Microarray analysis of cell-free fetal DNA in amniotic fluid: a prenatal molecular karyotype

Metaphase karyotype analysis of fetal cells obtained by amniocentesis or chorionic villus sampling is the current standard for prenatal cytogenetic diagnosis, particularly for the detection of trisomy 21. We previously demonstrated that large quantities of cell-free fetal DNA (cffDNA) are easily extracted from amniotic fluid (AF). In this study, we explored potential clinical applications of AF cffDNA by testing its ability to hybridize to DNA microarrays for comparative genomic hybridization (CGH) analysis. cffDNA isolated from 11 male fetuses showed significantly increased hybridization signals on SRY and decreased signals on X-chromosome markers, compared with female reference DNA. cffDNA isolated from six female fetuses showed the reverse when compared with male reference DNA. cffDNA from three fetuses with trisomy 21 had increased hybridization signals on the majority of the chromosome 21 markers, and cffDNA from a fetus with monosomy X (Turner syndrome) had decreased hybridization signals on most X-chromosome markers, compared with euploid female reference DNA. These results indicate that cffDNA extracted from AF can be analyzed using CGH microarrays to correctly identify fetal sex and aneuploidy. This technology facilitates rapid screening of samples for whole-chromosome changes and may augment standard karyotyping techniques by providing additional molecular information.     

In vitro protein microarrays for detecting protein-protein interactions: application of a new method for fluorescence labeling of proteins

Protein microarrays or proteome chips are potentially powerful tools for comprehensive analysis of protein-protein interactions. In interaction analysis, a set of immobilized proteins is arrayed on slides and each slide is probed with a set of fluorescently labeled proteins. Here we have developed and tested an in vitro protein microarray, in which both arraying and probing proteins were prepared by cell-free translation. The in vitro synthesis of fluorescently labeled proteins was accomplished by a new method: a fluorophore-puromycin conjugate was incorporated into a protein at the C-terminus on the ribosome. The resulting fluorescently labeled proteins were confirmed to be useful for probing protein-protein interactions on protein microarrays in model experiments. Since the in vitro protein microarrays can easily be extended to a high-throughput format and also combined with in vitro display technologies such as the streptavidin-biotin linkage in emulsions method (Doi and Yanagawa, FEBS Lett. 1999, 457, 227-230), our method should be useful for large-scale analysis of protein-protein interactions.     

Identifying protein interactions with metal-modified DNA using microarray technology

Protein microarrays have been used extensively to identify protein–protein interactions; however, this technology has not been widely applied to protein–DNA interactions. In particular, this work demonstrates the utility of this technique for rapidly identifying interactions of proteins with metal-modified DNA. Protein macroarray experiments were carried out with high mobility group protein 1 (HMG-1) and cisplatin- and chromium-modified 50-mer oligonucleotides to demonstrate “proof of principle.” Commercially available protein microarrays containing many different classes of human proteins were then employed to search for additional interactions with cisplatin-modified DNA. The results of the microarray experiments confirmed some known interactions and, more importantly, identified many novel protein interactions, demonstrating the utility of this method as a rapid, high-throughput technique to discover proteins that interact with metal-modified DNA. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

应用多重标记引物增强寡核苷酸芯片的荧光信号强度

寡核苷酸芯片技术是一种高通量发掘和采集生物信息的强大技术平台,目前已广泛应用于生物科学领域 . 为改善寡核苷酸芯片的分析性能,对影响芯片杂交结果的因素,如片基表面的化学处理、探针的长度、间隔臂的长度、杂交条件等,进行了深入的研究和优化 . 对寡核苷酸芯片而言,仍有待解决的问题是如何产生更强的荧光信号来改善其检测灵敏度 . 利用两种类型的多个荧光分子标记的引物,来增强二维寡核苷酸芯片平面上的荧光信号强度 . 两种引物分别命名为：多标记线性引物和多标记分支引物 . 通过增加标记在目标 DNA 片段上的荧光分子数,可以显著增强寡核苷酸芯片上相应捕获探针的信号强度 . 实验表明,使用多标记引物能将所用的寡核苷酸微阵列的检测限 ( 以能够检测的最低模板量计算 ) 降低至单荧光标记引物的 1/100 以下,多重标记技术是一种有效增强微型化探针矩阵检测灵敏度的信号放大方法 .     

Glycosylphosphatidylinositol-anchored proteins are not required for crosslinking-mediated endocytosis or transfection of avidin bioconjugates into biotinylated cells

Even though glycosylphosphatidylinositol (GPI)-anchored proteins lack direct structural contact with the intracellular space, these ubiquitously expressed surface receptors activate signaling cascades and endocytosis when crosslinked by extracellular ligands. Such properties may be due to their association with membrane microdomains composed of glycosphingolipids, cholesterol and some signaling proteins. In this study, we hypothesize that GPI proteins may be required for crosslinking-mediated endocytosis of extracellular bioconjugates. To test this hypothesis, we first biotinylated the surface membranes of native K562 erythroleukemia cells versus K562 cells incapable of surface GPI protein expression. We then compared the entry of fluorescently labeled avidin or DNA condensed on polyethylenimine-avidin bioconjugates into the two biotinylated cell populations. Using fluorescence microscopy, nearly 100% efficiency of fluorescent avidin endocytosis was demonstrated in both cell types over a 24 h period. Surprisingly, plasmid DNA transfer was slightly more efficient among the biotinylated GPI-negative cells as measured by the expression of green fluorescence protein. Our findings that GPI proteins are not required for the endocytosis of avidin bioconjugates into biotinylated cells suggest that endocytosis associated with general membrane crosslinking may be due to overall reorganization of the membrane domains rather than GPI protein-specific interactions.     

