Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry protocol for monitoring the progress of enzymatic 13C/15N-labeled DNA syntheses
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文摘
We demonstrate that a simple matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry protocol provides a rapid and accurate method for monitoring the stage and completeness of enzymatic DNA syntheses. A crucial step of these syntheses is to quench the reaction at the desired nucleotide length. This is especially important when expensive, e.g., 13C/15N-labeled DNA segments, are synthesized for multinuclear magnetic resonance purposes to reveal detailed structural information. The analyses of three templates for a human telomeric 22-mer, a wild type, and a mutant human c-MYC promoter (18- and 22-mer) DNA and their reactions with the 3′–5′ exo− Klenow fragment of DNA polymerase I demonstrate the usefulness of our protocol. Small amounts of samples (ca. 1–2 μl each) were taken from the reaction mixtures at different times and analyzed promptly by MALDI-TOF, applying our successive on-plate desalting method that eliminates the insensitivity of the MALDI technique at high salt content. The progress of the reaction was detected by monitoring the relative intensity ratios of ions corresponding to the desired products and the primer–template complexes. The effectiveness of NH3 cleavage leading to final products was also followed by MALDI-TOF in successful enzymatic reactions.