title: An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules.
abstract: Toward the expansion of the genetic alphabet, we present an unnatural base pair system for efficient PCR amplification, enabling the site-specific incorporation of extra functional components into DNA. This system can be applied to conventional PCR protocols employing DNA templates containing unnatural bases, natural and unnatural base triphosphates, and a 3'-->5' exonuclease-proficient DNA polymerase. For highly faithful and efficient PCR amplification involving the unnatural base pairing, we identified the natural-base sequences surrounding the unnatural bases in DNA templates by an in vitro selection technique, using a DNA library containing the unnatural base. The system facilitates the site-specific incorporation of a variety of modified unnatural bases, linked with functional groups of interest, into amplified DNA. DNA fragments (0.15 amol) containing the unnatural base pair can be amplified 10(7)-fold by 30 cycles of PCR, with <1% total mutation rate of the unnatural base pair site. Using the system, we demonstrated efficient PCR amplification and functionalization of DNA fragments for the extremely sensitive detection of zeptomol-scale target DNA molecules from mixtures with excess amounts (pmol scale) of foreign DNA species. This unnatural base pair system will be applicable to a wide range of DNA/RNA-based technologies.
authors.affiliation: Systems and Structural Biology Center, RIKEN, Yokohama, Kanagawa 230-0045, Japan.
authors.forename: Michiko
authors.initials: M
authors.lastname: Kimoto
authors.forename: Rie
authors.initials: R
authors.lastname: Kawai
authors.forename: Tsuneo
authors.initials: T
authors.lastname: Mitsui
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
authors.forename: Ichiro
authors.initials: I
authors.lastname: Hirao
mesh_terms: D000480:Alkynes / Q000737:chemistry*; D020029:Base Pairing; D004247:DNA / Q000032:analysis / Q000737:chemistry*; D007093:Imidazoles / Q000737:chemistry*; D009711:Nucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011725:Pyridines / Q000737:chemistry*; D011758:Pyrroles / Q000737:chemistry*
chemical_list: C528341:7-(2-thienyl)imidazo(4,5-b)pyridine; D000480:Alkynes; D007093:Imidazoles; D009711:Nucleotides; D011725:Pyridines; D011758:Pyrroles; D004247:DNA