Poly-L-Lysine玻片在寡核苷酸芯片制备中的改进

目的为了制得适合固定未修饰寡核苷酸的芯片,提高检测灵敏性,对Patrick Brown 实验室的多聚左旋赖氨酸包被玻片的方法进行改进。方法玻片经清洗后用缩水甘油-丙氧基三甲氧基硅烷进行硅烷化,然后应用Poly-L-Lysine在玻片表面形成聚合物涂层,经次亚苯基二异硫氰酸盐表面活化后可使寡核苷酸共价连接在芯片表面。设计了各种实验考察方法改进前后芯片表面的性能,并将改进后的玻片初步应用于SARS冠状病毒寡核苷酸芯片检测中。结果方法改进后芯片表面性能优良:固定效率高、点的同一性好、杂交效率和热稳定性好、寡核苷酸结合牢固、芯片可以重复利用。结论利用共价连接,方法改进后的芯片表面适合固定未修饰的寡核苷酸,解决了寡核苷酸与玻片之间物理结合不稳定、易剥离的缺陷,提高了芯片检测的灵敏性。     

基于核酸保护原理的DNA芯片检测技术

目的：制备出3－末端与载玻片交联,5－末端用32P标记的检测DNA的芯片。方法：以化学法合成了3－末端为尿嘧啶核糖的寡聚脱氧核糖核苷酸片段,用32P标记寡核苷酸的5－末端,经过高碘酸氧化后与玻璃基片表面的脂肪胺基缩合,并用硼氢化钠还原,制成寡核苷酸3－末端与玻片共价交联,5－末端为同位素标记的DNA芯片,将芯片与液相中的核酸片段杂交,再用酸酶S1酶切,结果,当液相中的核酸与玻片上共价交联的寡核苷酸片段产生特异性杂交配对时,核酸酶S1不能酶切玻片上的寡核苷酸片段,且玻片上被保护的寡核苷酸量与液相中的与核酸,由于该法制备的DNA芯片各个位点的DNA探针的含量为已知,待测的核酸样品无需进行同位素或荧光标记,操作简便,适用于对样品的DNA或RNA进行检测。     

两种氨基修饰的基因芯片表面制备方法比较

目的探讨两种氨基化方法修饰玻片表面的过程及其在基因芯片制备中应用的可行性。方法玻片经清洗、硅烷化后，一种用多组分多氨化合物包被，经次亚苯基二异硫氰酸盐表面活化；另一种方法采用的包被剂是丙烯酸-丙烯酰胺单体，活化剂是碳二业胺／N-羟基琥珀亚胺酯官能团分子。将被修饰好的玻片用于λ噬菌体基闲组DNA芯片的制备，并进行杂交检测分析结果经两种方法修饰后玻片表面分别形成分支结构和聚合物涂层，与商业化的芯片比较发现，两种芯片杂交、扫描检测后显示所得点阵的杂交信号均匀、稳定、清晰，杂交点饱满、同一性好。结论两种方法修饰的玻片用于制备基因芯片均取得了成功，其中丙烯酸-丙烯酰胺聚合物包被法处理过程简单、成本低廉、能大规模生产，是一种较理想的芯片表面制备方法。     

单克隆抗体修饰玻璃表面制备蛋白质芯片的初步研究

目的探讨应用单克隆抗体修饰玻璃表面制备蛋白质芯片的方法.方法用不同浓度的抗6X His单克隆抗体修饰玻璃表面,从蛋白质固定效率和固定蛋白质的反应活性两个方面,对修饰效果进行评价.结果初步观察到单克隆抗体修饰玻片对蛋白质的固定量较醛基化修饰的玻片没有明显增加,但其固定蛋白质中有反应活性的蛋白质的比例较高.结论单克隆抗体修饰的玻片也适合于制备蛋白质芯片.     

