Biomedical subjects

Erdogan Gulari

Publications and source records attributed to Erdogan Gulari.

15 recordsLinked to original sources

PubMed2006

Influence of dangling ends and surface-proximal tails of targets on probe-target duplex formation in 16S rRNA gene-based diagnostic arrays.

Dangling ends and surface-proximal tails of gene targets influence probe-target duplex formation and affect the signal intensity of probes on diagnostic microarrays. This phenomenon was evaluated using an oligonucleotide microarray containing 18-mer probes corresponding to the 16S rRNA genes of 10 waterborne pathogens and a number of synthetic and PCR-amplified gene targets. Signal intensities for Klenow/random primer-labeled 16S rRNA gene targets were dissimilar from those for 45-mer synthetic targets for nearly 73% of the probes tested. Klenow/random primer-labeled targets resulted in an interaction with a complex mixture of 16S rRNA genes (used as the background) 3.7 times higher than the interaction of 45-mer targets with the same mixture. A 7-base-long dangling end sequence with perfect homology to another single-stranded background DNA sequence was sufficient to produce a cross-hybridization signal that was as strong as the signal obtained by the probe-target duplex itself. Gibbs free energy between the target and a well-defined background was found to be a better indicator of hybridization signal intensity than the sequence or length of the dangling end alone. The dangling end (Gibbs free energy of -7.6 kcal/mol) was found to be significantly more prone to target-background interaction than the surface-proximal tail (Gibbs free energy of -64.5 kcal/mol). This study underlines the need for careful target preparation and evaluation of signal intensities for diagnostic arrays using 16S rRNA and other gene targets due to the potential for target interaction with a complex background.

Bacteria↗

PubMed2006

An open-access long oligonucleotide microarray resource for analysis of the human and mouse transcriptomes.

Two collections of oligonucleotides have been designed for preparing pangenomic human and mouse microarrays. A total of 148,993 and 121,703 oligonucleotides were designed against human and mouse transcripts. Quality scores were created in order to select 25,342 human and 24,109 mouse oligonucleotides. They correspond to: (i) a BLAST-specificity score; (ii) the number of expressed sequence tags matching each probe; (iii) the distance to the 3' end of the target mRNA. Scores were also used to compare in silico the two microarrays with commercial microarrays. The sets described here, called RNG/MRC collections, appear at least as specific and sensitive as those from the commercial platforms. The RNG/MRC collections have now been used by an Anglo-French consortium to distribute more than 3500 microarrays to the academic community. Ad hoc identification of tissue-specific transcripts and a approximately 80% correlation with hybridizations performed on Affymetrix GeneChiptrade mark suggest that the RNG/MRC microarrays perform well. This work provides a comprehensive open resource for investigators working on human and mouse transcriptomes, as well as a generic method to generate new microarray collections in other organisms. All information related to these probes, as well as additional information about commercial microarrays have been stored in a freely-accessible database called MEDIANTE.

Animals↗

PubMed2006

Fabrication of ordered catalytically active nanoparticles derived from block copolymer micelle templates for controllable synthesis of single-walled carbon nanotubes.

We report the use of the block copolymer micelle approach to produce various transition metal nanoparticles such as iron, cobalt, and nickel with precisely controlled size and spacing. These uniformly sized catalyst nanoparticles derived from the block copolymer micelle approach have enabled the synthesis of carbon nanotubes (CNTs) with narrow size distribution. Because of the excellent film forming ability of the polymeric material, metal-bearing surface micelles produced from the solution micelles can be distributed uniformly on a surface, resulting in evenly dispersed catalyst nanoparticles. As a result, high quality and uniformly distributed CNTs have been synthesized. Spatially selective growth of CNTs from a lithographically patterned metal-bearing micelle film has been achieved. The polymer template approach can potentially be extended to synthesize single-metallic and bimetallic catalytically active nanoparticles with uniform size and spacing and is fully compatible with conventional lithographic process. Additionally, catalyst nanoparticles produced from this method do not coalesce at high growth temperature. All these attributes make this approach a promising fabrication pathway for controllable synthesis of CNTs.

