well as in genes involved in calcium signaling. The current study extends our previous findings, showing that repeated instillations of PM2.5win trigger systemic adverse effects. PM2.5win thus likely poses an acute threat primarily to susceptible people, such as the elderly and those with unrecognized coronary artery or structural heart disease. The study of genomic responses will improve understanding of disease mechanisms and enable future clinical testing of interventions against the toxic effects of air pollutant.
PMCID: PMC4190364  PMID: 25296036
2.  Repeated Intratracheal Instillation of PM10 Induces Lipid Reshaping in Lung Parenchyma and in Extra-Pulmonary Tissues 
PLoS ONE  2014;9(9):e106855.
Adverse health effects of air pollution attributed mainly to airborne particulate matter have been well documented in the last couple of decades. Short term exposure, referring to a few hours exposure, to high ambient PM10 concentration is linked to increased hospitalization rates for cardiovascular events, typically 24 h after air pollution peaks. Particulate matter exposure is related to pulmonary and cardiovascular diseases, with increased oxidative stress and inflammatory status. Previously, we have demonstrated that repeated intratracheal instillation of PM10sum in BALB/c mice leads to respiratory tract inflammation, creating in lung a condition which could potentially evolve in a systemic toxic reaction. Additionally, plasma membrane and tissue lipids are easily affected by oxidative stress and directly correlated with inflammatory products. With this aim, in the present investigation using the same model, we analyzed the toxic potential of PM10sum exposure on lipid plasma membrane composition, lipid peroxidation and the mechanisms of cells protection in multiple organs such as lung, heart, liver and brain. Obtained results indicated that PM10 exposure led to lung lipid reshaping, in particular phospholipid and cholesterol content increases; concomitantly, the generation of oxidative stress caused lipid peroxidation. In liver we found significant changes in lipid content, mainly due to an increase of phosphatidylcholine, and in total fatty acid composition with a more pronounced level of docosahexaenoic acid; these changes were statistically correlated to lung molecular markers. Heart and brain were similarly affected; heart was significantly enriched in triglycerides in half of the PM10sum treated mice. These results demonstrated a direct involvement of PM10sum in affecting lipid metabolism and oxidative stress in peripheral tissues that might be related to the serious systemic air-pollution effects on human health.
PMCID: PMC4178018  PMID: 25259850
3.  Integrative Analysis of miRNA and Inflammatory Gene Expression After Acute Particulate Matter Exposure 
Toxicological Sciences  2013;132(2):307-316.
MicroRNAs (miRNAs) are environmentally sensitive inhibitors of gene expression that may mediate the effects of metal-rich particulate matter (PM) and toxic metals on human individuals. Previous environmental miRNA studies have investigated a limited number of candidate miRNAs and have not yet evaluated the functional effects on gene expression. In this study, we wanted to identify PM-sensitive miRNAs using microarray profiling on matched baseline and postexposure RNA from foundry workers with well-characterized exposure to metal-rich PM and to characterize miRNA relations with expression of candidate inflammatory genes. We applied microarray analysis of 847 human miRNAs and real-time PCR analysis of 18 candidate inflammatory genes on matched blood samples collected from foundry workers at baseline and after 3 days of work (postexposure). We identified differentially expressed miRNAs (fold change [FC] > 2 and p < 0.05) and correlated their expression with the inflammatory associated genes. We performed in silico network analysis in MetaCore v6.9 to characterize the biological pathways connecting miRNA-mRNA pairs. Microarray analysis identified four miRNAs that were differentially expressed in postexposure compared with baseline samples, including miR-421 (FC = 2.81, p < 0.001), miR-146a (FC = 2.62, p = 0.007), miR-29a (FC = 2.91, p < 0.001), and let-7g (FC = 2.73, p = 0.019). Using false discovery date adjustment for multiple comparisons, we found 11 miRNA-mRNA correlated pairs involving the 4 differentially expressed miRNAs and candidate inflammatory genes. In silico network analysis with MetaCore database identified biological interactions for all the 11 miRNA-mRNA pairs, which ranged from direct mRNA targeting to complex interactions with multiple intermediates. Acute PM exposure may affect gene regulation through PM-responsive miRNAs that directly or indirectly control inflammatory gene expression.
