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  • Title: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the detection of polymerase chain reaction products containing 7-deazapurine moieties.
    Author: Siegert CW, Jacob A, Köster H.
    Journal: Anal Biochem; 1996 Dec 01; 243(1):55-65. PubMed ID: 8954525.
    Abstract:
    Double-stranded DNA fragments of different length with and without the incorporation of 7-deazapurine deoxynucleotides have been prepared via the polymerase chain reaction (PCR) using exo(-)Pseudococcus furiosus DNA polymerase, unmodified primers, and c7-dATP and c7-dGTP as the only purine triphosphates. In spite of the presence of some unmodified purine moieties due to the primers, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed remarkable differences between the modified and unmodified DNA fragments. The incorporation of 7-deazapurine nucleotides resulted in a significant reduction of fragmentation and therefore in increased signal intensities and higher mass resolution. The molecular weights could be determined with an accuracy of up to 0.03%. Mass resolution was sufficient to resolve the (M + H)+ signals of the two single strands of a 7-deazapurine-containing 99-base-pair DNA duplex. Thus, MALDI-TOF mass spectrometry offers a very fast and accurate way to detect and analyze short PCR products sufficient in length for many diagnostic applications without gel electrophoresis and labeling.
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