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'-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).
Krahmer
M T
MT
Department of Microbiology & Immunobiology, University of South Carolina, School of Medicine, Columbia 29208, USA.
Walters
J J
JJ
Fox
K F
KF
Fox
A
A
Creek
K E
KE
Pirisi
L
L
Wunschel
D S
DS
Smith
R D
RD
Tabb
D L
DL
Yates
J R
JR
D013058:Mass Spectrometry; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic