Evaluation of trichloroethylene degradation by E. coli transformed with dimethyl sulfide monooxygenase genes and/or cumene dioxygenase genes

Pseudomonas fluorescens IP01 grown on isopropylbenzene (cumene) and Acinetobacter sp. 20B grown on dimethyl sulfide (DMS) degraded up to 90% and 25% of 1.5 mg trichloroethylene (TCE)/l, respectively. Escherichia coli harboring the DMS monooxygenase genes from strain 20B, the cumene dioxygenase genes from strain IP01 and both oxygenase genes, degraded up to 50%, 75% and 88% of 75 mg TCE/l, respectively. The growth rates of the E. coli recombinants remained nearly unaffected by TCE at 15 150 mg/l. Thus, the E. coli recombinants were indicated to degrade high concentrations of TCE efficiently at least up to 150 mg l–1.     

等Tm检测探针寡核苷酸芯片在p53突变热点检测中的应用

用等长探针检测基因的点突变,不同GC含量探针的碱基错分辨率很难均一。尝试利用探针近似等Tm的原则设计、制备了检测抑癌基因p53外显子7中密码子245、248、249单碱基突变及缺失的寡核苷酸芯片。实验得到较好的碱基错配分辨率,检测不同位点的碱基错配分辨率较为一致,芯片检测结果与测序结果一致。实验结果为制备检测p53常见热点突变的寡核酸芯片奠定了基础。     

Development and optimization of an 18S rRNA‐based oligonucleotide microarray for the fungal order Eurotiales

Aims: For identification of members of the fungal order Eurotiales, an 18S rRNA gene‐based oligonucleotide microarray was developed and optimized. Methods and Results: Eurotiales‐specific probes covering most members of the Eurotiales as well as species‐specific probes were designed and evaluated with three pure cultures (two fungi from the Eurotiales and one fungus from the Hypocreales). Nearly complete 18S rRNA genes of each reference culture were amplified and fluorescently labelled by random priming. Conclusions: Positive and negative hybridization results confirmed that the Eurotiales probes tested in this study could correctly identify members of the Eurotiales. The species‐specific probes were also capable of species‐level detection. Significance and Impact of the Study: These findings demonstrate the potential applications of a phylogenetic oligonucleotide microarray approach to characterizing fungal species and populations in environmental and other samples.     

Characterization of monooxygenase gene diversity in benzene‐amended soils

Aim: To understand soil benzene monooxygenase gene diversity by clone library construction and microarray profiling. Methods and Results: A primer set was designed, and benzene monooxygenase gene diversity was characterized in two benzene‐amended soils. The dominant sequence types in the clone libraries were distinct between the two soils, and both sequences were assigned to novel clusters. Monooxygenase gene richness and diversity increased after benzene degradation. Oligonucleotide probes for microarray analysis were designed to detect a number of sequenced clones and reported monooxygenase genes. The microarray detected several genes that were not detected in the clone libraries of the same samples. Six probes were detected in more than one soil. Conclusions: The primer set designed in this study successfully detected diverse benzene monooxygenase genes. The level of diversity may have increased because the degradation of benzene differed from soil to soil. Microarrays have great potential in the comprehensive detection of gene richness as well as the elucidation of key genes for degradation. Significance and Impact of the Study: This study introduces a new primer set that may be used to identify diverse benzene monooxygenase genes in the environment; moreover, it demonstrates the potential of microarray technology in the profiling of environmental samples.     

细小病毒B19 Oligo探针设计

利用BLAST软件对细小病毒B19的序列进行序列比对,获得特异序列;利用生物学软件Oligo6.40设计特异性高、Tm值接近、长度均一的Oligo探针。结果获得了13条70bp的Oligo探针,用于芯片打印及细小病毒B19的检测。表明利用BLAST系统和生物学软件Oligo6.40设计细小病毒B19诊断芯片的探针是一种简便而有效的方法。     

Development of a 60-mer oligonucleotide microarray on the basis of benzene monooxygenase gene diversity

We constructed a 60-mer oligonucleotide microarray on the basis of benzene monooxygenase gene diversity to develop a new technology for simultaneous detection of the functional gene diversity in environmental samples. The diversity of the monooxygenase genes associated with benzene degradation was characterized. A new polymerase chain reaction (PCR) primer set was designed using conserved regions of benzene monooxygenase gene (BO12 primer) and used for PCR-clone library analysis along with a previously designed RDEG primer which targeted the different types of benzene monooxygenase gene. We obtained 20 types of amino acid sequences with the BO12 primer and 40 with the RDEG primer. Phylogenetic analysis of the sequences obtained suggested the large diversity of the benzene monooxygenase genes. A total of 87 60-mer probes specific for each operational taxonomical unit were designed and spotted on a microarray. When genomic DNAs of single strains were used in microarray hybridization assays, corresponding sequences were successfully detected by the microarray without any false-negative signals. Hybridization with soil DNA samples showed that the microarray was able to detect sequences that were not detected in clone libraries. Constructed microarray can be a useful tool for characterizing monooxygenase gene diversity in benzene degradation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Quantitative oligonucleotide microarray data analysis with an artificial standard probe strategy

Quantitative data analysis is an important element in several applications of DNA microarray, including mRNA expression profiling and estimation of infectious doses for pathogens. Here, we introduce an artificial standard probe strategy for quantitative pathogen detection using an oligonucleotide chip as a model system. The standard capture probe sequence was artificially designed to prevent non-specific hybridization with bacterial targets. Based on the fluorescence intensities of artificial standard spots, the raw fluorescence intensity data for specific spots could be corrected to generate linear correlations with target concentrations. Therefore, our novel artificial standard probe may be effectively applied for the correction of chip-to-chip variations and quantitative data analysis of a one-color labeled DNA microarray system.     

Development and application of an oligonucleotide microarray for the detection of food-borne bacterial pathogens

The rapid and accurate detection and identification of food-borne pathogenic bacteria is critical for food safety. In this paper, we describe a rapid (<4 h) high-throughput detection and identification system that uses universal polymerase chain reaction (PCR) primers to amplify a variable region of bacterial the 16S rRNA gene, followed by reverse hybridization of the products to species-specific oligonucleotide probes on a chip. This procedure was successful in discriminating 204 strains of bacteria from pure culture belonging to 13 genera of bacteria. When this method was applied directly to 115 strains of bacteria isolated from foods, 112/115 (97.4%) were correctly identified; two strains were indistinguishable due to weak signal, while one failed to produce a PCR product. The array was used to detect and successfully identify two strains of bacteria from food poisoning outbreak samples, giving results through hybridization that were identical to those obtained by traditional methods. The sensitivity of the microarray assay was 10² CFU of bacteria. Thus, the oligonucleotide microarray is a powerful tool for the detection and identification of pathogens from foods. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

烟草细胞色素P450的基因组学分析

细胞色素P450是一类含血红素的单加氧酶超基因家族, 在植物多种代谢途径中起着重要作用。为了解烟草中的P450的种类和数量, 文章将植物代表性P450蛋白质序列与烟草基因组序列比对, 在烟草基因组中鉴定了44个P450家族共263个成员。将这些烟草P450基因与烟草表达序列标签(EST)比对, 发现173个成员有EST证据。通过与拟南芥中已知的P450蛋白序列比较, 分析了部分烟草P450蛋白序列的特征和二级结构。根据烟草基因芯片数据和部分基因的RT-PCR结果, 发现73个烟草P450基因能够在不同的生长发育时期表达, 其中部分基因具有组织特异性。这些研究结果为烟草P450基因功能的深入分析奠定了基础。     

Adaptive thresholds to detect differentially expressed genes in microarray data

To detect changes in gene expression data from microarrays, a fixed threshold for fold difference is used widely. However, it is not always guaranteed that a threshold value which is appropriate for highly expressed genes is suitable for lowly expressed genes. In this study, aiming at detecting truly differentially expressed genes from a wide expression range, we proposed an adaptive threshold method (AT). The adaptive thresholds, which have different values for different expression levels, are calculated based on two measurements under the same condition. The sensitivity, specificity and false discovery rate (FDR) of AT were investigated by simulations. The sensitivity and specificity under various noise conditions were greater than 89.7% and 99.32%, respectively. The FDR was smaller than 0.27. These results demonstrated the reliability of the method.     

Discovery of cross-reactive probes and polymorphic CpGs in the Illumina Infinium HumanMethylation450 microarray

DNA methylation, an important type of epigenetic modification in humans, participates in crucial cellular processes, such as embryonic development, X-inactivation, genomic imprinting and chromosome stability. Several platforms have been developed to study genome-wide DNA methylation. Many investigators in the field have chosen the Illumina Infinium HumanMethylation microarray for its ability to reliably assess DNA methylation following sodium bisulfite conversion. Here, we analyzed methylation profiles of 489 adult males and 357 adult females generated by the Infinium HumanMethylation450 microarray. Among the autosomal CpG sites that displayed significant methylation differences between the two sexes, we observed a significant enrichment of cross-reactive probes co-hybridizing to the sex chromosomes with more than 94% sequence identity. This could lead investigators to mistakenly infer the existence of significant autosomal sex-associated methylation. Using sequence identity cutoffs derived from the sex methylation analysis, we concluded that 6% of the array probes can potentially generate spurious signals because of co-hybridization to alternate genomic sequences highly homologous to the intended targets. Additionally, we discovered probes targeting polymorphic CpGs that overlapped SNPs. The methylation levels detected by these probes are simply the reflection of underlying genetic polymorphisms but could be misinterpreted as true signals. The existence of probes that are cross-reactive or of target polymorphic CpGs in the Illumina HumanMethylation microarrays can confound data obtained from such microarrays. Therefore, investigators should exercise caution when significant biological associations are found using these array platforms. A list of all cross-reactive probes and polymorphic CpGs identified by us are annotated in this paper.     

An oligonucleotide array to detect genetically modified events in potato

For rapid and simultaneous detection of transgenic elements in genetically modified (GM) food crops, we explored DNA array technology. Forty-four oligonucleotide 23-to 31-mers were selected to use in an array on the basis of melting temperature and sequence specificity. Selected oligonucleotides consisted of DNA fragments corresponding to structural and regulatory elements and selectable markers used in developing transgenic crops, such as potato. Other oligonucleotides represented endogenous genes from potato to serve as positive controls and from heterologous crops, such as soybean and canola, to serve as negative controls. Amino-terminated oligonucleotides were hand-spotted on activated nylon membrane with a commercial spotting device. Target DNA was isolated from foliage of transgenic and nontransgenic crops, including potato, and labeled with digoxigenin-dUTP by random priming following restriction digestion to reduce DNA fragment size. Hybridization signals were visualized by an alkaline phosphatase anti-DIG-Fab conjugate and the chemiluminescent substrate, CDP-star. We detected the presence or absence of transgenic elements in transgenic and nontransgenic potato samples. Preliminary studies demonstrated that more specific and sensitive hybridization signals were generated from an oligonucleotide probe array than from a PCR product array. We anticipate that oligonucleotide probe arrays will be useful for regulatory monitoring of transgenic events.     

Development of an enzyme-labeled oligonucleotide probe for detecting the Escherichia coli attaching and effacing A gene.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) can produce attaching and effacing (AE) lesions on intestinal epithelium in vitro and in vivo. A gene necessary to cause the AE lesion has been identified and designated Escherichia coli attaching and effacing A (eaeA) gene. In this study, an alkaline phosphatase (ALP)-conjugated oligonucleotide probe for the eaeA gene was developed and used to detect the eaeA gene among 163 strains of classical EPEC and 25 strains of EHEC O157. The prevalence rates of eaeA gene in the strains of classical EPEC and EHEC O157 were 51.5 and 100%, respectively. The eaeA-positive rate (60.0%) in strains of class I EPEC serogroups (O26, O55, O86, O111, O119, O125, O126, O127, O128ab, and O142) was significantly higher than that (22.9%) in strains of the class II EPEC serogroups (O18, O44, O114) (P<0.01). A total of 109 eaeA-positive classical EPEC and EHEC O157 were positive for fluorescent actin staining (FAS) assay, whereas 79 eaeA-negative classical EPEC were negative. Both the sensitivity and specificity of the eaeA probe versus the FAS assay positivity were 100%. Thus, use of the ALP-conjugated oligonucleotide probe for the eaeA gene would be specific and reliable in identifying the adherence capability of EPEC and EHEC.     