PMCID: PMC3595525  PMID: 23358196
miRNA expression; integrative analysis; mRNA expression; inflammation; metal-rich particulate matter; microarray.
4.  Transcriptomic Profiling of the Development of the Inflammatory Response in Human Monocytes In Vitro 
PLoS ONE  2014;9(2):e87680.
Monocytes/macrophages are key players in all phases of physiological and pathological inflammation. To understanding the regulation of macrophage functional differentiation during inflammation, we designed an in vitro model that recapitulates the different phases of the reaction (recruitment, initiation, development, and resolution), based on human primary blood monocytes exposed to sequential changes in microenvironmental conditions. All reaction phases were profiled by transcriptomic microarray analysis. Distinct clusters of genes were identified that are differentially regulated through the different phases of inflammation. The gene sets defined by GSEA analysis revealed that the inflammatory phase was enriched in inflammatory pathways, while the resolution phase comprised pathways related to metabolism and gene rearrangement. By comparing gene clusters differentially expressed in monocytes vs. M1 and vs. M2 macrophages extracted from an in-house created meta-database, it was shown that cells in the model resemble M1 during the inflammatory phase and M2 during resolution. The validation of inflammatory and transcriptional factors by qPCR and ELISA confirmed the transcriptomic profiles in the different phases of inflammation. The accurate description of the development of the human inflammatory reaction provided by this in vitro kinetic model can help in identifying regulatory mechanisms in physiological conditions and during pathological derangements.
PMCID: PMC3912012  PMID: 24498352
5.  EXCAVATOR: detecting copy number variants from whole-exome sequencing data 
Genome Biology  2013;14(10):R120.
We developed a novel software tool, EXCAVATOR, for the detection of copy number variants (CNVs) from whole-exome sequencing data. EXCAVATOR combines a three-step normalization procedure with a novel heterogeneous hidden Markov model algorithm and a calling method that classifies genomic regions into five copy number states. We validate EXCAVATOR on three datasets and compare the results with three other methods. These analyses show that EXCAVATOR outperforms the other methods and is therefore a valuable tool for the investigation of CNVs in largescale projects, as well as in clinical research and diagnostics. EXCAVATOR is freely available at
PMCID: PMC4053953  PMID: 24172663
6.  Whole Genome SNP Genotyping and Exome Sequencing Reveal Novel Genetic Variants and Putative Causative Genes in Congenital Hyperinsulinism 
PLoS ONE  2013;8(7):e68740.
Congenital hyperinsulinism of infancy (CHI) is a rare disorder characterized by severe hypoglycemia due to inappropriate insulin secretion. The genetic causes of CHI have been found in genes regulating insulin secretion from pancreatic β-cells; recessive inactivating mutations in the ABCC8 and KCNJ11 genes represent the most common events. Despite the advances in understanding the molecular pathogenesis of CHI, specific genetic determinants in about 50 % of the CHI patients remain unknown, suggesting additional locus heterogeneity. In order to search for novel loci contributing to the pathogenesis of CHI, we combined a family-based association study, using the transmission disequilibrium test on 17 CHI patients lacking mutations in ABCC8/KCNJ11, with a whole-exome sequencing analysis performed on 10 probands. This strategy allowed the identification of the potential causative mutations in genes implicated in the regulation of insulin secretion such as transmembrane proteins (CACNA1A, KCNH6, KCNJ10, NOTCH2, RYR3, SCN8A, TRPV3, TRPC5), cytosolic (ACACB, CAMK2D, CDKAL1, GNAS, NOS2, PDE4C, PIK3R3) and mitochondrial enzymes (PC, SLC24A6), and in four genes (CSMD1, SLC37A3, SULF1, TLL1) suggested by TDT family-based association study. Moreover, the exome-sequencing approach resulted to be an efficient diagnostic tool for CHI, allowing the identification of mutations in three causative CHI genes (ABCC8, GLUD1, and HNF1A) in four out of 10 patients. Overall, the present study should be considered as a starting point to design further investigations: our results might indeed contribute to meta-analysis studies, aimed at the identification/confirmation of novel causative or modifier genes.
PMCID: PMC3711910  PMID: 23869231
7.  Comprehensive Genomic Characterization of Cutaneous Malignant Melanoma Cell Lines Derived from Metastatic Lesions by Whole-Exome Sequencing and SNP Array Profiling 
PLoS ONE  2013;8(5):e63597.
