基因芯片技术及其在植物上的应用

基因芯片技术（gene chip technology)是采用光导原位合成或缩微印刷等方法,将大量特定的DNA探针片段有序地固定于固相载体的表面,形成DNA微阵列,然后与待测的标记样品靶DNA或RNA分子杂交,对杂交信号进行扫描及计算机检测分析,从而获取所需的生物信息。该技术在植物研究中广泛应用于寻找特异性相关基因和新基因,基因表达分析,基因突变和多态性检测,DNA测序等。     

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。     

Development of a novel DNA chip based on a bipolar semiconductor microchip system

We have applied an integrated circuit photodiode array (PDA) chip system to a DNA chip. The PDA chip system, constructed using conventional bipolar semiconductor technology, acts as a solid transducer surface as well as a two-dimensional photodetector. DNA hybridization was performed directly on the PDA chip. The target DNA, the Bacillus subtilis sspE gene, was amplified by polymerase chain reaction (PCR). The 340-bp PCR product was labeled using digoxigenin (DIG). A silicon nitride layer on the photodiode was treated with poly-L-lysine to immobilize the DNA on the surface of the photodiode detection elements. Consequently, the surface of the photodiode detector became positively charged. An anti-DIG-alkaline phosphatase conjugate was reacted with the hybridized DIG-labeled DNA. A color reaction was performed based on the enzymatic reaction between nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) staining solution and a DNA complex containing antibodies. A blue precipitate was formed on the surfaces of the photodiode detection elements. Successful quantitative analysis of the hybridized PCR products was achieved from the light absorption properties of the blue enzymatic reaction product that was produced after a series of reaction processes. Our DNA chip system avoids the complicated optical alignments and light-collecting optical components that are usually required for an optical DNA chip device. As a result, a simple, compact, portable and low-cost DNA chip is achieved. This system has great potential as an alternative system to the conventional DNA reader.     

乳酸菌基因芯片应用研究进展

基因芯片技术是上世纪90年代兴起的一种对成百上千甚至上万个基因同时进行检测的新技术,具有高通量、并行化的特点,广泛应用于基因表达谱测定、基因功能预测、基因突变检测和多态性分析等方面。多种乳酸菌基因组全序列以及其大量EST、16S rDNA、16S-23S基因间区和功能基因序列测定的完成,有力地推动了基因芯片技术在乳酸菌研究中的应用。介绍了基因芯片的基本原理及乳酸菌基因芯片在基因表达、种属鉴定等研究中的应用进展,以期更好地利用和开发乳酸菌基因芯片。     

采用分子生物学技术诊断隐球菌脑膜脑炎的研究

目的采用分子检测技术对疑似隐球菌感染的脑膜炎病例进行诊断。方法收集患者的脑脊液样本,提取DNA,设计引物进行PCR扩增,采用DNA芯片技术对扩增产物进行分子检测。结果显示样本新生隐球菌阳性。结论通过ITS保守序列设计引物进行PCR扩增和DNA芯片技术对常规真菌学检查不能确定的疑似隐球菌脑膜炎患者脑脊液样本进行非培养检测,具有实验室诊断参考价值。     

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.     

A simple and reliable assay for detecting specific nucleotide sequences in plants using optical thin-film biosensor chips

Here we report the adaptation and optimization of an efficient, accurate and inexpensive assay that employs custom-designed silicon-based optical thin-film biosensor chips to detect unique transgenes in genetically modified (GM) crops and SNP markers in model plant genomes. Briefly, aldehyde-attached sequence-specific single-stranded oligonucleotide probes are arrayed and covalently attached to a hydrazine-derivatized biosensor chip surface. Unique DNA sequences (or genes) are detected by hybridizing biotinylated PCR amplicons of the DNA sequences to probes on the chip surface. In the SNP assay, target sequences (PCR amplicons) are hybridized in the presence of a mixture of biotinylated detector probes and a thermostable DNA ligase. Only perfect matches between the probe and target sequences, but not those with even a single nucleotide mismatch, can be covalently fixed on the chip surface. In both cases, the presence of specific target sequences is signified by a color change on the chip surface (gold to blue/purple) after brief incubation with an anti-biotin IgG horseradish peroxidase (HRP) to generate a precipitable product from an HRP substrate. Highly sensitive and accurate identification of PCR targets can be completed within 30 min. This assay is extremely robust, exhibits high sensitivity and specificity, and is flexible from low to high throughput and very economical. This technology can be customized for any nucleotide sequence-based identification assay and widely applied in crop breeding, trait mapping, and other work requiring positive detection of specific nucleotide sequences.     

Microfluidic device reads up to four consecutive base pairs in DNA sequencing-by-synthesis

We have developed the first fully integrated microfluidic system for DNA sequencing-by-synthesis. Using this chip and fluorescence detection, we have reliably sequenced up to 4 consecutive bps. The described sequencer can be integrated with other microfluidic components on the same chip to produce true lab-on-a-chip technology. The surface chemistry that was designed to anchor the DNA to elastomeric microchannels is useful in a broad range of studies and applications.     

Design and Validation of DNA Libraries for Multiplexing Proximity Ligation Assays

Here, we present an in silico, analytical procedure for designing and testing orthogonal DNA templates for multiplexing of the proximity ligation assay (PLA). PLA is a technology for the detection of protein interactions, post-translational modifications, and protein concentrations. To enable multiplexing of the PLA, the target information of antibodies was encoded within the DNA template of a PLA, where each template comprised four single-stranded DNA molecules. Our DNA design procedure followed the principles of minimizing the free energy of DNA cross-hybridization. To validate the functionality, orthogonality, and efficiency of the constructed template libraries, we developed a high-throughput solid-phase rolling-circle amplification assay and solid-phase PLA on a microfluidic platform. Upon integration on a microfluidic chip, 640 miniaturized pull-down assays for oligonucleotides or antibodies could be performed in parallel together with steps of DNA ligation, isothermal amplification, and detection under controlled microenvironments. From a large computed PLA template library, we randomly selected 10 template sets and tested all DNA combinations for cross-reactivity in the presence and absence of antibodies. By using the microfluidic chip application, we determined rapidly the false-positive rate of the design procedure, which was less than 1%. The combined theoretical and experimental procedure is applicable for high-throughput PLA studies on a microfluidic chip.     

生物芯片与肿瘤研究

生物芯片技术是指通过微加工和微电子技术,在芯片表面构建微型生物化学分析系统,对组织细胞中的蛋白质、DNA或者其他生物组分进行高通量检测。生物芯片广泛应用于生命科学、司法鉴定、食品及营养科学、环境科学、农林科学、军事科学等多种领域。本文重点对其在肿瘤研究和诊断治疗中的应用做一简要综述。     

Oligonucleotide-arrayed TFT photosensor applicable for DNA chip technology

A thin film transistor (TFT) photosensor fabricated by semiconductor integrated circuit (IC) technology was applied to DNA chip technology. The surface of the TFT photosensor was coated with TiO2 using a vapor deposition technique for the fabrication of optical filters. The immobilization of thiolated oligonucleotide probes onto a TiO2-coated TFT photosensor using gamma-aminopropyltriethoxysilane (APTES) and N-(gamma-maleimidobutyloxy) sulfosuccinimide ester (GMBS) was optimized. The coverage value of immobilized oligonucleotides reached a plateau at 33.7 pmol/cm2, which was similar to a previous analysis using radioisotope-labeled oligonucleotides. The lowest detection limits were 0.05 pmol/cm2 for quantum dot and 2.1 pmol/cm2 for Alexa Fluor 350. Furthermore, single nucleotide polymorphism (SNP) detection was examined using the oligonucleotide-arrayed TFT photosensor. A SNP present in the aldehyde dehydrogenase 2 (ALDH2) gene was used as a target. The SNPs in ALDH2*1 and ALDH2*2 target DNA were detected successfully using the TFT photosensor. DNA hybridization in the presence of both ALDH2*1 and ALDH2*2 target DNA was observed using both ALDH2*1 and ALDH2*2 detection oligonucleotides-arrayed TFT photosensor. Use of the TFT photosensor will allow the development of a disposable photodetecting device for DNA chip systems.     