两种氨基修饰的基因芯片表面制备方法比较

目的 探讨两种氨基化方法修饰玻片表面的过程及其在基因芯片制备中应用的可行性。方法 玻片经清洗、硅烷化后,一种用多组分多氨化合物包被,经次亚苯基二异硫氰酸盐表面活化;另一种方法采用的包被剂是丙烯酸-丙烯酰胺单体,活化剂是碳二亚胺/N-羟基琥珀亚胺酯官能团分子。将被修饰好的玻片用于λ噬菌体基因组DNA芯片的制备,并进行杂交检测分析。结果 经两种方法修饰后玻片表面分别形成分支结构和聚合物涂层,与商业化的芯片比较发现,两种芯片杂交、扫描检测后显示所得点阵的杂交信号均匀、稳定、清晰,杂交点饱满、同一性好。结论 两种方法修饰的玻片用于制备基因芯片均取得了成功,其中丙烯酸-丙烯酰胺聚合物包被法处理过程简单、成本低廉、能大规模生产,是一种较理想的芯片表面制备方法。     

60 mer长寡核苷酸微阵列构建的优化

目的　优化长寡核苷酸芯片方阵的构建。方法　采用4种不同玻片表面,4种点样液溶解经设计高度特异性探针,制备长寡核苷酸芯片。样品经RD -梯度PCR扩增标记后与芯片杂交,扫描芯片后进行统计学分析。结果　点样液3×SSC与丙烯酰胺聚合物表面(国产玻片)联合使用杂交效果最佳。结论　该研究优化了长寡核苷酸芯片微阵列的构建,为芯片的下游技术发展提供支持。     

β-地中海贫血与先天性软骨发育不全诊断性基因芯片的构建

目的建立一种能同时快速、准确地检测β-地中海贫血与先天性软骨发育不全常见基因突变的技术。方法将针对2种疾病常见突变的特异性氨基修饰寡核苷酸探针点样于醛基修饰的玻片上，制成基因芯片，对β-地中海贫血、先天性软骨发育不全的病例和正常样本进行基因诊断。结果该基因芯片成功检测到相应的突变位点。结论该芯片制作成本低，重复性好，特异性高，能满足临床标本检测的要求，具有良好的临床应用前景。     

寡核苷酸阵列芯片快速鉴定大肠杆菌O157:H7初步研究

目的设计、制作一种微型化寡核苷酸阵列芯片，评价快速鉴定肠出血性大肠杆菌O157：H7的效果。方法多重PCR扩增大肠杆菌O157：H7七个特异性基因位点（rfbE、flicH7、intimin、Shiga-like toxins Ⅰ and Ⅱ、hemolysin A和uidA）．通过PCR反应掺入SpectrumOrang^TM-dUTP获取荧光标记的靶序列，与制备的芯片寡核苷酸探针杂交。结果寡核苷酸阵列芯片检测结果与试验预期相符，获取的杂交图分辨效果明显优于多重PCR琼脂糖凝胶电泳。结论基于玻片的寡核苷酸阵列芯片制作简便，鉴定病原菌检测细菌毒力因子快速、灵敏、特异，有良好的应用前景。     

结核杆菌检测基因芯片的制备研究

目的 建立结核杆菌检测基因芯片的制备技术。方法 制备和标记靶基因，用点样仪将靶基因点于玻片介质上，并给点样后处理制成基因芯片，用扫描仪测定处理前后芯片上荧光强度的变化。计算DNA固定率，同时测定和比较两种不同破片，不同点样液和三种不同点样后处理方法的DNA固定率。结果 制备结核杆菌检测基因芯片，用醛基修饰玻片作为介质，用DMSO溶液作为点样液，点样后芯片处理以水合1小时再干燥30分钟为佳。结论 本研究所建立的结核杆菌检测基因芯片制备技术具有较高的效率和可靠的实用性能。     

利用寡核苷酸芯片检测SARS病毒

目的：利用寡核苷酸芯片检测SARS病毒。方法：根据序列Blast比对分析结果，设计逆转录不对称PCR扩增引物和寡核苷酸检测探针。采用Trizol试剂从人静脉血中提取总RNA，经过逆转录和不对称PCR扩增得到的PCR产物与排列有寡核苷酸探针的芯片杂交，杂交后的芯片经激光共聚焦扫描分析。结果：SARS阳性样品与寡核苷酸芯片杂交后出现阳性杂交信号，具有不同二级结构的寡核苷酸探针杂交信号强度不一。结论：寡核苷酸芯片适于快速准确、平行大量地检测SARS病毒。     