Screening of obligate methanotrophs for soluble methane monooxygenase genes

A 5.8 kb fragment of chromosomal DNA from Methylococcus capsulatus (Bath) containing genes encoding the soluble methane monooxygenase enzyme complex was used as a probe for the detection of soluble monooxygenase genes in a number of representative strains of obligate methanotrophs. Only type II methanotrophs of the genus Methylosinus were found to contain homologues to the Methylococcus gene probe. This probe was also used successfully to detect soluble methane monooxygenase genes in a variety of methanotrophs by colony hybridizations.     

Development of an oligonucleotide microarray method for Salmonella serotyping

Adequate identification of Salmonella enterica serovars is a prerequisite for any epidemiological investigation. This is traditionally obtained via a combination of biochemical and serological typing. However, primary strain isolation and traditional serotyping is time‐consuming and faster methods would be desirable. A microarray, based on two housekeeping and two virulence marker genes (atpD, gyrB, fliC and fljB), has been developed for the detection and identification of the two species of Salmonella (S. enterica and S. bongori), the five subspecies of S. enterica (II, IIIa, IIIb, IV, VI) and 43 S. enterica ssp. enterica serovars (covering the most prevalent ones in Austria and the UK). A comprehensive set of probes (n = 240), forming 119 probe units, was developed based on the corresponding sequences of 148 Salmonella strains, successfully validated with 57 Salmonella strains and subsequently evaluated with 35 blind samples including isolated serotypes and mixtures of different serotypes. Results demonstrated a strong discriminatory ability of the microarray among Salmonella serovars. Threshold for detection was 1 colony forming unit per 25 g of food sample following overnight (14 h) enrichment.     

Oligonucleotide-based microarray for DNA methylation analysis: principles and applications

Gene silencing via promoter CpG island hypermethylation offers tumor cells growth advantages. This epigenetic event is pharmacologically reversible, and uncovering a unique set of methylation-silenced genes in tumor cells can bring a new avenue to cancer treatment. However, high-throughput tools capable of surveying the methylation status of multiple gene promoters are needed for this discovery process. Herein we describe an oligonucleotide-based microarray technique that is both versatile and sensitive in revealing hypermethylation in defined regions of the genome. DNA samples are bisulfite-treated and PCR-amplified to distinguish CpG dinucleotides that are methylated from those that are not. Fluorescently labeled PCR products are hybridized to arrayed oligonucleotides that can discriminate between methylated and unmethylated alleles in regions of interest. Using this technique, two clinical subtypes of non-Hodgkin's lymphomas, mantle cell lymphoma, and grades I/II follicular lymphoma, were further separated based on the differential methylation profiles of several gene promoters. Work is underway in our laboratory to extend the interrogation power of this microarray system in multiple candidate genes. This novel tool, therefore, holds promise to monitor the outcome of various epigenetic therapies on cancer patients.     

利用寡核苷酸微阵列技术对HBV、HCV、HIV、HAV、GBV-C/HGV和B19进行同时监测的初步研究

探讨研制能同时检测HBV、HCV、HIV、HAV、GBV-C/HGV和B19的微阵列监控芯片。根据病毒公开发表序列,序列比对,得出保守区域,设计病毒的特异性检测探针,同时设置阴性、阳性参照探针,制备监控微阵列。利用随机引物PCR方法标记样品中的病毒靶序列,标记产物与微阵列上的探针杂交,清洗、扫描后进行结果分析。通过对质粒或模式分子的检测以及经HBV、HCV、HIV临床标本的验证,发现该微阵列监控芯片具有良好的特异性。其对质粒的检测灵敏度可达102病毒拷贝数,对临床标本的检测灵敏度可达103病毒拷贝数。此外,该微阵列监控芯片可检测出病毒混合感染血清。为微阵列监控芯片应用于此六种血液病毒的检测打下一定的基础。