Beyond Affymetrix arrays: expanding the set of known hybridization isotherms and observing pre-wash signal intensities

Microarray hybridization studies have attributed the nonlinearity of hybridization isotherms to probe saturation and post-hybridization washing. Both processes are thought to distort ‘true’ target abundance because immobilized probes are saturated with excess target and stringent washing removes loosely bound targets. Yet the paucity of studies aimed at understanding hybridization and dissociation makes it difficult to align physicochemical theory to microarray results. To fill the void, we first examined hybridization isotherms generated on different microarray platforms using a ribosomal RNA target and then investigated hybridization signals at equilibrium and after stringent wash. Hybridization signal at equilibrium was achieved by treating the microarray with isopropanol, which prevents nucleic acids from dissolving into solution. Our results suggest that (i) the shape of hybridization isotherms varied by microarray platform with some being hyperbolic or linear, and others following a power-law; (ii) at equilibrium, fluorescent signal of different probes hybridized to the same target were not similar even with excess of target and (iii) the amount of target removed by stringent washing depended upon the hybridization time, the probe sequence and the presence/absence of nonspecific targets. Possible physicochemical interpretations of the results and future studies are discussed.     

基因芯片技术检测重要人兽共患病病毒方法的建立

为了建立能对25种重要人兽共患病病毒进行筛查及鉴定用的基因芯片技术,本实验首先设计针对每种病毒的寡核苷酸探针并进行探针特异性的生物信息学验证.然后探索病毒核酸随机扩增方法,优化杂交动力学条件,建立本芯片标准的数据处理分析方法.最后用细胞培养的病毒和模拟临床标本验证芯片的敏感性与特异性．结果表明,锚定随机PCR扩增法适合于本芯片病毒核酸的扩增;芯片杂交前用0.25% NaBH4进行封闭,最优杂交条件为51 ℃,2 h及50%甲酰胺浓度;芯片具有较好的敏感性及检测特异性．初步结果表明,本实验所建立的基因芯片技术可应用于对25种重要人兽共患病病毒进行筛查及鉴定．     

Properties of hybridization isotherms upon binding of ligands on microchips

A simple thermodynamic model describing microarray oligo-target hybridization has been constructed. The relationship between the hybridization signal intensity and Gibbs free energy for oligo-target duplex formation has been considered. The behavior of this function, which we called energy hybridization isotherm, in response to target concentration change was modeled at different ratios of oligo/target concentrations. The results of modeling were compared with the relevant and currently available data from microarray adsorption experiments.     

cDNA芯片表面核酸固定化的优化

cDNA芯片技术表面核酸固定化影响因素众多,其中涉及选择载体、固定于玻片的DNA片段浓度、玻片DNA片段的固定方法、玻片预处理方法、DNA片段的变性、溶解DNA片段的点样液等等.针对这些因素进行了优化筛选实验,以便于提高cDNA芯片技术检测基因表达的效率.     

A dimensionless number analysis of the hybridization process in diffusion- and convection-driven DNA microarray systems

The present theoretical analysis aims at providing a general understanding of the combined effect the many different process variables have on the hybridization rate in diffusion- and convection-driven DNA microarray systems. It is shown that all process variables can be grouped into only four different dimensionless numbers (the Damkohler number Da, the dimensionless association constant kappa(A), the dimensionless initial concentration C'(0) and a geometrical ratio alpha). These four numbers have a straightforward physical meaning and only contain easily measurable parameters. Reducing the solution space from 7D to 4D, the dimensionless number representation greatly facilitates the insight in the conditions leading to the occurrence of diffusion-limited hybridization rates in both diffusion- and convection-driven DNA microarray systems. This in turn simplifies their design and the interpretation of the experimental results that are obtained with these systems.     

Conditions to ensure competitive hybridization in two-color microarray: a theoretical and experimental analysis

We derived a theoretical model that explains certain biases observed in the two-color microarray hybridization experiments reported in the literature. We show that true competition is achieved only when the hybridization kinetics of the two differentially labeled probes are the same. If the hybridization kinetics of the two differentially labeled probes is different, which can occur when the labeling and hybridization conditions for the two probes are dissimilar, then differential expression observed becomes a function of the amount of the target (i.e., DNA spotted on the slide). We use this model to validate the microarray methodology by determining the differential expression of four select Arabidopsis genes and two human genes (beta-actin and GAPDH) as a function of the amount of target arrayed. We show through both modeling and experiments that the rate constants for Cy5- and Cy3-labeled probes are the same under our exrimental conditions. Therefore, the target concentrations need not greatly exceed the probe concentration. It is obvious from the data presented that a simple treatment of an individual hybridization rate calculation does notfully describe what is occuring in today's complex, multispecies experiments. The method of validation is easily implemented to ensure data reliability by two-color microarray.     

