title: Model of elongation of short DNA sequence by thermophilic DNA polymerase under isothermal conditions.
abstract: Short DNA sequences, especially those that are repetitive or palindromic, can be used as the seeds for synthesis of long DNA by some DNA polymerases in an unusual manner. Although several elongation mechanisms have been proposed, there is no well-established model that explains highly efficient elongation under isothermal conditions. In the present study, we analyzed the elongation of nonrepetitive sequences with distinct hairpins at each end. These DNAs were elongated efficiently under isothermal conditions by thermophilic Vent (exo(-)) DNA polymerase, and the products were longer than 10 kb within 10 min of the reaction. A 20-nucleotide DNA with only one hairpin was also elongated. Sequence analysis revealed that the long products are mainly tandem repeats of the short seed sequences. The thermal melting temperatures of the products were much higher than the reaction temperature, indicating that most DNAs form duplexes during the reaction. Accordingly, a terminal hairpin formation and self-priming extension model was proposed in detail, and the efficient elongation was explained. Formation of the hairpin at the 5' end plays an important role during the elongation.
authors.affiliation: Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8603, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Kato
authors.forename: Xingguo
authors.initials: X
authors.lastname: Liang
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Asanuma
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D055029:Inverted Repeat Sequences; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides; D013696:Temperature
chemical_list: D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D004259:DNA-Directed DNA Polymerase