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.     

On-chip hybridization kinetics for optimization of gene expression experiments

DNA microarray technology is a powerful tool for getting an overview of gene expression in biological samples. Although the successful use of microarray-based expression analysis was demonstrated in a number of applications, the main problem with this approach is the fact that expression levels deduced from hybridization experiments do not necessarily correlate with RNA concentrations. Moreover oligonucleotide probes corresponding to the same gene can give different hybridization signals. Apart from cross-hybridizations and differential splicing, this could be due to secondary structures of probes or targets. In addition, for low-copy genes, hybridization equilibrium may be reached after hybridization times much longer than the one commonly used (overnight, i.e., 15 h). Thus, hybridization signals could depend on kinetic properties of the probe, which may vary between different oligonucleotide probes immobilized on the same microarray. To validate this hypothesis, on-chip hybridization kinetics and duplex thermostability analysis were performed using oligonucleotide microarrays containing 50-mer probes corresponding to 10 mouse genes. We demonstrate that differences in hybridization kinetics between the probes exist and can influence the interpretation of expression data. In addition, we show that using on-chip hybridization kinetics, quantification of targets is feasible using calibration curves.     

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.     

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.     

Cooperativity of paired oligonucleotide probes for microarray hybridization assays

Synthetic DNA probes attached to microarrays usually range in length from 25 to 70 nucleotides. There is a compromise between short probes with lower sensitivity, which can be accurately synthesized in higher yields, and long probes with greater sensitivity but lower synthesis yields. Described here are microarrays printed with spots containing a mixture of two short probes, each designed to hybridize at noncontiguous sites in the same targeted sequence. We have shown that, for a printed microarray, mixed probe spots containing a pair of 30mers show significantly greater hybridization than spots containing a single 30mer and can approach the amount of hybridization to spots containing a 60mer or a 70mer. These spots with mixed oligonucleotide probes display cooperative hybridization signals greater than those that can be achieved by either probe alone. Both the higher synthesis yields of short probes and the greater sensitivity of long oligonucleotides can be utilized. This strategy provides new design options for microarray hybridization assays to detect RNA abundance, RNA splice variants, or sequence polymorphisms.     

Analysis of crucial factors resulting in microarray hybridization failure

The factors that affect the formation and stability of DNA/DNA duplexes are complicated and still mostly unknown. In this study attempts were made to look for the crucial factor affecting hybridization failure in DNA microarray assays. A comprehensive range of factors were investigated simultaneously using a 25-mer oligonucleotide Potyvirus microarray. These included steric hindrance, direct/indirect labelling types, distance of a probe to the fluorescent labelling end, target (the DNA fragment used to hybridize with microarray probes) strand types either single strand or double strand, probes without mismatch and with different numbers of mismatch nucleotides (up to 36%) and different mismatch locations (5' end, centre and 3' end), probe GC content and T(m), secondary structures of probes and targets, different target lengths (0.277 kb to ~1.3 kb) and concentrations (0.1-30 nM). The results showed that whilst most of these known factors were unlikely to be the main causes of failed hybridization, there was strong evidence suggesting that the viral amplicon target structure is the most crucial factor. However, computing predicted target secondary structures by Mfold showed no correlation with the hybridization results. One explanation is that the predicted target secondary structures are different from the real structures. Here we postulate that the real target structure might be a combination of secondary structures resulting in a three-dimensional structure from exposure to three types of sub-structures: (1) a completely exposed linear structure to allow probes access for the successful hybridization and showing strong fluorescent signals; (2) a partially exposed structure to allow unstable binding and showing weak fluorescent signals; (3) a closed structure resulting in failed hybridization. These results are very important for microarray based studies as they not only provide an explanation for some current controversial results, but also provide potential resolution for the future studies. Due to the lack of available software for predicting the true target structure, development of microarrays should conduct an initial oligonucleotide probe selection procedure and those probes with capacity to hybridize with the target should be considered for the microarray development.     

Modeling formamide denaturation of probe-target hybrids for improved microarray probe design in microbial diagnostics

Application of high-density microarrays to the diagnostic analysis of microbial communities is challenged by the optimization of oligonucleotide probe sensitivity and specificity, as it is generally unfeasible to experimentally test thousands of probes. This study investigated the adjustment of hybridization stringency using formamide with the idea that sensitivity and specificity can be optimized during probe design if the hybridization efficiency of oligonucleotides with target and non-target molecules can be predicted as a function of formamide concentration. Sigmoidal denaturation profiles were obtained using fluorescently labeled and fragmented 16S rRNA gene amplicon of Escherichia coli as the target with increasing concentrations of formamide in the hybridization buffer. A linear free energy model (LFEM) was developed and microarray-specific nearest neighbor rules were derived. The model simulated formamide melting with a denaturant m-value that increased hybridization free energy (ΔG°) by 0.173 kcal/mol per percent of formamide added (v/v). Using the LFEM and specific probe sets, free energy rules were systematically established to predict the stability of single and double mismatches, including bulged and tandem mismatches. The absolute error in predicting the position of experimental denaturation profiles was less than 5% formamide for more than 90 percent of probes, enabling a practical level of accuracy in probe design. The potential of the modeling approach for probe design and optimization is demonstrated using a dataset including the 16S rRNA gene of Rhodobacter sphaeroides as an additional target molecule. The LFEM and thermodynamic databases were incorporated into a computational tool (ProbeMelt) that is freely available at http://DECIPHER.cee.wisc.edu.     