硅酸修饰的抗体微阵列玻片对红细胞的血型抗体吸附能力的研究

目的探讨用硅酸修饰的微阵列玻璃基片,对红细胞血型抗体的吸附能力.方法将玻片用硅酸钠溶液处理,经酸化以形成硅酸,然后用抗红细胞血型抗体(抗-A、抗-B、抗-AB、抗-D)包被,以对应红细胞作反应指示物,以检测经硅酸修饰后的玻片对红细胞血型抗体的吸附能力,以及血型抗体的免疫活性.结果抗红细胞血型抗体可以牢固的结合在硅酸修饰过的玻片上,并且可以与其对应的红细胞抗原进行免疫学反应.结论经硅酸修饰的玻片对血型抗体有很高的结合效力,同时抗体仍保持很高的免疫活性,因而可进一步充当蛋白质抗体微阵列基片.     

痘苗病毒寡核苷酸检测芯片的设计及研制

目的 对痘苗病毒进行寡核苷酸检测芯片的初步研究，为建立寡核苷酸芯片检测该类病毒提供初步研究依据。方法 根据痘苗病毒特异基因设计寡核苷酸探针，人工合成探针后制备寡核苷酸芯片。在病毒感染的不同阶段提取病毒样品DNA及阴性样品DNA，采用限制性显示技术标记，标记样品与芯片杂交后，用Agilent芯片扫描仪检测杂交结果。结果 芯片与病毒样品杂交有较强的杂交信号，而与阴性样品杂交除阳性探针外均无信号。结论 病毒样品与阴性样品杂交信号区别明显，在病毒感染的各个时段也都有明显的杂交信号，反映了寡核苷酸芯片具有较高的特异性和灵敏度。     

Capture of cervical exfoliative cells on a glass slide coated by 3-glycidyloxypropyl trimethoxysilane and poly-L-lysine

A new modification method for glass slides was developed and applied to make ThinPrep Pap smears, in order to increase the adhesion ability of cervical exfoliative cells. 3-glycidyloxypropyl trimethoxysilane (GOPS) was coated on the glass slides firstly on the slides, then poly-L-lysine (PLL) was covalently modified onto the above epoxy-terminated slides to form GOPS-PLL double decorated slides. The modified slides were characterized using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The cell adhesion ability effect was tested and compared with traditional PLL coated slides by fixing the cervical exfoliative cells on the double adorned slides. The control test was conducted by the bare glass slides unmodified. The cell morphology of cervical exfoliative cells adhered on different slides was observed under the microscope after Papanicolaou staining. The number of cervical exfoliative cells on the unmodified slides, PLL coated slides and GOPS-PLL coated slides was 1030±300, 3283±226 and 4119±280 (n=12), respectively. The data among the three different modification methods showed significant differences (one-way analysis of variance, ANOVA test, P<0.05). The cell capturing effect of the GOPS-PLL slide was the best among the three different modified slides. In addition, the GOPS-PLL slide could enhance the uniformity of the adhered cells and be widely applied to the ThinPrep system for cervical carcinoma screening to increase the accuracy rate of diagnosis.     

痘苗病毒寡核苷酸检测芯片的设计及研制

目的对痘苗病毒进行寡核苷酸检测芯片的初步研究,为建立寡核苷酸芯片检测该类病毒提供初步研究依据。方法根据痘苗病毒特异基因设计寡核苷酸探针,人工合成探针后制备寡核苷酸芯片。在病毒感染的不同阶段提取病毒样品DNA及阴性样品DNA,采用限制性显示技术标记,标记样品与芯片杂交后,用Agilent芯片扫描仪检测杂交结果。结果芯片与病毒样品杂交有较强的杂交信号,而与阴性样品杂交除阳性探针外均无信号。结论病毒样品与阴性样品杂交信号区别明显,在病毒感染的各个时段也都有明显的杂交信号,反映了寡核苷酸芯片具有较高的特异性和灵敏度。     

