利用基因芯片技术筛选HIV-1F亚型基因限制性显示探针

为筛选限制性显示技术制备的HIV 1F亚型基因探针 ,应用基因芯片打印仪将其有序地打印在玻片上制备基因芯片 .在随机引物延伸的过程中进行HIV样品的荧光标记 ,然后与芯片进行杂交 .杂交后清洗玻片并干燥 ,对芯片进行扫描 ,分析各探针的杂交信号 .从中筛选了 14个基因片段作为芯片下一步研究的探针 .实验证明 ,限制性显示技术是一种制备基因芯片探针的实用方法     

制备炭疽芽胞杆菌检测基因芯片的初步研究

建立制备炭疽芽胞杆菌检测基因芯片的技术,并探讨研制检测炭疽芽胞杆菌基因芯片的方法。酶切炭疽芽胞杆菌的毒素质粒和荚膜质粒,通过建立质粒DNA文库的方法获取探针,并打印在经过氨基化修饰的玻片上,制成用于炭疽芽胞杆菌检测的基因芯片。收集了290个阳性克隆探针,制备了检测炭疽芽胞杆菌的基因芯片。提取炭疽芽胞杆菌质粒DNA与基因芯片杂交,经ScanArray Lite芯片阅读仪扫描得到初步的杂交荧光图像。通过分析探针的杂交信号初步筛选出273个基因片段作为芯片下一步研究的探针。     

乙型脑炎病毒可视化分型基因芯片的制备

目的:制备乙型脑炎病毒(JEV)可视化分型基因芯片。方法:根据JEV的基因组序列,应用生物学软件设计JEV分型引物及探针,制备其可视化分型基因芯片;用生物素标记的引物PCR扩增目的片段,并与固定于玻片上的探针杂交,加入链霉亲和素标记的纳米金,银增强实现可视化;进行特异性、灵敏性及重复性试验。结果:探针特异地与相应的标记目的基因片段杂交,并在芯片上呈现较强的阳性杂交信号;2号探针能特异性检出JEV,3、4号探针可分别对Ⅰ型和Ⅲ型JEV进行分型;芯片对JEV质粒检测的灵敏度达105拷贝/mL;以蓝耳病病毒等5种病毒为对照,芯片只对JEV响应,具有特异性;制备的基因芯片具有批间、批内重复性。结论:制备的基因芯片具有高特异性、灵敏性及重复性,可以快速、准确、高通量地对JEV进行可视化分型检测。     

炭疽芽胞杆菌基因芯片探针文库的构建

为制备炭疽芽胞杆菌的基因芯片探针文库,以炭疽芽胞杆菌毒素质粒pX01和荚膜质粒pX02为原材料,用Sau3A I酶切pX01和pX02质粒DNA,Taq DNA聚合酶72℃补平加A,经AT克隆,PCR初步鉴定筛选出炭疽质粒片段的阳性克隆．DNA自动分析仪对克隆片段进行序列测定;用生物信息学方法对其片段进行同源性分析;并将克隆的探针打印于玻片上,制备成炭疽芽胞杆茵基因芯片,与炭疽杆茵质粒DNA样品进行初步芯片杂交的实验,杂交实验的阳性率达到了90％以上,证明大部分克隆探针属于炭疽芽胞杆菌．炭疽芽胞杆菌基因芯片探针文库的构建为基因芯片探针的制备摸索出一条简便、高效、可行的方法．     

基因导流杂交法于低密度基因芯片平台上检测乙肝病毒

运用基因导流杂交法在低密度基因芯片平台上检测乙肝病毒DNA（HBV DNA）。设计特异性引物,对HBV基因组DNA中的编码HBV多聚酶蛋白的一段序列进行PCR扩增;根据被扩增片段,设计保守的特异性探针,并将该探针固定在杂交膜上,制备低密度基因芯片：使用导流杂交法将上述扩增产物和低密度基因芯片进行杂交,根据显色反应判断被检测样本有无HBV DNA。基因导流杂交法在低密度基因芯片平台上可以方便、快速、准确地检测乙肝病毒。     

表达序列标签及基因芯片技术在植物抗性基因研究中的应用

表达序列标签和基因芯片技术是基因组学研究的重要手段。表达序列标签是cDNA的3’或5’端的一段序列,通过表达序列标签可以寻找在某种胁迫条件下特异表达的基因并推测其可能的功能。基因芯片技术是指将大量基因探针分子固定于载体上并与标记的样品分子进行杂交,通过检测每个探针分子的杂交信号强度获取样品分子数量和序列信息,通过基因芯片技术,可以研究基因在不同的条件下的表达量,进而研究植物抗性机理。     

消减杂交技术结合限制性显示技术制备K562细胞特异基因探针

建立一种简便、快速、特异的制备基因芯片探针的方法.以K562细胞和正常人淋巴细胞作为消减对象,利用自行建立的消减方法进行消减杂交,结合限制性显示技术,分组扩增差异cDNA,回收K562细胞特异基因片段,制作基因芯片探针.结果显示,分离到400个K562特异的基因,片段大小均一,适于制作cDNA芯片.消减杂交技术结合限制性显示技术制备基因芯片探针,具有快速、简便、特异的特点,降低了芯片制作成本,可加速芯片的推广应用.     