Cutaneous malignant melanoma is the most fatal skin cancer and although improved comprehension of its pathogenic pathways allowed to realize some effective molecular targeted therapies, novel targets and drugs are still needed. Aiming to add genetic information potentially useful for novel targets discovery, we performed an extensive genomic characterization by whole-exome sequencing and SNP array profiling of six cutaneous melanoma cell lines derived from metastatic patients. We obtained a total of 3,325 novel coding single nucleotide variants, including 2,172 non-synonymous variants. We catalogued the coding mutations according to Sanger COSMIC database and to a manually curated list including genes involved in melanoma pathways identified by mining recent literature. Besides confirming the presence of known melanoma driver mutations (BRAFV600E, NRASQ61R), we identified novel mutated genes involved in signalling pathways crucial for melanoma pathogenesis and already addressed by current targeted therapies (such as MAPK and glutamate pathways). We also identified mutations in four genes (MUC19, PAICS, RBMXL1, KIF23) never reported in melanoma, which might deserve further investigations. All data are available to the entire research community in our Melanoma Exome Database (at In summary, these cell lines are valuable biological tools to improve the genetic comprehension of this complex cancer disease and to study functional relevance of individual mutational events, and these findings could provide insights potentially useful for identification of novel therapeutic targets for cutaneous malignant melanoma.
PMCID: PMC3660556  PMID: 23704925
8.  Milano Summer Particulate Matter (PM10) Triggers Lung Inflammation and Extra Pulmonary Adverse Events in Mice 
PLoS ONE  2013;8(2):e56636.
Recent studies have suggested a link between particulate matter (PM) exposure and increased mortality and morbidity associated with pulmonary and cardiovascular diseases; accumulating evidences point to a new role for air pollution in CNS diseases. The purpose of our study is to investigate PM10sum effects on lungs and extra pulmonary tissues. Milano PM10sum has been intratracheally instilled into BALB/c mice. Broncho Alveolar Lavage fluid, lung parenchyma, heart and brain were screened for markers of inflammation (cell counts, cytokines, ET-1, HO-1, MPO, iNOS), cytotoxicity (LDH, ALP, Hsp70, Caspase8-p18, Caspase3-p17) for a putative pro-carcinogenic marker (Cyp1B1) and for TLR4 pathway activation. Brain was also investigated for CD68, TNF-α, GFAP. In blood, cell counts were performed while plasma was screened for endothelial activation (sP-selectin, ET-1) and for inflammation markers (TNF-α, MIP-2, IL-1β, MPO). Genes up-regulation (HMOX1, Cyp1B1, IL-1β, MIP-2, MPO) and miR-21 have been investigated in lungs and blood. Inflammation in the respiratory tract of PM10sum-treated mice has been confirmed in BALf and lung parenchyma by increased PMNs percentage, increased ET-1, MPO and cytokines levels. A systemic spreading of lung inflammation in PM10sum-treated mice has been related to the increased blood total cell count and neutrophils percentage, as well as to increased blood MPO. The blood-endothelium interface activation has been confirmed by significant increases of plasma ET-1 and sP-selectin. Furthermore PM10sum induced heart endothelial activation and PAHs metabolism, proved by increased ET-1 and Cyp1B1 levels. Moreover, PM10sum causes an increase in brain HO-1 and ET-1. These results state the translocation of inflammation mediators, ultrafine particles, LPS, metals associated to PM10sum, from lungs to bloodstream, thus triggering a systemic reaction, mainly involving heart and brain. Our results provided additional insight into the toxicity of PM10sum and could facilitate shedding light on mechanisms underlying the development of urban air pollution related diseases.
