MS for Identification of Single Nucleotide Polymorphisms and MS/MS for Discrimination of Isomeric PCR Products

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• 作者：Mark T. Krahmer ; James J. Walters ; Karen F. Fox ; Alvin Fox ; Kim E. Creek ; Lucia Pirisi ; David S. Wunschel ; Richard D. Smith ; David L. Tabb ; and John R. Yates ; III
• 刊名：Analytical Chemistry
• 出版年：2000
• 出版时间：September 1, 2000
• 年：2000
• 卷：72
• 期：17
• 页码：4033 - 4040
• 全文大小：118K
• 年卷期：v.72,no.17(September 1, 2000)
• ISSN：1520-6882

文摘

ESI (electrospray ionization) MS and tandem mass spectrometry (MS/MS) were used for the analysis of singlenucleotide polymorphisms (SNPs) and more complexgenetic variations. Double-stranded (ds) PCR productswere studied. PCR products of the proline [5'-x(G₁₇)x(C₃₈)x-3'] and arginine variants [(5'-x(G₁₇)-x(G₃₈)x-3']of the p53 gene are distinguished by an SNP (cytosine orguanine) and were discriminated using both quadrupoleand quadrupole ion trap MS analysis. A 69 bp argininemutant PCR product [5'-x(C₁₇ )-x(G₃₈)x-3'] with a negating switch has the same mass as the proline variant butwas readily distinguishable on ion trap MS/MS analysis;fragments containing the mutation site, but not the polymorphism, were identified. The 69 bp PCR products wererestriction-enzyme-digested, to create 43 bp fragments.ESI quadrupole ion trap MS/MS analysis of the 43 bpproduct-ion spectra readily demonstrated both polymorphism and negating switch sites. MS and MS/MS arepowerful and complementary techniques for analysis ofDNA. MS can readily distinguish SNPs but MS/MS isrequired to differentiate isomeric PCR products (samenucleotide composition but different sequence).