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.     

Microarray gene expression analysis free of reverse transcription and dye labeling

A new microarray system has been developed for gene expression analysis using cationic gold nanoparticles with diameters of 250 nm as a target detection reagent. The approach utilizes nonlabeled target molecules hybridizing with complementary probes on the array, followed by incubation in a colloidal gold solution. The hybridization signal results from the precipitation of nanogold particles on the hybridized spots due to the electrostatic attraction of the cationic gold particles and the anionic phosphate groups in the target DNA backbone. In contrast to conventional fluorescent detection, this nanoparticle-based detection system eliminates the target labeling procedure. The visualization of hybridization signals can be accomplished with a flatbed scanner instead of a confocal laser scanner, which greatly simplifies the process and reduces the cost. The sensitivity is estimated to be less than 2 pg of DNA molecules captured on the array surface. The signal from hybridized spots quantitatively represents the amount of captured target DNA and therefore permits quantitative gene expression analysis. Cross-array reproducibility is adequate for detecting twofold or less signal changes across two microarray experiments.     

Colorimetric silver detection of DNA microarrays

Development of microarrays has revolutionized gene expression analysis and molecular diagnosis through miniaturization and the multiparametric features. Critical factors affecting detection efficiency of targets hybridization on microarray are the design of capture probes, the way they are attached to the support, and the sensitivity of the detection method. Microarrays are currently detected in fluorescence using a sophisticated confocal laser-based scanner. In this work, we present a new colorimetric detection method which is intented to make the use of microarray a powerful procedure and a low-cost tool in research and clinical settings. The signal generated with this method results from the precipitation of silver onto nanogold particles bound to streptavidin, the latter being used for detecting biotinylated DNA. This colorimetric method has been compared to the Cy-3 fluorescence method. The detection limit of both methods was equivalent and corresponds to 1 amol of biotinylated DNA attached on an array. Scanning and data analysis of the array were obtained with a colorimetric-based workstation.     

百合病毒的DNA芯片检测技术研究

根据已知的黄瓜花叶病毒,百合无症病毒、百合斑驳病毒基因核苷酸序列,设计引物和探针,制备寡核苷酸芯片。用Cy3标记核苷酸引物,不对称RT-PCR扩增产物与芯片上的寡核苷酸探针杂交,荧光扫描仪检测并分析信号。研究制备的基因芯片能够检测侵染百合的3种重要病毒核酸的特异性荧光信号,该项技术具有特异、灵敏、快速的优点。     

Visual DNA microarrays for simultaneous detection of Ureaplasma urealyticum and Chlamydia trachomatis coupled with multiplex asymmetrical PCR

Visual DNA microarrays, based on gold label silver stain (GLSS) and coupled with multiplex asymmetrical PCR, were developed for simultaneous, sensitive and specific detection of Ureaplasma urealyticum and Chlamydia trachomatis. 5'-end-amino-modified oligonucleotides, which were immobilized on glass surface, acted as capturing probes that were designed to bind complementary biotinylated targets DNA. The gold-conjugated streptavidins were introduced to the microarray for specific binding to biotin. The black image of microarray spots, resulting from the precipitation of silver onto nanogold particles bound to streptavidins, were used to detect biotinylated targets DNA visually or with a visible light scanner. Multiplex asymmetrical PCR of U. urealyticum, C. trachomatis and Bacillus subtilis (used as positive control) was performed to prepare abundant biotinylated single-stranded targets DNA, which affected detection efficiency and sensitivity of hybridization on microarray. Plenty of clinical samples of U. urealyticum and C. trachomatis from infected patients were tested using home-made DNA microarrays. For its high sensitivity, good specificity, simplicity, cheapness and speed, the present visual gene-detecting technique has potential applications in clinical fields.     