PMCID: PMC3581503  PMID: 23451061
9.  Profiling, Bioinformatic, and Functional Data on the Developing Olfactory/GnRH System Reveal Cellular and Molecular Pathways Essential for This Process and Potentially Relevant for the Kallmann Syndrome 
During embryonic development, immature neurons in the olfactory epithelium (OE) extend axons through the nasal mesenchyme, to contact projection neurons in the olfactory bulb. Axon navigation is accompanied by migration of the GnRH+ neurons, which enter the anterior forebrain and home in the septo-hypothalamic area. This process can be interrupted at various points and lead to the onset of the Kallmann syndrome (KS), a disorder characterized by anosmia and central hypogonadotropic hypogonadism. Several genes has been identified in human and mice that cause KS or a KS-like phenotype. In mice a set of transcription factors appears to be required for olfactory connectivity and GnRH neuron migration; thus we explored the transcriptional network underlying this developmental process by profiling the OE and the adjacent mesenchyme at three embryonic ages. We also profiled the OE from embryos null for Dlx5, a homeogene that causes a KS-like phenotype when deleted. We identified 20 interesting genes belonging to the following categories: (1) transmembrane adhesion/receptor, (2) axon-glia interaction, (3) scaffold/adapter for signaling, (4) synaptic proteins. We tested some of them in zebrafish embryos: the depletion of five (of six) Dlx5 targets affected axonal extension and targeting, while three (of three) affected GnRH neuron position and neurite organization. Thus, we confirmed the importance of cell–cell and cell-matrix interactions and identified new molecules needed for olfactory connection and GnRH neuron migration. Using available and newly generated data, we predicted/prioritized putative KS-disease genes, by building conserved co-expression networks with all known disease genes in human and mouse. The results show the overall validity of approaches based on high-throughput data and predictive bioinformatics to identify genes potentially relevant for the molecular pathogenesis of KS. A number of candidate will be discussed, that should be tested in future mutation screens.
PMCID: PMC3876029  PMID: 24427155
olfactory development; GnRH neuron; Kallmann syndrome; extracellular matrix; transcription profiling; disease gene prediction
10.  Renal cell carcinoma primary cultures maintain genomic and phenotypic profile of parental tumor tissues 
BMC Cancer  2011;11:244.
Clear cell renal cell carcinoma (ccRCC) is characterized by recurrent copy number alterations (CNAs) and loss of heterozygosity (LOH), which may have potential diagnostic and prognostic applications. Here, we explored whether ccRCC primary cultures, established from surgical tumor specimens, maintain the DNA profile of parental tumor tissues allowing a more confident CNAs and LOH discrimination with respect to the original tissues.
We established a collection of 9 phenotypically well-characterized ccRCC primary cell cultures. Using the Affymetrix SNP array technology, we performed the genome-wide copy number (CN) profiling of both cultures and corresponding tumor tissues. Global concordance for each culture/tissue pair was assayed evaluating the correlations between whole-genome CN profiles and SNP allelic calls. CN analysis was performed using the two CNAG v3.0 and Partek software, and comparing results returned by two different algorithms (Hidden Markov Model and Genomic Segmentation).
A very good overlap between the CNAs of each culture and corresponding tissue was observed. The finding, reinforced by high whole-genome CN correlations and SNP call concordances, provided evidence that each culture was derived from its corresponding tissue and maintained the genomic alterations of parental tumor. In addition, primary culture DNA profile remained stable for at least 3 weeks, till to third passage. These cultures showed a greater cell homogeneity and enrichment in tumor component than original tissues, thus enabling a better discrimination of CNAs and LOH. Especially for hemizygous deletions, primary cultures presented more evident CN losses, typically accompanied by LOH; differently, in original tissues the intensity of these deletions was weaken by normal cell contamination and LOH calls were missed.
ccRCC primary cultures are a reliable in vitro model, well-reproducing original tumor genetics and phenotype, potentially useful for future functional approaches aimed to study genes or pathways involved in ccRCC etiopathogenesis and to identify novel clinical markers or therapeutic targets. Moreover, SNP array technology proved to be a powerful tool to better define the cell composition and homogeneity of RCC primary cultures.
PMCID: PMC3141767  PMID: 21668985
11.  A computational procedure to identify significant overlap of differentially expressed and genomic imbalanced regions in cancer datasets† 
Nucleic Acids Research  2009;37(15):5057-5070.
