MATRIX-ASSISTED LASER DESORPTION IONIZATION TIME-OF-FLIGHT MASS-SPECTROMETRY FOR THE DETECTION OF POLYMERASE CHAIN-REACTION PRODUCTS CONTAINING 7-DEAZAPURINE MOIETIES/

Double-stranded DNA fragments of different length with and without the incorporation of 7-deazapurine deoxynucleotides have been prepared vi a the polymerase chain reaction (PCR) using exo(-)Pseudococcus furiosu s DNA polymerase, unmodified primers, and c(7)-dATP and c(7)-dGTP as t he only purine triphosphates. In spite of the presence of some unmodif ied purine moieties due to the primers, matrix-assisted laser desorpti on/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed re markable differences between the modified and unmodified DNA fragments . The incorporation of 7-deazapurine nucleotides resulted in a signifi cant reduction of fragmentation and therefore in increased signal inte nsities and higher mass resolution. The molecular weights could be det ermined with an accuracy of up to 0.03%. Mass resolution was sufficien t to resolve the (M+H)(+) signals of the two single strands of a 7-dea zapurine-containing 99-base-pair DNA duplex. Thus, MALDI-TOF mass spec trometry offers a very fast and accurate way to detect and analyze sho rt PCR products sufficient in length for many diagnostic applications without gel electrophoresis and labeling. (C) 1996 Academic Press, Inc .