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 complex genetic variations. Double-stranded (ds) PCR products were studied. PCR products of the proline [5'-(mathematical expression not reproducible in ASCII) (G(sub 17))- (mathematical expression not reproducible in ASCII) (C(sub 38)) (mathematical expression not reproducible in ASCII)-3'] and arginine variants [(5'-(mathematical expression not reproducible in ASCII)(G(sub 17))- (mathematical expression not reproducible in ASCII) (G(sub 38)) (mathematical expression not reproducible in ASCII)-3'] of the p53 gene are distinguished by an SNP (cytosine or guanine) and were discriminated using both quadrupole and quadrupole ion trap MS analysis. A 69 bp arginine mutant PCR product [5'-(mathematical expression not reproducible in ASCII)(C(sub 17))- (mathematical expression not reproducible in ASCII)(G(sub 38)) (mathematical expression not reproducible in ASCII)-3'] with a negating switch has the same mass as the proline variant but was readily distinguishable on ion trap MS/MS analysis; fragments containing the mutation site, but not the polymorphism, were identified. The 69 bp PCR products were restriction-enzyme-digested, to create 43 bp fragments. ESI quadrupole ion trap MS/MS analysis of the 43 bp product-ion spectra readily demonstrated both polymorphism and negating switch sites. MS and MS/MS are powerful and complemenmor techniques for analysis of DNA. MS can readily distinguish SNPs but MS/MS is required to differentiate isomeric PCR products (same nucleotide composition but different sequence).