The integration of high-throughput genomic data represents an opportunity for deciphering the interplay between structural and functional organization of genomes and for discovering novel biomarkers. However, the development of integrative approaches to complement gene expression (GE) data with other types of gene information, such as copy number (CN) and chromosomal localization, still represents a computational challenge in the genomic arena. This work presents a computational procedure that directly integrates CN and GE profiles at genome-wide level. When applied to DNA/RNA paired data, this approach leads to the identification of Significant Overlaps of Differentially Expressed and Genomic Imbalanced Regions (SODEGIR). This goal is accomplished in three steps. The first step extends to CN a method for detecting regional imbalances in GE. The second part provides the integration of CN and GE data and identifies chromosomal regions with concordantly altered genomic and transcriptional status in a tumor sample. The last step elevates the single-sample analysis to an entire dataset of tumor specimens. When applied to study chromosomal aberrations in a collection of astrocytoma and renal carcinoma samples, the procedure proved to be effective in identifying discrete chromosomal regions of coordinated CN alterations and changes in transcriptional levels.
PMCID: PMC2731905  PMID: 19542187
12.  Using Pathway Signatures as Means of Identifying Similarities among Microarray Experiments 
PLoS ONE  2009;4(1):e4128.
Widespread use of microarrays has generated large amounts of data, the interrogation of the public microarray repositories, identifying similarities between microarray experiments is now one of the major challenges. Approaches using defined group of genes, such as pathways and cellular networks (pathway analysis), have been proposed to improve the interpretation of microarray experiments. We propose a novel method to compare microarray experiments at the pathway level, this method consists of two steps: first, generate pathway signatures, a set of descriptors recapitulating the biologically meaningful pathways related to some clinical/biological variable of interest, second, use these signatures to interrogate microarray databases. We demonstrate that our approach provides more reliable results than with gene-based approaches. While gene-based approaches tend to suffer from bias generated by the analytical procedures employed, our pathway based method successfully groups together similar samples, independently of the experimental design. The results presented are potentially of great interest to improve the ability to query and compare experiments in public repositories of microarray data. As a matter of fact, this method can be used to retrieve data from public microarray databases and perform comparisons at the pathway level.
PMCID: PMC2610483  PMID: 19125200
13.  Genome-wide screening of copy number alterations and LOH events in renal cell carcinomas and integration with gene expression profile 
Molecular Cancer  2008;7:6.
Clear cell renal carcinoma (RCC) is the most common and invasive adult renal cancer. For the purpose of identifying RCC biomarkers, we investigated chromosomal regions and individual genes modulated in RCC pathology. We applied the dual strategy of assessing and integrating genomic and transcriptomic data, today considered the most effective approach for understanding genetic mechanisms of cancer and the most sensitive for identifying cancer-related genes.
We performed the first integrated analysis of DNA and RNA profiles of RCC samples using Affymetrix technology. Using 100K SNP mapping arrays, we assembled a genome-wide map of DNA copy number alterations and LOH areas. We thus confirmed the typical genetic signature of RCC but also identified other amplified regions (e.g. on chr. 4, 11, 12), deleted regions (chr. 1, 9, 22) and LOH areas (chr. 1, 2, 9, 13). Simultaneously, using HG-U133 Plus 2.0 arrays, we identified differentially expressed genes (DEGs) in tumor vs. normal samples. Combining genomic and transcriptomic data, we identified 71 DEGs in aberrant chromosomal regions and observed, in amplified regions, a predominance of up-regulated genes (27 of 37 DEGs) and a trend to clustering. Functional annotation of these genes revealed some already implicated in RCC pathology and other cancers, as well as others that may be novel tumor biomarkers.
By combining genomic and transcriptomic profiles from a collection of RCC samples, we identified specific genomic regions with concordant alterations in DNA and RNA profiles and focused on regions with increased DNA copy number. Since the transcriptional modulation of up-regulated genes in amplified regions may be attributed to the genomic alterations characteristic of RCC, these genes may encode novel RCC biomarkers actively involved in tumor initiation and progression and useful in clinical applications.
PMCID: PMC2253555  PMID: 18194544
14.  Development of a Universal Microarray Based on the Ligation Detection Reaction and 16S rRNA Gene Polymorphism To Target Diversity of Cyanobacteria 
Applied and Environmental Microbiology  2004;70(12):7161-7172.
