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1.
Pelizzari CA Khodarev NN Gupta N Calvin DP Weichselbaum RR 《Nucleic acids research》2000,28(22):4577-4581
DNA arrays and chips are powerful new tools for gene expression profiling. Current arrays contain hundreds or thousands of probes and large scale sequencing and screening projects will likely lead to the creation of global genomic arrays. DNA arrays and chips will be key in understanding how genes respond to specific changes of environment and will also greatly assist in drug discovery and molecular diagnostics. To facilitate widespread realization of the quantitative potential of this approach, we have designed procedures and software which facilitate analysis of autoradiography films with accuracy comparable to phosphorimaging devices. Algorithms designed for analysis of DNA array autoradiographs incorporate 3-D peak fitting of features on films and estimation of local backgrounds. This software has a flexible grid geometry and can be applied to different types of DNA arrays, including custom arrays. 相似文献
2.
It is a common practice to use resampling methods such as the bootstrap for calculating the p-value for each test when performing large scale multiple testing. The precision of the bootstrap p-values and that of the false discovery rate (FDR) relies on the number of bootstraps used for testing each hypothesis. Clearly, the larger the number of bootstraps the better the precision. However, the required number of bootstraps can be computationally burdensome, and it multiplies the number of tests to be performed. Further adding to the computational challenge is that in some applications the calculation of the test statistic itself may require considerable computation time. As technology improves one can expect the dimension of the problem to increase as well. For instance, during the early days of microarray technology, the number of probes on a cDNA chip was less than 10,000. Now the Affymetrix chips come with over 50,000 probes per chip. Motivated by this important need, we developed a simple adaptive bootstrap methodology for large scale multiple testing, which reduces the total number of bootstrap calculations while ensuring the control of the FDR. The proposed algorithm results in a substantial reduction in the number of bootstrap samples. Based on a simulation study we found that, relative to the number of bootstraps required for the Benjamini-Hochberg (BH) procedure, the standard FDR methodology which was the proposed methodology achieved a very substantial reduction in the number of bootstraps. In some cases the new algorithm required as little as 1/6th the number of bootstraps as the conventional BH procedure. Thus, if the conventional BH procedure used 1,000 bootstraps, then the proposed method required only 160 bootstraps. This methodology has been implemented for time-course/dose-response data in our software, ORIOGEN, which is available from the authors upon request. 相似文献
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4.
PCR amplification and simultaneous digoxigenin incorporation of long DNA probes for fluorescence in situ hybridization. 总被引:4,自引:0,他引:4
Nonradioactive in situ hybridization has found widespread applications in cytogenetics. Basic requirements are DNA probes in sufficient amounts and of high specificity as well as a labeling protocol of good reproducibility. The PCR has been of fundamental importance for the amplification of DNA sequences and thus for the production of DNA probes. Meanwhile, PCR protocols for amplification of DNA have reached a high degree of automation. So far, incorporation of labeled nucleotides into these DNA probes has normally been done by nick translation. Here we show that in using the PCR, amplification of a DNA probe larger than one kilobase accompanied by simultaneous incorporation of digoxigenin-11-dUTP can be performed for in situ hybridization experiments. As an example, the DNA probe pUC 1.77 specific for the subcentromeric region q12 of chromosome number 1 was used and hybridized against metaphase chromosomes from human lymphocytes. The labeled chromosome region was detected by anti-digoxigenin-fluorescein, Fab fragments. The experiments were evaluated by digital image analysis of microphotographs. 相似文献
5.
H Yu J Chao D Patek R Mujumdar S Mujumdar A S Waggoner 《Nucleic acids research》1994,22(15):3226-3232
Fluorescence in situ hybridization (FISH) has become and indispensable tool in a variety of areas of research and clinical diagnostics. Many applications demand an approach for simultaneous detection of multiple target sequences that is rapid and simple, yet sensitive. In this work, we describe the synthesis of two new cyanine dye-labeled dUTP analogs, Cy3-dUTP and Cy5-dUTP. They are efficient substrates for DNA polymerases and can be incorporated into DNA probes by standard nick translation, random priming and polymerase chain reactions. Optimal labeling conditions have been identified which yield probes with 20-40 dyes per kilobase. The directly labeled DNA probes obtained with these analogs offer a simple approach for multicolor multisequence analysis that requires no secondary detection reagents and steps. 相似文献
6.
