Critical factors for the performance of chip array-based electrical detection of DNA for analysis of pathogenic bacteria

Different factors influencing chip array-based electrical detection of DNA for analysis of pathogenic bacteria were examined. Both rehydration of capture probe layer of functionalized chip arrays and efficient hybridization of targets irrespective of their length resulted in signal enhancement when high-ionic phosphate-buffered saline (i.e., 600 mM sodium chloride and 40 mM disodium hydrogen phosphate) was used. Similarly, placement of two adjacent capture and detection probe-binding sites at a terminal part of the target strand resulted in significant signal increase. Moreover, 10-min ultrasonic fragmentation of targets amplified the signals up to twofold for longer DNA strands (i.e., >300 bp). No obvious effects on signals were visible for shorter than 400-bp PCR amplicons subjected to ultrasonication. For DNA strands of all sizes, more than 10 min ultrasonication diminished the specific electrical responses. Our results also demonstrate that target analytes are detected with discrimination against mismatches even for single nucleotide sequence alteration. The mismatch detection appeared in order of ease of recognition as follows: triple random > quintuple middle > triple middle > single middle mismatch. Among the three variants of one-base mismatches, a sequence variation was most remarkable for adenine. On the other hand, no benefits in assay sensitivity were recognized by the use of longer capture probe linkers as the 6-C linker.     

Transformation of maize embryogenic calli mediated by ultrasonication and regeneration of fertile transgenic plants

An insecticidal protein gene isolated fromBacillus thuringiensis was transferred into maize by using ultrasonication. The fertile transgenic plants and their progeny were obtained. The Southern hybridization results indicated that the foreign gene had integrated into the maize genome. It has been found that the acoustic intensity and the duration of treatment are the important parameters influencing transformation efficiency by ultrasonication. The maximum relative transformation frequency of 34.1 % was achieved after 30 min of sonication at 0. 5 W/cm² acoustic intensity. With appropriate parameters the ultrasonication can make a number of micropores formed on the cell surface and minimize the treatment damage to the foreign DNA molecules, thus facilitating the DNA molecules to enter the cells. Project supported by “863” State High Technology Development Program.     

Ultrasonication as a rapid and high yield DNA extraction method for bacterial gene quantification by NanoGene assay

We demonstrated ultrasonication as a rapid and high yield DNA extraction method suitable for bacterial gene quantification by NanoGene assay. The NanoGene assay utilizes DNA hybridization in solution and a combination of magnetic beads and quantum dot nanoparticles. Unlike the existing gene quantification assays, the NanoGene method is capable of quantifying genes in the presence of environmental inhibitors and cell materials. The performance of the ultrasonication was compared with heating and freeze-thaw. They first were evaluated for their cell lysis capability in humic acids laden sand samples, via EtBr assay. Using autoclaved samples as a bench mark, their cell lysis capability were 106 ± 3, 68 ± 5, and 48 ± 15%, respectively. Morphological changes of cells for each method were also observed by FE-SEM. More importantly, ultrasonication performed significantly better (more than 3× fluorescence signal) than commercial DNA extraction methods during bacterial gene quantification in humic acids laden sand samples.     

Reduction of autofluorescence on DNA microarrays and slide surfaces by treatment with sodium borohydride

Microarray technology is currently being used extensively in functional genomics research and modern drug discovery and development. Henceforward, tremendous application potential for this technology exists in the fields of clinical diagnostics and prognostics, pathology, and toxicology for high-throughput analysis of "disease" gene expression. However, the major hurdle now in this technology is not the performance of the arrays but rather the efficient reproducibility of the hybridization signal intensity in a fluorescence-based analysis. The sensitivity of fluorescence detection on an array is to a large extent limited by the amount of background signal arising due to nonspecifically bound probes and fluorescence that is intrinsically associated with the chip substrate and/or the attached target DNA, the so-called autofluorescence. Here, we describe a simple and efficient method to reduce autofluorescence from undetermined sources on coated glass slides with and without DNA arrays. This sodium borohydride-mediated reduction process resulted in significantly lower and more even background fluorescence. This in turn extended the dynamic range of detection and reduced the average coefficient of variation of fluorescent signal ratios on DNA microarrays in addition to improving the detection of genes that are expressed at a low level.     

