Genotyping single nucleotide polymorphisms using intact polymerase chain reaction products by electrospray quadrupole mass spectrometry.

G (G <--> C on the opposite strand), were each detected by a 40.0 Da change upon ESI quadrupole MS analysis. Using known PCR products as standards, the genotypes determined for 10 human samples corresponded with restriction fragment length polymorphism (RFLP) analysis. Cytosine/thymine (T) transitions, designated C <--> T (G <--> A on the opposite strand), were also genotyped by ESI-MS. This SNP is discriminated by a 15.0 Da change on one strand (C <--> T) and a 16.0 Da change on the other (G <--> A). Appropriate sample preparation and instrumental configuration (including heated sample inlet syringe and MS source), to limit adducts, are both vital for successful ESI quadrupole MS analysis of intact PCR products.
(Copyright 2001 John Wiley & Sons, Ltd.) -->
Grant Information: R21 HG01810-01 United States HG NHGRI NIH HHS
Substance Nomenclature: 0 (Nucleotides)
9007-49-2 (DNA)
Entry Date(s): Date Created: 20010914 Date Completed: 20011101 Latest Revision: 20071114
Update Code: 20221213
DOI: 10.1002/rcm.435
PMID: 11555877
Autor: Walters JJ; Department of Microbiology and Immunology, University of South Carolina, School of Medicine, Columbia, SC 29208, USA., Muhammad W, Fox KF, Fox A, Xie D, Creek KE, Pirisi L
Jazyk: angličtina
Zdroj: Rapid communications in mass spectrometry : RCM [Rapid Commun Mass Spectrom] 2001; Vol. 15 (18), pp. 1752-9.
DOI: 10.1002/rcm.435
Abstrakt: Both single nucleotide polymorphisms (SNPs) and mutations are commonly observed in the gene encoding the tumor suppressor protein, p53. SNPs occur at specific locations within genes whereas mutations may be distributed across large regions of genes. When determining nucleotide differences, mass spectrometry is the only method other than Sanger sequencing which offers direct structural information. Electrospray ionization (ESI) quadrupole mass spectrometry (MS) analysis of intact polymerase chain reaction (PCR) products was performed following a simple purification and on-line heating to limit ion adduction. The PCR products were amplified directly from genomic DNA rather than plasmids, as in our previous work. Two known polymorphisms of the p53 gene were genotyped. A cytosine (C) or guanine (G) transversion, designated C <--> G (G <--> C on the opposite strand), were each detected by a 40.0 Da change upon ESI quadrupole MS analysis. Using known PCR products as standards, the genotypes determined for 10 human samples corresponded with restriction fragment length polymorphism (RFLP) analysis. Cytosine/thymine (T) transitions, designated C <--> T (G <--> A on the opposite strand), were also genotyped by ESI-MS. This SNP is discriminated by a 15.0 Da change on one strand (C <--> T) and a 16.0 Da change on the other (G <--> A). Appropriate sample preparation and instrumental configuration (including heated sample inlet syringe and MS source), to limit adducts, are both vital for successful ESI quadrupole MS analysis of intact PCR products.
(Copyright 2001 John Wiley & Sons, Ltd.)
Databáze: MEDLINE
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