质粒pCAMBIA1301的检测

用引物延伸芯片法实现对转基因水稻中 生物芯片技术是生物技术和微制造技术的融合, 已广泛用于生命科学的研究及实践、医学科研及临床、药物设计、环境保护、农业、军事等各个领域。而基因芯片是生物芯片中的一种,是指将大量基因探针分子固定于支持物上,然后与标记的样品进行杂交,所以一次可对大量核酸分子进行检测分析,从而解决了传统核酸印迹杂交技术操作复杂、自动化程度低、检测目的分子数量少、效率低等不足。文章探讨了用基因芯片这一新的检测手段对转基因植物的初步检测,采用一种新的反应机制－引物延伸芯片法（arrayed primer extension）,实现了样品扩增和杂交的一步化,而在传统的基因芯片检测中要需要两步来完成,从而为目前基因芯片中大片段样品的检测提供了一种可能性。 Abstract: Biochip technology which had emerged from the fusion of biotechnology and micro/nanofabrication technology at the end of 1980s has been widely used in life science ,medicine,clinical diagnosis,durg design,agricμLture,envioment pretection and strategics. DNA microarray (also call gene chip,DNA chip),one kind of biochips,is small chip containing many oligonucleotide probe .It can hybridize with labelled sample which makes it possible to detect large numbers of oligonucleotides at one time.So DNA microarray can overcome the disadvantage of traditional hybridization technology such as complexity,low automatization,poor efficiency and amount of detcting molecμLes. This paper describes a new method to detect transgenic plant with gene chip.We have developed a novel arrayed-primer extension technique. It combines hybridization and PCR at one step, while two separate steps are needed in the ordinary DNA microarray, therefore our method provide a feasibility to detect long DNA fragment .     

基于微流控芯片的细胞-细菌相互作用光电检测技术

细胞/细菌及其相互作用研究对于生命科学、药物研发、医学诊疗等领域的研究具有重要意义。微流控芯片分析技术因微环境可控、生物相容性好、检测并行性、微型化等特性，正发展成为细胞/细菌及其相互作用研究的高效手段。本文在简要介绍基于微流控芯片分析技术的细胞-细菌分析方法和技术基础之上，对微流控芯片上细胞-细菌相互作用模型的建立进行了讨论，重点针对细胞-细菌及其相互作用过程的芯片检测进行了综述，尤其对芯片集成的光电检测技术及其测试效果进行总结和比较。通过芯片集成微流体控制、多种光电传感监测模块，使微流控芯片分析技术成为细胞/细菌及其相互作用过程分析和检测的支撑平台和优势手段。最后，对微流控光电检测技术在细胞-细菌相互作用检测中面临的挑战及发展趋势进行了讨论和展望。     

The BARC biosensor applied to the detection of biological warfare agents

The Bead ARray Counter (BARC) is a multi-analyte biosensor that uses DNA hybridization, magnetic microbeads, and giant magnetoresistive (GMR) sensors to detect and identify biological warfare agents. The current prototype is a table-top instrument consisting of a microfabricated chip (solid substrate) with an array of GMR sensors, a chip carrier board with electronics for lock-in detection, a fluidics cell and cartridge, and an electromagnet. DNA probes are patterned onto the solid substrate chip directly above the GMR sensors, and sample analyte containing complementary DNA hybridizes with the probes on the surface. Labeled, micron-sized magnetic beads are then injected that specifically bind to the sample DNA. A magnetic field is applied, removing any beads that are not specifically bound to the surface. The beads remaining on the surface are detected by the GMR sensors, and the intensity and location of the signal indicate the concentration and identity of pathogens present in the sample. The current BARC chip contains a 64-element sensor array, however, with recent advances in magnetoresistive technology, chips with millions of these GMR sensors will soon be commercially available, allowing simultaneous detection of thousands of analytes. Because each GMR sensor is capable of detecting a single magnetic bead, in theory, the BARC biosensor should be able to detect the presence of a single analyte molecule.     

