GFP质控芯片的初步研究

目的：建立一种质量控制芯片来监测样品标记、杂交和检测过程中的失误。方法：针对GFP基因设计的4条60mer寡核苷酸探针和1条阳性对照探针polv（U）与流感寡核苷酸探针一起打印在DAKO玻片上,并构建了GFP基因的克隆载体和体外表达载体,将从这两种重组载体上获得的绿色荧光蛋白（Green Fluorescent Protein,GFP）基因的ILNA、DNA片段和人的全血样品中的DNA用限制性显示技术（Restriction Display technology,RD）扩增标记,将标记的样品和荧光标记的通用引物U分别与芯片杂交、检测,并对扫描的结果进行统计分析。结果：GFP探针与相应的样品杂交时出现阳性信号,阳性对照探针在所有的杂交中均出现阳性信号,而空白对照则未检测荧光信号。结论：建立的质控芯片具有较好的敏感性和特异性,可以用于基因芯片中的质量监控。     

Reproducible and inexpensive probe preparation for oligonucleotide arrays

We present a new protocol for the preparation of nucleic acids for microarray hybridization. DNA is fragmented quantitatively and reproducibly by using a hydroxyl radical-based reaction, which is initiated by hydrogen peroxide, iron(II)-EDTA and ascorbic acid. Following fragmentation, the nucleic acid fragments are densely biotinylated using a biotinylated psoralen analog plus UVA light and hybridized on microarrays. This non-enzymatic protocol circumvents several practical difficulties associated with DNA preparation for microarrays: the lack of reproducible fragmentation patterns associated with enzymatic methods; the large amount of labeled nucleic acids required by some array designs, which is often combined with a limited amount of starting material; and the high cost associated with currently used biotinylation methods. The method is applicable to any form of nucleic acid, but is particularly useful when applying double-stranded DNA on oligonucleotide arrays. Validation of this protocol is demonstrated by hybridizing PCR products with oligonucleotide-coated microspheres and PCR amplified cDNA with Affymetrix Cancer GeneChip microarrays.     

Position of the fluorescent label is a crucial factor determining signal intensity in microarray hybridizations

A key issue in applications of short oligonucleotide-based microarrays is how to design specific probes with high sensitivity. Some details of the factors affecting microarray hybridization remain unclear, hampering a reliable quantification of target nucleic acids. We have evaluated the effect of the position of the fluorescent label [position of label (POL)] relative to the probe-target duplex on the signal output of oligonucleotide microarrays. End-labelled single-stranded DNA targets of different lengths were used for hybridization with perfect-match oligonucleotide probe sets targeting different positions of the same molecule. Hybridization results illustrated that probes targeting the labelled terminus of the target showed significantly higher signals than probes targeting other regions. This effect was independent of the target gene, the fluorophore and the slide surface chemistry. Comparison of microarray signal patterns of fluorescently end-labelled, fluorescently internally random-labelled and radioactively end-labelled target-DNAs with the same set of oligonucleotide probes identified POL as a critical factor affecting signal intensity rather than binding efficiency. Our observations define a novel determinant for large differences of signal intensities. Application of the POL effect may contribute to better probe design and data interpretation in microarray applications.     

Evaluation of thin films of agarose on glass for hybridization of DNA to identify plant pathogens with microarray technology

Agarose-coated glass slides, after activation, were spotted with amine-modified oligonucleotide probes using a manual eight-pin arraying device. Two probes, designed to identify two common greenhouse fungal plant pathogens, Didymella bryoniae and Botrytis cinerea, were hybridized with polymerase chain reaction (PCR)-amplified fluorescently labeled DNA extracted from pure culture and from diseased plant tissue. The probes easily distinguished these pathogens from each other without cross reaction. Thickness of the agarose layer and length of the sample DNA were important factors affecting hybridization efficiency of immobilized probe to PCR product. These factors did not affect hybridization with short complementary oligonucleotide. Probes fixed on agarose-coated slides could differentiate samples as readily as probes on nylon but with potentially higher spot density and gave much better signal than probes on silylated slides. The use of plain glass slides, agarose, and a manual arrayer makes this technique useful for developing specialized and inexpensive DNA microarrays on a solid rigid substrate.     