“缺口翻译”法制备~(32)P 标记DNA探针和几种核酸杂交技术

定性或定量检测天然DNA或重组DNA中某些特殊的顺序片段,分子杂交是最常用的方法。近几年,核酸的分子杂交技术日趋完善,以不同材料为支持物的固相杂交技术取得了更为迅速的发展。核酸分子杂交方法的灵敏性主要取决于同位素标记杂交探针的比放射性强度。因此制备高比放射性探针是提高灵敏性的关键。用缺口翻译方法制备较稳定的高比放射性强度的杂交探针,大肠杆菌DNA聚合酶Ⅰ是一个理想的工具。本文介绍我们建立并改进“缺口翻译”制备P标记的探针方法,以及几种重要的核酸固相杂交技术。     

基于表面等离子共振扫描成像的DNA芯片的研究

基于表面等离子共振(surface plasmon resonance,SPR)技术的检测方法是一种灵敏度极高的光学检测方法,用于基因芯片的检测时具有高灵敏、免标记、无污染等优点,是一种很有发展潜力的芯片检测方法。将酵母Y5基因的特异性片段5′端修饰巯基后作为探针,利用单分子层自组装法把探针固定在金膜表面,应用列扫描表面等离子共振成像检测系统研究和分析DNA芯片上探针点阵的杂交信息,从而建立一套基于列扫描表面等离子共振检测系统的DNA芯片的制备和检测分析技术。实验结果表明:37℃条件下探针的最佳固定时间为5～7h,杂交特异性良好,杂交的最佳时间为5～30min。探针浓度低于0.5μmol/L时,杂交效率高而且SPR信号变化明显,探针浓度达到20μmol/L时SPR响应达到最大。     

用巢式PCR方法制备基因芯片的探针

制备出高纯度的探针,用于诊断基因芯片的打印。采用巢式PCR技术,M13作为外侧引物并自行设计内侧引物,扩增克隆在T载体上的基因片段。可制备出成分单一,上下游仅含19bp和20bp的短载体序列的探针,能使打印出的芯片得到较好的杂交效果。该法充分利用巢式PCR的优点,对制备探针的方法进行改进,且简便快速,能得到更高质量的探针,满足打印芯片的要求。     

生物信息学在基因芯片中的应用

生物信息学和基因芯片是生命科学研究领域中的两种新方法和新技术,生物信息学与基因芯片密切相关,生物信息学促进了基因芯片的研究与应用,而基因芯片则丰富了生物信息学的研究内容。本论文探讨生物信息学在基因芯片中的应用,将生物信息学方法运用到高密度基因芯片设计和芯片实验数据管理及分析。从信息学的角度提出基因芯片设计准则,提出寡核苷酸探针的优化设计方法,将该方法运用于再测序型芯片和基因表达型芯片的设计,在此基础上研制出高密度基因芯片设计软件系统和实验结果分析系统。     

一种高密度基因芯片的优化方法

基因芯片 (ｇｅｎｅｃｈｉｐ) ,又称ＤＮＡ微阵列(ｍｉｃｒｏａｒｒａｙ) ,是分子生物学和微电子、微机械学科交叉的产物。基因芯片技术将生命科学研究中所涉及的许多不连续的分析过程 ,如探针制备、杂交反应和检测分析等 ,通过采用微电子、微机械等工艺集成到芯片中 ,使之连续化、集成化和微型化。这一技术的成熟和应用将在新世纪里给遗传研究、疾病诊断和治疗、新药发现和环境保护等生命科学相关领域带来一场革命。本文探讨高密度基因芯片的优化技术。1　高密度基因芯片高密度基因芯片是由大量ＤＮＡ或寡核苷酸探针密集排列所形成的探针阵…     

Spreadsheet software for thermodynamic melting point prediction of oligonucleotide hybridization with and without mismatches

The use of thermodynamic parameters for the calculation of oligonucleotide duplex stability provides the best estimates of oligonucleotide melting temperatures (Tm). Such estimates can be used for evidence-based design of molecular biological experiments in which oligonucleotide melting behavior is a critical issue, such as temperature or denaturing gradient gel electrophoreses, Southern blotting or hybridization probe assays on the LightCycler. We have developed a user friendly program for Tm calculation of matched and mismatched probes using the spreadsheet software Microsoft Excel. The most recently published values for entropy and enthalpy of Watson-Crick paris are used, and salt and oligonucleotide concentrations are considered. The 5' and 3' end stability is calculated for the estimation of primer specificity. In addition, the influence of all possible mutations under a given probe can be calculated automatically. The experimental evaluation of predicted Tm with the LightCycler, based on 14 hybridization probes for different gene loci, showed an excellent fit between measured results and values predicted with the thermodynamic model in 14 matched, 25 single mismatched and 8 two-point mismatched assays (r = 0.98; Sy. x = 0.90; y = 1.01 x -0.38). This program is extremely useful for the design of oligonucleotide probes because the use of probes that do not discriminate with a reasonable Tm difference between wild-type and mutation can be avoided in advance.     