Improved In Situ Hybridization Efficiency with Locked-Nucleic-Acid-Incorporated DNA Probes 总被引:2,自引:0,他引:2
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Kengo Kubota Akiyoshi Ohashi Hiroyuki Imachi Hideki Harada 《Applied microbiology》2006,72(8):5311-5317
Low signal intensity due to poor probe hybridization efficiency is one of the major drawbacks of rRNA-targeted in situ hybridization. There are two major factors affecting the hybridization efficiency: probe accessibility and affinity to the targeted rRNA molecules. In this study, we demonstrate remarkable improvement in in situ hybridization efficiency by applying locked-nucleic-acid (LNA)-incorporated oligodeoxynucleotide probes (LNA/DNA probes) without compromising specificity. Fluorescently labeled LNA/DNA probes with two to four LNA substitutions exhibited strong fluorescence intensities equal to or greater than that of probe Eub338, although these probes did not show bright signals when they were synthesized as DNA probes; for example, the fluorescence intensity of probe Eco468 increased by 22-fold after three LNA bases were substituted for DNA bases. Dissociation profiles of the probes revealed that the dissociation temperature was directly related to the number of LNA substitutions and the fluorescence intensity. These results suggest that the introduction of LNA residues in DNA probes will be a useful approach for effectively enhancing probe hybridization efficiency. 相似文献
7.
Low signal intensity due to poor probe hybridization efficiency is one of the major drawbacks of rRNA-targeted in situ hybridization. There are two major factors affecting the hybridization efficiency: probe accessibility and affinity to the targeted rRNA molecules. In this study, we demonstrate remarkable improvement in in situ hybridization efficiency by applying locked-nucleic-acid (LNA)-incorporated oligodeoxynucleotide probes (LNA/DNA probes) without compromising specificity. Fluorescently labeled LNA/DNA probes with two to four LNA substitutions exhibited strong fluorescence intensities equal to or greater than that of probe Eub338, although these probes did not show bright signals when they were synthesized as DNA probes; for example, the fluorescence intensity of probe Eco468 increased by 22-fold after three LNA bases were substituted for DNA bases. Dissociation profiles of the probes revealed that the dissociation temperature was directly related to the number of LNA substitutions and the fluorescence intensity. These results suggest that the introduction of LNA residues in DNA probes will be a useful approach for effectively enhancing probe hybridization efficiency. 相似文献
8.
Rapid and accurate identification of species is required for the biological control of pest Noctuoidea moths. DNA barcodes and thin‐film biosensor chips are two molecular approaches that have gained wide attention. Here, we compare these two methods for the identification of a limited number of Noctuoidea moth species. Based on the commonly used mitochondrial gene cytochrome c oxidase I (the standard DNA barcode for animal species), 14 probes were designed and synthesized for 14 species shared by two national nature reserves in Beijing and Hebei, China. Probes ranged in length from 18 to 27 bp and were designed as mismatch probes to guarantee that there were at least three base differences between the probe and nontarget sequences. The results on the chip could be detected by the naked eye without needing special equipment. No cross‐hybridizations were detected although we tested all probes on the 14 target and 24 nontarget Noctuoidea species. The neighbour‐joining tree of the 38 species based on COI sequences gave 38 highly supported independent groups. Both DNA barcoding and thin‐film biosensor chips, based on the COI gene, are able to accurately identify and discriminate the 14 targeted moth species in this study. Because of its speed, high accuracy and low cost, the thin‐film biosensor chip is a very practical means of species identification. Now, a more comprehensive chip will be developed for the identification of additional Noctuoidea moths for pest control and ecological protection. 相似文献
9.
