Effect of sample storage time on detection of hybridization signals in Checkerboard DNA-DNA hybridization

Long-term sample storage can affect the intensity of the hybridization signals provided by molecular diagnostic methods that use chemiluminescent detection. The aim of this study was to evaluate the effect of different storage times on the hybridization signals of 13 bacterial species detected by the Checkerboard DNA-DNA hybridization method using whole-genomic DNA probes. Ninety-six subgingival biofilm samples were collected from 36 healthy subjects, and the intensity of hybridization signals was evaluated at 4 different time periods: (1) immediately after collecting (n?= 24) and (2) after storage at -20?°C for 6 months (n?= 24), (3) for 12 months (n?= 24), and (4) for 24 months (n?= 24). The intensity of hybridization signals obtained from groups 1 and 2 were significantly higher than in the other groups (p?< 0.001). No differences were found between groups 1 and 2 (p?> 0.05). The Checkerboard DNA-DNA hybridization method was suitable to detect hybridization signals from all groups evaluated, and the intensity of signals decreased significantly after long periods of sample storage.     

Rapid Decalcification Using Microwaves for in Situ Hybridization in Skeletal Tissues

In situ hybridization histochemistry is the sole tool available for detecting the localization and expression of specific RNA on histological sections under various in vivo conditions. For this paper, we examined the effect of microwave exposure on the time needed for decalcification of skeletal tissues and on the preservation of sensitivity for hybridization signals. Our data show that the use of microwave decalcification reduces the decalcification period while preserving intense hybridization signals for mouse al chain of procollagen type I mRNA in osteogenic cells in bone. The use of microwave treatment to decalcify skeletal tissues may prevent delay in obtaining experimental results or the loss of signals during in situ hybridization.     

Strategies for covalent attachment of DNA to beads

Several covalent attachment chemistries were tested for the immobilization of DNA onto glass beads. The comparison was based on the ability of these chemistries to produce derivatized beads that give good hybridization signals. Cyanuric chloride, isothiocyanate, nitrophenyl chloroformate, and hydrazone chemistries gave us the best (yet comparable) hybridization signals. We further characterized the cyanuric chloride method for the number of attachment sites, number of hybridizable sites, hybridization kinetics, effect of linker length on hybridization intensity and stability of the derivatized beads.     

利用栽培稻C0t-1 DNA和基因组DNA比较分析宽叶野生稻基因组

利用AA染色体组栽培稻的中高度重复序列C0t-1 DNA和基因组DNA作为探针,通过荧光原位杂交技术对宽叶野生稻（Oryza latifolia）（CCDD染色体组）进行了比较基因组分析。结果显示,在宽叶野生稻染色体上,C0t-1 DNA的杂交信号没有基因组DNA的杂交信号明显;杂交信号主要分布在着丝粒、近着丝粒及端粒区域;随着洗脱严谨度的不同,杂交信号呈现出较高的种特异性。本研究以不同洗脱严谨度下的荧光原位杂交结果为依据,对宽叶野生稻进行的核型分析,可进一步提高稻属染色体识别的准确性。     

Kinetics of hybridization on surface oligonucleotide microchips: theory, experiment, and comparison with hybridization on gel-based microchips

