title: MS for identification of single nucleotide polymorphisms and MS/MS for discrimination of isomeric PCR products.
abstract: 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'-x(G17)-x(C38)x-3'] and arginine variants [(5'-x(Gl7)-x(G38)x-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'-x(C17)-x(G38)x-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 complementary 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).
authors.affiliation: Department of Microbiology & Immunobiology, University of South Carolina, School of Medicine, Columbia 29208, USA.
authors.forename: M T
authors.initials: MT
authors.lastname: Krahmer
authors.forename: J J
authors.initials: JJ
authors.lastname: Walters
authors.forename: K F
authors.initials: KF
authors.lastname: Fox
authors.forename: A
authors.initials: A
authors.lastname: Fox
authors.forename: K E
authors.initials: KE
authors.lastname: Creek
authors.forename: L
authors.initials: L
authors.lastname: Pirisi
authors.forename: D S
authors.initials: DS
authors.lastname: Wunschel
authors.forename: R D
authors.initials: RD
authors.lastname: Smith
authors.forename: D L
authors.initials: DL
authors.lastname: Tabb
authors.forename: J R
authors.initials: JR
authors.lastname: Yates
mesh_terms: D013058:Mass Spectrometry; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic