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     

寡核苷酸探针制备的优化

目的:探讨寡核苷酸微阵列制备中适合使用的探针浓度、探针缓冲液pH值、离子强度、优化杂交条件。方法:选取人白细胞抗原DQA1位点,针对多态性集中的外显子2设计一对保守引物及16条特异性分型探针;分别用ddH2O和0.1、0.2mol/L碳酸盐缓冲液(pH=7)稀释探针至100μmol/L;选取合适的缓冲液浓度后,调碳酸盐缓冲液为5.0、6.0、7.0、8.0、9.0、10.0等6种pH值,选取最优pH值及离子强度,分别溶解探针至20、50、100、200μmol/L,比较上述不同条件的杂交结果。结果:用0.1mol/L、pH9的碳酸盐缓冲液溶解探针杂交效果最佳;探针浓度为20μmol/L时信号弱,其他浓度下无显著差别。结论:通过探针制备的优化可以提高杂交效率,探针浓度与杂交信号强度无明显正相关。     

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。     

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．     

用氨基修饰的载玻片制作cDNA微阵列

cDNA微阵列已在基因差异表达、寻找新基因等研究方面获得广泛应用,但有关cDNA微阵列的制作,目前多采用多聚赖氨酸修饰的载玻片为探针固定载体,固定效果较差.用氨基硅烷处理的载玻片为载体制作cDNA微阵列,然后考察其固定效率、检测灵敏度、稳定性、实用性等指标.结果表明,用氨基硅烷处理的载玻片具有比多聚赖氨酸更令人满意的核酸固定效率、检测灵敏度,且稳定实用.因此,用氨基硅烷修饰的载玻片为探针固定载体制作cDNA微阵列较为理想.     

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。     

蛋白芯片生产用醛基化玻片制备的条件优化及初步检定

建立一种以玻片为基质的蛋白芯片制备的优化及检定方法。将经过清洗处理的玻片,用不同浓度的氨基硅烷试剂和戊二醛试剂,在不同的时间内,进行氨基化和醛基化处理,通过蛋白结合强度与处理时间及溶剂浓度的动力学相关性分析,确定最佳的优化条件,将不同浓度梯度的人IgG抗体结合于该玻片表面,经过洗涤、封闭,再加入Cy3荧光标记的二抗,孵育,洗涤后检测各点的荧光强度,确定其线性范围及其检测灵敏度。氨基硅烷试剂浓度为5%,作用时间为30min;戊二醛试剂浓度为2.5%,作用时间为60min时,蛋白结合强度达到饱和。蛋白芯片在2～2×4-7mg/ml浓度范围内有良好的线性,能够检测出人IgG的最低浓度为2×4-8mg/ml。     

荧光原位杂交法检测双歧杆菌

检验荧光原位杂交法在双歧杆菌属鉴定方面的调途。方法：采用对数生长期的８株双歧杆菌和１０析其他厌氧、需氧杆菌在相同的条件下分别与双歧杆菌属特异性１６ＳｒＲＮＡ寡核苷酸基因探针和细菌界通用１６ＳｒＲＮＡ寡核苷酸基因探针在载玻片上进行原位杂交,在荧光显微镜下观察杂交结果,拍摄同一视野的荧光显微镜照片和相关显微镜照片,计算杂交率。结果所用的双歧杆菌菌株均与两种基因探针杂交,在荧光显微镜下发校菌与黑暗背景对     

HPV病毒检测分型芯片的研制和优化

研制和优化寡核苷酸芯片以初步实现对多种常见HPV(Human papillomavirus)病毒的分型检测.应用生物学软件对四型常见HPV病毒(6、11、16、18型)的全基因组序列进行分析,设计具有型特异性、熔解温度(Tm)相近的～60 mer寡核苷酸探针,对玻片片基进行优化处理后,点样制备成寡核苷酸基因芯片.将含HPV全长基因序列的质粒作为阳性标准品,利用梯度限制性荧光标记技术对其进行荧光标记,标记好的样品与芯片杂交.结果显示HPV样品与相应的型特异性探针杂交有明显的荧光信号,而与阴性对照探针和空白对照探针没有杂交信号.通过对芯片片基处理和样品荧光标记方法的优化,可以提高芯片检测的杂交特异性和荧光信号强度.     

Assessing unmodified 70-mer oligonucleotide probe performance on glass-slide microarrays

Background

Long oligonucleotide microarrays are potentially more cost- and management-efficient than cDNA microarrays, but there is little information on the relative performance of these two probe types. The feasibility of using unmodified oligonucleotides to accurately measure changes in gene expression is also unclear.

Results

Unmodified sense and antisense 70-mer oligonucleotides representing 75 known rat genes and 10 Arabidopsis control genes were synthesized, printed and UV cross-linked onto glass slides. Printed alongside were PCR-amplified cDNA clones corresponding to the same genes, enabling us to compare the two probe types simultaneously. Our study was designed to evaluate the mRNA profiles of heart and brain, along with Arabidopsis cRNA spiked into the labeling reaction at different relative copy number. Hybridization signal intensity did not correlate with probe type but depended on the extent of UV irradiation. To determine the effect of oligonucleotide concentration on hybridization signal, 70-mers were serially diluted. No significant change in gene-expression ratio or loss in hybridization signal was detected, even at the lowest concentration tested (6.25 μm). In many instances, signal intensity actually increased with decreasing concentration. The correlation coefficient between oligonucleotide and cDNA probes for identifying differentially expressed genes was 0.80, with an average coefficient of variation of 13.4%. Approximately 8% of the genes showed discordant results with the two probe types, and in each case the cDNA results were more accurate, as determined by real-time PCR.

Conclusions

Microarrays of UV cross-linked unmodified oligonucleotides provided sensitive and specific measurements for most of the genes studied.     

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.     

应用双色荧光原位杂交技术检测克氏综合征

探讨用双色荧光原位杂交技术（dual-color fluorescence in situ hybridization,D-FISH）检测性染色体数目异常克氏综合征的应用价值,建立常规分裂期染色体和间期细胞FISH技术的实验方法。以Biotin标记的X染色体α-卫星DNA（pBamX7）探针和以Digoxigenin标记的Y染色体长臂末端重复序列（pY3.4）探针对19例克氏综合征标本同时进行外周血染色体及其间期细胞核的原位杂交,分别用Avidin-FITC和Rhodamine-FITC及其Anti-avidin进行信号的检测与放大,DAPI复染。于Olympus AX-70型荧光显微镜下,分别通过WIB、WIG及其WU滤光镜观察杂交信号及其染色体或间期核背景,并统计外周血中期染色体及其间期细胞核的杂交信号颗粒数量。在显微镜下可见以Biotin标记的pBamX7探针显示2个绿色杂交信号,以Digoxigenin标记的pY3.4探针显示1个红色杂交信号,染色体或间期核背景经DAPI复染显示蓝色;18例出现XXY杂交信号的细胞,染色体及其间期细胞核杂交平均出现率分别为95.89%和95%,明显大于正常对照标准值2.75%,证实核型为47,XXY,与染色体检测的结果一致;其余1例染色体核型检测为嵌合体,XXY杂交信号细胞出现率为92%,同时检出6.7%的XY杂交信号细胞（>正常对照标准值4.17%）。用FISH 技术检测性染色体数目异常克氏综合征具有快速、敏感度高、信号强、背景低、多色等优点,故FISH 技术在产前诊断检测领域中显示其重要的应用价值和发展前景。 Abstract:The objective of the work is to study the technique of dual-color fluorescence in situ hybridization(D-FISH) and its application value in the diagnosis of sex chromosomal count abnormality Klinefelter syndrome and establish an experimental approach to metaphase chromosome and interphase nucleus FISH technique.Biotin labeled alpha satellite X-chromosome DNA(pBamX7) probe and Digoxigenin labeled Y-chromosome long arm terminal repetitive sequence (pY3.4) probe were hybridized with pre-treated slides of peripheral blood chromosome and interphase nucleus in 19 cases of Klinefelter syndrome specimens.After being washed,the slides were treated with Avidin-FITC,Rhodamine-FITC and Anti-avidin,amplified with an additional layer and counter-stained with DAPI in an antifade solution.The hybridization signals,chromosomal or interphase nucleus settings were observed respectively with WIB,WIG and WU filters under fluorescence microscope Olympus AX-70,and the number of metaphase chromosome and interphase nucleus in the peripheral blood was counted.It was observed under the microscope that the Biotin labeled pBamX7 probe showed 2 green hybridization signals and that the Digoxigenin labeled pY3.4 probe showed 1 red hybridization signal.Chromosome or interphase nucleus counter-stained with DAPI showed blue.The average signal rate of chromosome and interphase nucleus hybridization was 95.89% and 95% respectively,significantly higher than the normal control (2.75%).Karyotype 47,XXY was confirmed,which agrees with the chromosomal findings.One case showed mosaic nuclei.XXY chromosome hybridization signal rate was 92% and XY hybridization signal rate was 6.7%,higher than the normal control rate of 4.17%.FISH is a valuable technique in diagnosing sex chromosomal count abnormality Klinefelter syndrome with the merits of fast speed,high sensitivity,strong signal,low background and multiple color.Therefore,FISH technique can find wide application and potential in prenatal diagnosis.     