蛋白质芯片技术进展

人类基因组测序工作的完成 ,引起人们对蛋白质组研究的热忱。蛋白质作为生命活动的执行者 ,种类繁多 ,结构复杂 ,并且其活性与空间结构密切相关 ,需要更为先进的技术去研究和探索。近来出现的蛋白质芯片以并行、高通量检测、分析和处理蛋白质样品 ,发展迅速 ,应用前景广泛。介绍蛋白质芯片的种类、蛋白质固定的表面化学以及不同的检测方法 ,简述蛋白质芯片在不同领域的应用 ,并讨论蛋白质芯片目前存在的问题。     

DNA芯片技术与基因表达研究

随着基因组计划的顺利实施,大量的生物信息被解析,基因表达及基因功能的研究将成为生命科学研究的热点。ＤＮＡ世片技术是近年来出现的分子生物学与微电子技术相结合的最新ＤＮＡ分析检测技术。该技术将在生命科学与信息科学之间架起一道桥梁,因而成为后基因组时代基因功能分析撮重要的技术之一。目前ＤＮＡ芯片技术已在基因保得到广泛的应用。     

RCA combined nanoparticle-based optical detection technique for protein microarray: a novel approach

Developing a readily available biosensor with excellent performances is the main focus of many research groups. Recently, major breakthroughs in miniaturization of molecular analysis have produced DNA and protein microarrays. The aim of our group is to develop a sensitive technique for analyzing signals on protein microarray by applying the surface plasmon resonance (SPR) method. This new detection technique for specific molecular binding utilizes rolling circles amplification (RCA) post-signal processing method [Nat. Genet. 19 (1998) 225-232] and optical visualization by nanogold particle-labeled molecules on a micro-structured chip surface. By covalent bonding of the RCA primer to the detection antibody guarantees that the linkage between the analyte and the amplified RCA product is maintained during the assay. Experimental results show that RCA has significantly enhanced sensitivity compared to conventional methods. This combination of an easily detectable signal with chip technology should have the potential to become a successful commercial application.     

A duplex approach for immunochemical staining and typing of proteins in Western blots

In this study, we describe a detection system for the indirect detection of vaccinia virus by DNA analysis. The system uses quartz crystal microbalance (QCM) as the detection technique and polymerase chain reaction (PCR) for amplification. Different immobilization strategies for the capture probe on the quartz chip are studied. For the QCM detection of hybridisation, the influence of the structure and length of target DNA is analyzed. For the detection of DNA from an amplification product, an efficient denaturation procedure is developed. On the basis of these investigations, vaccinia virus DNA is detected with only a low number of amplification rounds and a short analysis time. Specificity can be clearly shown. To enhance the signal strength and to have a further proof of specificity, a gold nanoparticle-tagged enhancer sequence can be used.     

DNA芯片技术研究进展及其在分子营养上的应用

DNA芯片技术是近年发展起来的又一新分子生物学研究工具,可使研究者得以自动化、快速、平行地对大量的生物信息加以分析,在基因组水平上研究基因表达。这种技术为从基因组水平研究基因表达水平与生理反应及生理状况的改变之间的关系提供了强有力的手段。通过比较不同营养水平或不同环境条件下的组织细胞基因达到表达谱差异,可以从基因组水平阐明各种营养成分或环境因素对动物机体的基因表达的影响,从而进一步揭示营养生理的机制和环境对动物影响的机理。DNA芯片技术为分子营养的研究开辟了一条崭新的道路,在从DNA芯片的原理、种类、实验设计、统计方法及在分子营养上的应用作一综述。     

分子生态学技术及其在环境微生物研究中的应用

分子生态学作为一门新兴的学科已经成为国内外科学家关注和研究的热点。目前的分子生态学技术主要有核酸杂交分析技术、特异性PCR扩增技术、DNA序列分析、基因芯片技术等。这些技术在环境微生物研究中的应用主要包括对微生物多样性的研究、种群结构和动力学的研究、代谢活性的研究以及在全球气候变化中对微生物影响的研究。最后,对环境微生物的分子生态学研究进行了展望。