Stable-isotope-assisted MALDI-TOF mass spectrometry for accurate determination of nucleotide compositions of PCR products

In parallel with a large-scale sequencing effort, the human genome project will need next-generation tools for accurate and efficient analyses of the enormous pool of DNA sequences. Such analyses are required for (a) validati on of DNA sequences, (b) comparison of a parent (known) sequence with a rel ated (unknown) sequence, and (c) characterization of sequence polymorphisms in various genes, especially those associated with genetically inherited h uman diseases. Here, we report a novel method that combines stable isotope C-13/N-15 labeling of PCR products of the target sequences with analysis of the mass shifts by mass spectrometry (MS). The mass shift due to the label ing of a single type of nucleotide (i.e., A, T, G, or C) reveals the number of that type of nucleotide in a given DNA fragment. Using this technique, we have accurately determined nucleotide compositions of DNA fragments. The method has also been applied to score a known single-nucleotide polymorphi sm (SNP), The comparisons of nucleotide compositions determined by our meth od among homologous sequences are useful in sequence validation, sequence c omparison, and characterizations of sequence polymorphisms.