Nanospheres for DNA separation chips

We report here a technology to carry out separations of a wide range of DNA fragments with high speed and high resolution. The approach uses a nanoparticle medium, core-shell type nanospheres, in conjunction with a pressurization technique during microchip electrophoresis. DNA fragments up to 15 kilobase pairs (kbp) were successfully analyzed within 100 s without observing any saturation in migration rates. DNA fragments migrate in the medium while maintaining their characteristic molecular structure. To guarantee effective DNA loading and electrofocusing in the nanosphere solution, we developed a double pressurization technique. Optimal pressure conditions and concentrations of packed nanospheres are critical to achieve improved DNA separations.     

DNA芯片技术在植物功能基因组研究中的应用

DNA芯片技术是功能基因组学研究中应用非常广泛的高通量方法之一。随着该技术的不断发展,DNA芯片技术可广泛应用于植物重要性状的基因克隆,鉴别和功能分析,植物抗性机理,重要性状的遗传及杂种优势遗传机理等的研究,同时在诸如植物病原菌转基因产品检测等应用方面也将发挥重要作用。     

DNA芯片技术在微生物学研究中的应用

DNA芯片技术作为一种高通量的核酸分析方法,已经成为“后基因组时代”中研究海量序列信息的重要分析工具之一。本简述了目前一些常用以及和新出现的DNA芯片的技术原理,并从微生物基因表达谱研究,微生物基因组学研究以及微生物检测鉴定研究等多个方面概述了DNA芯片技术在微生物学中的应用,同时在对DNA芯片技术的不足进行简要分析的基础上,展望了其进一步应用的前景。     

Impact of pharmacogenomics on clinical practice in oncology

Multiple drug strategies for many cancer types are now readily available and there is a clear need for tools to inform decision making on therapy selection. Although there is still a long way to go before pharmacogenomics achieves the goal of individualized selection of cancer treatment, promising progress is being made. Genetic testing for thiopurine methyltransferase (TPMT) variant alleles in patients prior to mercaptopurine administration, and for UGT1A1*28 in patients prior to administration of irinotecan therapy, along with the instigation of genotype-guided clinical trials (e.g. TYMS) are important advances in cancer pharmacogenomics. Markers for the toxicity and efficacy of many oncology drugs remain unknown; however, the examples highlighted here suggest progress is being made towards the incorporation of pharmacogenomics into clinical practice in oncology.     

From DNA biosensors to gene chips

Wide-scale DNA testing requires the development of small, fast and easy-to-use devices. This article describes the preparation, operation and applications of biosensors and gene chips, which provide fast, sensitive and selective detection of DNA hybridization. Various new strategies for DNA biosensors and gene chips are examined, along with recent trends and future directions. The integration of hybridization detection schemes with the sample preparation process in a ‘Lab-on-a-Chip’ format is also covered. While the use of DNA biosensors and gene chips is at an early stage, such devices are expected to have an enormous effect on future DNA diagnostics.     

DNA diagnostics in oncology

The most topical areas of oncological molecular diagnostics are reviewed with reference to DNA diagnostics for syndromes and malignancies known to be of hereditary predisposition. The data on some prognostically important tumor-specific markers are summarized. The possible implications of micrometastasis diagnosis are presented, and its essential role is shown. Some DNA polymorphisms predisposing to tumorigenesis are described. Consideration is given to a new aspect of carcinogenesis, epigenetic regulation of the genes involved in the malignant process. Highlighted is the need to develop these basically and practically important studies. Diagnostic protocols for various forms of malignancies are shown to practically result from the tumor cell genome studies.     

Optimal chips for megabase DNA sequencing

The SHOM method (Sequencing by Hybridization with Oligonucleotide Matrix) developed in 1988 is a new approach to nucleic acid sequencing by hybridization to a octanucleotide matrix composed of an array of immobilized oligonucleotides. The original matrix proposed for sequencing by SHOM had to contain at least 65,536 octanucleotides. The present work describes a new family of matrices for sequencing, which allows one to reduce the number of synthesized oligonucleotides 5-15 times without essentially decreasing the resolving power of the method.     