Development of a high-throughput DNA microarray for drug-resistant gene detection and its preliminary application

Most bacteria are resistant to a wide variety of antibiotics and other drugs, which decrease the effectiveness of clinical drug therapies. The present study developed a high-throughput DNA microarray for drug-resistant gene detection. A total of 115 specific oligonuclieotide probes with lengths of 42 nt to 45 nt and comparable Tm values were selected from 17 categories of drug-resistant genes in the National Center for Biotechnology Information database and were chemically synthesized. The entire bacterial DNA was extracted, randomly amplified, and labeled using Cy3-dCTP. The hybridization conditions of the microarray test were optimized to improve sensitivity and specificity. The drug-resistant genes were detected and genotyped using microarray analysis after hydration at 42°C for 4h with 2× hybridization solution. The microarray test sensitivity was 20ng/μL DNA. The performance of the microarray was validated using reference strains and clinical isolates. The results were consistent with direct DNA sequence analysis and drug susceptibility tests. The developed DNA microarray could be used to detect and screen drug-resistant bacteria rapidly and simultaneously. Thus, the present study could be helpful in effectively using antibiotics and controlling infectious diseases.     

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。     

重要肠道病原菌多重PCR-基因芯片检测方法研究

目的:建立并初步评价一种针对重要肠道病原菌的多重PCR 基因芯片检测方法。方法：对筛选出的特异引物进行多重PCR优化,将引物分别按种属内混合和种属间混合的方案排查引物间的竞争性抑制现象,再将不同菌属的模板混合,用相对应的混合引物扩增,探寻高效特异的引物组合。分别掺入和不掺入荧光素,验证其对混合PCR反应的影响,并与芯片杂交,探寻多重PCR扩增效率对芯片杂交的影响。分析不同数量引物组合产生的杂交结果,筛选出无交叉反应的最优引物组合。结果:种属内引物混合均得到特异性扩增结果。种属间混合霍乱弧菌和空肠弯曲菌得到部分预期条带,随着混合引物数量的增加,交叉抑制现象也增多。杂交信号强度随多重PCR扩增效率的增加而增强。反应中掺入荧光素的扩增条带产量低于无荧光素的产物。可将35对混合引物拆成3个体系分别标记样品,以避免假阴性结果。结论:PCR反应中掺入荧光素降低扩增效率和杂交效率,但并不影响对杂交结果的判读和数据分析。基因芯片杂交信号强度取决于多重PCR的扩增效率。肠道病原菌多重PCR 基因芯片检测方法具有较高的特异性,混合PCR可以分别按照种属内和种属间的引物组合方案用于多病原的筛检。该基因芯片检测可以采用3个引物体系完成样品标记。     

Microarray results improve significantly as hybridization approaches equilibrium

Dual-channel long oligonucleotide microarrays are in widespread use. Although much attention has been given to proper experimental design and analysis regarding long oligonucleotide microarrays, relatively little information is available concerning the optimization of protocols. We carried out a series of microarray experiments designed to investigate the effects of different levels of target concentration and hybridization times using a long oligonucleotide library. Based on principles developed from nucleic acid renaturation kinetics studies, we show that increasing the time of hybridization from 18 h to 42 h and 66 h, especially when lower than optimal concentrations of target were used, significantly improved the quality of the microarray results. Longer hybridization times significantly increased the number of spots detected, signal-to-noise ratios, and the number of differentially expressed genes and correlations among replicate arrays. We conclude that at 18 h of incubation, target-to-probe hybridization has not reached equilibrium and that a relatively high proportion of nonspecific hybridization occurs. This result is striking, given that most, if not all, published microarray protocols stipulate 8-24 h for hybridization. Using shorter than optimal hybridization times (i.e., not allowing hybridization to reach equilibrium) has the consequence of underestimating the fold change of differentially expressed genes and of missing less represented sequences.     

Development and validation of a prototype 16S rRNA-based taxonomic microarray for Alphaproteobacteria

The microarray approach has been proposed for high throughput analysis of the microbial community by providing snapshots of the microbial diversity under different environmental conditions. For this purpose, a prototype of a 16S rRNA-based taxonomic microarray was developed and evaluated for assessing bacterial community diversity. The prototype microarray is composed of 122 probes that target bacteria at various taxonomic levels from phyla to species (mostly Alphaproteobacteria). The prototype microarray was first validated using bacteria in pure culture. Differences in the sequences of probes and potential target DNAs were quantified as weighted mismatches (WMM) in order to evaluate hybridization reliability. As a general feature, probes having a WMM > 2 with target DNA displayed only 2.8% false positives. The prototype microarray was subsequently tested with an environmental sample, which consisted of an Agrobacterium-related polymerase chain reaction amplicon from a maize rhizosphere bacterial community. Microarray results were compared to results obtained by cloning-sequencing with the same DNA. Microarray analysis enabled the detection of all 16S rRNA gene sequences found by cloning-sequencing. Sequences representing only 1.7% of the clone library were detected. In conclusion, this prototype 16S rRNA-based taxonomic microarray appears to be a promising tool for the analysis of Alphaproteobacteria in complex ecosystems.     

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.     

