开发可设计灵活粘性末端的 PCR 引物,实现长 DNA 片段的高效连接

IF 4.2 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY RSC Chemical Biology Pub Date : 2024-02-26 DOI:10.1039/D3CB00212H
Kohei Nomura, Kaoru Onda, Hirotaka Murase, Fumitaka Hashiya, Yukiteru Ono, Goro Terai, Natsuhisa Oka, Kiyoshi Asai, Daisuke Suzuki, Naho Takahashi, Haruka Hiraoka, Masahito Inagaki, Yasuaki Kimura, Yoshihiro Shimizu, Naoko Abe and Hiroshi Abe
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引用次数: 0

摘要

我们开发了经过化学修饰的 PCR 引物,通过在磷酸分子上引入光可分解基团,可以设计出灵活的粘性末端。在寡核苷酸合成的强碱处理和 PCR 反应的热循环过程中,含有邻硝基苄基光可切除基团和位于苄基位置的叔丁基的核酸衍生物是稳定的。使用含有这些核酸衍生物的引物进行的聚合酶链式反应证实,在引入光可裂解基团位点前后,链延伸反应在第 1 位完全停止。粘端为 50 个碱基的 2 kbp 和 3 kbp DNA 片段被成功连接,连接率高达 77%。用这种方法构建了一个质粒。最后,我们用这种方法构建了 48.5 kbp 的λ噬菌体 DNA,这是使用基于限制性酶的方法难以实现的。7 天后,我们确认生成了所需长度的 DNA。虽然效率还有待提高,但化学修饰的 PCR 引物有可能补充酶法的不足,并成为一种 DNA 连接技术。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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Development of PCR primers enabling the design of flexible sticky ends for efficient concatenation of long DNA fragments†

We developed chemically modified PCR primers that allow the design of flexible sticky ends by introducing a photo-cleavable group at the phosphate moiety. Nucleic acid derivatives containing o-nitrobenzyl photo-cleavable groups with a tert-butyl group at the benzyl position were stable during strong base treatment for oligonucleotide synthesis and thermal cycling in PCR reactions. PCR using primers incorporating these nucleic acid derivatives confirmed that chain extension reactions completely stopped at position 1 before and after the site of the photo-cleavable group was introduced. DNA fragments of 2 and 3 kbp, with sticky ends of 50 bases, were successfully concatenated with a high yield of 77%. A plasmid was constructed using this method. Finally, we applied this approach to construct a 48.5 kbp lambda phage DNA, which is difficult to achieve using restriction enzyme-based methods. After 7 days, we were able to confirm the generation of DNA of the desired length. Although the efficiency is yet to be improved, the chemically modified PCR primer offers potential to complement enzymatic methods and serve as a DNA concatenation technique.

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来源期刊
CiteScore
6.10
自引率
0.00%
发文量
128
审稿时长
10 weeks
期刊最新文献
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