Fundamentals of DNA hybridization arrays for gene expression analysis

DNA hybridization arrays [also known as macroarrays, microarrays and/or high-density oligonucleotide arrays (Gene Chips)] bring gene expression analysis to a genomic scale by permitting investigators to simultaneously examine changes in the expression of literally thousands of genes. For hybridization arrays, the general approach is to immobilize gene-specific sequences (probes) on a solid state matrix (nylon membranes, glass microscope slides, silicon/ceramic chips). These sequences are then queried with labeled copies of nucleic acids from biological samples (targets). The underlying theory is that the greater the expression of a gene, the greater the amount of labeled target, and hence, the greater output signal. In spite of the simplicity of the experimental design, there are at least four different platforms and several different approaches to processing and labeling the biological samples. Moreover, investigators must also determine whether they will utilize commercially available arrays or generate their own. This review will cover the status of the hybridization array field with an eye toward underlying principles and available technologies. Future developments and technological trends will also be evaluated.     

DNA microarrays with unimolecular hairpin double-stranded DNA probes: fabrication and exploration of sequence-specific DNA/protein interactions

We have fabricated double-stranded DNA (dsDNA) microarrays containing unimolecular hairpin dsDNA probes immobilized on glass slides. The unimolecular hairpin dsDNA microarrays were manufactured by four steps: Firstly, synthesizing single-stranded DNA (ssDNA) oligonucleotides with two reverse-complementary sequences at 3' hydroxyl end and an overhang sequence at 5' amino end. Secondly, microspotting ssDNA on glutaraldehyde-derived glass slide to form ssDNA microarrays. Thirdly, annealing two reverse-complementary sequences to form hairpin primer at 3' end of immobilized ssDNA and thus to create partial-dsDNA microarray. Fourthly, enzymatically extending hairpin primer to convert partial-dsDNA microarrays into complete-dsDNA microarray. The excellent efficiency and high accuracy of the enzymatic synthesis were demonstrated by incorporation of fluorescently labeled dUTPs in Klenow extension and digestion of dsDNA microarrays with restriction endonuclease. The accessibility and specificity of the DNA-binding proteins binding to dsDNA microarrays were verified by binding Cy3-labeled NF-kappaB to dsDNA microarrays. The dsDNA microarrays have great potential to provide a high-throughput platform for investigation of sequence-specific DNA/protein interactions involved in gene expression regulation, restriction and so on.     

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 .     

Portable System for Microbial Sample Preparation and Oligonucleotide Microarray Analysis

We have developed a three-component system for microbial identification that consists of (i) a universal syringe-operated silica minicolumn for successive DNA and RNA isolation, fractionation, fragmentation, fluorescent labeling, and removal of excess free label and short oligonucleotides; (ii) microarrays of immobilized oligonucleotide probes for 16S rRNA identification; and (iii) a portable battery-powered device for imaging the hybridization of fluorescently labeled RNA fragments with the arrays. The minicolumn combines a guanidine thiocyanate method of nucleic acid isolation with a newly developed hydroxyl radical-based technique for DNA and RNA labeling and fragmentation. DNA and RNA can also be fractionated through differential binding of double- and single-stranded forms of nucleic acids to the silica. The procedure involves sequential washing of the column with different solutions. No vacuum filtration steps, phenol extraction, or centrifugation is required. After hybridization, the overall fluorescence pattern is captured as a digital image or as a Polaroid photo. This three-component system was used to discriminate Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, and human HL60 cells. The procedure is rapid: beginning with whole cells, it takes approximately 25 min to obtain labeled DNA and RNA samples and an additional 25 min to hybridize and acquire the microarray image using a stationary image analysis system or the portable imager.     

Portable system for microbial sample preparation and oligonucleotide microarray analysis

We have developed a three-component system for microbial identification that consists of (i) a universal syringe-operated silica minicolumn for successive DNA and RNA isolation, fractionation, fragmentation, fluorescent labeling, and removal of excess free label and short oligonucleotides; (ii) microarrays of immobilized oligonucleotide probes for 16S rRNA identification; and (iii) a portable battery-powered device for imaging the hybridization of fluorescently labeled RNA fragments with the arrays. The minicolumn combines a guanidine thiocyanate method of nucleic acid isolation with a newly developed hydroxyl radical-based technique for DNA and RNA labeling and fragmentation. DNA and RNA can also be fractionated through differential binding of double- and single-stranded forms of nucleic acids to the silica. The procedure involves sequential washing of the column with different solutions. No vacuum filtration steps, phenol extraction, or centrifugation is required. After hybridization, the overall fluorescence pattern is captured as a digital image or as a Polaroid photo. This three-component system was used to discriminate Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, and human HL60 cells. The procedure is rapid: beginning with whole cells, it takes approximately 25 min to obtain labeled DNA and RNA samples and an additional 25 min to hybridize and acquire the microarray image using a stationary image analysis system or the portable imager.     

