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Abstract

A method for determining the presence of polymorphisms, including mutations, in nucleic acids by using mass spectrometry is presented. The method requires amplification of the nucleic acid region to be analyzed followed by analysis by mass spectrometry and comparison of the obtained spectrum with spectra obtained from wild-type sequences and/or sequences known to contain the polymorphism. Differences between the spectra, either the appearance or disappearance of one or more peaks indicating a change in mass or a change in the height of one or more peaks indicating a change in the amount of nucleic acid of a specific mass, indicate the presence of a polymorphism. Variations of the method involve digestion of the amplified nucleic acid, e.g., by using restriction enzymes, nucleases or chemical methods, prior to analysis by mass spectrometry. The method can be applied to any type of nucleic acid including genomic DNA, CDNA and RNA. The method is especially well suited for performing routine genetic screening on a large scale for mutations known to be associated with a disease. The method is also appropriate for determining the presence of polymorphisms for other purposes, e.g., for genotyping or screening for mutations in a positional cloning project. A preferred approach is to amplify then digest the nucleic acid and then to analyze it via matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using a neodymium-garnet laser and a 3-hydroxypicolinic acid matrix.

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