Enhancement of DNA immobilization and hybridization on gold electrode modified by nanogold aggregates

Gold electrodes modified by nanogold aggregates (nanogold electrode) were obtained by the electrodeposition of gold nanoparticles onto planar gold electrode. The Electrochemical response of single-stranded DNA (ssDNA) probe immobilization and hybridization with target DNA was measured by cyclic voltammograms (CV) using methylene blue (MB) as an electroactive indicator. An improving method using long sequence target DNA, which greatly enhanced the response signal during hybridization, was studied. Nanogold electrodes could largely increase the immobilization amount of ssDNA probe. The hybridization amount of target DNA could be increased several times for the manifold nanogold electrodes. The detection limit of nanogold electrode for the complementary 16-mer oligonucleotide (target DNA1) and long sequence 55-mer oligonucleotide (target DNA2) could reach the concentration of 10(-9) mol/L and 10(-11) mol/L, respectively, which are far more sensitive than that of the planar electrode.     

Evaluation of thin films of agarose on glass for hybridization of DNA to identify plant pathogens with microarray technology

Agarose-coated glass slides, after activation, were spotted with amine-modified oligonucleotide probes using a manual eight-pin arraying device. Two probes, designed to identify two common greenhouse fungal plant pathogens, Didymella bryoniae and Botrytis cinerea, were hybridized with polymerase chain reaction (PCR)-amplified fluorescently labeled DNA extracted from pure culture and from diseased plant tissue. The probes easily distinguished these pathogens from each other without cross reaction. Thickness of the agarose layer and length of the sample DNA were important factors affecting hybridization efficiency of immobilized probe to PCR product. These factors did not affect hybridization with short complementary oligonucleotide. Probes fixed on agarose-coated slides could differentiate samples as readily as probes on nylon but with potentially higher spot density and gave much better signal than probes on silylated slides. The use of plain glass slides, agarose, and a manual arrayer makes this technique useful for developing specialized and inexpensive DNA microarrays on a solid rigid substrate.     

Sensitive detection of unlabeled oligonucleotides using a paired surface plasma waves biosensor

Detection of unlabeled oligonucleotides using surface plasmon resonance (SPR) is difficult because of the oligonucleotides' relatively lower molecular weight compared with proteins. In this paper, we describe a method for detecting unlabeled oligonucleotides at low concentration using a paired surface plasma waves biosensor (PSPWB). The biosensor uses a sensor chip with an immobilized probe to detect a target oligonucleotide via sequence-specific hybridization. PSPWB measures the demodulated amplitude of the heterodyne signal in real time. In the meantime, the ratio of the amplitudes between the detected output signal and reference can reduce the excess noise from the laser intensity fluctuation. Also, the common-path propagation of p and s waves cancels the common phase noise induced by temperature variation. Thus, a high signal-to-noise ratio (SNR) of the heterodyne signal is detected. The sequence specificity of oligonucleotide hybridization ensures that the platform is precisely discriminating between target and non-target oligonucleotides. Under optimized experimental conditions, the detected heterodyne signal increases linearly with the logarithm of the concentration of target oligonucleotide over the range 0.5-500 pM. The detection limit is 0.5 pM in this experiment. In addition, the non-target oligonucleotide at concentrations of 10 pM and 10nM generated signals only slightly higher than background, indicating the high selectivity and specificity of this method. Different length of perfectly matched oligonucleotide targets at 10-mer, 15-mer and 20-mer were identified at the concentration of 150 pM.     

Increased concentrations of radioisotopically-labeled complementary ribonucleic acid probe, dextran sulfate, and dithiothreitol in the hybridization buffer can improve results of in situ hybridization histochemistry.

The goal of the present studies was to optimize mRNA detection with radioisotopic in situ hybridization histochemistry (ISHH). Test experiments performed on sections of rat brain tissue used computer-assisted image analysis to compare autoradiographic signals resulting when varying concentrations of (35)S-labeled cRNA probes, dextran sulfate (DS), and dithiothreitol (DTT) were used for ISHH. We found that greatly enhanced corrected signal density (total density of signal area minus background density) was obtained using concentrations of probe and/or DS that were several-fold higher than those widely recommended in published ISHH procedures (probe concentration >4 x 10(4) cpm/microl; DS concentration >10%). Extended hybridization reaction (>16 hr) also significantly augmented the corrected signal density. Finally, nonspecific probe binding was greatly reduced and corrected signal density enhanced by including 750-1000 mM, rather than the widely used 10-200 mM DTT, in the hybridization buffer. These observations indicate that the low efficiency of hybridization and the formation of high background may largely compromise the sensitivity of routine ISHH procedures. We suggest that the new method using increased concentrations of (35)S-labeled cRNA probe, DS, and DTT will be especially important for the cellular localization of rare mRNA species.