基因芯片技术筛选家蝇抗菌肽相关基因

用生物学软件对GenBank中部分昆虫抗菌肽基因编码区保守域设计探针, 用直接点样法将探针点印在特制玻片上构建寡核苷酸(Oligonucleotide, oligo)探针微阵列; 提取诱导后24 h的家蝇三龄幼虫脂肪体总RNA, 逆转录成cDNA并标记上荧光标记物Cy3, 与构建的oligo探针微阵列杂交, 经洗片、扫描处理后进行数据分析。结果在两次重复实验中均检测到有效杂交信号的基因点有15个(不包括阳性对照基因), 为进一步发现其新基因提供了依据。     

Simple and effective method for generating single-stranded DNA targets and probes

A simple and efficient PCR method was developed for generating dye- or radiolabeled single-stranded DNA targets or probes used for hybridization studies. The method involved the use of a pair of long primers with high annealing temperatures and a short, labeled primer with a low annealing temperature in a PCR consisting of two cycles at different temperatures. We used this method to generate dye Cy 5-labeled and [32P]-radiolabeled single-stranded DNA targets and probes. These labeled probes were used successfully for the microarray identification of point mutations in Mycobacterium tuberculosis genes and for the Northern blot detection of expression changes of the GATA-2 gene in Pneumocystis carinii-infected rat lungs.     

Design and application of an oligonucleotide microarray for the investigation of compost microbial communities

A microarray consisting of oligonucleotide probes targeting variable regions of the 16S rRNA gene was designed and tested for the investigation of microbial communities in compost. Probes were designed for microorganisms that have been previously reported in the composting process and for plant, animal and human pathogens. The oligonucleotide probes were between 17 and 25 bp in length and included mostly species-specific sequences. Validation of probe specificity and optimization of hybridization conditions were conducted using fluorescently labeled 16S rRNA gene PCR products of pure culture strains. A labeling method employing a Cy3 or Cy5-labeled forward primer together with a phosphate-conjugated reverse primer for the production of single stranded DNA after a digestion step was optimised and used to label target DNA. A combination of two different DNA extraction methods using both physical and chemical lysis was found to give the best DNA yields. Increased hybridization signal intensities were obtained for probes modified with a 12 mer T-spacer. The microarray was found to have a detection limit of 10(3) cells, although in compost spiking experiments, the detection limit was reduced to 10(5) cells. The application of the microarray to compost samples indicated the presence of Streptococcus, Acinetobacter lwoffii, and Clostridium tetani in various compost samples. The presence of A. lwoffii in those compost samples was confirmed by PCR using primers specific for the organism. The aim of this study was to develop a molecular tool that would allow screening for the presence or absence of different microorganisms within compost samples.     

The influence of RNA integrity,purity and cDNA labelling on glass slide cDNA microarray image quality

The accuracy of gene expression measurements generated using cDNA microarrays is dependent on the quality of the image generated following hybridization of fluorescently labelled cDNA. It is not known how this image is influenced by sample preparation factors which such as RNA quality, cDNA synthesis and labelling efficiency. In this study we used a simple metric based on the ratio of the total feature (F) and background (B) fluorescence, which correlates with the visual assessment of 60 microarray images, to determine the influence of sample preparation on image quality. Results indicate that RNA purity (A260/A280) and integrity (18S:28S ratio) do not strongly influence microarray image quality. cDNA having an nucleotide to dye ratio greater than 100 produced poor microarray images, however, cDNA labelled more efficiently was not a guarantee of a better image. The data also indicate that the array image quality is not improved by loading more cDNA into the hybridization mixture however poor image quality did result from a disproportionate amounts of Cy5 and Cy3 labelled cDNA. This study provides insight into the source of variation in microarray image analysis introduced during sample preparation and will assist in the standardisation of cDNA glass slide microarray protocols.     

Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis

We describe here a protocol for the representative amplification of global mRNAs from typical single mammalian cells to provide a template for high-density oligonucleotide microarray analysis. A single cell is lysed in a tube without purification and first-strand cDNAs are synthesized using a poly(dT)-tailed primer. Unreacted primer is specifically eliminated by exonuclease treatment and second strands are generated with a second poly(dT)-tailed primer after poly(dA) tailing of the first-strand cDNAs. The cDNAs are split into four tubes, which are independently directionally amplified by PCR, and then recombined. The amplified products (approximately 100 ng) show superior representation and reproducibility of original gene expression, especially for genes expressed in more than 20 copies per cell, compared with those obtained by a conventional PCR protocol, and can effectively be used for quantitative PCR and EST analyses. The cDNAs are then subjected to another PCR amplification with primers bearing the T7 promoter sequence. The resultant cDNA products are gel purified, amplified by one final cycle and used for isothermal linear amplification by T7 RNA polymerase to synthesize cRNAs for microarray hybridization. This protocol yields cDNA templates sufficient for more than 80 microarray hybridizations from a single cell, and can be completed in 5-6 days.     

