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 Bpml. Bpml 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.
- Single nucleotide polymorphisms
- Type IIS restriction endonuclease
ASJC Scopus subject areas
- Applied Microbiology and Biotechnology
- Molecular Medicine
- Biomedical Engineering