Abstract
A method has been developed to produce small DNA fragments from PCR products for analysis of defined DNA variations by mass spectrometry. The genomic region to be analyzed is PCR-amplified with primers containing a sequence for the type IIS restriction endonuclease BpmI. BpmI digestion of the resultant PCR products yields fragments as small as seven bases, which are then analyzed by electrospray ionization mass spectrometry. The approach was validated using seven different variants within the APC tumor suppressor gene, in which a perfect correlation was obtained with DNA sequencing. Both the sense and antisense strands were analyzed independently, and several variants can be analyzed simultaneously. These results provide the basis for a generally applicable and highly accurate method that directly queries the mass of variant DNA sequences.
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Acknowledgements
Financial support for this work was provided by the Clayton Fund; grants P01 ES06052, NIEHS Center P30 ES03819, NCI Center P30 CA06973; NCI grants CA43460, CA57345, and CA 62924; the Lucille P. Markey Foundation in Cellular and Molecular Medicine; and the Association for International Cancer Research grant 94275. Contributions from P.E.J. and M.D.F. were carried out in the Department of Environmental Health Sciences, Johns Hopkins University, during a leave of absence from the International Agency for Research on Cancer, from whom P.E.J. was in receipt of a Special Training Award. K.W.K. received research funding from Genzyme Molecular Oncology (Genzyme). K.W.K. and B.V. are consultants to Genzyme. This University and researchers (K.W.K. and B.V.) own Genzyme stock, which is subject to certain restrictions under University policy. The terms of this arrangement are being managed by the University in accordance with its conflict of interest policies.
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Laken, S., Jackson, P., Kinzler, K. et al. Genotyping by mass spectrometric analysis of short DNA fragments. Nat Biotechnol 16, 1352–1356 (1998). https://doi.org/10.1038/4333
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DOI: https://doi.org/10.1038/4333
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