Test of the potential of a dATP surrogate for sequencing via MALDI-MS.
1-(2'-Deoxy-beta-d-ribofuranosyl)-3-nitropyrrole phosphate was incorporated into a DNA decamer and analyzed via matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The extent and composition of the various fragment peaks were compared with those in the MALDI-MS spectrum of dT4AT5. The nitropyrrole-containing oligomer proved to be more robust. Two different DNA template assays were then used to attempt to identify DNA replicating enzymes that would incorporate the corresponding triphosphate, i.e. 1-(2'-deoxy-beta-d-ribofuranosyl)-3-nitropyrrole triphosphate (dXTP). It was shown that dXTP was not incorporated by some enzymes and it inhibited others. However, DNA polymerase I Klenow fragment and avian myeloblastosis virus reverse transcriptase incorporated dXTP in place of dATP and then replicated the template overhang in the usual way. The potential of dXTP as a surrogate for dATP in DNA sequencing with MALDI-MS analysis is discussed.
Jacutin
S
S
Department of Chemistry, Texas A&M University, College Station, TX 77843-3255, USA.
Zhang
A J
AJ
Russell
D H
DH
Gibbs
R A
RA
Burgess
K
K
D004256:DNA Polymerase I / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D009711:Nucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D011758:Pyrroles / Q000138:chemical synthesis / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D012190:Retroviridae / Q000201:enzymology; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D019914:Taq Polymerase / Q000378:metabolism; D013698:Templates, Genetic
C110411:1-(2'-deoxyribofuranosyl)-3-nitropyrrole triphosphate; D003838:Deoxyadenine Nucleotides; D009711:Nucleotides; D011758:Pyrroles; D019914:Taq Polymerase; C034970:bacteriophage T7 induced DNA polymerase; D012194:RNA-Directed DNA Polymerase; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; C026600:2'-deoxyadenosine triphosphate