Characterization of DNA chips by nanogold staining

DNA microarray is an important tool in biomedical research. Up to now, there are no chips that can allow both quality analysis and hybridization using the same chip. It is risky to draw conclusions from results of different chips if there is no knowledge of the quality of the chips before hybridization. In this article, we report a colorimetric method to do quality control on an array. The quality analysis of probe spots can be obtained by using gold nanoparticles with positive charges to label DNA through electrostatic attraction. The probe spots can also be detected by a simple personal computer scanner. Gold nanoparticles deposited on a glass surface can be dissolved in bromine-bromide solution. The same microarray treated with gold particles staining and destaining can still be used for hybridization with nearly the same efficiency. This approach makes quality control of a microarray chip feasible and should be a valuable tool for biomarker discovery in the future.     

DNA chips: the future of biomarkers

DNA chips are small, solid supports such as microscope slides onto which thousands of cDNAs or oligonucleotides are arrayed, representing known genes or simply EST clones, or covering the entire sequence of a gene with all its possible mutations. Fluorescently labeled DNA or RNA extracted from tissues is hybridized to the array. Laser scanning of the chip permits quantitative evaluation of each individual complementary sequence present in the sample. DNA chip technology is currently being proposed for qualitative and quantitative applications, firstly for the detection of point mutations, small deletions and insertions in genes involved in human diseases or affected during cancer progression; secondly, to determine on a genome-wide basis the pattern of gene expression in tumors, as well as in a number of experimental situations. The extraordinary power of DNA chips will have a strong impact on medicine in the near future, both in the molecular characterization of tumors and genetic diseases and in drug discovery and evaluation. Quantitative applications will soon spread through all fields of biology.     

High-throughput site-directed mutagenesis using oligonucleotides synthesized on DNA chips

Site-directed mutagenesis has greatly helped researchers both to understand the precise role of specific residues in coding sequences and to generate variants of proteins that have acquired new characteristics. Today's demands for more complete functional cartographies of proteins and advances in selection and screening technologies require that site-directed mutagenesis be adapted for high-throughput applications. We describe here the first generation of a library of single and multiple site-directed mutants using a mixture of oligonucleotides synthesized on DNA chips. We have used the human interleukin 15 (IL15) gene as a model, of which 37 codons were simultaneously targeted for substitution by any of eight possible codons. Ninety-six clones were sequenced, exhibiting a broad spectrum of targeted substitutions over the whole gene length with no unwanted mutations. Libraries produced using such pools of oligonucleotides open new perspectives to direct the evolution of proteins in vitro, by enabling the simple, rapid, and cost-effective generation of large tailor-made genetic diversities from any gene.     

Cohort analysis of a single nucleotide polymorphism on DNA chips

A method has been developed to determine SNPs on DNA chips by applying a flow-through bioscanner. As a practical application we demonstrated the fast and simple SNP analysis of 24 genotypes in an array of 96 spots with a single hybridisation and dissociation experiment. The main advantage of this methodical concept is the parallel and fast analysis without any need of enzymatic digestion. Additionally, the DNA chip format used is appropriate for parallel analysis up to 400 spots. The polymorphism in the gene of the human phenol sulfotransferase SULT1A1 was studied as a model SNP. Biotinylated PCR products containing the SNP (The SNP summary web site: ) (mutant) and those containing no mutation (wild-type) were brought onto the chips coated with NeutrAvidin using non-contact spotting. This was followed by an analysis which was carried out in a flow-through biochip scanner while constantly rinsing with buffer. After removing the non-biotinylated strand a fluorescent probe was hybridised, which is complementary to the wild-type sequence. If this probe binds to a mutant sequence, then one single base is not fully matching. Thereby, the mismatched hybrid (mutant) is less stable than the full-matched hybrid (wild-type). The final step after hybridisation on the chip involves rinsing with a buffer to start dissociation of the fluorescent probe from the immobilised DNA strand. The online measurement of the fluorescence intensity by the biochip scanner provides the possibility to follow the kinetics of the hybridisation and dissociation processes. According to the different stability of the full-match and the mismatch, either visual discrimination or kinetic analysis is possible to distinguish SNP-containing sequence from the wild-type sequence.     