Application of sonication to release DNA from Bacillus cereus for quantitative detection by real-time PCR

A rapid sonication method for lysis of Gram-positive bacteria was evaluated for use in combination with quantitative real-time polymerase chain reaction (PCR) analyses for detection. Other criteria used for evaluation of lysis were microscopic cell count, colony forming units (cfu), optical density at 600 nm and total yield of DNA measured by PicoGreen fluorescence. The aim of this study was complete disruption of cellular structures and release of DNA without the need for lysing reagents and time-consuming sample preparation. The Gram-positive bacterium Bacillus cereus was used as a model organism for Gram-positive bacteria. It was demonstrated by real-time PCR that maximum yield of DNA was obtained after 3 to 5 min of sonication. The yield of DNA was affected by culture age and the cells from a 4-h-old culture in the exponential phase of growth gave a higher yield of DNA after 5 min of sonication than a 24-h-old culture in the stationary phase of growth. The 4-h-old culture was also more sensitive for lysis caused by heating. The maximum yield of DNA, evaluated by real-time PCR, from a culture of the Gram-negative bacterium Escherichia coli, was obtained after 20 s of sonication. However, the yield of target DNA from E. coli rapidly decreased after 50 s of sonication due to degradation of DNA. Plate counting (cfu), microscopic counting and absorbance at 600 nm showed that the number of viable and structurally intact B. cereus cells decreased rapidly with sonication time, whereas the yield of DNA increased as shown by PicoGreen fluorescence and real-time PCR. The present results indicate that 3-5 min of sonication is sufficient for lysis and release of DNA from samples of Gram-positive bacteria.     

基因芯片在衣原体研究中的应用

基因芯片又称为DNA微阵列,是指将大量核酸片段以预先设计的方式固定在载体上组成密集分子阵列,与荧光素或其他方式标记的样品进行杂交,通过检测杂交信号的强弱来判断样品中有无靶分子以及对靶分子进行定量,是一种研究生物大分子功能的新技术。在衣原体研究方面,基因芯片主要应用于衣原体的检测与分型、感染机制的研究、特定基因作用分析、毒力及耐药基因的筛选等。     

Isolation of pathogen-induced Chinese cabbage genes by subtractive hybridization employing selective adaptor ligation

We have developed a subtractive cloning method in which target sequences are effectively enriched by selective adaptor ligation and PCR after hybridization. In this method both tester and driver DNAs are digested with RsaI, ligated with the linker DNA containing a KpnI recognition site, and amplified by PCR. The tester DNA samples are divided into two aliquots, each digested with either RsaI or KpnI. The two DNA samples are then combined and hybridized with an excess of the driver DNA retaining the linker. After hybridization, the DNA mixture is ligated to a new adaptor compatible only with double-stranded tester/tester DNAs. Therefore, only the tester/tester is selectively amplified in subsequent PCR. This also leads to complete elimination of the tester DNA hybridized with driver DNA from the tester DNA population. Although our protocol employs enzymatic treatments, the efficiency of the enzymatic treatments does not affect the subtraction efficiency. This new subtractive enrichment method was applied to isolate Chinese cabbage defense-related genes induced by Pseudomonas syringae pv. tomato (Pst), which elicits a hypersensitive response in Chinese cabbage. After two or three rounds of subtractive hybridization, the sequences of enriched DNAs were determined and examined by BLAST analysis. Northern blot hybridization showed that 12 of the 19 genes analyzed were strongly induced by Pst treatment. Among the 12 Pst-induced genes five represent pathogenesis-related genes encoding PR1a, two chitinases, a thaumatin-like protein, and a PR4 protein. Other Pst-induced genes include two cytochrome P450 genes responsible for glucosinolate biosynthesis, a disease resistance gene homolog, and several genes encoding proteins with unknown functions.     

