Format

Send to

Choose Destination
See comment in PubMed Commons below
Nucleic Acids Res. 2009 Feb;37(2):e14. doi: 10.1093/nar/gkn956. Epub 2008 Dec 10.

An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules.

Author information

1
Systems and Structural Biology Center, RIKEN, Yokohama, Kanagawa 230-0045, Japan.

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.

PMID:
19073696
PMCID:
PMC2632903
DOI:
10.1093/nar/gkn956
[Indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Silverchair Information Systems Icon for PubMed Central
    Loading ...
    Support Center