The effects of mismatches on hybridization in DNA microarrays: determination of nearest neighbor parameters

Quantifying interactions in DNA microarrays is of central importance for a better understanding of their functioning. Hybridization thermodynamics for nucleic acid strands in aqueous solution can be described by the so-called nearest neighbor model, which estimates the hybridization free energy of a given sequence as a sum of dinucleotide terms. Compared with its solution counterparts, hybridization in DNA microarrays may be hindered due to the presence of a solid surface and of a high density of DNA strands. We present here a study aimed at the determination of hybridization free energies in DNA microarrays. Experiments are performed on custom Agilent slides. The solution contains a single oligonucleotide. The microarray contains spots with a perfect matching (PM) complementary sequence and other spots with one or two mismatches (MM) : in total 1006 different probe spots, each replicated 15 times per microarray. The free energy parameters are directly fitted from microarray data. The experiments demonstrate a clear correlation between hybridization free energies in the microarray and in solution. The experiments are fully consistent with the Langmuir model at low intensities, but show a clear deviation at intermediate (non-saturating) intensities. These results provide new interesting insights for the quantification of molecular interactions in DNA microarrays.     

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.     

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.     

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。     

Characterization of an inexpensive, nontoxic, and highly sensitive microarray substrate

An agarose film has been proposed as an efficient substrate for producing microarrays. The original film preparation procedure was simplified significantly by grafting the agarose layer directly onto unmodified microscope glass slides instead of aminated glass slides, and the blocking procedure was replaced with a wash in 0.1x standard saline citrate (SSC) and 0.5% sodium dodecyl sulfate (SDS) without decreasing the performance of the produced microarrays. Characterization of the grafted agarose film using atomic force microscopy (AFM) and scanning electron microscopy (SEM) showed that the agarose film had a 10-fold increase in surface roughness compared to glass and that the interior of the agarose film was porous, with pore sizes between 100-500 nm. A comparison of hybridization on aldehyde-activated agarose-coated microarray slides and commercial amino-reactive microarray slides showed that aldehyde-activated agarose-coated slides had the highest signal-to-noise ratio of 850, suggesting that the aldehyde-activated agarose microarray slides are suitable in applications where analytes have a wide concentration range. By immobilizing the DNA probes using ultraviolet (UV) light, the signal-to-noise ratio was further increased to 3000 on the agarose microarray slides. The specificity of the UV cross-linked DNA probes was demonstrated using 21 and 25 bp long capture probes, enabling discrimination of target molecules differing in only one base.     

Oligonucleotide Microarray with RD-PCR Labeling Technique for Detection and Typing of Human Papillomavirus

Currently, screening for high-risk human papillomavirus (HPV) infection remains an important health concern throughout the world, because of the close association between certain types of HPV and cervical cancer. In this study, we explore the possibility of using ∼70mer oligonucleotide microarray for detection and genotyping of HPV. The ∼70mer type-specific oligonucleotide probes of four different types HPV were designed by using biological software Arraydesigner 2.0, which analyzed the whole genome sequences of HPV and selected optimal probes. These probes were synthesized and printed onto the surface of glass slides in order to prepare a low-density microarray. HPV samples were labeled with fluorescence dyes Cy3 using a method of restriction display polymerase chain reaction (RD-PCR). HPV plasmid DNA was restricted with Sau3A I to produce multiple fragments that were ligated to adaptors subsequently and used as PCR template. PCR labeling was performed with the fluorescently labeled universal primer (Cy3-UP) whose sequence is designed according to the adaptor of the RD-PCR approaches. The labeled samples were hybridized with the oligonucleotide microarray. The scanning results showed that HPV DNA hybridized specifically with multiple spots correspondingly to show positive signals, whereas no signals were detected of all the negative and blank controls. These results demonstrated that ∼70mer oligonucleotide microarray can be applied to HPV detection and genotyping. The application of RD-PCR in the sample labeling can increase significantly the sensitivity of the assay and will be especially useful for the discriminate diagnosis of multiple pathogens.     

微阵列（microarrays）技术及其应用

微阵列分为cDNA微阵列和寡聚核苷酸微阵列,微阵列上“印”有大量已知部分序列的DNA探针,微阵列技术就是利用分子杂交原理,使同时被比较的标本（用同位素或荧光素标记）与微阵列杂交,通过检测杂交信号强度及数据处理,把他们转化成不同标本中特异基因的丰度,从而全国比较不同标本的基因表达水平的差异,微阵列技术是一种探索基因组功能的有力手段。     

Enzymatic labeling of nucleic acids

Enzymatic labeling of nucleic acids is a fundamental tool in molecular biology with virtually every aspect of nucleic acid hybridization technique involving the use of labeled probes. Different methods for enzymatic labeling of DNA, RNA and oligonucleotide probes are available today. In this review, we will describe both radioactive and nonradioactive labeling methods, yet the choice of system for labeling the probe depends on the application under study.