Method of preparing an equimolar DNA mixture for one-step DNA assembly of over 50 fragments

Kenji Tsuge, Yukari Sato, Yuka Kobayashi, Maiko Gondo, Masako Hasebe, Takashi Togashi, Masaru Tomita, Mitsuhiro Itaya

研究成果: Article査読

13 被引用数 (Scopus)

抄録

In the era of synthetic biology, techniques for rapidly constructing a designer long DNA from short DNA fragments are desired. To realize this, we attempted to establish a method for one-step DNA assembly of unprecedentedly large numbers of fragments. The basic technology is the Ordered Gene Assembly in Bacillus subtilis (OGAB) method, which uses the plasmid transformation system of B. subtilis. Since this method doesn' t require circular ligation products but needs tandem repeat ligation products, the degree of deviation in the molar concentration of the material DNAs is the only determinant that affects the efficiency of DNA assembly. The strict standardization of the size of plasmids that clone the DNA block and the measurement of the block in the state of intact plasmid improve the reliability of this step, with the coefficient of variation of the molar concentrations becoming 7%. By coupling this method with the OGAB method, one-step assembly of more than 50 DNA fragments becomes feasible.

本文言語English
論文番号10655
ジャーナルScientific reports
5
DOI
出版ステータスPublished - 2015 5月 20

ASJC Scopus subject areas

  • 一般

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