结合SSH和cDNA芯片技术在植物研究中的应用

抑制性差减杂交(Suppression Subtractive Hybridization,SSH)技术是分离差异表达基因的一种新方法。cDNA芯片也是近年来发展起来的一种新技术,它是指将大量的特定的寡核苷酸片段或基因片段作为探针,有规律地排列固定于硅片、玻片、塑料片等固相支持物上制成的芯片。本文主要介绍抑制差减杂交和cDNA芯片技术原理及其在植物研究中的应用。     

基于纳米金复合探针的基因芯片膜转印检测法

建立了一种基于纳米金复合探针的基因芯片膜转印核酸检测新方法。首先,用纳米金颗粒同时标记检测探针P2和两种长短不同且生物素化的信号探针 (T10,T40),其中检测探针与靶DNA 5￠端互补,两种信号探针起信号放大作用。当靶DNA分子存在时,芯片表面捕捉探针P1 (与靶DNA分子3￠端互补) 通过碱基互补配对原则结合靶DNA分子,将其固定于芯片上,同时检测探针通过与靶DNA 5￠端互补配对将纳米金复合探针结合于芯片表面,结果在芯片表面形成“三明治”结构,后通过链霉亲和素-生物素反应,使芯片表面对应有靶DNA分子的部位结合上碱性磷酸酶,最后利用BCIP/NBT显色系统使芯片表面信号结果镜面转印至尼龙膜表面。当检测探针和信号探针摩尔比为1∶10,T10和T40摩尔比为9:1时可以检测1 pmol/L合成靶DNA分子或0.23 pmol/L结核分枝杆菌16S rDNA PCR扩增产物,检测结果通过普通的光学扫描仪读取或肉眼直接判读信号有无。本芯片检测系统灵敏度高,操作方法简单、快速,不需要特殊仪器设备,在生物分子的检测方面具有较高的应用价值。     

Quantitative detection of microbial genes by using DNA microarrays

To quantify target genes in biological samples using DNA microarrays, we employed reference DNA to normalize variations in spot size and hybridization. This method was tested using nitrate reductase (nirS), naphthalene dioxygenase (nahA), and Escherichia coli O157 O-antigen biosynthesis genes as model genes and lambda DNA as the reference DNA. We observed a good linearity between the log signal ratio and log DNA concentration ratio at DNA concentrations above the method's detection limit, which was approximately 10 pg. This approach for designing quantitative microarrays and the inferred equation from this study provide a simple and convenient way to estimate the target gene concentration from the hybridization signal ratio.     

DNA Fragmentation Induced by Cytotoxic T Lymphocytes Can Result in Target Cell Death

Cytotoxic T lymphocyte (CTL)-mediated lysis is accompanied by fragmentation of target cell DNA into an oligonucleosome ladder, a hallmark of apoptosis. Is this a fortuitous coincidence, or could CTL be inducing lysis by activation of the suicide signal? In this report we demonstrate that CTL-mediated target cell death can be blocked with the drug aurintricarboxylic acid (ATA). The abrogation of death correlates with the inhibition of DNA fragmentation. While ATA prevented DNA fragmentation, it failed to significantly alter protein, RNA, or DNA synthesis in the cell lines over the dose range used. In addition, there was no inhibition of cell-cell interaction or granule exocytosis during CTL-mediated killing. ATA also significantly inhibited the cytolysis and DNA fragmentation mediated by isolated cytolytic granules, as well as the granular protein fragmentin. We developed an assay in which target cells could be separated from CTL after binding and programming for lysis. Once they had received the "kiss of death," target cells could be rescued from lysis (as indicated by inhibition of DNA fragmentation and increased target cell viability) by treatment with ATA. These results suggest that ATA blocks target cell death by inhibition of DNA fragmentation, and further, that chromatin degradation is a cause rather than a result of cell death in CTL-mediated lysis.     

