An efficient unnatural base pair for PCR amplification.
Expansion of the genetic alphabet by an unnatural base pair system provides a powerful tool for modern biotechnology. As an alternative to previous unnatural base pairs, we have developed a new pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitropyrrole (Pn), which functions in DNA amplification. Pn more selectively pairs with Ds in replication than another previously reported pairing partner, pyrrole-2-carbaldehyde (Pa). The nitro group of Pn efficiently prevented the mispairing with A. High efficiency and selectivity of the Ds-Pn pair in PCR amplification were achieved by using a substrate mixture of the gamma-amidotriphosphate of Ds and the usual triphosphates of Pn and the natural bases, with Vent DNA polymerase as a 3' to 5' exonuclease-proficient polymerase. After 20 cycles of PCR, the total mutation rate of the Ds-Pn site in an amplified DNA fragment was approximately 1%. PCR amplification of DNA fragments containing the unnatural Ds-Pn pair would be useful for expanded genetic systems in DNA-based biotechnology.
Hirao
Ichiro
I
Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan. ihirao@riken.jp
Mitsui
Tsuneo
T
Kimoto
Michiko
M
Yokoyama
Shigeyuki
S
D020029:Base Pairing; D001483:Base Sequence; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009705:Nucleosides / Q000737:chemistry; D009711:Nucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*
D009705:Nucleosides; D009711:Nucleotides