The optimal design of oligonucleotide microchips and efficient discrimination between perfect and mismatch duplexes strongly depend on the external transport of target DNA to the cells with immobilized probes as well as on respective association and dissociation rates at the duplex formation. In this paper we present the relevant theory for hybridization of DNA fragments with oligonucleotide probes immobilized in the cells on flat substrate. With minor modifications, our theory also is applicable to reaction-diffusion hybridization kinetics for the probes immobilized on the surface of microbeads immersed in hybridization solution. The main theoretical predictions are verified with control experiments. Besides that, we compared the characteristics of the surface and gel-based oligonucleotide microchips. The comparison was performed for the chips printed with the same pin robot, for the signals measured with the same devices and processed by the same technique, and for the same hybridization conditions. The sets of probe oligonucleotides and the concentrations of probes in respective solutions used for immobilization on each platform were identical as well. We found that, despite the slower hybridization kinetics, the fluorescence signals and mutation discrimination efficiency appeared to be higher for the gel-based microchips with respect to their surface counterparts even for the relatively short hybridization time about 0.5-1 hour. Both the divergence between signals for perfects and the difference in mutation discrimination efficiency for the counterpart platforms rapidly grow with incubation time. In particular, for hybridization during 3 h the signals for gel-based microchips surpassed their surface counterparts in 5-20 times, while the ratios of signals for perfect-mismatch pairs for gel microchips exceeded the corresponding ratios for surface microchips in 2-4 times. These effects may be attributed to the better immobilization efficiency and to the higher thermodynamic association constants for duplex formation within gel pads.     

利用栽培稻C_0t-1 DNA和基因组DNA比较分析宽叶野生稻基因组

利用AA染色体组栽培稻的中高度重复序列C0t-1 DNA和基因组DNA作为探针,通过荧光原位杂交技术对宽叶野生稻(Oryza latifolia)(CCDD染色体组)进行了比较基因组分析。结果显示,在宽叶野生稻染色体上,C0t-1 DNA的杂交信号没有基因组DNA的杂交信号明显;杂交信号主要分布在着丝粒、近着丝粒及端粒区域;随着洗脱严谨度的不同,杂交信号呈现出较高的种特异性。本研究以不同洗脱严谨度下的荧光原位杂交结果为依据,对宽叶野生稻进行的核型分析,可进一步提高稻属染色体识别的准确性。     

原位分子杂交法检测恙螨体内肾综合征出血热病毒核酸的研究

为探讨肾综合征出血热病毒（ＨＦＲＳＶ）在恙螨体内的分布和定位,采用原位分子杂交技术检测恙螨体内ＨＦＲＳＶＲＮＡ。结果发现：原位分子杂交技术的检出阳性率较ＩＦＡＴ高;ＨＦＲＳＶ阳性信号颗粒呈弥散分布,多见于恙螨幼虫和若虫的腹部组织细胞内,该部位是恙螨的卵巢细胞、中肠及支囊上皮细胞。前体组织细胞内少见;个别幼虫和若虫的前足和中足细胞内亦可见到ＨＦＲＳＶＲＮＡ阳性信号。在原位分子杂交中,若虫组织细胞内的ＨＦＲＳＶＲＮＡ阳性信号较幼虫密集而且量多,表明ＨＦＲＳＶ在恙螨体内可传递并有增殖现象     

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)     

Interaction of chromosomal and plasmid DNA in Acidithiobacillus ferrooxidans strains adapted to different oxidation substrates

Restriction analysis of plasmids pTFK1 and pTFK2 of the Acidithiobacillus ferrooxidans strain TFBk was carried out, and the sizes of these plasmids were determined (13.5 and 30 kb, respectively). A macrorestriction map was built for plasmid pTFK1. DNA-DNA hybridization revealed that the plasmids contained homologous nucleotide sequences. Plasmid pTFK2 labeled with 32P was used as a probe for Southern hybridization with blots of XbaI-generated fragments of the chromosomal DNA of A. ferrooxidans strains grown on a medium containing Fe2+ or adapted to different oxidation substrates. Low-intensity hybridization signals were observed for many fragments of the chromosomal DNA of the strains studied. In the process of adaptation to new oxidation substrates, the localization of bands producing the low-intensity hybridization signals changed in a number of cases. Certain fragments of the chromosomal DNA of the strains adapted to different oxidation substrates produced strong hybridization signals with pTFK2. The data obtained are discussed in terms of the possible role of IST elements and plasmids in the adaptation of A. ferrooxidans to new energy substrates, microevolution, and strain polymorphism.     

