A mass spectrometry-based approach for identifying novel DNA polymerase substrates from a pool of dNTP analogues.
There has been a long-standing interest in the discovery of unnatural nucleotides that can be incorporated into DNA by polymerases. However, it is difficult to predict which nucleotide analogs will prove to have biological relevance. Therefore, we have developed a new screening method to identify novel substrates for DNA polymerases. This technique uses the polymerase itself to select a dNTP from a pool of potential substrates via incorporation onto a short oligonucleotide. The unnatural nucleotide(s) is then identified by high-resolution mass spectrometry. By using a DNA polymerase as a selection tool, only the biologically relevant members of a small nucleotide library can be quickly determined. We have demonstrated that this method can be used to discover unnatural base pairs in DNA with a detection threshold of < or =10% incorporation.
Kincaid
Kristi
K
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA.
Kuchta
Robert D
RD
D020029:Base Pairing; D002853:Chromatography, Liquid; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D009705:Nucleosides / Q000302:isolation & purification; D009838:Oligodeoxyribonucleotides / Q000032:analysis / Q000737:chemistry*; D013698:Templates, Genetic
D009705:Nucleosides; D009838:Oligodeoxyribonucleotides; D004259:DNA-Directed DNA Polymerase