Automated two-column purification of iminobiotin and BrdU-labeled PCR products for rapid cloning: application to genes synthesized by polymerase chain assembly.
Automated two-column purification of iminobiotin and BrdU-labeled PCR products for rapid cloning: application to genes synthesized by polymerase chain assembly.
复制标题
用于快速克隆的亚氨基生物素和 BrdU 标记 PCR 产物的自动两柱纯化：应用于聚合酶链组装合成的基因。
DOI：
10.1093/chromsci/48.2.120
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发表时间：
2010
影响因子：
1.3
通讯作者：
Subramanian,Anuradha
中科院分区：
文献类型：
--
作者：
TerMaat,JoelR;Mamedov,TarlanG;Pienaar,Elsje;Whitney,ScottE;Subramanian,Anuradha
聚合酶链组装（PCA）是基础生物学研究和生物技术应用的有力工具。在过去的几年中，主要进展已经在novogene合成。然而，仍然需要快速和可重复的方法来自动纯化合成的基因。PCA完成后，随后的PCR扩增产物混合物仍含有不需要的较短DNA片段，这阻碍了克隆工作。为了避免繁琐的凝胶纯化，已经开发了自动化的双柱纯化，并与快速PCA结合使用。该系统能够快速合成和分离感兴趣的全长DNA，这对于容易克隆所需DNA片段很重要。在PCR扩增步骤期间，分别使用用亚氨基生物素和溴脱氧尿苷标记物标记的正向和反向引物。然后使用串联的两个亲和/免疫捕获柱对PCR混合物进行自动纯化，以仅分离所需的全长产物。该方法已应用于pUC 19 β-内酰胺酶基因（929 bp）。纯化产物的后续PCR、克隆和测序证明了该技术在获得纯全长基因方面的有效性。纯化也已在其他合成基因上进行，表明其作为一般方法的实用性。
Polymerase chain assembly (PCA) is a powerful tool for basic biological research and biotechnology applications. During the last several years, major advances have been made inde novogene synthesis. However, there is still a need for fast and reproducible methods to automatically purify the synthesized genes. Upon completion of PCA, the subsequent PCR-amplified product mixture still contains undesired shorter DNA fragments that hinder cloning efforts. To avoid tedious gel purification, an automated two-column purification has been developed and used in conjunction with rapid PCA. The system enables fast synthesis and isolation of the full-length DNA of interest, important for facile cloning of desired DNA fragments. During the PCR amplification step, forward and reverse primers tagged with iminobiotin and bromodeoxyuridine labels, respectively, were used. The automated purification was then performed on the PCR mixture using two affinity/immunocapture columns in series to isolate only the desired full-length product. The procedure has been applied to the pUC19 β-lactamase gene (929 bp). Follow-up PCR of the purified product, cloning, and sequencing demonstrated the technique's effectiveness in obtaining the pure full-length gene. The purification has also been performed on other synthesized genes, indicating its utility as a general approach.