原位杂交技术在检测染色体畸变中的研究

实验运用原位杂交技术检测肿瘤组织的染色体畸变。用3、7、8、9、12号染色体着丝粒特异性DNA探针,分别对外周静脉血的淋巴细胞、新鲜实体瘤组织、瘤组织石蜡切片进行染色体检测。二个染色体杂交点代表一对正常的染色体、三个染色体杂交点显示该细胞有染色体三体。整个实验过程简单、快速、准确、易重复。此方法和传统的细胞遗传学方法相结合,对探讨肿瘤的发生、发展有很大的意义。     

Silicon nanopillar substrates for enhancing signal intensity in DNA microarrays

The use of ordered, high-aspect ratio nanopillar arrays on the surface of silicon-based chips to enhance signal intensity in DNA microarrays is reported. These nanopillars consisting either of a single silicon dioxide substrate or a dual silicon/silicon dioxide substrate are fabricated using deep-UV lithography followed by reactive ion etching. These pillar type arrays provide a three-dimensional high surface-density platform that increases the immobilization capacity of captured probes, enhances target accessibility and reduces background noise interference in DNA microarrays, leading to improved signal-to-noise ratios, sensitivity and specificity. Consequently, it was found that the use of such nanopillars enhanced the hybridization signals by up to seven times as compared to silicon dioxide thin film substrates. In addition, hybridization of synthetic targets to capture probes that contained a single-base variation showed that the perfect matched duplex signals on dual-substrate nanopillars can be up to 23 times higher than the mismatched duplex signals, allowing the targets to be unambiguously identified. These results suggest that the nanopillars, particularly the dual-substrate pillars, are able to enhance the hybridization signals and discrimination power in nucleic acids-based detection, providing an alternative platform for improving the performance of DNA microarrays.     

A method for cross-species gene expression analysis with high-density oligonucleotide arrays

DNA microarrays have been widely used in gene expression analysis of biological processes. Due to a lack of sequence information, the applications have been largely restricted to humans and a few model organisms. Presented within this study are results of the cross-species hybridization with Affymetrix human high-density oligonucleotide arrays or GeneChip® using distantly related mammalian species; cattle, pig and dog. Based on the unique feature of the Affymetrix GeneChip® where every gene is represented by multiple probes, we hypothesized that sequence conservation within mammals is high enough to generate sufficient signals from some of the probes for expression analysis. We demonstrated that while overall hybridization signals are low for cross-species hybridization, a few probes of most genes still generated signals equivalent to the same-species hybridization. By masking the poorly hybridized probes electronically, the remaining probes provided reliable data for gene expression analysis. We developed an algorithm to select the reliable probes for analysis utilizing the match/mismatch feature of GeneChip®. When comparing gene expression between two tissues using the selected probes, we found a linear correlation between the cross-species and same-species hybridization. In addition, we validated cross-species hybridization results by quantitative PCR using randomly selected genes. The method shown herein could be applied to both plant and animal research.     

Linking probe thermodynamics to microarray quantification

Understanding the difference in probe properties holds the key to absolute quantification of DNA microarrays. So far, Langmuir-like models have failed to link sequence-specific properties to hybridization signals in the presence of a complex hybridization background. Data from washing experiments indicate that the post-hybridization washing has no major effect on the specifically bound targets, which give the final signals. Thus, the amount of specific targets bound to probes is likely determined before washing, by the competition against nonspecific binding. Our competitive hybridization model is a viable alternative to Langmuir-like models.     

玉米有丝分裂染色体上玉米BAC探针的FISH杂交体系的构建

本研究分别探讨了玉米和水稻基因组c 0t DNA对探针的封阻、杂交后洗脱的严谨度、杂交液中FAD的浓度变化对BAC-FISH杂交的影响;探讨了玉米BAC探针中重复序列含量对FISH信号的影响.初步形成了一套以玉米BAC探针在玉米有丝分裂染色体上进行FISH杂交的优化技术体系.结果表明,玉米基因组c 0t DNA对探针封阻的c 0t值应小于50;而降低杂交液中FAD浓度和适度控制杂交后洗脱的严谨度,尤其是使用水稻基因组的c 0t 100 DNA封阻探针重复序列对BAC-FISH杂交信号特异性的改善具有明显的效果;同时,验证了选择重复序列含量较少的玉米BAC作为FISH杂交的探针也是获得特异性杂交信号的重要条件.     