PCR amplification on magnetic nanoparticles: application for high-throughput single nucleotide polymorphism genotyping

A novel approach for the genotyping of single nucleotide polymorphisms (SNPs) based on solidphase PCR on magnetic nanoparticles (MNPs) is described. PCR products were amplified directly on MNPs. The genotypes of a given SNP were differentiated by hybridization with a pair of allele-specific probes labeled with dual-color fluorescence (Cy3, Cy5). The results were analyzed by scanning the microarray printed with the denatured fluorescent probes on an unmodified glass slide. Electrophoresis analysis indicated that PCR could proceed successfully when MNPs-bound primers were used. Furthermore, nine different samples were genotyped and their fluorescent signals were quantified. Genotyping results showed that three genotypes for the locus were very easily discriminated. The fluorescent ratios (match probe:mismatch probe signal) of homozygous samples were over 9.3, whereas heterozygous samples had ratios near 1.0. Without any purification and concentration of PCR products, this new MNP-PCR based genotyping assay potentially provides a rapid, labor-saving method for genotyping of a large number of individuals.     

问号钩端螺旋体基因组DNA芯片的研制

目的 研制并初步评估问号钩端螺旋体(简称钩体)赖型赖株的基因组DNA芯片。方法 利用Primegens引物设计软件筛选出问号钩体赖型赖株全基因组中的特异性基因进行引物设计。对成功设计出相应引物的3 290个基因用聚合酶链反应方法进行扩增,以纯化后的产物点样制备芯片。并用双色荧光杂交策略对芯片质量进行了初步平估。结果 共获得3 290个基因产物用于点样。参考株自身杂交实验结果表明:该芯片有较高的点一致性、信噪比和较低的假阳性率。结论 成功制备了包含问号钩体赖型赖株3 290个目的基因的基因组DNA芯片,并可用于基于该芯片的问号钩体比较基因组学的研究。     

Oligonucleotide conjugates with minor groove ligands as probes for hybridization microarray chips

A possibility of using oligonucleotide conjugates with minor groove ligands as probes for hybridization microarray chips was studied. The oligonucleotide conjugates contain a hairpin ligand (MGB) composed of two tripyrrolcarboxamide residues with an aminocaproic acid residue as a linker and bound to the oligonucleotide duplex AT tract in a site-specific manner. We used as (5′-3′)-probes: GACAAGAp, GACAAAAp, GACAAGA-MGB, and GACAAAA-MGB. The oligonucleotides labeled with the Cy3 cyanine dye, Cy3-ACTAATTTTGTC and Cy3-ACTAATCTTGTC, were used as targets. The maximal MGB effect on the fluorescence level of microarray chip spots, which caused its fourfold increase as compared with the initial unmodified duplex, was observed for the duplex containing only AT pairs in the ligand binding site. The presence of AC and GT mutations in the binding site (imperfect duplexes) or a CG pair (perfect duplex) affect the change in fluorescence level to a considerably lesser degree.     

Oligonucleotide conjugates with minor groove ligands as probes for hybridization microarray chips

A possibility of using oligonucleotide conjugates with minor groove ligands as probes for hybridization microarray chips was studied. The oligonucleotide conjugates contain a hairpin ligand (MGB) composed of two tripyrrolcarboxamide residues with an aminocaproic acid residue as a linker and bound to the oligonucleotide duplex AT tract in a site-specific manner. We used as (5'-3') probes GACAAGAp, GACAAAAp, GACAAGA-MGB, and GACAAAA-MGB. The oligonucleotides labeled with Cy3 cyanine dye, Cy3-ACTAATTTTGTC and Cy3-ACTAATCTTGTC, were used as targets. The maximal MGB effect on the fluorescence level of microarray chip spots, which caused its fourfold increase as compared with the initial unmodified duplex, was observed for the duplex containing only AT pairs in the ligand binding site. The presence of A-C and G-T mutations in the binding site (imperfect duplexes) or a C-G pair (perfect duplex) affects the change in fluorescence level to a considerably lesser degree.     

AIV血凝素亚型同步检测基因芯片技术研究

对流感病毒14个血凝素亚型的基因芯片检测技术进行了初步研究。通过RT-PCR克隆禽流感病毒血凝素基因片段,获得重组质粒。从重组质粒扩增大约500bp的DNA片段,浓缩后点到氨基化玻璃载体上,制成芯片。待检病毒样品用TRIzolLS提取RNA,反转录过程中用Cy5标记样品cDNAs。将标记样品与芯片杂交,扫描芯片上待检样品与芯片上捕捉探针的结合位点,杂交信号与预期设想一致。结果显示,DNA芯片技术可以提供一种有效的AIV血凝素亚型鉴别诊断方法。     

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.     

