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.     

Parallel gene analysis with allele-specific padlock probes and tag microarrays

Parallel, highly specific analysis methods are required to take advantage of the extensive information about DNA sequence variation and of expressed sequences. We present a scalable laboratory technique suitable to analyze numerous target sequences in multiplexed assays. Sets of padlock probes were applied to analyze single nucleotide variation directly in total genomic DNA or cDNA for parallel genotyping or gene expression analysis. All reacted probes were then co-amplified and identified by hybridization to a standard tag oligonucleotide array. The technique was illustrated by analyzing normal and pathogenic variation within the Wilson disease-related ATP7B gene, both at the level of DNA and RNA, using allele-specific padlock probes.     

Single molecule linear analysis of DNA in nano-channel labeled with sequence specific fluorescent probes

An array of nano-channels was fabricated from silicon based semiconductor materials to stretch long, native dsDNA. Here we present a labeling scheme in which it is possible to identify the location of specific sequences along the stretched DNA molecules. The scheme proceeds by first using the strand displacement activity of the Vent (exo-) polymerase to generate single strand flaps on nicked dsDNA. These single strand flaps are hybridized with sequence specific fluorophore-labeled probes. Subsequent imaging of the DNA molecules inside a nano-channel array device allows for quantitative identification of the location of probes. The highly efficient DNA hybridization on the ss-DNA flaps is an excellent method to identify the sequence motifs of dsDNA as it gives us unique ability to control the length of the probe sequence and thus the frequency of hybridization sites on the DNA. We have also shown that this technique can be extended to a multi color labeling scheme by using different dye labeled probes or by combining with a DNA- polymerase-mediated incorporation of fluorophore-labeled nucleotides on nicking sites. Thus this labeling chemistry in conjunction with the nano-channel platform can be a powerful tool to solve complex structural variations in DNA which is of importance for both research and clinical diagnostics of genetic diseases.     

电场驱动微悬臂生物传感器及对DNA 的快速、高灵敏度检测

微悬臂列阵传感器在生物检测方面具有快速、痕量和非标记的特性. 我们以镀金并在其上固定了 DNA 探针的微悬臂为正极,在靶杂交液槽内引入另一电极作为负极,构成电场驱动微悬臂 DNA 生物传感器. 对该传感器系统施加静电场,驱动 DNA 分子朝正极迁移,使溶液中的 DNA 分子富集在微悬臂上,促进 DNA 分子的杂交. 结果表明： a. DNA 在微悬臂上的杂交时间仅需 3 min,加快了微悬臂生物传感器对 DNA 分子的检测速度; b. 提高了微悬臂生物传感器的灵敏度,可以检测到皮克级的 DNA 分子.     

A novel method for determining linkage between DNA sequences: hybridization to paired probe arrays

Cooperative hybridization has been used to establish physical linkage between two loci on a DNA strand. Linkage was detected by hybridization to a new type of high-density oligonucleotide array. Each synthesis location on the array contains a mixture of two different probe sequences. Each of the two probes can hybridize independently to a different target sequence, but if the two target sequences are physically linked there is a cooperative increase in hybridization yield. The ability to create and control non-linear effects raises a host of possibilities for applications of oligonucleotide array hybridization. The method has been used to assign linkage in 50:50 mixtures of DNA containing single nucleotide polymorphisms (SNPs) separated by 17, 693, 1350 and 2038 bp and to reconstruct haplotypes. Other potential uses include increasing the specificity of hybridization in mutation detection and gene expression monitoring applications, determining SNP haplotypes, characterizing repetitive sequences, such as short tandem repeats, and aiding contig assembly in sequen-cing by hybridization.     

