cDNA微阵列与寡核苷酸芯片的制备方法

cDNA微阵列和寡核苷酸芯片是常见的合成后点样的DNA微阵列。点样方法主要是通过物理吸附或共价结合的方式将探针固定于载体上,本总结了近年来国内外献报道的cDNA微阵列制备方法;在多聚赖氨酸包被的玻璃基片表面制备cDNA微阵列;用琼脂糖包被的玻璃基片制备cDNA微阵列;在氨基或醛基修饰的玻璃基片表面制备cDNA微阵列;寡核苷酸芯片的制备方法;氨基修饰的玻片与5′末端带氨基的寡核苷酸探针通过不同的linker连接;硅烷化寡核苷酸直接点样于玻片上制成寡核苷酸微阵列;硫代寡核苷酸通过二硫键与巯基修饰的玻片连接;水凝胶芯片固定寡核苷酸。丙烯酰胺硅烷化的基片与5′丙烯酰胺修饰的寡核苷酸连接。并展望了基因芯片的应用前景。     

用于寡核苷酸芯片制备载体的醛基载玻片的制备方法优化及性质研究

优化了醛基载玻片的制备方法 ,探讨了醛基修饰载玻片固定寡核苷酸探针的性质。研究发现氨基硅烷试剂的浓度是影响载玻片荧光背景的主要因素 ;2 %氨基化试剂处理 16min、戊二醛处理 30min可以得到荧光背景较低、固定效果较好的醛基载玻片。寡核苷酸固定过程中 ,末端氨基修饰没有明显的特异性 ,但是可以提高被固定探针的杂交容量。在较低的浓度 (小于 10 μmol L)时 ,探针的浓度与杂交信号趋近线性关系 ,浓度为 2 0 μmol L时杂交信号达到饱和     

蛋白芯片载体表面修饰对探针固定影响的研究

采用3-氨基丙基-三甲氧基硅烷((3-aminopropyl)trimethoxysilane,APTES)、戊二醛(glutaraldehyde,GA)、多聚-L-赖氨酸(poly-L-lysine,PLL)修饰芯片载体表面,对3种不同修饰方法制备的蛋白质芯片进行对比研究。将Cy3标记羊抗鼠IgG固定在修饰后片基上,选择蛋白探针的固定率作为检测指标;将小鼠IgG作为探针固定在芯片上,靶蛋白为Cy3标记羊抗鼠IgG,通过生物芯片扫描仪检测反应后荧光强度,选择蛋白探针的反应性作为检测指标,探讨制备蛋白质芯片较佳的表面修饰方法。结果显示,戊二醛修饰玻片对蛋白固定较好,有较高的反应活性,检测限较宽,但背景噪声较高。     

文摘

30 0 12用于寡核苷酸芯片制备载体的醛基载玻片的制备方法优化及性质研究〔中〕/邹宗亮… //生物工程学报 .- 2 0 0 1,17(5 ) .- 4 98～ 5 0 2优化了醛基载玻片的制备方法 ,探讨了醛基修饰载玻片固定寡核苷酸探针的性质。研究发现氨基硅烷试剂的浓度是影响载玻片荧光背景的主要     

蛋白质微阵列生产用琼脂糖修饰玻片制备的条件优化

目的:建立一种以琼脂糖修饰的玻片为载体的蛋白质微阵列制备的优化方法,比较琼脂糖修饰玻片和醛基修饰玻片及氨基修饰玻片对蛋白质固定效率的优劣。方法:将羊IgG固定在载体表面,经过洗涤、封闭,再加入Cy3标记的兔抗羊IgG,孵育,洗涤后用共聚焦激光扫描仪获取图像,检测各点的荧光强度,根据荧光强度确定最佳琼脂糖浓度,最佳NaIO4浓度,最佳固定时间以及封闭时间等实验条件。结果:琼脂糖浓度为1.2％、NaIO4浓度为20mmol／L、固定时间为1h、孵育时间为45min时,蛋白质在载体上的固定效率和反应活性最高。在固定的抗体浓度相同的情况下,琼脂糖修饰玻片荧光强度是醛基修饰玻片的2.6倍,是氨基修饰玻片的9倍。结论:确立了蛋白质微阵列生产用琼脂糖修饰玻片制备的优化条件,用该优化条件制备的琼脂糖玻片更适合用于蛋白质微阵列载体。     

三种寡核苷酸芯片片基的比较

将荧光标记的寡核苷酸打印在Corning、多聚赖氨酸包被的DAKO玻片和多聚赖氨酸处理的一种显微镜载玻片上,并按常规方法进行洗脱,通过GenePix4100A扫描并以Genepix6．0软件分析点的大小、荧光强度和背景荧光强度来衡量3种片基的均一性和固定效率,并通过不同浓度梯度的60bp寡核苷酸探针的杂交实验来衡量片基对杂交效率的影响．结果显示,3种片基的均一性都较好,而多聚赖氨酸包被的DAKO和Cornning芯片的固定效率和杂交效率优于国产芯片．     

介质表面修饰对蛋白质芯片固定率和反应性的影响

评价最常用的二种玻璃表面修饰方法对蛋白质芯片质量的影响．选择蛋白质的固定效率、反应性作为检测指标,对戊二醛修饰法和多聚赖氨酸修饰法进行比较,由机械手将探针蛋白质分别固定在两种玻片上,靶蛋白用荧光染料Cy3标记,两种修饰方法的芯片均可使蛋白质保持较好的固定效率和反应活性．由共价键偶联的醛基修饰玻片制备的蛋白质芯片不仅有更高的反应活性,而且图象佳,但背景偏高、用醛基修饰的玻片制备蛋白质芯片是较理想的选择、     

聚丙烯酰胺凝胶固定核酸方法及其应用

固相核酸已被广泛用于DNA/cDNA微阵列、固相PCR及其它核酸与生物分子检测的传感技术中.和硬质玻璃载片相比,三维聚丙烯酰胺凝胶作为固定核酸的载体具有结合核酸容量高、利于反应的类似液相环境和较少的空间效应等优点.综述了丙烯酰胺凝胶作为固定核酸载体的发展历史.着重介绍了丙烯酰胺修饰核酸直接聚合固定的方法以及在DNA芯片、焦测序、固相PCR(克隆)、及全基因组测序等核酸分析中的应用.     

糖蛋白结合疫苗制备中载体蛋白琥珀酰化程度定量检测方法的建立

糖蛋白结合疫苗制备中赖氨酸单位减少率（或赖氨酸单位替代率）是衡量载体蛋白琥珀酰化程度的一个重要指标。本文建立了一种方便易行的化学检测方法,即氨基定量测定法（简称改良Fields法）,它是通过对琥珀酰化处理前和处理后载体蛋白中赖氨酸的ε－氨基的定量测定,间接计算出赖氨酸单位减少率大小的。经测试方法中标准赖氨酸浓度的测试灵敏度范围在0.5-40μg。同时用已知不同浓度赖氨酸和三批待测琥珀酰化载体蛋白进行验证,显示该方法具有良好的稳定性和重复性。     

基因芯片的制备方法

合成后点样的DNA微阵列分析cDNA微阵列和寡核苷酸芯片,点样方法主要是通过物理吸附或共价结合的方式将探针固定于载体上。本文归纳了近年来国内外文献报道的基因芯片的制备方法,展望了基因芯片的应用前景。     

Dendrimeric coating of glass slides for sensitive DNA microarrays analysis

Successful use and reliability of microarray technology is highly dependent on several factors, including surface chemistry parameters and accessibility of cDNA targets to the DNA probes fixed onto the surface. Here, we show that functionalisation of glass slides with homemade dendrimers allow production of more sensitive and reliable DNA microarrays. The dendrimers are nanometric structures of size-controlled diameter with aldehyde function at their periphery. Covalent attachment of these spherical reactive chemical structures on amino-silanised glass slides generates a reactive ~100 Å layer onto which amino-modified DNA probes are covalently bound. This new grafting chemistry leads to the formation of uniform and homogenous spots. More over, probe concentration before spotting could be reduced from 0.2 to 0.02 mg/ml with PCR products and from 20 to 5 µM with 70mer oligonucleotides without affecting signal intensities after hybridisation with Cy3- and Cy5-labelled targets. More interestingly, while the binding capacity of captured probes on dendrimer-activated glass surface (named dendrislides) is roughly similar to other functionalised glass slides from commercial sources, detection sensitivity was 2-fold higher than with other available DNA microarrays. This detection limit was estimated to 0.1 pM of cDNA targets. Altogether, these features make dendrimer-activated slides ideal for manufacturing cost-effective DNA arrays applicable for gene expression and detection of mutations.     

cDNA文库法在HCV-1b检测芯片研制中的应用

制备丙型肝炎病毒(HCV) 1b亚型诊断芯片并进行初步验证评价.采用cDNA文库法制备探针,用限制性内切酶Sau3AⅠ消化HCV 1b全长cDNA ,所得的酶切片段72℃补平加A ,AT克隆,PCR初步鉴定,并测序.将筛选出的片段打印在氨基修饰的玻片上制备成检测芯片并进行杂交验证分析.运用cDNA文库法,得到2 2个大小相对一致(2 5 0～75 0bp)的基因片段.序列分析表明,均属于HCV 1b基因,可以作为诊断芯片探针;样品标记采用限制性显示(restrictiondisplay ,RD)技术,标记后进行杂交.杂交结果显示,样品和诊断基因芯片杂交的敏感性和特异性均佳.批内和批间精密度CV值分别为5 4 %和6 8% ,表明用cDNA文库法收集片段是一种快速、简便制备芯片探针的实用方法.     

Protein microarrays on hybrid polymeric thin films prepared by self-assembly of polyelectrolytes for multiple-protein immunoassays

We report here the development and characterization of protein microarrays fabricated on nanoengineered 3-D polyelectrolyte thin films (PET) deposited on glass slide by consecutive adsorption of polyelectrolytes via self-assembly technique. Antibodies or antigens were immobilized in the PET-coated glass slides by electrostatic adsorption and entrapment of porous structure of the 3-D polymer film and thus establishing a platform for parallel analysis. Both antigen and antibody microarrays were fabricated on the PET-coated slides, and direct and indirect immunoassays on protein microarrays for multiple-analyte detection were demonstrated. Microarrays produced on these PET-coated slides have consistent spot morphology and provide performance features needed for proteomic analysis. The protein microarrays on the PET films provide LOD as low as 6 pg/mL and dynamic ranges up to three orders of magnitude, which are wider than the protein microarrays fabricated on aldehyde and poly-L-lysine functionalized slides. The PET films constructed by self-assembly technique in aqueous solution is green chemistry based, cost-effective method to generate 3-D thin film coatings on glass surface, and the coated slide is well suited for immobilizing many types of biological molecules so that a wide variety of microarray formats can be developed on this type of slide.     

Impact of surface chemistry and blocking strategies on DNA microarrays

The surfaces and immobilization chemistries of DNA microarrays are the foundation for high quality gene expression data. Four surface modification chemistries, poly-L-lysine (PLL), 3-glycidoxypropyltrimethoxysilane (GPS), DAB-AM-poly(propyleminime hexadecaamine) dendrimer (DAB) and 3-aminopropyltrimethoxysilane (APS), were evaluated using cDNA and oligonucleotide sub-arrays. Two un-silanized glass surfaces, RCA-cleaned and immersed in Tris-EDTA buffer were also studied. DNA on amine-modified surfaces was fixed by UV (90 mJ/cm(2)), while DNA on GPS-modified surfaces was immobilized by covalent coupling. Arrays were blocked with either succinic anhydride (SA), bovine serum albumin (BSA) or left unblocked prior to hybridization with labeled PCR product. Quality factors evaluated were surface affinity for cDNA versus oligonucleotides, spot and background intensity, spotting concentration and blocking chemistry. Contact angle measurements and atomic force microscopy were preformed to characterize surface wettability and morphology. The GPS surface exhibited the lowest background intensity regardless of blocking method. Blocking the arrays did not affect raw spot intensity, but affected background intensity on amine surfaces, BSA blocking being the lowest. Oligonucleotides and cDNA on unblocked GPS-modified slides gave the best signal (spot-to-background intensity ratio). Under the conditions evaluated, the unblocked GPS surface along with amine covalent coupling was the most appropriate for both cDNA and oligonucleotide microarrays.     

An Activated GOPS‐poly‐L‐Lysine‐ Coated Glass Surface for the Immobilization of 60mer Oligonucleotides

To explore a method for enhancing the immobilization and hybridization efficiency of oligonucleotides on DNA microarrays, conventional protocols of poly‐L‐lysine coating were modified by means of surface chemistry, namely, the slides were prepared by the covalently coupling of poly‐L‐lysine to a glycidoxy‐modified glass surface. The modified slides were then used to print microarrays for the detection of the SARS coronavirus by means of 60mer oligonucleotide probes. The characteristics of the modified slides concerning immobilization efficiency, hybridization dynamics, and probe stripping cycles were determined. The improved surface exhibited high immobilization efficiency, a good quality uniformity, and satisfactory hybridization dynamics. The spotting concentration of 10 μmol/L can meet the requirements of detection; the spots were approximately 170 nm in diameter; the mean fluorescence intensity of the SARS spots were between 3.2 × 10⁴ and 5.0 × 10⁴ after hybridization. Furthermore, the microarrays prepared by this method demonstrated more resistance to consecutive probe stripping cycles. The activated GOPS‐PLL slide could undergo hybridization stripping cycles for at least three cycles, and the highest loss in fluorescence intensity was found to be only 11.9 % after the third hybridization. The modified slides using the above‐mentioned method were superior to those slides treated with conventional approaches, which theoretically agrees with the fact that modification by surface chemistry attaches the DNA covalently firmly to the slides. This protocol may have great promise in the future for application in large‐scale manufacture.     