Journal Article↗

PubMed2006

On-chip non-equilibrium dissociation curves and dissociation rate constants as methods to assess specificity of oligonucleotide probes.

Nucleic acid hybridization serves as backbone for many high-throughput systems for detection, expression analysis, comparative genomics and re-sequencing. Specificity of hybridization between probes and intended targets is always critical. Approaches to ensure and evaluate specificity include use of mismatch probes, obtaining dissociation curves rather than single temperature hybridizations, and comparative hybridizations. In this study, we quantify effects of mismatch type and position on intensity of hybridization signals and provide a new approach based on dissociation rate constants to evaluate specificity of hybridized signals in complex target mixtures. Using an extensive set of 18mer oligonucleotide probes on an in situ synthesized biochip platform, we demonstrate that mismatches in the center of the probe are more discriminating than mismatches toward the extremities of the probe and mismatches toward the attached end are less discriminating than those toward the loose end. The observed destabilizing effect of a mismatch type agreed in general with predictions using the nearest neighbor model. Use of a new parameter, specific dissociation temperature (T(d-w), temperature of maximum specific dissociation rate constant), obtained from probe-target duplex dissociation profiles considerably improved the evaluation of specificity. These results have broad implications for hybridization data obtained from complex mixtures of nucleic acids.

Base Pair Mismatch↗

PubMed2005

Layer-by-layer assembly of nacre-like nanostructured composites with antimicrobial properties.

In a recent report, we have presented the layer-by-layer (LBL) assembly of a biomimetic nanostructured composite from Na(+)-montmorillonite clay nanosheets and poly(diallylmethylammonium chloride) (Tang, Z.; Kotov, N.; Magonov, S.; Ozturk, B. Nat. Mater. 2003, 2, 413). The structure, deformation mechanism, and mechanical properties of the material are very similar to those of natural nacre and lamellar bones. This fact prompts further investigation of these composites as potential bone implants. LBL assembly affords preparation of multifunctional composites, and here we demonstrate that not only mechanical strength, but also antibacterial activity, can be introduced in these implantable materials by alternating clay layers with starch-stabilized silver nanoparticles. The resulting composite showed excellent structural stability with no detectable levels of silver lost over a 1 month period. Evaluation of the antibacterial properties showed almost complete growth inhibition of E. coli over an 18 h period. The amount of silver eluted from the LBL composite over a 1 month period was determined to be only 0.5-3.0 microg/L. This concentration of silver did not prevent the growth of the mammalian tissue cultures. The LBL composite has shown biocompatibility with the human osteoblast cell line.

Animals↗

PubMed2004

Accurate multiplex gene synthesis from programmable DNA microchips.

Testing the many hypotheses from genomics and systems biology experiments demands accurate and cost-effective gene and genome synthesis. Here we describe a microchip-based technology for multiplex gene synthesis. Pools of thousands of 'construction' oligonucleotides and tagged complementary 'selection' oligonucleotides are synthesized on photo-programmable microfluidic chips, released, amplified and selected by hybridization to reduce synthesis errors ninefold. A one-step polymerase assembly multiplexing reaction assembles these into multiple genes. This technology enabled us to synthesize all 21 genes that encode the proteins of the Escherichia coli 30S ribosomal subunit, and to optimize their translation efficiency in vitro through alteration of codon bias. This is a significant step towards the synthesis of ribosomes in vitro and should have utility for synthetic biology in general.

Escherichia coli↗

PubMed2004

Potential of DNA microarrays for developing parallel detection tools (PDTs) for microorganisms relevant to biodefense and related research needs.

Development of parallel detection tools using microarrays is critically reviewed in view of the need for screening multiple microorganisms in a single test. Potential research needs with respect to probe design and specificity, validation, sample concentration, selective target enrichment and amplification, and data analysis are discussed. Data illustrating selected probe design issues for detecting multiple targets in mixed microbial systems is presented. Challenges with respect to cost, time, and ease of use compared to other methods are also summarized.

Animals↗

PubMed2004

Microfluidic PicoArray synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences.

