Efficient One-Step Fusion PCR Based on Dual-Asymmetric Primers and Two-Step Annealing
Efficient One-Step Fusion PCR Based on Dual-Asymmetric Primers and Two-Step Annealing
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基于双不对称引物和两步退火的高效一步融合PCR
DOI：
10.1007/s12033-017-0050-7
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发表时间：
2018
影响因子：
2.6
通讯作者：
A. Thygesen
中科院分区：
文献类型：
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作者：
Yilan Liu;Jinjin Chen;A. Thygesen
融合PCR基因剪接是一种用途广泛的方法，特别是在合成生物学中。本文描述了一种利用双不对称引物和两步退火（ODT）方法，通过一轮融合PCR快速拼接两个片段的方法。在此过程中，不对称中间片段在早期产生。然后，在随后的循环中杂交，作为目标全长产物的模板。对引物比、延伸温度、循环次数等工艺参数进行了优化。此外，该方法产生的融合产物已成功应用于无缝基因组编辑。用这种方法融合两个碎片的时间不到0.5天。该方法有望以更高的效率促进各种复杂的基因工程项目。
Gene splicing by fusion PCR is a versatile and widely used methodology, especially in synthetic biology. We here describe a rapid method for splicing two fragments by one-round fusion PCR with a dual-asymmetric primers and two-step annealing (ODT) method. During the process, the asymmetric intermediate fragments were generated in the early stage. Thereafter, they were hybridized in the subsequent cycles to serve as template for the target full-length product. The process parameters such as primer ratio, elongation temperature and cycle numbers were optimized. In addition, the fusion products produced with this method were successfully applied in seamless genome editing. The fusion of two fragments by this method takes less than 0.5 day. The method is expected to facilitate various kinds of complex genetic engineering projects with enhanced efficiency.
DOI：
10.1007/978-1-62703-764-8_12
发表时间：
2014-01-01
期刊：
DNA CLONING AND ASSEMBLY METHODS
影响因子：
--
作者：
Nakamura, Mikiko;Suzuki, Ayako;Akada, Rinji
通讯作者：
Akada, Rinji