GENOTYPING BY MASS-SPECTROMETRIC ANALYSIS OF SHORT DNA FRAGMENTS

A method has been developed to produce small DNA fragments from PCR pr oducts for analysis of defined DNA variations by mass spectrometry. Th e genomic region to be analyzed is PCR-amplified with primers containi ng a sequence for the type IIS restriction endonuclease Bpml. Bpml dig estion of the resultant PCR products yields fragments as small as seve n bases, which are then analyzed by electrospray ionization mass spect rometry. The approach was validated using seven different variants wit hin the APC tumor suppressor gene, in which a perfect correlation was obtained with DNA sequencing. Both the sense and antisense strands wer e analyzed independently, and several variants can be analyzed simulta neously. These results provide the basis for a generally applicable an d highly accurate method that directly queries the mass of variant DNA sequences.