基因芯片技术检测肾综合征出血热病毒核酸的研究

To establish a rapid, sensitive and specific diagnostic assay for Hantavirus with microarray techniques, specific primers and probes were designed according to the conservative and specific DNA sequence of 76-118 strain and R22 strain. The probes were spotted on glass slides to form microarrays.The Cy3-1abled single stranded DNA fragments prepared by dissymmetical PCR were hybridized with the probes on the glass slides. The microarrays were scanned and analyzed with a scanner. The results showed that the DNA microarray could detect the different typed DNA of HTN and SEO with adequate specificity and sensitivity. The developed DNA microarray and techniques might be a very useful method for diagnosis and prevention, and could be widely applied in specific pathogens detection ofinfectious diseases such as hemorrhagic fever with renal syndrome.     

Real-time detection of DNA hybridization on microarray using a CCD-based imaging system equipped with a rotated microlens array disk

This work describes a novel charge-coupled device (CCD)-based imaging system (MB Biochip Reader?) for real-time detection of DNA hybridization to DNA microarrays. The MB Biochip Reader? consisted of a laser light source (532 nm), a microlens array for generation of a multi-beam laser, and a CCD for 2-D signal imaging. The MB Biochip Reader? with a rotated microlens array, allowed large-field imaging (6.2 mm × 7.6 mm with 6.45 μm resolution) with fast time-resolution at 0.2 s without speckle noise. Furthermore, real-time detection of DNA hybridization, which is sufficient to obtain accurate data from tens of thousands of array element per field, was successfully performed without the need for laser scanning. The performance of the MB Biochip Reader? for DNA microarray imaging was similar to the commercially available photomultiplier tube (PMT)-based microarray scanner, ScanArray Lite. The system potentially could be applied toward real-time analysis in many other fluorescent techniques in addition to real-time DNA microarray analysis.     

Stripping custom microRNA microarrays and the lessons learned about probe-slide interactions

Microarrays have been used extensively in gene expression profiling and genotyping studies. To reduce the high cost and enhance the consistency of microarray experiments, it is often desirable to strip and reuse microarray slides. Our genome-wide analysis of microRNA expression involves the hybridization of fluorescently labeled nucleic acids to custom-made, spotted DNA microarrays based on GAPSII-coated slides. We describe here a simple and effective method to regenerate such custom microarrays that uses a very low-salt buffer to remove labeled nucleic acids from microarrays. Slides can be stripped and reused multiple times without significantly compromising data quality. Moreover, our analyses of the performance of regenerated slides identifies parameters that influence the attachment of oligonucleotide probes to GAPSII slides, shedding light on the interactions between DNA and the microarray surface and suggesting ways in which to improve the design of oligonucleotide probes.     

TaqMan probe array for quantitative detection of DNA targets

To date real-time quantitative PCR and gene expression microarrays are the methods of choice for quantification of nucleic acids. Herein, we described a unique fluorescence resonance energy transfer-based microarray platform for real-time quantification of nucleic acid targets that combines advantages of both and reduces their limitations. A set of 3′ amino-modified TaqMan probes were designed and immobilized on a glass slide composing a regular microarray pattern, and used as probes in the consecutive PCR carried out on the surface. During the extension step of the PCR, 5′ nuclease activity of DNA polymerase will cleave quencher dyes of the immobilized probe in the presence of nucleic acids targets. The increase of fluorescence intensities generated by the change in physical distance between reporter fluorophore and quencher moiety of the probes were collected by a confocal scanner. Using this new approach we successfully monitored five different pathogenic genomic DNAs and analyzed the dynamic characteristics of fluorescence intensity changes on the TaqMan probe array. The results indicate that the TaqMan probe array on a planar glass slide monitors DNA targets with excellent specificity as well as high sensitivity. This set-up offers the great advantage of real-time quantitative detection of DNA targets in a parallel array format.     

Comprehensive quality control utilizing the prehybridization third-dye image leads to accurate gene expression measurements by cDNA microarrays

Background  

Gene expression profiling using microarrays has become an important genetic tool. Spotted arrays prepared in academic labs have the advantage of low cost and high design and content flexibility, but are often limited by their susceptibility to quality control (QC) issues. Previously, we have reported a novel 3-color microarray technology that enabled array fabrication QC. In this report we further investigated its advantage in spot-level data QC.     