DNA chips have proven to be effective tools in detecting gene expression levels. Compared with DNA chips using complementary DNA as probes, oligonucleotide microarrays using oligonucleotides as probes have attracted great attention because of their well known advantages. The design of gene-specific probes for each target is essential to the development of oligonucleotide microarrays. We have previously reported the development of a probe design software termed Mprobe 1.0. Here, we present a new version of this software, termed Mprobe 2.0. Several new features are included in Mprobe 2.0. Firstly, a paradox-based sequence database management system has been developed and integrated into the software, which consequently allows interoperability with sequences in GenBank, EMBL, and FASTA formats. Secondly, in contrast to setting a fixed threshold for the secondary structure of probes in Mprobe 1.0 and other related software, Mprobe 2.0 employs a different method. After parameters such as GC type, probe melting temperature and GC contents have been evaluated, candidate probes are sorted by the free energy from high to low value, followed by specificity analysis. Thirdly, Mprobe 2.0 provides users with substantial parameter options in the visual mode. Mprobe 2.0 possesses an easier interface for users to manage sequences annotated in different formats and design the optimal probes for oligonucleotide microarrays and other applications. AVAILABILITY: The program is free for non-commercial users and can be downloaded from the web page http://www.biosun.org.cn/mprobe/ CONTACT: Wuju Li (wujuli@yahoo.com or liwj@nic.bmi.ac.cn). 相似文献
10.
文章讨论了DNA芯片的制作原理和杂交信号的检测方法。依其结构,DNA芯片可分为两种形式,DNA阵列和寡核苷酸微芯片。DNA芯片的制作方法主要有光导原位合成法和自动化点样法。DNA芯片与标记的探针或DNA样品杂交,并通过探测杂交信号谱型来实现DNA序列或基因表达的分析。适应于DNA芯片的发展,同时出现了许多新型的杂交信号检测方法。主要有激光荧光扫描显微镜、激光扫描共焦显微镜、结合使用CCD相机的荧光显微镜、光纤生物传感器、化学发生法、光激发磷光物质存储屏法、光散射法等。 相似文献
11.
Despite the fact that many genomes have been decoded, proteome chips comprising individually purified proteins have been reported only for budding yeast, mainly because of the complexity and difficulty of high-throughput protein purification. To facilitate proteomics studies in prokaryotes, we have developed a high-throughput protein purification protocol that allowed us to purify 4,256 proteins encoded by the Escherichia coli K12 strain within 10 h. The purified proteins were then spotted onto glass slides to create E. coli proteome chips. We used these chips to develop assays for identifying proteins involved in the recognition of potential base damage in DNA. By using a group of DNA probes, each containing a mismatched base pair or an abasic site, we found a small number of proteins that could recognize each type of probe with high affinity and specificity. We further evaluated two of these proteins, YbaZ and YbcN, by biochemical analyses. The assembly of libraries containing DNA probes with specific modifications and the availability of E. coli proteome chips have the potential to reveal important interactions between proteins and nucleic acids that are time-consuming and difficult to detect using other techniques. 相似文献
12.
S Singh-Gasson R D Green Y Yue C Nelson F Blattner M R Sussman F Cerrina 《Nature biotechnology》1999,17(10):974-978
Oligonucleotide microarrays, also called "DNA chips," are currently made by a light-directed chemistry that requires a large number of photolithographic masks for each chip. Here we describe a maskless array synthesizer (MAS) that replaces the chrome masks with virtual masks generated on a computer, which are relayed to a digital micromirror array. A 1:1 reflective imaging system forms an ultraviolet image of the virtual mask on the active surface of the glass substrate, which is mounted in a flow cell reaction chamber connected to a DNA synthesizer. Programmed chemical coupling cycles follow light exposure, and these steps are repeated with different virtual masks to grow desired oligonucleotides in a selected pattern. This instrument has been used to synthesize oligonucleotide microarrays containing more than 76,000 features measuring 16 microm 2. The oligonucleotides were synthesized at high repetitive yield and, after hybridization, could readily discriminate single-base pair mismatches. The MAS is adaptable to the fabrication of DNA chips containing probes for thousands of genes, as well as any other solid-phase combinatorial chemistry to be performed in high-density microarrays. 相似文献
13.