Optimization strategies for DNA microarray-based detection of bacteria with 16S rRNA-targeting oligonucleotide probes

The usability of the DNA microarray format for the specific detection of bacteria based on their 16S rRNA genes was systematically evaluated with a model system composed of six environmental strains and 20 oligonucleotide probes. Parameters such as secondary structures of the target molecules and steric hindrance were investigated to better understand the mechanisms underlying a microarray hybridization reaction, with focus on their influence on the specificity of hybridization. With adequate hybridization conditions, false-positive signals could be almost completely prevented, resulting in clear data interpretation. Among 199 potential nonspecific hybridization events, only 1 false-positive signal was observed, whereas false-negative results were more common (17 of 41). Subsequent parameter analysis revealed that this was mainly an effect of reduced accessibility of probe binding sites caused by the secondary structures of the target molecules. False-negative results could be prevented and the overall signal intensities could be adjusted by introducing a new optimization strategy called directed application of capture oligonucleotides. The small number of false-positive signals in our data set is discussed, and a general optimization approach is suggested. Our results show that, compared to standard hybridization formats such as fluorescence in situ hybridization, a large number of oligonucleotide probes with different characteristics can be applied in parallel in a highly specific way without extensive experimental effort.     

Optimization Strategies for DNA Microarray-Based Detection of Bacteria with 16S rRNA-Targeting Oligonucleotide Probes

The usability of the DNA microarray format for the specific detection of bacteria based on their 16S rRNA genes was systematically evaluated with a model system composed of six environmental strains and 20 oligonucleotide probes. Parameters such as secondary structures of the target molecules and steric hindrance were investigated to better understand the mechanisms underlying a microarray hybridization reaction, with focus on their influence on the specificity of hybridization. With adequate hybridization conditions, false-positive signals could be almost completely prevented, resulting in clear data interpretation. Among 199 potential nonspecific hybridization events, only 1 false-positive signal was observed, whereas false-negative results were more common (17 of 41). Subsequent parameter analysis revealed that this was mainly an effect of reduced accessibility of probe binding sites caused by the secondary structures of the target molecules. False-negative results could be prevented and the overall signal intensities could be adjusted by introducing a new optimization strategy called directed application of capture oligonucleotides. The small number of false-positive signals in our data set is discussed, and a general optimization approach is suggested. Our results show that, compared to standard hybridization formats such as fluorescence in situ hybridization, a large number of oligonucleotide probes with different characteristics can be applied in parallel in a highly specific way without extensive experimental effort.     

Optimizing stringency for expression microarrays

While several studies have reported methods to optimize expression microarray protocols, none have dealt directly with hybridization wash stringency. We designed a series of experiments to determine the optimal stringency conditions for microarray experiments, using reproducibility and magnitudes of log2 (test/reference) ratio values as measures of quality. Low-stringency wash conditions of cell line hybridizations led to nonspecific binding, resulting in increased intensities, decreased magnitude of ratios, and poor reproducibility. Relatively high-stringency wash conditions were found to give the best reproducibility and large magnitude ratio changes, although increasing the stringency beyond this point led to lower magnitude ratios and poorer reproducibility. The expression levels of the ERBB2 oncogene in the BT474 versus MCF7 cell lines showed that high-stringency wash conditions gave the best agreement with real-time quantitative PCR, although the magnitude of the changes by microarray was smaller than for real-time quantitative PCR. Analysis of a series of cell lines washed at the optimized stringency indicated that the rank order of relative expression levels for ERBB2 microarray clones agreed well with the rank order of ERBB2 levels, as measured by quantitative PCR. These results indicate that the optimization of stringency conditions will improve microarray reproducibility and give more representative expression values.     

Identification of HCV-1b by Low-Density cDNA Microarray-Based Assay

A rapid and sensitive microarray assay for the detection of HCV-1b was developed in our laboratory and a cDNA fragment library for HCV-1b cDNA microarray probes was constructed. The full-length cDNAs of HCV-1b were digested with restriction endonuclease Sau3A I and the fragments were cloned with the pMD18-T vectors. Positive clones were isolated and identified by sequencing. The cDNA microarray was prepared by spotting the gene fragment on the surface of an amido-modified glass slide using the robotics system and samples were fluorescent labeled by the restriction display PCR (RD-PCR) technique, In the present study, modified protocols were used for probe selection and hybridization temperature. The detection of a microarray was validated by the hybridization and the sequence analysis. A total of 22 different specific gene fragments of HCV-1b ranging from 250 to 750 bp were isolated and sequenced, and these fragments were further used as probes in the microarray preparation. The diagnostic validity of the microarray method was evaluated after the washing and scanning process. The results of hybridization and sequence data analysis showed a significant specificity and sensitivity in the detection of HCV-1b RNA. The method of preparing microarray probes by construction of cDNA fragments library was effective, rapid, and simple; the optimized microarray was sensitive in the clinical detection of HCV-1b. The RD-PCR technique for the sample labeling was useful for significantly increasing the sensitivity of the assay. The cDNA microarray assay can be widely used in the clinical diagnosis of HCV-1b.     

Direct detection of 16S rRNA in soil extracts by using oligonucleotide microarrays

We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 microg of total RNA, representing approximately 7.5 x 10(6) Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR.