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Eur. J. Mass Spectrom. 1, 479 - 485 (1995) |
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DNA base-pair substitutions detected in double-stranded DNA with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry | ||
W.H. Benner and J.M. Jaklevic |
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ABSTRACT: | ||
We demonstrate the utility of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for identifying the presence or absence of single base-pair substitutions in a short section of the cystic fibrosis gene. The analysis was performed in two ways: (i) the molecular weight of known lengths of single-stranded DNA from a section of this gene was determined by mass spectrometry, and (ii) short complementary strands from the identical section of the gene were denatured and the difference in molecular weight of the strands was determined. Oligonucleotides corresponding to the wild type sequence were synthesized. The G551D or the R553X mutations were created by introducing single-base substitutions in this oligonucleotide. We recreated double-stranded DNA by annealing each of these three oligonucleotides to complementary oligonucleotides. The MALDI mass spectrometric technique, operating at a mass resolution approaching 1000, correctly identified five of these six oligonucleotides. The most reliable identification was obtained by measuring the difference in molecular weight of the denatured strands. The difference in molecular weight of the complementary strands comprising the wild type, the G551D and the R553X is 52.2 Da, 83.2 Da and 21.2 Da, respectively. The MALDI analysis shows promise as a rapid way to detect the presence of base-pair substitutions without the need for sequencing. | ||
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