We report a rapid method for the direct multicolor imaging of multiple subnuclear genetic sequences using novel quantum dot-based fluorescence in situ hybridization (FISH) probes (QD-FISH). Short DNA oligonucleotides were attached on QDs and used in a single hybridization/detection step of target sites in situ. QD-FISH probes penetrate both intact interphase nuclei and metaphase chromosomes and showed good targeting of dense chromatin domains with minimal steric hindrances. We further demonstrated that QD's broad absorption spectra allowed different colored probes specific for distinct subnuclear genetic sequences to be simultaneously excited with a single excitation wavelength and imaged free of chromatic aberrations in a single exposure. Thus, these results demonstrate that QD-FISH probes are very effective in multicolor FISH applications. This work also documents new possibilities of using QD-FISH probes detection down to the single molecule level. 相似文献
14.
Nonradioactive in situ hybridization techniques are becoming increasingly important tools for rapid analysis of the topological organization of DNA and RNA sequences within cells. Prerequisite for further advances with these techniques are multiple labeling and detection systems for different probes. Here we summarize our results with a recently developed labeling and detection system. The DNA probe for in situ hybridization is modified with digoxigenin-labeled deoxyuridine-triphosphate. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Labeled DNA probes were hybridized in situ to chromosome preparations. The hybridization signal was detected using digoxigenin-specific antibodies covalently coupled to enzyme markers (alkaline phosphatase or peroxidase) or to fluorescent dyes. Color reactions catalyzed by the enzymes resulted in precipitates located on the chromosomes at the site of probe hybridization. This was verified by hybridizing DNA probes of known chromosomal origin. The signals were analyzed by bright field, reflection contrast and fluorescence microscopy. The results indicate that the new technique gives strong signals and can also be used in combination with other systems (e.g., biotin) to detect differently labeled DNA probes on the same metaphase plate. 相似文献
15.
B R Jordan 《BioEssays : news and reviews in molecular, cellular and developmental biology》1991,13(5):243-251
Fragile X-linked mental retardation is an enigmatic inheritable syndrome in which severe mental retardation, a cytogenetically detectable fragile site at Xq27.3 (FraX) and a number of dysmorphic features are associated. Genetic analysis shows that the mode of inheritance is more complex than a straightforward X-linked recessive trait and probably involves a two-step process for which several models have been proposed. Early attempts at 'cloning the fragile site' provided several DNA segments lying in its general vicinity, and large scale DNA mapping methods were extensively applied in an effort to generate maps including this region. These efforts were complemented by more focussed methods such as microdissection; together these approaches have now provided a number of DNA segments within a 5 cM interval around FraX, and with the help of these new probes the site is indeed being cloned. Unravelling the nature of the sequence(s) responsible for the mental retardation syndrome will probably take some time, however. 相似文献
16.
基因芯片技术和蛋白质组技术在神经科学中的应用及其研究进展 总被引:2,自引:0,他引:2
基因芯片技术和蛋白质组技术是最近发展起来的高通量技术,二者的出现使同时分析神经系统的大量基因的表达和基因产物蛋白质及其相互作用网络成为可能。它们在神经科学中的应用为了解脑功能提供了前所未有的机会。一个典型的基因芯片实验包括:芯片的准备或购买、靶DNA和探针的准备或标记、标记探针与靶DNA的杂交、芯片扫描和影象信息的数据分析。蛋白质组技术较为复杂,包括蛋白质分离、鉴定和信息分析三方面的内容。其中,分离技术多种多样。若分离技术以二维电泳为基础,则该实验通常由以下步骤组成:蛋白质样品的准备、电泳分离、染胶、分离蛋白点的切除、蛋白质的酶解(常用胰蛋白酶)、质谱分析(鉴定)和数据的信息处理。本文综述这两项技术的内容和实验步骤,然后着重叙述它们在神经科学中的应用,讨论其优缺点和面临的挑战,展望其发展前景。 相似文献
17.
