从头合成DNA过程中DNA延伸的机制。

Mechanism of DNA elongation during de novo DNA synthesis.

作者信息

Liang Xingguo, Kato Tomohiro, Asanuma Hiroyuki

机构信息

Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Chikusa, Nagoya 464-8603, Japan.

出版信息

Nucleic Acids Symp Ser (Oxf). 2008(52):411-2. doi: 10.1093/nass/nrn209.

DOI:10.1093/nass/nrn209

PMID:18776428

Abstract

Under isothermal conditions, short oligodeoxynucleotides (ODNs) were elongated to long DNA by Vent(exo(-)), a thermophilic DNA polymerase, in the presence of dNTPs. Short ODNs (14-28 nt) were designed to form hairpin structures based on the sequence we obtained from de novo DNA synthesis in the presence of restriction enzyme Tsp509I. As short as 14-nt-long DNA could be elongated to longer than 20000 nucleotides by Vent(exo(-)) at 65 degrees C in 1 h. The high efficiency of elongation at very low concentration (<1 nM) supported the THF-SPE (terminal hairpin formation and self-priming extension) mechanism we purposed for DNA elongation during de novo DNA synthesis. The hairpin structure forms at a DNA duplex end as a self-priming complex, followed by strand displacement extension to longer DNA. The highly efficient elongation attributes to the successive repetition of the process of THF-SPE.

摘要

在等温条件下，嗜热DNA聚合酶Vent(exo(-))在脱氧核苷三磷酸（dNTPs）存在的情况下，将短链寡脱氧核苷酸（ODNs）延伸成长链DNA。短链ODNs（14 - 28个核苷酸）基于我们在限制性内切酶Tsp509I存在下从头DNA合成所获得的序列设计形成发夹结构。在65摄氏度下，1小时内，短至14个核苷酸长的DNA能被Vent(exo(-))延伸至超过20000个核苷酸。在极低浓度（<1 nM）下的高效延伸支持了我们提出的在从头DNA合成过程中DNA延伸的THF-SPE（末端发夹形成和自我引发延伸）机制。发夹结构在DNA双链末端作为自我引发复合物形成，随后通过链置换延伸成长链DNA。高效延伸归因于THF-SPE过程的连续重复。