title: Highly specific unnatural base pair systems as a third base pair for PCR amplification.
abstract: Toward the expansion of the genetic alphabet of DNA, we present highly efficient unnatural base pair systems as an artificial third base pair for PCR. Hydrophobic unnatural base pair systems between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) were fine-tuned for efficient PCR, by assessing the amplification efficiency and fidelity using different polymerases and template sequence contexts and modified Px bases. Then, we found that some modifications of the Px base reduced the misincorporation rate of the unnatural base substrates opposite the natural bases in templates without reducing the Ds-Px pairing selectivity. Under optimized conditions using Deep Vent DNA polymerase, the misincorporation rate was extremely low (0.005%/bp/replication), which is close to that of the natural base mispairings by the polymerase. DNA fragments with different sequence contexts were amplified ∼10(10)-fold by 40 cycles of PCR, and the selectivity of the Ds-Px pairing was >99.9%/replication, except for 99.77%/replication for unfavorable purine-Ds-purine motifs. Furthermore, >97% of the Ds-Px pair in DNA survived in the 10(28)-fold amplified products after 100-cycle PCR (10 cycles repeated 10 times). This highly specific Ds-Px pair system provides a framework for new biotechnology.
authors: RIKEN Systems and Structural Biology Center (SSBC), 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
authors: Rie
authors: R
authors: Yamashige
authors: Michiko
authors: M
authors: Kimoto
authors: Yusuke
authors: Y
authors: Takezawa
authors: Akira
authors: A
authors: Sato
authors: Tsuneo
authors: T
authors: Mitsui
authors: Shigeyuki
authors: S
authors: Yokoyama
authors: Ichiro
authors: I
authors: Hirao
mesh_terms: D020029:Base Pairing; D007093:Imidazoles / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011725:Pyridines / Q000737:chemistry*; D011758:Pyrroles / Q000737:chemistry*; D017422:Sequence Analysis, DNA
chemical_list: C528341:7-(2-thienyl)imidazo(4,5-b)pyridine; D007093:Imidazoles; D011725:Pyridines; D011758:Pyrroles