The cyanobacteria are photosynthetic prokaryotes of significant ecological and biotechnological interest, since they strongly contribute to primary production and are a rich source of bioactive compounds. In eutrophic fresh and brackish waters, their mass occurrences (water blooms) are often toxic and constitute a high potential risk for human health. Therefore, rapid and reliable identification of cyanobacterial species in complex environmental samples is important. Here we describe the development and validation of a microarray for the identification of cyanobacteria in aquatic environments. Our approach is based on the use of a ligation detection reaction coupled to a universal array. Probes were designed for detecting 19 cyanobacterial groups including Anabaena/Aphanizomenon, Calothrix, Cylindrospermopsis, Cylindrospermum, Gloeothece, halotolerants, Leptolyngbya, Palau Lyngbya, Microcystis, Nodularia, Nostoc, Planktothrix, Antarctic Phormidium, Prochlorococcus, Spirulina, Synechococcus, Synechocystis, Trichodesmium, and Woronichinia. These groups were identified based on an alignment of over 300 cyanobacterial 16S rRNA sequences. For validation of the microarrays, 95 samples (24 axenic strains from culture collections, 27 isolated strains, and 44 cloned fragments recovered from environmental samples) were tested. The results demonstrated a high discriminative power and sensitivity to 1 fmol of the PCR-amplified 16S rRNA gene. Accurate identification of target strains was also achieved with unbalanced mixes of PCR amplicons from different cyanobacteria and an environmental sample. Our universal array method shows great potential for rapid and reliable identification of cyanobacteria. It can be easily adapted to future development and could thus be applied both in research and environmental monitoring.
PMCID: PMC535161  PMID: 15574913
15.  Bacterial discrimination by means of a universal array approach mediated by LDR (ligase detection reaction) 
BMC Microbiology  2002;2:27.
PCR amplification of bacterial 16S rRNA genes provides the most comprehensive and flexible means of sampling bacterial communities. Sequence analysis of these cloned fragments can provide a qualitative and quantitative insight of the microbial population under scrutiny although this approach is not suited to large-scale screenings. Other methods, such as denaturing gradient gel electrophoresis, heteroduplex or terminal restriction fragment analysis are rapid and therefore amenable to field-scale experiments. A very recent addition to these analytical tools is represented by microarray technology.
Here we present our results using a Universal DNA Microarray approach as an analytical tool for bacterial discrimination. The proposed procedure is based on the properties of the DNA ligation reaction and requires the design of two probes specific for each target sequence. One oligo carries a fluorescent label and the other a unique sequence (cZipCode or complementary ZipCode) which identifies a ligation product. Ligated fragments, obtained in presence of a proper template (a PCR amplified fragment of the 16s rRNA gene) contain either the fluorescent label or the unique sequence and therefore are addressed to the location on the microarray where the ZipCode sequence has been spotted. Such an array is therefore "Universal" being unrelated to a specific molecular analysis. Here we present the design of probes specific for some groups of bacteria and their application to bacterial diagnostics.
The combined use of selective probes, ligation reaction and the Universal Array approach yielded an analytical procedure with a good power of discrimination among bacteria.
PMCID: PMC128835  PMID: 12243651
16.  Investigation of the multiple anchors approach in oligonucleotide microarray preparation using linear and stem–loop structured probes 
Nucleic Acids Research  2002;30(8):e34.
Enzyme-mediated reactions are a useful tool in mutation detection when using a microarray format. Discriminating probes attached to the surface of a DNA chip have to be accessible to target DNA and to the enzyme (ligase or polymerase) that catalyses the formation of a new phosphodiester bond. This requires an appropriate chemical platform. Recently, an oligonucleotide hairpin architecture incorporating multiple phosphorothioate moieties along the loop has been proposed as an effective approach to solid-phase minisequencing. We have explored in depth several variables (stem length, number of phosphorothioates, stem–loop architecture versus linear structure) involved in this strategy by using a solid-phase ligation reaction. Microarrays were fabricated either from aminosilyl-modified glass or from aminated polymeric surfaces made of poly-lysine. Both platforms were bromoacetylated and reacted with thiophosphorylated oligonucleotides. The resulting microarrays were tested using either a synthetic template or a PCR-amplified 16S rRNA genomic region as the target sequence. Our results confirm the robustness of the proposed chemistry. We extend its range of application to solid-phase ligation, demonstrating the effectiveness of multiple anchors and suggest that linear oligonucleotides incorporating multiple phosphorothioates are equivalent to their hairpin-structured counterparts.
PMCID: PMC113229  PMID: 11937642

Results 1-16 (16)