Fabrication of high quality microarrays

Fabrication of DNA microarray demands that between ten (diagnostic microarrays) and many hundred thousands of probes (research or screening microarrays) are efficiently immobilised to a glass or plastic surface using a suitable chemistry. DNA microarray performance is measured by parameters like array geometry, spot density, spot characteristics (morphology, probe density and hybridised density), background, specificity and sensitivity. At least 13 factors affect these parameters and factors affecting fabrication of microarrays are used in this review to compare different fabrication methods (spotted microarrays and in situ synthesis of microarrays) and immobilisation chemistries.     

Monitoring of cDNA microarray with common primer target and hybridization specificity with selected targets

Academic researchers are increasingly producing and using cDNA microarrays. Their quality and hybridization specificity are crucial in determining whether the generated data are accurate and interpretable. Here, we describe two methods of monitoring microarray production, the sustainability of DNA attachment, and the specificity of hybridization. The first method consists of labeling an oligonucleotide, which is one of the primers used to amplify all cDNA probes on the array (except for beta-actin and GAPDH) with fluorescent dye and hybridize it to the cDNA microarray. Attachment of the cDNAs on the array after the hybridization procedure was monitored by visualizing fluorescent signals from the spots on the array. In the second method, two selected DNA targets, beta-actin and GAPDH, were labeled with fluorescent dye to hybridize to the cDNA array. Hence, hybridization specificity was demonstrated by obtaining fluorescent signals solely from the genes corresponding to the target.     

Efficient oligonucleotide probe selection for pan-genomic tiling arrays

Background  

Array comparative genomic hybridization is a fast and cost-effective method for detecting, genotyping, and comparing the genomic sequence of unknown bacterial isolates. This method, as with all microarray applications, requires adequate coverage of probes targeting the regions of interest. An unbiased tiling of probes across the entire length of the genome is the most flexible design approach. However, such a whole-genome tiling requires that the genome sequence is known in advance. For the accurate analysis of uncharacterized bacteria, an array must query a fully representative set of sequences from the species' pan-genome. Prior microarrays have included only a single strain per array or the conserved sequences of gene families. These arrays omit potentially important genes and sequence variants from the pan-genome.     

Optical readout of gold nanoparticle-based DNA microarrays without silver enhancement

We present a novel readout scheme for gold nanoparticle-based DNA microarrays relying on "Laser-Induced Scattering around a NanoAbsorber". It provides direct counting of individual nanoparticles present on each array spot and stable signals, without any silver enhancement. Given the detection of nanometer-sized particles, which minimize the steric hindrance, the linear dynamic range of the method is particularly large and well suited for microarray detection.     

Comparison between different strategies of covalent attachment of DNA to glass surfaces to build DNA microarrays

DNA microarray is a powerful tool allowing simultaneous detection of many different target molecules present in a sample. The efficiency of the array depends mainly on the sequence of the capture probes and the way they are attached to the support. The coupling procedure must be quick, covalent, and reproducible in order to be compatible with automatic spotting devices dispensing tiny drops of liquids on the surface. We compared several coupling strategies currently used to covalently graft DNA onto a glass surface. The results indicate that fixation of aminated DNA to an aldehyde-modified surface is a choice method to build DNA microarrays. Both the coupling procedure and the hybridization efficiency have been optimized. The detection limit of human cytomegalovirus target DNA amplicons on such DNA microarrays has been estimated to be 0.01 nM by fluorescent detection.     

Analysis of DNA microarrays by non-destructive fluorescent staining using SYBR green II

A simple, non-destructive procedure is described to determine the quality of DNA arrays before they are used. It consists of a preliminary staining step of the DNA microarray by using SYBR green II, a fluorophore with specific affinity for ssDNA, followed by a laser scan analysis. The surface quality, integrity and homogeneity of each DNA spot of the array can thus be assessed. After this preliminary control, which may avoid further analytical steps that lead to the waste of precious biological samples, a fully reversible staining procedure is performed that produces an array ready for subsequent use.     