Molecular research technologies in mitochondrial diseases: the microarray approach

Mitochondria are ubiquitous in eukaryotic cells where they generate much of the cellular energy by the process of oxidative phosphorylation (OXPHOS). The approximately 1500 genes of the mitochondrial genome are distributed between the cytoplasmic, maternally-inherited, mitochondrial DNA (mtDNA) which encodes 37 genes and the nuclear DNA (nDNA) which encompasses the remaining mitochondrial genes. The interplay between the mtDNA and nDNA encoded mitochondrial genes and their role in mitochondrial disorders is still largely unclear. One approach for elucidating the pathophysiology of mitochondrial diseases has been to look at changes in the expression of mtDNA and nDNA-encoded genes in response to specific mitochondrial genetic defects. Initial studies of gene expression changes in response to mtDNA defect employed blot technologies to analyze changes in the expression of individual genes one at a time. While Southern/Northern blot experiments confirmed the importance of nDNA-mtDNA interactions in the pathophysiology of mitochondrial myopathy, the methodology used limited the number of genes that could be analyzed from each patient. This barrier has been overcome, in part by the advent of DNA microarray technology. In DNA microarrays gene sequences or oligonucleotides homologous to gene sequences are arrayed on a solid support. The RNA from the subject is then isolated, the mRNA converted to cDNA and the cDNA labeled with a fluorescent probe. The labeled cDNA is hybridized on the microarray and the fluorescence bound to each array is then quantified. Recently, these technologies have been applied to mitochondrial disease patient tissues and the presence of coordinate changes in mitochondrial gene expression confirmed.     

Detection of differentially expressed genes in primary tumor tissues using representational differences analysis coupled to microarray hybridization.

The identification of differential gene expressionbetween cells is a frequent goal in modern biological research. Here we demonstrate the coupling of representational difference analysis (RDA) of cDNA with microarray analysis of the output for high throughput screening. Two primary Ewing's sarcoma tissue samples with different biological behavior in vivo were compared by RDA: one which was metastatic and progressed rapidly; the other localized and successfully treated. A modified RDA protocol that minimizes the necessary starting material was employed. After a reduced number of subtractive rounds, the output of RDA was shotgun cloned into a plasmid vector. Inserts from individual colonies from the subtracted library were amplified with vector-specific primers and arrayed at high density on glass slides. The arrays were then hybridized with differentially fluorescently labeled starting amplicons from the two tissues and fluorescent signals were measured at each DNA spot. We show that the relative amounts of fluorescent signal correlate well with the abundance of fragments in the RDA amplicon and in the starting mRNA. In our system, we analyzed 192 products and 173 (90%) were appropriately detected as being >2-fold differentially expressed. Fifty unique, differentially expressed clones were identified. Therefore, the use of RDA essentially provides an enriched library of differentially expressed genes, while analysis of this library with microarrays allows rapid and reproducible screening of thousands of DNA molecules simultaneously. The coupling of these two techniques in this system resulted in a large pool of differentially expressed genes.     

Detection of in vivo protein-DNA interactions using DamID in mammalian cells

Understanding gene regulatory networks in mammalian cells requires detailed knowledge of protein-DNA interactions. Commonly used methods for genome-wide mapping of these interactions are based on chromatin immunoprecipitation. However, these methods have some drawbacks, such as the use of crosslinking reagents, the need for highly specific antibodies and relatively large amounts of starting material. We present DamID, an alternative technique to map genome-wide occupancy of interaction sites in vivo, that bypasses these limitations. DamID is based on the expression of a fusion protein consisting of a protein of interest and DNA adenine methyltransferase (Dam). This leads to methylation of adenines near sites where the protein of interest interacts with the DNA. These methylated sequences are subsequently amplified by a methylation-specific PCR protocol and identified by hybridization to microarrays. Using DamID, genome-wide maps of the binding of DNA-interacting proteins in mammalian cells can be constructed efficiently. Depending on the strategy used for expression of the Dam-fusion proteins, genome-wide binding maps can be obtained in as little as 2 weeks.     