Genome-directed primers for selective labeling of bacterial transcripts for DNA microarray analysis

DNA microarrays have the ability to analyze the expression of thousands of the same set of genes under at least two different experimental conditions. However, DNA microarrays require substantial amounts of RNA to generate the probes, especially when bacterial RNA is used for hybridization (50 microg of bacterial total RNA contains approximately 2 microg of mRNA). We have developed a computer-based algorithm for prediction of the minimal number of primers to specifically anneal to all genes in a given genome. The algorithm predicts, for example, that 37 oligonucleotides should prime all genes in the Mycobacterium tuberculosis genome. We tested the usefulness of the genome-directed primers (GDPs) in comparison to random primers for gene expression profiling using DNA microarrays. Both types of primers were used to generate fluorescent-labeled probes and to hybridize to an array of 960 mycobacterial genes. Compared to random-primer probes, the GDP probes were more sensitive and more specific, especially when mammalian RNA samples were spiked with mycobacterial RNA. The GDPs were used for gene expression profiling of mycobacterial cultures grown to early log or stationary growth phases. This approach could be useful for accurate genome-wide expression analysis, especially for in vivo gene expression profiling, as well as directed amplification of sequenced genomes.     

Characterization and application of soybean YACs to molecular cytogenetics

Yeast artificial chromosomes (YACs) are widely used in the physical analysis of complex genomes. In addition to their value in chromosome walking for map-based cloning, YACs represent excellent probes for chromosome mapping using fluorescence in situ hybridization (FISH). We have screened such a library for low-copy-number clones by hybridization to total genomic DNA. Four clones were chosen for chromosome tagging based upon their low or moderate signal. By using degenerate oligonucleotide-primed PCR (DOP-PCR), we were able to use relatively small amounts of soybean YAC DNA, isolated directly by preparative pulsed-field gel electrophoresis, as FISH probes for both metaphase chromosome spreads and interphase nuclei. FISH chromosomal analysis using the three of the clones as probes resulted in relatively simple hybridization patterns consistent with a single homologous locus or two homoeologous loci. The fourth YAC probe resulted in a diffuse hybridization pattern with signal on all metaphase chromosomes. We conclude that YACs represent a valuable source of probes for chromosomal analysis in soybean.     

七株昆虫核型多角体病毒基因组同源性的测定

应用限制性内切酶图谱分析法,结合Southern印迹法和核酸杂交技术,对茶毛虫、棉蛉虫,油桐尺蠖、斜纹夜蛾以及蓖麻蚕等5种昆虫的7株核型多角体病毒DNA,进行了基因组同源性测定。结果表明,不同种昆虫多角体病毒DNA的酶切图谱不相同,DNA片段与不同源的DNA标记探针之间无杂交带出现。而同种昆虫病毒的不同分离株间,除少数DNA片段的电泳迁移率稍有不同,以及出现一些互不相同的亚克分子带之外,它们的DNA酶切图谱基本一致,並且几乎所有片段都可与同种的标记探钟杂交。对一些DNA片段迁移率的改变及亚克分子带出现的原因进行了讨论。     

Oligonucleotide microarrays with stem-loop probes: enhancing the hybridization of nucleic acids for sensitive analysis

We have demonstrated that the dynamics of nucleic acid hybridization in microarrays depend on the physical structure of immobilized probes. We have immobilized oligonucleotide-3'-phosphates with and without stem-loop structure on epoxylated glass surface, followed by hybridization under different conditions, viz., hybridization buffer, pH condition, temperature and ionic strength. In a comparative study, we have established that array constructed using probes with stem-loop structure displayed approximately 2.2 times higher hybridization signals than the probes without it. The stem-loop DNA array format is simple and flexible in design and thus potentially useful in various DNA diagnostic tests.     