High reproducibility using sodium hydroxide-stripped long oligonucleotide DNA microarrays

Recently, long oligonucleotide (60- to 70-mer) microarrays for two-color experiments have been developed and are gaining widespread use. In addition, when there is limited availability of mRNA from tissue sources, RNA amplification can and is being used to produce sufficient quantities of cRNA for microarray hybridization. Taking advantage of the selective degradation of RNA under alkaline conditions, we have developed a method to "strip" glass-based oligonucleotide microarrays that use fluorescent RNA in the hybridization, while leaving the DNA oligonucleotide probes intact and usable for a second experiment. Replicate microarray experiments conducted using stripped arrays showed high reproducibility, however, we found that arrays could only be stripped and reused once without compromising data quality. The intraclass correlation (ICC) between a virgin array and a stripped array hybridized with the same sample showed a range of 0.90-0.98, which is comparable to the ICC of two virgin arrays hybridized with the same sample. Using this method, once-stripped oligonucleotide microarrays are usable, reliable, and help to reduce costs.     

Oligonucleotide microarrays: immobilization of phosphorylated oligonucleotides on epoxylated surface

A facile and efficient method for direct immobilization of phosphorylated oligonucleotides on an epoxy-activated glass surface is described. The new immobilization strategy has been analyzed for its performance in DNA microarray under both microwave and thermal conditions. It reflects high immobilization efficiency ( approximately 23%), and signal-to-noise ratio ( approximately 98) and resulted in high hybridization efficiency ( approximately 36%) in comparison to those obtained with standard methods, viz., NTMTA ( approximately 9.76%) and epoxide-amine ( approximately 9.82%). The probes immobilized through the new strategy were found to be heat-stable, since the performance of microarray decreased by only approximately 7% after subjecting it to 20 PCR-like heat cycles, suggesting that the chemistry could be used in integrated PCR/microarray devices. The immobilization of probes following the proposed chemistry resulted in spots of superior quality in terms of spot morphology, spot homogeneity, and signal reproducibility. The constructed microarrays have been successfully used for the discrimination of nucleotide mismatches. In conclusion, these features make the new immobilization strategy ideal for facile, efficient, and cost-effective manufacturing of DNA microarrays.     

cDNA文库法在HCV-1b检测芯片研制中的应用

制备丙型肝炎病毒(HCV) 1b亚型诊断芯片并进行初步验证评价.采用cDNA文库法制备探针,用限制性内切酶Sau3AⅠ消化HCV 1b全长cDNA ,所得的酶切片段72℃补平加A ,AT克隆,PCR初步鉴定,并测序.将筛选出的片段打印在氨基修饰的玻片上制备成检测芯片并进行杂交验证分析.运用cDNA文库法,得到2 2个大小相对一致(2 5 0～75 0bp)的基因片段.序列分析表明,均属于HCV 1b基因,可以作为诊断芯片探针;样品标记采用限制性显示(restrictiondisplay ,RD)技术,标记后进行杂交.杂交结果显示,样品和诊断基因芯片杂交的敏感性和特异性均佳.批内和批间精密度CV值分别为5 4 %和6 8% ,表明用cDNA文库法收集片段是一种快速、简便制备芯片探针的实用方法.     

应用多重标记引物增强寡核苷酸芯片的荧光信号强度

寡核苷酸芯片技术是一种高通量发掘和采集生物信息的强大技术平台,目前已广泛应用于生物科学领域 . 为改善寡核苷酸芯片的分析性能,对影响芯片杂交结果的因素,如片基表面的化学处理、探针的长度、间隔臂的长度、杂交条件等,进行了深入的研究和优化 . 对寡核苷酸芯片而言,仍有待解决的问题是如何产生更强的荧光信号来改善其检测灵敏度 . 利用两种类型的多个荧光分子标记的引物,来增强二维寡核苷酸芯片平面上的荧光信号强度 . 两种引物分别命名为：多标记线性引物和多标记分支引物 . 通过增加标记在目标 DNA 片段上的荧光分子数,可以显著增强寡核苷酸芯片上相应捕获探针的信号强度 . 实验表明,使用多标记引物能将所用的寡核苷酸微阵列的检测限 ( 以能够检测的最低模板量计算 ) 降低至单荧光标记引物的 1/100 以下,多重标记技术是一种有效增强微型化探针矩阵检测灵敏度的信号放大方法 .