Large DNA constructs of arbitrary sequences can currently be assembled with relative ease by joining short synthetic oligodeoxynucleotides (oligonucleotides). The ability to mass produce these synthetic genes readily will have a significant impact on research in biology and medicine. Presently, high-throughput gene synthesis is unlikely, due to the limits of oligonucleotide synthesis. We describe a microfluidic PicoArray method for the simultaneous synthesis and purification of oligonucleotides that are designed for multiplex gene synthesis. Given the demand for highly pure oligonucleotides in gene synthesis processes, we used a model to improve key reaction steps in DNA synthesis. The oligonucleotides obtained were successfully used in ligation under thermal cycling conditions to generate DNA constructs of several hundreds of base pairs. Protein expression using the gene thus synthesized was demonstrated. We used a DNA assembly strategy, i.e. ligation followed by fusion PCR, and achieved effective assembling of up to 10 kb DNA constructs. These results illustrate the potential of microfluidics-based ultra-fast oligonucleotide parallel synthesis as an enabling tool for modern synthetic biology applications, such as the construction of genome-scale molecular clones and cell-free large scale protein expression.

Base Sequence↗

PubMed2004

Gene2Oligo: oligonucleotide design for in vitro gene synthesis.

There is substantial interest in implementing a bioinformatics tool that allows the design of oligonucleotides to support the development of in vitro gene synthesis. Current protocols to make long synthetic DNA molecules rely on the in vitro assembly of a set of short oligonucleotides, either by ligase chain reaction (LCR) or by assembly PCR. Ideally, such oligonucleotides should represent both strands of the final DNA molecule. They should be adjacent on the same strand and overlap the complementary oligonucleotides from the second strand to ensure good hybridization during assembly. This implies that the thermodynamic properties of each oligonucleotide have to be consistent across the set. Furthermore, any given oligonucleotide has to be totally specific to its target to avoid the creation of incorrectly assembled sequences. We have developed Gene2Oligo (http://berry.engin.umich.edu/gene2oligo/), a web-based tool that divides a long input DNA sequence into a set of adjacent oligonucleotides representing both DNA strands. The length of the oligonucleotides is dynamically optimized to ensure both the specificity and the uniform melting temperatures necessary for in vitro gene synthesis. We have successfully designed and used a set of oligonucleotides to synthesize the Saccharomyces cerevisiae cytochrome b5 by using both LCR and assembly PCR.

Algorithms↗

PubMed2004

In situ synthesis of oligonucleotide microarrays.

This contribution presents a brief overall look of the methods for the preparation of various types of DNA microarrays and a thorough examination of the methods for in situ synthesis of oligonucleotide microarrays.

Biotechnology↗

PubMed2004

High density peptide microarrays. In situ synthesis and applications.

The technologies enabling the creation of large scale, miniaturized peptide or protein microarrays are emerging. The focuses of this review are the synthesis and applications of peptide and peptidomimetic microarrays, especially the light directed parallel synthesis of individually addressable high density peptide microarrays using a novel photogenerated reagent chemistry and digital photolithography (Gao et al., 1998, J. Am. Chem. Soc. 120, 12698; Pellois et al. 2002, Nat. Biotechnol. 20, 922). Concepts related to the synthesis are discussed, such as the reactions of photogenerated acids in the deprotection step of peptide synthesis or oligonucleotide synthesis, and the applications of high density peptide chips in antibody binding assays are discussed. Peptide chips provide versatile tools for probing antigen-antibody, protein-protein, peptide-ligand interactions and are basic components for miniaturization, automation, and system integration in research and clinical diagnosis applications.

Peptide Library↗

PubMed2003

OligoArray 2.0: design of oligonucleotide probes for DNA microarrays using a thermodynamic approach.

