构建单链 DNA 分离系统。

Construction of a system for single-stranded DNA isolation.

机构信息

School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China.

Key Laboratory of Systems Bioengineering of Ministry of Education, Tianjin University, Tianjin, 300072, China.

出版信息

Biotechnol Lett. 2020 Sep;42(9):1663-1671. doi: 10.1007/s10529-020-02905-1. Epub 2020 May 5.

DOI:10.1007/s10529-020-02905-1

PMID:32372267

Abstract

OBJECTIVE

The system of Strep-Tactin and StrepII tag-SSB proteins binding (ST-SSB) was established to isolate the purified single-stranded DNA in a single step with low cost and high efficiency.

RESULTS

We demonstrate that in the presence of large amounts of dsDNA, the ssDNA binding specificity of Escherichia coli (E. coli) single stranded DNA binding (EcSSB) protein was stronger than gene-5-protein (g5p). ST-SSB system relies on the affinity between Strep-Tactin, StrepII tag-SSB protein and ssDNA in binding buffer. Here, we successfully isolated the purified ssDNA from mixed DNA (ds- and ss-DNA form) samples and asymmetric polymerase chain reaction (aPCR) products. This system can purify ssDNA in a single tube within 1 h, and the recovery efficiency of purified ssDNA was around 60%.

CONCLUSIONS

The ST-SSB system has obvious advantages of high efficiency and one-step purification to recycle any ssDNA.

摘要

目的

建立链霉亲和素和 StrepII 标签-单链结合蛋白（ST-SSB）系统，以低成本、高效率的方式一步法分离纯化单链 DNA。

结果

我们证明，在大量 dsDNA 存在的情况下，大肠杆菌（E. coli）单链 DNA 结合蛋白（EcSSB）的 ssDNA 结合特异性强于基因 5 蛋白（g5p）。ST-SSB 系统依赖于链霉亲和素、StrepII 标签-SSB 蛋白和结合缓冲液中 ssDNA 之间的亲和力。在这里，我们成功地从混合 DNA（ds 和 ss-DNA 形式）样品和不对称聚合酶链反应（aPCR）产物中分离出纯化的 ssDNA。该系统可以在 1 小时内于单管内纯化 ssDNA，纯化 ssDNA 的回收率约为 60%。