Mechanism of DNA elongation during de novo DNA synthesis.
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.
Liang
Xingguo
X
Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Chikusa, Nagoya 464-8603, Japan. liang@mol.nagoya-u.ac.jp
Kato
Tomohiro
T
Asanuma
Hiroyuki
H
D004247:DNA / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000096:biosynthesis* / Q000737:chemistry*
D009838:Oligodeoxyribonucleotides; D004247:DNA; D004259:DNA-Directed DNA Polymerase