Apoptosis: Programmed cell death in health and disease

Apoptosis is a normal physiological cell death process of eliminating unwanted cells from living organisms during embryonic and adult development. Apoptotic cells are characterised by fragmentation of nuclear DNA and formation of apoptotic bodies. Genetic analysis revealed the involvement of many death and survival genes in apoptosis which are regulated by extracellular factors. There are multiple inducers and inhibitors of apoptosis which interact with target cell specific surface receptors and transduce the signal by second messengers to programme cell death. The regulation of apoptosis is elusive, but defective regulation leads to aetiology of various ailments. Understanding the molecular mechanism of apoptosis including death genes, death signals, surface receptors and signal pathways will provide new insights in developing strategies to regulate the cell survival/death. The current knowledge on the molecular events of apoptotic cell death and their significance in health and disease is reviewed.     

DNA fiber-FISH staining mechanism.

Fluorescence in situ hybridization to DNA fibers (Fiber-FISH) is a high-resolution, wide-ranging physical DNA mapping method that finds increasing application in the study of pathological gene rearrangements. Here we present experiments designed to understand the nature of the discontinuous FISH signal patterns seen after Fiber-FISH. Use of a novel cisplatin-based chemical labeling method enabled us to produce intact biotin-labeled cosmid target DNA molecules. We monitored by immunofluorescence the fate of such cosmid targets during denaturation and hybridization. The same cosmid DNA labeled with digoxigenin by nick-translation was used to analyze the FISH probe signal distribution in a different color. The probe signals proved to be a subset of the target signals remaining after denaturation and hybridization. We argue that the discontinuity of probe signals in Fiber-FISH is mainly caused by loss of target DNA and limited accessibility due to in situ renaturation and attachment. Furthermore, we conclude that FISH sensitivity is determined by hybridization efficiency and not the ability to generate sufficient signal from small probes. (J Histochem Cytochem 48:743-745, 2000)     

Quantitative Detection of Microbial Genes by Using DNA Microarrays

To quantify target genes in biological samples using DNA microarrays, we employed reference DNA to normalize variations in spot size and hybridization. This method was tested using nitrate reductase (nirS), naphthalene dioxygenase (nahA), and Escherichia coli O157 O-antigen biosynthesis genes as model genes and lambda DNA as the reference DNA. We observed a good linearity between the log signal ratio and log DNA concentration ratio at DNA concentrations above the method's detection limit, which was approximately 10 pg. This approach for designing quantitative microarrays and the inferred equation from this study provide a simple and convenient way to estimate the target gene concentration from the hybridization signal ratio.     

Identification and characterization of genes responsive to apoptosis: application of DNA chip technology and mRNA differential display

Apoptosis (programmed cell death) is a genetically programmed active cell death process for maintaining homeostasis under physiological conditions and for responding to various stimuli. Many human diseases have been associated with either increased apoptosis (such as AIDS and neurodegenerative disorders) or decreased apoptosis (such as cancer and autoimmune disorders). In an attempt to understand apoptosis signaling pathway and genes associated with apoptosis, we established two cell model systems on which apoptosis is induced either by DNA damaging agent, etoposide or by redox agent, 1,10-phenanthroline (OP). DNA chip profiling or mRNA differential display (DD) was utilized to identify genes responsive to apoptosis induced by these two agents. In etoposide model with chip hybridization, we defined signaling pathways that mediate apoptosis in p53 dependent manner (through activation of p53 target genes such as Waf-1/p21, PCNA, GPX, S100A2 and PTGF-beta) as well as in p53-independent manner (through activation of ODC and TGF-beta receptor, among others). In OP model with DD screening, we cloned and characterized two genes: glutathione synthetase, encoding an enzyme involved in glutathione synthesis and Sensitive to Apoptosis Gene (SAG), a novel evolutionarily conserved gene encoding a zinc RING finger protein. Both genes appear to protect cells from apoptosis induced by redox agents. Further characterization of SAG revealed that it is a growth essential gene in yeast and belongs to a newly identified gene family that promotes protein ubiquitination and degradation. Through this activity, SAG regulates cell cycle progression and many other key biological processes. Thus, SAG could be a valid drug target for anti-cancer and anti-inflammation therapies.     

