MS for identification of single nucleotide polymorphisms and MS/MS for discrimination of isomeric PCR products

ESI (electrospray ionization) MS and tandem mass spectrometry (MS/MS) were used for the analysis of single nucleotide polymorphisms (SNPs) and more co mplex genetic variations. Double-stranded (ds) PCR products were studied. P CR products of the proline [5'-x(G(17))- alpha(C-38) x-3'] and arginine var iants [(5'-x(G(17))-x(G(38))x-3'] of the p53 gene are distinguished by an S NP (cytosine or guanine) and were discriminated using both quadrupole and q uadrupole ion trap MS analysis. A 69 bp arginine mutant PCR product [5)-x(C -17)-x(G(38))x-3'] With a negating switch has the same mass as the proline variant but was readily distinguishable on ion trap MS/MS analysis; fragmen ts containing the mutation site, but not the polymorphism, were identified. The 69 bp PCR products were restriction-enzyme-digested, to create 43 bp f ragments. ESI quadrupole ion trap MS/MS analysis of the 43 bp product-ion s pectra readily demonstrated both polymorphism and negating switch sites. MS and MS/MS are powerful and complementary techniques for analysis of DNA. M S can readily distinguish SNPs but MS/MS is required to differentiate isome ric PCR products (same nucleotide composition but different sequence).