应用限制性显示技术制备HCV cDNA诊断基因芯片的初步研究

制备丙型肝炎病毒 (HCV)检测芯片并进行验证、初步检测质量评价。采用限制性显示 (Restrictiondisplay ,RD)技术制备芯片探针 ,从载体pCV_J4L6S中切出HCV全长cDNA ,Sau3AⅠ酶消化 ,所得的限制性片段进行RD_PCR扩增 ,经聚丙烯酰胺电泳 (PAGE)结合银染法进行分离。切胶回收后作 3次PCR ,得到较纯净的HCVcDNA限制性片段。扩增后的产物克隆至pMD18_T载体进行快速鉴定。将筛选出的限制性片段打印在氨基修饰的玻片上制备成检测芯片进行杂交验证分析 ,对芯片检测进行优化、初步的质量评估。运用RD技术 ,得到 2 4个 2 0 0～ 80 0bp、大小均一的基因片段 ,序列分析表明 ,均属于HCV特异基因 ,可以作为诊断芯片探针 ;杂交、测序结果显示 ,芯片检测的敏感性、特异性、准确度、重复性、线性等指标均佳。利用RD技术制备基因芯片探针是一种快速、简便的实用方法 ;制备的诊断芯片可以用于检测HCVRNA ,具有敏感、检测结果较为可靠的优点。     

Attachment of oligonucleotide probes to poly carbodiimide-coated glass for microarray applications

Oligonucleotide-based DNA microarrays are becoming increasingly useful tools for the analysis of gene expression and single nucleotide polymorphisms (SNPs). Here, we present a method that permits the manufacture of microarrays from non-modified oligonucleotides on a poly carbodiimide-coated glass surface by UV-irradiation. The use of UV-irradiation facilitates an increase in the level of signal intensity, but it does not affect signal discrimination by the oligonucleotides immobilized on the surface. The signal intensity obtained for an array fabricated using non-modified oligonucleotides with UV-irradiation is ~7-fold greater than that without UV-irradiation. The detection of SNPs was tested to ascertain whether this technique could discriminate specific hybridization signals without causing significant UV-irradiation-induced damage to the immobilized oligonucleotides. We found that this immobilization method provides greater hybridization signals and a better match/mismatch ratio of SNPs than do the established aminosilane techniques. Application of this technology to manufacturing DNA microarrays for sequence analysis is discussed.     

Manufacturing DNA microarrays of high spot homogeneity and reduced background signal

Analyses on DNA microarrays depend considerably on spot quality and a low background signal of the glass support. By using betaine as an additive to a spotting solution made of saline sodium citrate, both the binding efficiency of spotted PCR products and the homogeneity of the DNA spots is improved significantly on aminated surfaces such as glass slides coated with the widely used poly-L-lysine or aminosilane. In addition, non-specific background signal is markedly diminished. Concomitantly, during the arraying procedure, the betaine reduces evaporation from the microtitre dish wells, which hold the PCR products. Subsequent blocking of the chip surface with succinic anhydride was improved considerably in the presence of the non-polar, non-aqueous solvent 1,2-dichloroethane and the acylating catalyst N:-methylimidazole. This procedure prevents the overall background signal that occurs with the frequently applied aqueous solvent 1-methyl-2-pyrrolidone in borate buffer because of DNA that re-dissolves from spots during the blocking process, only to bind again across the entire glass surface.     

DNA microarrays with unimolecular hairpin double-stranded DNA probes: fabrication and exploration of sequence-specific DNA/protein interactions

We have fabricated double-stranded DNA (dsDNA) microarrays containing unimolecular hairpin dsDNA probes immobilized on glass slides. The unimolecular hairpin dsDNA microarrays were manufactured by four steps: Firstly, synthesizing single-stranded DNA (ssDNA) oligonucleotides with two reverse-complementary sequences at 3' hydroxyl end and an overhang sequence at 5' amino end. Secondly, microspotting ssDNA on glutaraldehyde-derived glass slide to form ssDNA microarrays. Thirdly, annealing two reverse-complementary sequences to form hairpin primer at 3' end of immobilized ssDNA and thus to create partial-dsDNA microarray. Fourthly, enzymatically extending hairpin primer to convert partial-dsDNA microarrays into complete-dsDNA microarray. The excellent efficiency and high accuracy of the enzymatic synthesis were demonstrated by incorporation of fluorescently labeled dUTPs in Klenow extension and digestion of dsDNA microarrays with restriction endonuclease. The accessibility and specificity of the DNA-binding proteins binding to dsDNA microarrays were verified by binding Cy3-labeled NF-kappaB to dsDNA microarrays. The dsDNA microarrays have great potential to provide a high-throughput platform for investigation of sequence-specific DNA/protein interactions involved in gene expression regulation, restriction and so on.