Sequencing-independent method to generate oligonucleotide probes targeting a variable region in bacterial 16S rRNA by PCR with detachable primers

Oligonucleotide probes targeting the small-subunit rRNA are commonly used to detect and quantify bacteria in natural environments. We developed a PCR-based approach that allows synthesis of oligonucleotide probes targeting a variable region in the 16S rRNA without prior knowledge of the target sequence. Analysis of all 16S rRNA gene sequences in the Ribosomal Database Project database revealed two universal primer regions bracketing a variable, population-specific region. The probe synthesis is based on a two-step PCR amplification of this variable region in the 16S rRNA gene by using three universal bacterial primers. First, a double-stranded product is generated, which then serves as template in a linear amplification. After each of these steps, products are bound to magnetic beads and the primers are detached through hydrolysis of a ribonucleotide at the 3' end of the primers. This ultimately produces a single-stranded oligonucleotide of about 30 bases corresponding to the target. As probes, the oligonucleotides are highly specific and could discriminate between nucleic acids from closely and distantly related bacterial strains, including different species of VIBRIO: The method will facilitate rapid generation of oligonucleotide probes for large-scale hybridization assays such as screening of clone libraries or strain collections, ribotyping microarrays, and in situ hybridization. An additional advantage of the method is that fluorescently or radioactively labeled nucleotides can be incorporated during the second amplification, yielding intensely labeled probes.     

植物病毒检测芯片的杂交条件优化

利用芯片点样仪将5种侵染马铃薯的病毒/类病毒(苜蓿花叶病毒、黄瓜花叶病毒、黄瓜花叶病毒-卫星病毒、马铃薯病毒Y、马铃薯块茎纺锤状类病毒)的保守区寡核苷酸(Oligonucleotide,oligo)探针和PCR探针点样于玻片,并以植物18S rRNA作为内参照制成基因芯片。研究探针浓度、杂交时间、杂交温度以及点样液对芯片杂交的影响,并验证优化后病毒检测芯片的特异性。结果表明,寡核苷酸探针浓度介于5-20 ?mol/L之间对杂交信号强度影响不大,PCR探针浓度与杂交信号强度间呈线性关系;在45℃杂交4 h时,芯片的杂交信号最强,且该条件下进行杂交对两种探针芯片的影响趋势一致;点样液中以DMSO的杂交效果最好。经过整体条件优化后的两种探针芯片在杂交检测上具有较高的特异性,适于检测植物病毒。     

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 .     

Sequence versus structure for the direct detection of 16S rRNA on planar oligonucleotide microarrays

A two-probe proximal chaperone detection system consisting of a species-specific capture probe for the microarray and a labeled, proximal chaperone probe for detection was recently described for direct detection of intact rRNAs from environmental samples on oligonucleotide arrays. In this study, we investigated the physical spacing and nucleotide mismatch tolerance between capture and proximal chaperone detector probes that are required to achieve species-specific 16S rRNA detection for the dissimilatory metal and sulfate reducer 16S rRNAs. Microarray specificity was deduced by analyzing signal intensities across replicate microarrays with a statistical analysis-of-variance model that accommodates well-to-well and slide-to-slide variations in microarray signal intensity. Chaperone detector probes located in immediate proximity to the capture probe resulted in detectable, nonspecific binding of nontarget rRNA, presumably due to base-stacking effects. Species-specific rRNA detection was achieved by using a 22-nt capture probe and a 15-nt detector probe separated by 10 to 14 nt along the primary sequence. Chaperone detector probes with up to three mismatched nucleotides still resulted in species-specific capture of 16S rRNAs. There was no obvious relationship between position or number of mismatches and within- or between-genus hybridization specificity. From these results, we conclude that relieving secondary structure is of principal concern for the successful capture and detection of 16S rRNAs on planar surfaces but that the sequence of the capture probe is more important than relieving secondary structure for achieving specific hybridization.     

Sequence versus Structure for the Direct Detection of 16S rRNA on Planar Oligonucleotide Microarrays

A two-probe proximal chaperone detection system consisting of a species-specific capture probe for the microarray and a labeled, proximal chaperone probe for detection was recently described for direct detection of intact rRNAs from environmental samples on oligonucleotide arrays. In this study, we investigated the physical spacing and nucleotide mismatch tolerance between capture and proximal chaperone detector probes that are required to achieve species-specific 16S rRNA detection for the dissimilatory metal and sulfate reducer 16S rRNAs. Microarray specificity was deduced by analyzing signal intensities across replicate microarrays with a statistical analysis-of-variance model that accommodates well-to-well and slide-to-slide variations in microarray signal intensity. Chaperone detector probes located in immediate proximity to the capture probe resulted in detectable, nonspecific binding of nontarget rRNA, presumably due to base-stacking effects. Species-specific rRNA detection was achieved by using a 22-nt capture probe and a 15-nt detector probe separated by 10 to 14 nt along the primary sequence. Chaperone detector probes with up to three mismatched nucleotides still resulted in species-specific capture of 16S rRNAs. There was no obvious relationship between position or number of mismatches and within- or between-genus hybridization specificity. From these results, we conclude that relieving secondary structure is of principal concern for the successful capture and detection of 16S rRNAs on planar surfaces but that the sequence of the capture probe is more important than relieving secondary structure for achieving specific hybridization.     

Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes

We describe here a new method for highly efficient detection of microRNAs by northern blot analysis using LNA (locked nucleic acid)-modified oligonucleotides. In order to exploit the improved hybridization properties of LNA with their target RNA molecules, we designed several LNA-modified oligonucleotide probes for detection of different microRNAs in animals and plants. By modifying DNA oligonucleotides with LNAs using a design, in which every third nucleotide position was substituted by LNA, we could use the probes in northern blot analysis employing standard end-labelling techniques and hybridization conditions. The sensitivity in detecting mature microRNAs by northern blots was increased by at least 10-fold compared to DNA probes, while simultaneously being highly specific, as demonstrated by the use of different single and double mismatched LNA probes. Besides being highly efficient as northern probes, the same LNA-modified oligonucleotide probes would also be useful for miRNA in situ hybridization and miRNA expression profiling by LNA oligonucleotide microarrays.     

Empirical Evaluation of Oligonucleotide Probe Selection for DNA Microarrays

DNA-based microarrays are increasingly central to biomedical research. Selecting oligonucleotide sequences that will behave consistently across experiments is essential to the design, production and performance of DNA microarrays. Here our aim was to improve on probe design parameters by empirically and systematically evaluating probe performance in a multivariate context. We used experimental data from 19 array CGH hybridizations to assess the probe performance of 385,474 probes tiled in the Duchenne muscular dystrophy (DMD) region of the X chromosome. Our results demonstrate that probe melting temperature, single nucleotide polymorphisms (SNPs), and homocytosine motifs all have a strong effect on probe behavior. These findings, when incorporated into future microarray probe selection algorithms, may improve microarray performance for a wide variety of applications.     

Computer simulation study of molecular recognition in model DNA microarrays

DNA microarrays have been widely adopted by the scientific community for a variety of applications. To improve the performance of microarrays there is a need for a fundamental understanding of the interplay between the various factors that affect microarray sensitivity and specificity. We use lattice Monte Carlo simulations to study the thermodynamics and kinetics of hybridization of single-stranded target genes in solution with complementary probe DNA molecules immobilized on a microarray surface. The target molecules in our system contain 48 segments and the probes tethered on a hard surface contain 8-24 segments. The segments on the probe and target are distinct and each segment represents a sequence of nucleotides ( approximately 11 nucleotides). Each probe segment interacts exclusively with its unique complementary target segment with a single hybridization energy; all other interactions are zero. We examine how the probe length, temperature, or hybridization energy, and the stretch along the target that the probe segments complement, affect the extent of hybridization. For systems containing single probe and single target molecules, we observe that as the probe length increases, the probability of binding all probe segments to the target decreases, implying that the specificity decreases. We observe that probes 12-16 segments ( approximately 132-176 nucleotides) long gave the highest specificity and sensitivity. This agrees with the experimental results obtained by another research group, who found an optimal probe length of 150 nucleotides. As the hybridization energy increases, the longer probes are able to bind all their segments to the target, thus improving their specificity. The hybridization kinetics reveals that the segments at the ends of the probe are most likely to start the hybridization. The segments toward the center of the probe remain bound to the target for a longer time than the segments at the ends of the probe.     

Discrimination between perfect and mismatched duplexes with oligonucleotide gel microchips: role of thermodynamic and kinetic effects during hybridization

The efficiency of discrimination between perfect and mismatched duplexes during hybridization on microchips depends on the concentrations of target DNA in solution and immobilized probes, buffer composition, and temperature of hybridization and is determined by both thermodynamic relationships and hybridization kinetics. In this work, optimal conditions of discrimination were studied using hybridization of fluorescently labeled target DNA with custom-made gel-based oligonucleotide microchips. The higher the concentration of immobilized probes and the higher the association constant, the higher the concentration of the formed duplexes and the stronger the corresponding fluorescence signal, but, simultaneously, the longer the time needed to reach equilibrium. Since mismatched duplexes hybridize faster than their perfect counterparts, perfect-to-mismatch signal ratio is lower in transient regime, and short hybridization times may hamper the detection of mutations. The saturation time can be shortened by decreasing the probe concentration or augmenting the gel porosity. This improves the detection of mutations in transient regime. It is shown that the decrease in the initial concentration of oligonucleotide probes by an order of magnitude causes only 1.5-2.5-fold decrease of fluorescence signals after hybridization of perfect duplexes for 3-12 h. At the same time, these conditions improve the discrimination between perfect and mismatched duplexes more than two-fold. A similar improvement may be obtained using an optimized dissociation procedure.