The detection of HBV DNA with gold nanoparticle gene probes

Objective:Gold nanoparticle Hepatitis B virus (HBV) DNA probes were prepared, and their application for HBV DNA measurement was studied. Methods:Alkanethiol modified oligonucleotide was bound with self-made Au nanoparticles to form nanoparticle HBV DNA gene probes, through covalent binding of Au-S. By using a fluorescence-based method, the number of thiol-derivatized, single-stranded oligonucleotides and their hybridization efficiency with complementary oligonucleotides in solution was determined. With the aid of Au nanoparticle-supported mercapto-modified oligonucleotides serving as detection probes, and oligonucleotides immobilized on a nylon membrane surface acting as capturing probes,HBV DNA was detected visually by sandwich hybridization based on highly sensitive aggregation and silver staining. The modified nanoparticle HBV DNA gene probes were also used to detect the HBV DNA extracted from serum in patients with hepatitis B. Results:Compared with bare Au nanoparticles, oligonucleotide modified nanoparticles had a higher stability in NaCl solution or under high temperature environment and the absorbance peak of modified Au nanoparticles shifted from 520nm to 524nm. For Au nanoparticles, the maximal oligonucleotide surface coverage of hexaethiol 30-mer oligonucleotide was (132 ± 10) oligonucleotides per nanoparticle, and the percentage of hybridization strands on nanoparticles was (22 ± 3% ). Based on a two-probe sandwich hybridization/nanoparticle amplification/silver staining enhancement method, Au nanoparticle gene probes could detect as low as 10-11 mol/L composite HBV DNA molecules on a nylon membrane and the PCR products of HBV DNA visually. As made evident by transmission electron microscopy, the nanoparticles assembled into large network aggregates when nanoparticle HBV DNA gene probes were applied to detect HBV DNA molecules in liquid. Conclusion:Our results showed that successfully prepared Au nanoparticle HBV DNA gene probes could be used to detect HBV DNA directly. The detection-visuallized method has many advantages, including high sensitivity,simple operation and low cost. This technique has potential applications in many fields, especially in multi-gene detection chips.     

用于HIV诊断的Oligo基因芯片研制

目的研制快速筛查诊断HIV病毒的Oligo芯片.方法以HIV-1B亚型U26942全基因组序列为靶序列,利用 DNA Club、Oligo 6.0、BLAST、Alignment等生物信息学软件,设计高度特异、长度均一、具近似熔解温度(Tm)的Oligo探针,并制备成Oligo芯片.B(U26942)、C(U46016)、F(AF075703)、G(AF061640)四种亚型质粒分别经RD-梯度PCR与随机特异性引物PCR,经荧光标记后与芯片杂交,扫描芯片后进行统计学分析.结果与结论获得22条60 mer探针,Oligo芯片特异性、敏感性及稳定性好,为进一步应用提供了条件.     

Research on preparation of mycobacterium tuberculosis DNA microarray]

OBJECTIVE: To optimize and develop the technique for mycobacterium tuberculosis DNA microarray. METHODS: The process included preparation of DNA samples, spotting and past-spotting treatment of arrays. DNA microarrays were prepared by spotting fluorescence labeled PCR products of target genes onto specially treated glass slides with robotics. The fluorescent signals before and after treatment were scanned with a scanner, and the DNA attachment rate was calculated from the obtained data by software. RESULTS: A foundation for optimizing the conditions of Mycobacterium tuberculosis DNA microarrays has been laid. The support aldehyde-modified glass slide is useful for anchoring DNA at Some distance. DMSO as spotting solution is of benefit to preparation of Mycobacterium tuberculosis DNA microarray. Drying the chip at 37 degrees C temperature after spotting can enhance the DNA combination rate. CONCLUSION: Several key steps of this technique have been optimized. This study has provided a foundation for optimizing the DNA attachment conditions in creating mycobacterium tuberculosis DNA microarray.