There is a substantial interest in implementing bioinformatics technologies that allow the design of oligonucleotides to support the development of microarrays made from short synthetic DNA fragments spotted or in situ synthesized on slides. Ideally, such oligonucleotides should be totally specific to their respective targets to avoid any cross-hybridization and should not form stable secondary structures that may interfere with the labeled probes during hybridization. We have developed OligoArray 2.0, a program that designs specific oligonucleotides at the genomic scale. It uses a thermodynamic approach to predict secondary structures and to calculate the specificity of targets on chips for a unique probe in a mixture of labeled probes. Furthermore, OligoArray 2.0 can adjust the oligonucleotide length, according to user input, to fit a narrow T(m) range compatible with hybridization requirements. Combined with on chip oligonucleotide synthesis, this program makes it feasible to perform expression analysis on a genomic scale for any organism for which the genome sequence is known. This is without relying on cDNA or oligonucleotide libraries. OligoArray 2.0 was used to design 75 764 oligonucleotides representing 26 140 transcripts from Arabidopsis thaliana. Among this set, we provide at least one specific oligonucleotide for 93% of these transcripts.

Algorithms↗

PubMed2003

Light directed massively parallel on-chip synthesis of peptide arrays with t-Boc chemistry.

Peptide and peptidomimetic molecule arrays are emerging powerful tools for parallel screening of binding in proteomics and pharmaceutical discovery research. Up to now the common method of preparing peptide arrays was based on spotting on glass using a library of presynthesized peptides. However, due to the large number of monomers (amino acids) it is not possible to have combinatorial libraries which include all combinations of natural and synthetic amino acids. We describe a very flexible on-chip oligopeptide synthesis method which uses the well developed t-Boc based solid state synthesis chemistry. A very high degree of flexibility is achieved by using light photo generated acids and maskless projection lithography for spatially directed deprotection. Use of microfluidic chips enables moderately high densities, short reaction times and off-the-shelf chemicals. Examples are given from synthesis of metal ion binding peptides and epitope binding assays.

Combinatorial Chemistry Techniques↗

PubMed2002

Individually addressable parallel peptide synthesis on microchips.

Miniaturized, spatially addressable microchips of peptides and peptidomimetics are powerful tools for high-throughput biomedical and pharmaceutical research and the advancement of proteomics. Here we report an efficient and flexible method for the parallel synthesis of peptides on individually addressable microchips, using digital photolithography and photogenerated acid in the deprotection step. We demonstrate that we are able to synthesize thousands of peptides in a 1 cm(2) area on a microchip using 20 natural amino acids as well as synthetic amino acid analogs, with high stepwise yields and short reaction-cycle times. Epitope screening experiments using a p53 antibody (PAb240) produced clearly defined binding patterns. The peptidomimetic sequences on the microchip show specific antibody binding and provide insights into the molecular details responsible for specificity of epitope binding. Our approach requires just a conventional synthesizer and a computer-controllable optical module, thereby allowing potential development of peptide microchips for various pharmaceutical and proteomic applications in routine research laboratories.

Amino Acid Sequence↗

PubMedDate not supplied

Light-directed simultaneous synthesis of oligopeptides on microarray substrate using a photogenerated acid.

Photogenerated acid (PGA) was used as the acid to remove the protection group from amino acids or peptide oligomers. Comparative study of the deprotection using a PGA, trisarylsulfonium antimonyhexafluoride (SSb), and trifluoroacetic acid (TFA) was performed on glass microscope slides. The results showed that PGA can replace TFA in the deprotection step of oligopeptide synthesis with comparable efficiencies. Acids needed for the deprotection step were generated in situ by light activation of the precursor molecule on the microwell substrate. A mask-less laser light illumination system was used to activate the precursor. The accuracy of the amino acid sequence of the synthesized oligopeptide and the location of the synthesis was illustrated by the specific recognition binding of two different models: lead(II) ion-peptide biosensor for lead(II) and human protein p53 (residue 20-25)-mouse MAb DO1. After parallel synthesis of the target peptide models and their analogues based on the predetermined pattern, specific binding treatment, and fluorescence labeling, the fluorescence emission images of the oligopeptide microarray showed fluorescence intensity as a result of specific binding at the correct locations of the array. The stepwise synthesis efficiencies of pentapeptide synthesis on the microwell substrate range are approximately 96-100% and do not decrease with respect to the chain length of the peptide.

Acids↗