Molecular-beacon-based array for sensitive DNA analysis

Molecular beacon (MB) DNA probes provide a new way for sensitive label-free DNA/protein detection in homogeneous solution and biosensor development. However, a relatively low fluorescence enhancement after the hybridization of the surface-immobilized MB hinders its effective biotechnological applications. We have designed new molecular beacon probes to enable a larger separation between the surface and the surface-bound MBs. Using these MB probes, we have developed a DNA array on avidin-coated cover slips and have improved analytical sensitivity. A home-built wide-field optical setup was used for imaging the array. Our results show that linker length, pH, and ionic strength have obvious effects on the performance of the surface-bound MBs. The fluorescence enhancement of the new MBs after hybridization has been increased from 2 to 5.5. The MB-based DNA array could be used for DNA detection with high sensitivity, enabling simultaneous multiple-target bioanalysis in a variety of biotechnological applications.     

Sub-array normalization subject to differentiation

From microarray measurement, we seek differentiation of mRNA expressions among different biological samples. However, each array has a ‘block effect’ due to uncontrolled variation. The statistical treatment of reducing the block effect is usually referred to as normalization. Our perspective is to find a transformation that matches the distributions of hybridization levels of those probes corresponding to undifferentiated genes between arrays. We address two important issues. First, array-specific spatial patterns exist due to uneven hybridization and measurement process. Second, in some cases a substantially large portion of genes are differentially expressed between a target and a reference array. For the purpose of normalization we need to identify a subset that exclude those probes corresponding to differentially expressed genes and abnormal probes due to experimental variation. Least trimmed squares (LTS) is a natural choice to achieve this goal. Substantial differentiation is protected in LTS by setting an appropriate trimming fraction. To take into account any spatial pattern of hybridization, we divide each array into sub-arrays and normalize probe intensities within each sub-array. We illustrate the problem and solution through an Affymetrix spike-in dataset with defined perturbation and a dataset of primate brain expression.     

'FloraArray' for screening of specific DNA probes representing the characteristics of a certain microbial community

To investigate uncharacterized microbial communities, a custom DNA microarray named 'FloraArray' was developed for screening specific probes that would represent the characteristics of a microbial community. The array was prepared by spotting 2000 plasmid DNAs from a genomic shotgun library of a sludge sample on a DNA microarray. By comparative hybridization of the array with two different samples of genomic DNA, one from the activated sludge and the other from a nonactivated sludge sample of an anaerobic ammonium oxidation (anammox) bacterial community, specific spots were visualized as a definite fluctuating profile in an MA (differential intensity ratio vs. spot intensity) plot. About 300 spots of the array accounted for the candidate probes to represent anammox reaction of the activated sludge. After sequence analysis of the probes and examination of the results of blastn searches against the reported anammox reference sequence, complete matches were found for 161 probes (58.3%) and >90% matches were found for 242 probes (87.1%). These results demonstrate that 'FloraArray' could be a useful tool for screening specific DNA molecules of unknown microbial communities.     

Optimal sequencing by hybridization in rounds.

Sequencing by hybridization (SBH) is a method for sequencing DNA. The Watson-Crick complementarity of DNA can be used to determine whether the DNA contains an oligonucleotide substring. A large number of oligonucleotides can be arranged on an array (SBH chip). A combinatorial method is used to construct the sequence from the collection of probes that occur in it. We develop an idea of Margaritis and Skiena and propose an algorithm that uses a series of small SBH chips to sequence long strings. The total number of probes used by our method matches the information theoretical lower bound up to a constant factor.     

DNA polymorphism in the living fossil Ginkgo biloba from the eastern United States.

Random amplified polymorphic DNA (RAPD) analysis is a valuable tool in studying inter- and intra-specific genetic variations, patterns of gene expression, and for the identification of specific genes using nearly isogenic variants. Here we used RAPD analysis to study the genetic variation in Ginkgo biloba grown in the eastern United States. Our results support the evidence that Southern blot hybridization of RAPD using probes made from cloned DNA fragments allows a more accurate analysis of the RAPD pattern than dye-stained gels or Southern blot hybridization of RAPD blots using probes made from purified PCR products. Using these techniques, we observed a high degree of relatedness among plants grown in certain localities although significant genetic variation may exist in the species, and could be a possible explanation for the observed variations in the efficacy of medications derived from G. biloba extract.     

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.     

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.