Detection of DNA Sequence Variations in Homo- and Heterozygous Samples via Molecular Mass Measurements by Electrospray Ionization Time-of-Flight Mass Spectrometry

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• 作者：Herbert Oberacher ; Harald Niederstä ; tter ; Bruno Casetta ; and Walther Parson
• 刊名：Analytical Chemistry
• 出版年：2005
• 出版时间：August 1, 2005
• 年：2005
• 卷：77
• 期：15
• 页码：4999 - 5008
• 全文大小：281K
• 年卷期：v.77,no.15(August 1, 2005)
• ISSN：1520-6882

文摘

The potential of ion-pair reversed-phase high-performanceliquid chromatography on-line hyphenated to electrosprayionization time-of-flight mass spectrometry for the characterization of polymerase chain reaction (PCR) amplifiednucleic acids was evaluated. For that purpose, a "SNPtoolbox" was constructed by cloning and PCR-mediatedsite-directed in vitro mutagenesis at nucleotide position(ntp) 16 519 of a sequence-verified fragment of thehuman mitochondrial genome (ntps 15 900-599). Confirmatory sequencing demonstrated that within the sequences of the clones one and the same base was mutatedto all other bases. Using these clones or equimolarmixtures of these clones as PCR templates, 51-401-bp-long amplicons were generated, which were used todetermine the upper size limits of PCR products for theunequivocal detection of sequence variations in homo-and heterozygous samples. Based on the high massspectrometric performance of the applied time-of-flightmass spectrometer, the unequivocal genotyping of allkinds of single base exchanges in PCR amplicons fromheterozygous samples with lengths up to 254 base pairs(bp) was demonstrated. Considering homozygous samples,the successful genotyping of single base substitutions inup to 401-bp-long PCR products was possible. Consequently, the described hyphenated technique representsone of the most powerful mass spectrometric genotypingassays available today.