In situ hybridization of albumin mRNA in normal liver and liver tumors: Identification of hepatocellular origin

In situ hybridization was performed to detect albumin mRNA in normal liver, liver cirrhosis, primary liver tumors and secondary liver neoplasms. In areas of normal liver, and liver cirrhosis, signals for albumin mRNA were present in hepatocytes, whereas no signals were seen in other cells such as endothelial and Küpffer cells, bile duct epithelium and smooth muscle cells. In 53 of 56 hepatocellular carcinomas signals were present in tumor cells but in eight cholangiocarcinomas and 14 metastatic adenocarcinomas from large bowel or pancreas, carcinoma cells were negative for albumin mRNA. In three metastatic tumors (from two neuroendocrine carcinomas and one gastric leiomyosarcoma), tumor cells contained no signals, while the surrounding hepatocytes showed diffuse grains. In 15 of the 84 specimens examined in situ hybridization was applied to routine formalin-fixed and paraffin-embedded blocks and strong signals were obtained for albumin mRNA. We conclude that in situ hybridization of human albumin is a valid tool in the differential diagnosis of hepatocellular carcinoma from cholangiocarcinomas and tumors metastatic to the liver.     

Reliability and efficiency of interphase-fish with alpha-satellite probe for detection of aneuploidy

Early diagnosis is very important in pre- and postnatal diagnosis of Down syndrome. This study examines the use of fluorescence in situ hybridization (FISH) to detect trisomy 21 in interphase nuclei and metaphase chromosome obtained from fifty-four Down syndrome patients with a regular type trisomy 21. Three of them showed six hybridization signals on both interphase nuclei and metaphase spreads instead of five signals corresponding to two chromosomes 13 and three chromosomes 21 although they were cytogenetically trisomy 21. Simultaneous application of probe combination revealed that one of the extra signals of chromosomes 13/21 a-satellite probe was located on chromosome 22 in two cases and one extra signal on chromosomes 15 in one case. In addition, another case showed four hybridization signals on both interphase nuclei and metaphase spreads instead of five signals, indicating deletion of the chromosome specific alpha-satellite DNA sequence of chromosome 13/21. These centromeric sequence changes may have pathological significance in the appearance of aneuploidy because they may be involved in the important centromere function.     

Improved procedure for fluorescence in situ hybridization on tissue microarrays

BACKGROUND: The recently developed tissue microarray (TMA) technology allows the arrangement of up to a thousand tissue specimens on a single microscope slide. This technology enables researchers to perform gene copy number studies on very large series of archival formalin-fixed tissues using fluorescence in situ hybridization (FISH). However, the hybridization properties of individual archival specimens can vary considerably. Therefore a highly optimized protocol is needed to fulfill the task of producing evaluable hybridization signals simultaneously in hundreds of specimens in a TMA. METHODS: The performance of two different FISH protocols, the standard protocol for paraffin embedded tissues and our new optimized protocol, was tested on TMAs using probes for the HER-2 and ZNF217 genes as well as the chromosome 17 centromere. RESULTS: The new protocol resulted in greatly increased signal intensity and an almost 30% increase in the number of tissue samples with evaluable hybridization signals. CONCLUSIONS: Our improved protocol for FISH on TMAs provides standardized hybridization conditions leading to high-quality hybridization signals in the majority of specimens. The increases in the signal intensity and the number of evaluable samples are extremely important for the successful analyses of TMAs by FISH and will allow the utilization of the TMA technology in its full potential.     

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。     

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.