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Poly-L-Lysine玻片在寡核苷酸芯片制备中的改进
引用本文:吴清华 马文丽 石嵘 郭秋野 张宝 李凌 张海燕 郑文岭. Poly-L-Lysine玻片在寡核苷酸芯片制备中的改进[J]. 第一军医大学学报, 2004, 24(11): 1236-1241
作者姓名:吴清华 马文丽 石嵘 郭秋野 张宝 李凌 张海燕 郑文岭
作者单位:[1]第一军医大学分子生物学研究所,广东广州510515 [2]广州总医院分子肿瘤学研究所,广东广州510010
摘    要: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。

关 键 词:寡核苷酸芯片 制备工艺 多聚左旋赖氨酸包被玻片 芯片检测

Oligonucleotide microarray preparation using enhanced poly-L-lysine glass slides]
Qing-hua Wu,Wen-li Ma,Rong Shi,Qiu-ye Guo,Bao Zhang,Ling Li,Hai-yan Zhang,Wen-ling Zheng. Oligonucleotide microarray preparation using enhanced poly-L-lysine glass slides][J]. Journal of First Military Medical University, 2004, 24(11): 1236-1241
Authors:Qing-hua Wu  Wen-li Ma  Rong Shi  Qiu-ye Guo  Bao Zhang  Ling Li  Hai-yan Zhang  Wen-ling Zheng
Affiliation:Institute of Molecular Biology, Southern Medical University, Guangzhou 510515, China. wuqh@fimmu.com
Abstract:OBJECTIVE: To modify conventional poly-L-lysine coating for oligonucleotide microarray preparation so as to enhance the sensitivity of the microarray. METHOD: The proposed chemical approaches included silanizing the slides with 3-glycid-oxypropyltrimethoxysilane (GOPS) after cleaning, followed by slide coating with polymers (poly-L-lysine) that was covalently bound to the modified glass. Subsequent attachment of the oligonucleotide to the modified slide surface was achieved after 1,4-phenylene diisothiocyanate (PDITC) activation of the surface. Various experiments were carried out, such as the immobilization efficiency and hybridization assays to test the modified slides, which were then used tentatively in the preparation of microarrays for SARS coronavirus detection. RESULS: The improved surface had high immobilization efficiency, good uniformity and satisfactory hybridization efficiency, better than those slides with conventional poly-L-lysine coating. In addition, such modified slides rendered the microarrays more resistant to consecutive probing/stripping cycles. CONCLUSION: The modified slide surface is satisfactory to immobilize unmodified oligonucleotide by covalent binding, which enhances not only the sensitivity of the prepared oligonucleotide microarray but also the binding of the oligonucleotide to the slide surface.
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