Stripping custom microRNA microarrays and the lessons learned about probe-slide interactions

Microarrays have been used extensively in gene expression profiling and genotyping studies. To reduce the high cost and enhance the consistency of microarray experiments, it is often desirable to strip and reuse microarray slides. Our genome-wide analysis of microRNA expression involves the hybridization of fluorescently labeled nucleic acids to custom-made, spotted DNA microarrays based on GAPSII-coated slides. We describe here a simple and effective method to regenerate such custom microarrays that uses a very low-salt buffer to remove labeled nucleic acids from microarrays. Slides can be stripped and reused multiple times without significantly compromising data quality. Moreover, our analyses of the performance of regenerated slides identifies parameters that influence the attachment of oligonucleotide probes to GAPSII slides, shedding light on the interactions between DNA and the microarray surface and suggesting ways in which to improve the design of oligonucleotide probes.     

ROLE OF MULTIPLE FTSZ RINGS IN CHLOROPLAST DIVISION UNDER OLIGOTROPHIC AND EUTROPHIC CONDITIONS IN THE UNICELLULAR GREEN ALGA NANNOCHLORIS BACILLARIS (CHLOROPHYTA,TREBOUXIOPHYCEAE)1

Chloroplasts of the unicellular green alga Nannochloris bacillaris Naumann cultured under nutrient‐enriched conditions have multiple rings of FtsZ, a prokaryote‐derived chloroplast division protein. We previously reported that synthesis of excess chloroplast DNA and formation of multiple FtsZ rings occur simultaneously. To clarify the role of multiple FtsZ rings in chloroplast division, we investigated chloroplast DNA synthesis and ring formation in cells cultured under various culture conditions. Cells transferred from a nutrient‐enriched medium to an inorganic medium in the light showed a drop in cell division rate, a reduction in chloroplast DNA content, and changes in the shape of chloroplast nucleoids as cells divided. We then examined DNA synthesis by immunodetecting BrdU incorporated into DNA strands using the anti‐BrdU antibody. BrdU‐labeled nuclei were clearly observed in cells 48 h after transfer into the inorganic medium, while only weak punctate signals were visible in the chloroplasts. In parallel, the number of FtsZ rings decreased from 6 to only 1. When the cells were transferred from an inorganic medium to a nutrient‐enriched medium, the number of cells increased only slightly in the first 12 h after transfer; after this time, however, they started to divide more quickly and increased exponentially. Chloroplast nucleoids changed from punctate to rod‐like structures, and active chloroplast DNA synthesis and FtsZ ring formation were observed. On the basis of our results, we conclude that multiple FtsZ ring assembly and chloroplast DNA duplication under nutrient‐rich conditions facilitate chloroplast division after transfer to oligotrophic conditions without further duplication of chloroplast DNA and formation of new FtsZ rings.     

基于TurboID的植物蛋白邻近标记实验方法

邻近标记作为近些年发展起来的一项检测活细胞内蛋白互作关系和亚细胞结构蛋白组的新型技术,已成功应用于多种动植物体系的研究。该技术通过给诱饵蛋白融合一个具有特定催化连接活性的酶,在酶的催化作用下将小分子底物(如生物素)共价连接到酶邻近的内源蛋白,通过富集和分析被标记的蛋白可获得与诱饵互作的蛋白组。经定向进化产生的生物素连接...     

In vivo protein-protein and protein-DNA crosslinking for genomewide binding microarray

Chromatin immunoprecipitation (ChrIP or ChIP) has commonly been used to map protein-DNA interaction sites at specific genomic loci through use of formaldehyde-induced crosslinking. However, formaldehyde alone has proved inadequate for crosslinking of certain proteins such as the yeast histone deacetylase Rpd3. We report here a modified crosslinking procedure that includes a protein-protein crosslinking agent in addition to formaldehyde. Using this double crosslinking method, we have successfully mapped Rpd3 binding sites in vivo. We also describe the use of ChrIP in combination with DNA microarrays (ChrIP-array) to determine the pattern of Rpd3 binding genomewide. This approach couples the versatility of ChrIP with that of microarrays to identify binding patterns that would otherwise be hidden in a gene-by-gene survey.     

ProCAT: a data analysis approach for protein microarrays

Protein microarrays provide a versatile method for the analysis of many protein biochemical activities. Existing DNA microarray analytical methods do not translate to protein microarrays due to differences between the technologies. Here we report a new approach, ProCAT, which corrects for background bias and spatial artifacts, identifies significant signals, filters nonspecific spots, and normalizes the resulting signal to protein abundance. ProCAT provides a powerful and flexible new approach for analyzing many types of protein microarrays.     

Comparison of fluorescent tag DNA labeling methods used for expression analysis by DNA microarrays

Gene expression profiling by DNA microarrays has found wide application in many fields of biomedical research. The protocols for this technique are not yet standardized, and for each given step in microarray analysis a number of different protocols are in use. As a consequence, results obtained in different laboratories can be difficult to compare. Of particular importance in this respect are the methods for the preparation of fluorescent cDNA probes that should quantitatively reflect the abundance of different mRNAs in the two samples to be compared. Here we systematically evaluate and compare five different published and/or commercial principles for the synthesis offluorescently labeled probes for microarray analysis (direct labeling, 77 RNA polymerase amplification, aminoallyl labeling, hapten-antibody enzymatic labeling, and 3-D multi-labeled structures). We show that individual labeling methods can significantly influence the expression pattern obtained in a microarray experiment and discuss the respective benefits and limitations of each method.