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.     

Chemical derivatization of compact disc polycarbonate surfaces for SNPs detection

Compact discs have been proposed as an efficient analytical platform, with potential to develop high-throughput affinity assays for genomics, proteomics, clinics, and health monitoring. Chemical derivatization of CD surfaces is one of the keys to developing highly efficient microarraying-based assays on discs. Approaches for mild chemical modification of polycarbonate (PC) disc surface based on nitration, reduction, and chloromethylation reactions have been developed. Derivatized surfaces as amino and thiol are obtained for PC, maintaining unchanged the mechanical and optical properties of the discs. Studies of covalent attachment of oligonucleotide probes (5' Cy5-labeled, 3' NH 2-ended) on the modified surfaces have been performed to develop microarraying assays based on hybridization of cDNA strands and single nucleotide polymorphism discrimination (SNPs). A demonstration of the applicability to the compact disc audio/video technology for its use as analytical system is performed, including the employment of a commercial CD player to read the results on disc.     

Dendrimeric coating of glass slides for sensitive DNA microarrays analysis

Successful use and reliability of microarray technology is highly dependent on several factors, including surface chemistry parameters and accessibility of cDNA targets to the DNA probes fixed onto the surface. Here, we show that functionalisation of glass slides with homemade dendrimers allow production of more sensitive and reliable DNA microarrays. The dendrimers are nanometric structures of size-controlled diameter with aldehyde function at their periphery. Covalent attachment of these spherical reactive chemical structures on amino-silanised glass slides generates a reactive ~100 Å layer onto which amino-modified DNA probes are covalently bound. This new grafting chemistry leads to the formation of uniform and homogenous spots. More over, probe concentration before spotting could be reduced from 0.2 to 0.02 mg/ml with PCR products and from 20 to 5 µM with 70mer oligonucleotides without affecting signal intensities after hybridisation with Cy3- and Cy5-labelled targets. More interestingly, while the binding capacity of captured probes on dendrimer-activated glass surface (named dendrislides) is roughly similar to other functionalised glass slides from commercial sources, detection sensitivity was 2-fold higher than with other available DNA microarrays. This detection limit was estimated to 0.1 pM of cDNA targets. Altogether, these features make dendrimer-activated slides ideal for manufacturing cost-effective DNA arrays applicable for gene expression and detection of mutations.     

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.     

Quantitative microarray-based DNA-DNA hybridization assay for measuring genetic distances among bacterial species and its application to the identification of family Enterobacteriaceae

Quantitative DNA-DNA hybridization to measure the genetic distances among bacterial species is indispensable for taxonomical determination. In the current studies, we developed a method to determine bacterial DNA relatedness on a glass microarray. Reference DNAs representing a total 93 species of Enterobacteriaceae were arrayed on a glass microplate, and signal intensities were measured after 2 hr of hybridization with Cy3-labeled bacterial DNAs. All immobilized DNAs from members of the family Enterobacteriaceae were identified by this method except for DNAs from Yersinia pseudotuberculosis and Y. pestis. These results suggest that quantitative microarray hybridization could be an alternative to conventional DNA-DNA hybridization for measuring chromosome relatedness among bacterial species.     

Dramatic increase in the signal and sensitivity of detection <Emphasis Type="Italic">via</Emphasis> self-assembly of branched DNA

In molecular testing using PCR, the target DNA is amplified via PCR and the sequence of interest is investigated via hybridization with short oligonucleotide capture probes that are either in a solution or immobilized on solid supports such as beads or glass slides. In this report, we report the discovery of assembly of DNA complex(es) between a capture probe and multiple strands of the PCR product. The DNA complex most likely has branched structure. The assembly of branched DNA was facilitated by the product of asymmetric PCR. The amount of branched DNA assembled was increased five fold when the asymmetric PCR product was denatured and hybridized with a capture probe all in the same PCR reaction mixture. The major branched DNA species appeared to contain three reverse strands (the strand complementary to the capture probe) and two forward strands. The DNA was sensitive to S1 nuclease suggesting that it had single-stranded gaps. Branched DNA also appeared to be assembled with the capture probes immobilized on the surface of solid support when the product of asymmetric PCR was hybridized. Assembly of the branched DNA was also increased when hybridization was performed in complete PCR reaction mixture suggesting the requirement of DNA synthesis. Integration of asymmetric PCR, heat denaturation and hybridization in the same PCR reaction mixture with the capture probes immobilized on the surface of solid support achieved dramatic increase in the signal and sensitivity of detection of DNA. Such a system should be advantageously applied for development of automated process for detection of DNA.