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。     

A Sexually Transmitted Disease (STD) DNA chip for the diagnosis of genitourinary infections

The results of an investigation aimed at the development of a DNA chip for the detection of genitourinary infections are described. Through analysis of over 35,000 clinical cases, 14 pathogens which are most abundantly found among Koreans were selected and candidate sequences for capture probes were accordingly chosen by considering their sequences and β-globin house-keeping gene. Among this group, the most suitable capture probe sequences were selected by employing repeated chip tests in which they are immobilized on a glass chip by using a recently developed novel gold nanoparticles-based method. A multiplex PCR method was established to generate fluorescence-labeled sequences for all 14 pathogens along with the β-globin gene. By using optimized hybridization conditions, the final chip was constructed and employed to diagnose reliably both single and multiple infections in clinical human samples for 14 target pathogens. The results show that the novel chip methodology serves as a highly reliable and convenient tool for the diagnosis of Sexually Transmitted Diseases (STDs). Furthermore, this study has its great significance in that it demonstrates the entire process from statistical analysis of a large number of clinical cases to the final development of STD DNA chip just ready to be applied or commercialized in the clinical diagnostic field.     

The detection of a transgenic soybean biochip using gold label silver stain technology

A method for the rapid detection of transgenic soybean crops based on a combination of gene chip and "gold label silver stain" (GLSS) technologies has been established. To ensure the specificity of this method, the CaMV35S promoter and Nos terminator were selected as probes because they are both exogenous genes that are specific to transgenic soybean plants. The addition of biotin-modified dUTPs to a polymerase chain reaction (PCR) system can produce amplified nucleic acid segments containing biotin. These labeled PCR products then hybridize with specific probes on the chip and are subsequently bound by streptavidin-modified gold nanoparticles (GNPs). Due to the catalytic nature of the GNPs, silver staining can be used to visualize the hybridized probes, which appear as signals in varying shades of gray. The intensity value of the gray signals can be obtained using a general scanner. Silver staining for 10 min was determined to produce the optimal signal-to-noise ratio. In addition, this method was shown to be highly specific and had a detection sensitivity of 288.57 pg/μL.     

A study of the relationships between oligonucleotide properties and hybridization signal intensities from NimbleGen microarray datasets

Well-defined relationships between oligonucleotide properties and hybridization signal intensities (HSI) can aid chip design, data normalization and true biological knowledge discovery. We clarify these relationships using the data from two microarray experiments containing over three million probes from 48 high-density chips. We find that melting temperature (T_m) has the most significant effect on HSI while length for the long oligonucleotides studied has very little effect. Analysis of positional effect using a linear model provides evidence that the protruding ends of probes contribute more than tethered ends to HSI, which is further validated by specifically designed match fragment sliding and extension experiments. The impact of sequence similarity (SeqS) on HSI is not significant in comparison with other oligonucleotide properties. Using regression and regression tree analysis, we prioritize these oligonucleotide properties based on their effects on HSI. The implications of our discoveries for the design of unbiased oligonucleotides are discussed. We propose that isothermal probes designed by varying the length is a viable strategy to reduce sequence bias, though imposing selection constraints on other oligonucleotide properties is also essential.     

Fluorescein-labeled oligonucleotides for real-time pcr: using the inherent quenching of deoxyguanosine nucleotides

Fluorescein-labeled oligonucleotide probes can be used to continuously monitor the polymerase chain reaction. Depending on the sequence, the fluorescence intensity of the probe is either increased or decreased by hybridization. The greatest effect is probe quenching by hybridization to amplicons containing deoxyguanosine nucleotides (Gs), giving a sequence-specific decrease in fluorescence as product accumulates. Quenching of the probes by Gs is position dependent. A 25% decrease in fluorescence of 5'-labeled probes was observed with a G at the first position of the 3'-dangling end. Additional Gs can increase quenching to about 40%. This change in fluorescence with hybridization allows real-time quantification and mutation detection with a simple single labeled probe. Quantification of the initial template copy number is possible by monitoring fluorescence at each cycle at a constant temperature. Mutation detection by Tm estimates from melting curve analysis for factor V Leiden, hemoglobin C, hemoglobin S, the thermolabile mutation of methylenetetrahydrofolate reductase, and the cystic fibrosis-associated deletion F508del is demonstrated. By using the inherent quenching of deoxyguanosine nucleotides in the amplicon, complicated probe designs involving internal quenching can be avoided.     

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。