A new generation of scanners for DNA chips

Today, most of the DNA chips are used with fluorescent markers. Associated with fluorescence confocal scanners, this technology achieves remarkable performances in terms of sensitivity and accuracy. The main technical issues related to these scanners have already been reviewed. However, these scanners are costly, especially when high density chips are used. In this case, a mechanical precision of 1 microm or less is required to achieve the measurement precision required. This cost level prevents the spread of this technology in the diagnostic market. We will present a new concept for scanners with equivalent or superior performances, with a cost cut of 5-10. This concept is inspired from the field of optical disk and reader. Basically, an optical format is added to the chip, before DNA deposition. This format contains tracks which are superimposed to the DNA features. These tracks define the path that an optical head of a CD player must follow in order to scan the surface of the DNA chip. Such a head is a very cheap component, and has a precision of less than 100 nm thanks to real-time focus and tracking. These functions are fulfilled by electromagnetic actuators mounted on the support of the frontal lens. We show here that it is possible to use such a head to build a fluorescence confocal scanner with equivalent or even better performances than conventional scanners.     

Self-assembling protein arrays on DNA chips by auto-labeling fusion proteins with a single DNA address

The high-throughput deposition of recombinant proteins on chips, beads or biosensor devices would be greatly facilitated by the implementation of self-assembly concepts. DNA-directed immobilization via conjugation of proteins to an oligonucleotide would be preeminently suited for this purpose. Here, we present a unique method to attach a single DNA address to proteins in one step during the purification from the E. coli lysate by fusion to human O6-alkylguanine-DNA-alkyltransferase (SNAP-tag) and the Avitag. Use of the conjugates in converting a DNA chip into a protein chip by self assembly is demonstrated.     

Aryldiazomethanes for universal labeling of nucleic acids and analysis on DNA chips

DNA and RNA labeling and detection are key steps in nucleic acid-based technologies, used in medical research and molecular diagnostics. We report here the synthesis, reactivity, and potential of a new type of labeling molecule, m-(N-Biotinoylamino)phenylmethyldiazomethane (m-BioPMDAM), that reacts selectively and efficiently with phosphates in nucleotide monomers, oligonucleotides, DNA, and RNA. This molecule contains a biotin as detectable unit and a diazomethyl function as reactive moiety. We demonstrate that this label fulfills the requirements of stability, solubility, reactivity, and selectivity for hybridization-based analysis and especially for detection on high-density DNA chips.     

Neonatal screening by DNA microarray: spots and chips

Newborn screening (NBS) is a public-health genetic screening programme aimed at early detection and treatment of pre-symptomatic children affected by specific disorders. It currently involves protein-based assays and PCR to confirm abnormal results. We propose that DNA microarray technology might be an improvement over protein assays in the first stage of NBS. This approach has important advantages, such as multiplex analysis, but also has disadvantages, which include a high initial cost and the analysis/storage of large data sets. Determining the optimal technology for NBS will require that technical, public health and ethical considerations are made for the collection and extent of analysis of paediatric genomic data, for privacy and for parental consent.     

Quantification of DNA methylation in electrofluidics chips (Bio-COBRA)

Alterations of normal gene expression patterns are a hallmark of human cancers. It is now clear that the dysregulation of epigenetic modifications of the DNA and surrounding histones contributes to aberrant gene silencing, thus being major participants not only in the progression but also the initiation of the disease phenotype. The best-studied epigenetic modification is DNA methylation, which converts cytosine to 5-methylcytosine. Aberrant hypermethylation of the promoter is frequently observed in cancer and is generally associated with gene silencing. Currently, accurate and reproducible quantification of DNA methylation remains challenging. Here, we describe Bio-COBRA, a modified protocol for Combined Bisulfite Restriction Analysis (COBRA), that incorporates an electrophoresis step in microfluidics chips. Microfluidics technology involves the handling of small amounts of liquid in miniaturized systems. In the life sciences, microfluidics usually entails the scaling down of at least one application, such as electrophoresis, to chip format, which often results in increased efficiency and reliability. Bio-COBRA provides a platform for the rapid and quantitative assessment of DNA methylation patterns in large sample sets. Its sensitivity and reproducibility also makes it a tool for the analysis of DNA methylation in clinical samples. The Bio-COBRA assay can be performed on 12 samples in less than 1 h. If the protocol is started at the DNA isolation step, however, approximately 48 h would be required to complete the entire procedure.