The influence of CpG (5′-d(CpG)-3′ dinucleotides) methylation on ultrasonic DNA fragmentation

Abstract

DNA methylation is an important way of gene regulation. The variety of methods for DNA methylation analysis based on chemical modification or enzyme digestion has been proposed. However, DNA is able to undergo transformations under physical power. Here, we report that the cytosine methylation in CpG dinucleotides determines the difference in fragmentation rate of methylated and unmethylated DNA under sonication. We found that at the beginning of sonication, methylated DNAs are degraded faster than unmethylated one, and the difference in fragmentation degree can be evaluated with high reliability by quantitative polymerase chain reaction (qPCR). The optimal parameters that provide the greatest difference in amount of amplifiable DNA targets corresponding to fragmentation degree are the following: moderate amplicon size (about 150–250?bp), medium CpG sparseness (one CpG dinucleotide per ～12–14 nucleotides of the chain), and short sonication time (less than 5?min). Along with CpG, the CpA and CpT contents of amplified regions should be taken into account for proper DNA fragmentation by ultrasound as well. The obtained data could be used for elaboration of a method for comparative methylation testing, when there is no need to detect methylation of certain CpG dinucleotides. This method will be simple (can be used by any technician familiar with PCR), low cost (no need to use an expensive reagents), and fast (only brief DNA sonication and conventional qPCR are carried out).

Communicated by Ramaswamy H. Sarma     

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.     

Hot-Alkaline DNA Extraction Method for Deep-Subseafloor Archaeal Communities

A prerequisite for DNA-based microbial community analysis is even and effective cell disruption for DNA extraction. With a commonly used DNA extraction kit, roughly two-thirds of subseafloor sediment microbial cells remain intact on average (i.e., the cells are not disrupted), indicating that microbial community analyses may be biased at the DNA extraction step, prior to subsequent molecular analyses. To address this issue, we standardized a new DNA extraction method using alkaline treatment and heating. Upon treatment with 1 M NaOH at 98°C for 20 min, over 98% of microbial cells in subseafloor sediment samples collected at different depths were disrupted. However, DNA integrity tests showed that such strong alkaline and heat treatment also cleaved DNA molecules into short fragments that could not be amplified by PCR. Subsequently, we optimized the alkaline and temperature conditions to minimize DNA fragmentation and retain high cell disruption efficiency. The best conditions produced a cell disruption rate of 50 to 80% in subseafloor sediment samples from various depths and retained sufficient DNA integrity for amplification of the complete 16S rRNA gene (i.e., ∼1,500 bp). The optimized method also yielded higher DNA concentrations in all samples tested compared with extractions using a conventional kit-based approach. Comparative molecular analysis using real-time PCR and pyrosequencing of bacterial and archaeal 16S rRNA genes showed that the new method produced an increase in archaeal DNA and its diversity, suggesting that it provides better analytical coverage of subseafloor microbial communities than conventional methods.     

Heterogeneity of mitochondrial DNA from Saccharomyces cerevisiae and genetic information for tRNA.

Mitochondrial DNA from wild-type Saccharomyces cerevisiae and from an "extreme" petite mutant were analyzed by hybridization of several tRNAs on DNA fragments of different buoyant density, obtained by sonication and fractionation on a CsCl gradient. The hybridization patterns show that the genes for tRNAser, tRNAphe, tRNAhis, tRNAval, tRNAileu are present on wild-type mitochondrial DNA, while only genes for tRNAser and tRNAhis are present on petite mitochondrial DNA; moreover the hybridization patterns indicate that these genes are not clustered and suggest that more than one gene might exist for tRNAser and tRNAhis.