Molecular beacons (MBs) are hairpin-like fluorescent DNA probes that have single-mismatch detection capability. Although they are extremely useful for many solution-based nucleic acid detections, MBs are expensive probes for applications that require the use of a large number of different DNA probes due to the high cost and tedious procedures associated with probe synthesis and purification. In addition, since both ends of MB probes are covalently modified with chromophores, they do not offer the flexibility for fluorophore change and the capability for surface immobilization through free DNA ends. In this report, we describe an alternative form of MB, denoted tripartite molecular beacon (TMB), that may help overcome these problems. A TMB uses an unmodified oligodeoxyribonucleotide that forms a MB-like structure with two universal single-stranded arms to bring on a universal pair of oligodeoxyribonucleotides modified separately with a fluorophore and a quencher. We found that TMBs are as effective as standard MBs in signaling the presence of matching nucleic acid targets and in precisely discriminating targets that differ by a single nucleotide. TMBs have the necessary flexibility that may make MBs more affordable for various nucleic acid detection applications. 相似文献
18.
Xenopus 5S RNA genes exhibit a pattern of differential expression during development in which some members (oocyte-type) are transcribed only in oocytes, while others (somatic-type) are expressed in both oocytes and somatic cells. Using cloned DNA probes specific for each gene type, we determined the positions of these genes on Xenopus metaphase chromosomes by in situ hybridization. Somatic-type 5S genes in both X. laevis and X. borealis are located at the distal end of the long arm of only one chromosome (number 9). The oocyte-type 5S RNA genes are found at the distal ends of the long arms of most Xenopus chromosomes, including chromosome 9. Thus, large scale differences in chromosomal location cannot explain the selective expression of these genes, as suggested previously. 相似文献
19.
《Journal of biochemical and biophysical methods》1995,30(2-3):91-102
The growth of analytical methods for the detection of nucleic acid from various biological samples reflects recent advances in biotechnology development especially in the areas of genetic, infections and cancer diagnosis. The target DNA is detected by hybridization techniques derived from Southern's blotting. However such assays, based on the use of 32P labelled DNA probes, bring with them the associated problems of handling radioactive materials. In order to overcome these difficulties, a number of chemiluminescent detection methods have recently been developed.These new, alternative probe labelling procedures and chemiluminescent detection methods are easy to use in routine assays performed in research laboratories as well as for medical applications, and can reach the level of sensitivity found in classical radiolabelling techniques.The techniques investigated include peroxydase, biotin 16-dUTP or digoxigenin 11-dUTP probe labelling. The target DNAs are transferred onto nitrocellulose or nylon membranes and further fixed by heat or UV crosslinking. Specific hybridization on the target DNA is finally revealed by the use of chemiluminescent substrates. For all these techniques the detection limit is 10 aM (attomol) of a 561 bp target DNA. However for the probes labelled with peroxydase and with digoxigenin the detection limit drops to 1.0 aM of the target DNA. In the present paper we shall compare several of these DNA labelling and detection procedures and show that the detection threshold can vary by as much as a factor of 20 from method to method. This is the first time that various chemiluminescent methods for label and detection of DNA are compared and evaluated in order to determine the best protocol. 相似文献
20.
Zhuo ZHOU Zhi-xun DOU Chen ZHANG Hou-qing YU Yi-jie LIU Cui-zhu ZHANG You-jia CAO 《Virologica Sinica》2007,22(4):326-335
DNA microarrays have been acknowledged to represent a promising approach for the detection of viral pathogens. However, the probes designed for current arrays could cover only part of the given viral variants, that could result in false-negative or ambiguous data. If all the variants are to be covered, the requirement for more probes would render much higher spot density and thus higher cost of the arrays. Here we have developed a new strategy for oligonucleotide probe design. Using type I human immunodeficiency virus (HIV-1) tat gene as an example, we designed the array probes and validated the optimized parameters in silico. Results show that the oligo number is significantly reduced comparing with the existing methods, while specificity and hybridization efficiency remain intact. The adoption of this method in reducing the oligo numbers could increase the detection capacity for DNA microarrays, and would significantly lower the manufacturing cost for making array chips. 相似文献