Advantages of Thermococcus kodakaraenis (KOD) DNA Polymerase for PCR-mass spectrometry based analyses.
The advantages of the thermostable DNA polymerase from Thermococcus kodakaraensis (KOD) are demonstrated for PCR amplification with subsequent detection by mass spectrometry. Commonly used DNA polymerases for PCR amplification include those from Thermus aquaticus (Taq) and Pyrococcus furiosus (Pfu). A 116 base-pair PCR product derived from a vWA locus was amplified by Taq, Pfu, or KOD DNA polymerase and compared by agarose gel electrophoresis and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). KOD DNA polymerase demonstrated a 2- to 3-fold increase in PCR product formation compared to Pfu or Taq, respectively, and generated blunt-ended PCR product which allows facile interpretation of the mass spectrum. Additionally, we demonstrate the advantage of using high magnetic fields to obtain unit resolution of the same 116 base pair (approximately 72 kDa) PCR product at high m/z.
Benson
Linda M
LM
Department of Biochemistry and Molecular Biology and the W. M. Keck FT-ICR Mass Spectrometry Laboratory, Mayo Proteomics Research Center, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Null
Allison P
AP
Muddiman
David C
DC
D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D005583:Fourier Analysis; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization; D019914:Taq Polymerase / Q000737:chemistry; D019712:Thermococcus / Q000201:enzymology*; D014841:von Willebrand Factor / Q000737:chemistry
D014841:von Willebrand Factor; C072146:Pfu DNA polymerase; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase