Use of immobilized PCR primers to generate covalently immobilized DNAs for in vitro transcription/translation reactions.
Use of immobilized PCR primers to generate covalently immobilized DNAs for in vitro transcription/translation reactions.
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DOI：
10.1093/nar/28.2.e5
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发表时间：
2000-01-15
影响因子：
14.9
通讯作者：
Chrisey, L A
中科院分区：
文献类型：
--
作者：
Andreadis, J D;Chrisey, L A
我们开发了一种新的生化方法，利用聚合酶链式反应同时扩增并固定目的基因到不溶性颗粒上。这种方法通过使用化学交联剂，在DNA合成过程中或DNA合成后直接将两个PCR引物中的一个共价连接到颗粒表面。在PCR扩增过程中可以直接实现目的基因的固定化，只需一条珠状结合的引物和一条可溶的引物。或者，这可以在聚合酶链式反应后，通过将加入到扩增子中的化学修饰的引物共价连接到活化的颗粒上来实现。所有含有适当调节区的固定化DNA模板都完全可以进行转录和翻译反应，其中一些可以在系列反应中重复使用。最成功的策略是使用氨基硅烷化的可控孔玻璃微珠，通过碳二亚胺化学将其偶联到磷酸化的底物上。在聚合酶链式反应过程中，这些珠状结合的引物被用来产生附加的DNA模板，这些模板可以被收集并重新用于至少七次顺序转录反应，而不会显著降低效率。这种方法还成功地应用于使用单一的固定引物对多个DNA模板进行扩增、转录和翻译。结果表明，基于聚合酶链式反应的聚合酶链式反应/固定化方法比聚合酶链式反应后的化学固定化方法更持久，而且便于在单管中进行连续的聚合酶链式反应、转录和翻译反应。
We have developed a novel biochemical method to simultaneously amplify and immobilize a target gene onto insoluble particles using PCR. This method employs the covalent attachment of one of two PCR primers to a particle surface either directly during DNA synthesis of the primer or post-DNA synthesis, through the use of chemical crosslinkers. Immobilization of the target gene can be achieved directly during PCR amplification, with one bead-bound primer and one soluble primer. Alternatively, this can be achieved post-PCR, through covalent attachment of a chemically modified primer incorporated into the amplicon to an activated particle. All of the immobilized DNA templates containing appropriate regulatory regions were fully competent for transcription and translation reactions and several could be re-used in serial reactions. The most successful strategy utilized amino-silanized controlled pore glass beads, which were coupled to phosphorylated primers using carbodiimide chemistry. These bead-bound primers were used during PCR to generate attached DNA templates that could be collected and re-used for at least seven sequential transcription reactions without significant loss in efficiency. This method has also been successfully applied to the amplification, transcription and translation of multiple DNA templates using a single, immobilized primer. The combined PCR-based amplification/immobilization method was shown to be more durable than post-PCR chemical immobilization and affords the convenience of performing sequential PCR amplification, transcription and translation reactions in a single tube.