Directed covalent immobilization of aminated DNA probes on aminated plates
Directed covalent immobilization of aminated DNA probes on aminated plates
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DOI：
10.1021/bm0343949
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发表时间：
2004-05-01
影响因子：
6.2
通讯作者：
Fern치ndez-Lafuente, R
中科院分区：
文献类型：
--
作者：
Fuentes, M;Mateo, C;Fern치ndez-Lafuente, R
本文报道了一种新的方法，通过在寡核苷酸探针的3'端引入一个氨基，使DNA探针固定在氨基化的微量滴定板上，该微量滴定板用葡聚糖活化。该方法是基于使用的异功能的葡聚糖作为一个长的和多功能的间隔臂共价连接到胺化的表面能够固定DNA寡核苷酸。固定仅通过引入探针3'端的氨基发生，而不涉及固定中的DNA碱基，确保探针的全长可用于杂交。这些具有固定的寡核苷酸探针的板能够与互补DNA靶分子杂交。特制的异功能性天冬氨酸-天冬氨酸-葡聚糖与使用乙酸酐对载体上剩余的伯氨基进行化学封闭一起避免了DNA在板表面上的非特异性吸附。因此，使用这些活化的板（研究探针浓度、温度和板活化时间对所获得的信号的影响）。优化了胺化DNA探针的共价固定，获得的灵敏度与使用商业生物素-链霉亲和素系统获得的灵敏度相似。新的DNA平板在非常剧烈的实验条件下（90%甲酰胺，100 ℃，100 mM NaOH中30分钟）稳定。
A new protocol that enables the immobilization of DNA probes on aminated micro-titer plates activated with aldehyde-dextran via an amino group artificially introduced in the 3' end of the oligonucleotide probe is reported in this work. The method is based on the use of hetero-functional-dextran as a long and multifunctional spacer arm covalently attached to an aminated surface capable of immobilizing DNA oligonucleotides. The immobilization occurred only via the amino introduced in the 3' end of the probe, with no implication of the DNA bases in the immobilization, ensuring that the full length of the probe is available for hybridization. These plates having immobilized oligonucleotide probes are able to hybridize complementary DNA target molecules. The tailor-made hetero-functional aldehyde-aspartic-dextran together with the chemical blocking of the remaining primary amino groups on the support using acetic anhydride avoid the nonspecific adsorption of DNA on the surface of the plates. Using these activated plates, (studying the effect of the probe concentration, temperature, and time of the plate activation on the achieved signal), thus. the covalent immobilization of the aminated DNA probe was optimized, and the sensitivity obtained was similar to that achieved using commercial biotin-streptavidin systems. The new DNA plates are stable under very drastic experimental conditions (90% formamide, at 100degreesC for 30 minor in 100 mM NaOH).