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• W2153123986 endingPage "5325" @default.
• W2153123986 startingPage "5325" @default.
• W2153123986 abstract "Among numerous available genotyping techniques, mass spectrometry (MS) based methods play a major role in providing high quality genotype data at reasonable costs for research and diagnostics, e.g. for pharmacogenetic applications. Ion-pair reversed-phase liquid chromatography hyphenated to electrospray ionization time-of-flight MS (ICEMS) is, for example, a powerful instrument that allows a direct characterization of complex mixtures of polymerase chain reaction (PCR) amplified DNA fragments. Current limitations of PCR-ICEMS genotyping are mainly concerned with the multiplex PCR set-up. Assay development often requires time-consuming primer design and intensive optimization of PCR conditions. To overcome this restraint, a robust amplification strategy originally combined with arrayed primer extension genotyping was transferred and adapted to ICEMS genotyping. The modifications involved limitation of the primer length, application of two universal sequences and amplification with an appropriate DNA polymerase. To demonstrate the applicability of the novel amplification strategy for ICEMS, a 23-plex pharmacogenetic genotyping assay was developed. After slight optimization steps, an efficient and quantitatively balanced amplification of all targeted markers was achieved, resulting in a convenient characterization of the multiplexed PCR fragments with ICEMS. Expenditure of time, costs and hands-on work associated with assay design and optimization was dramatically lowered compared to previous multiplex PCR-ICEMS assays. The developed 23-plex assay was applied in a pharmacogenetic study including 284 individuals (genotype call rate 99.0%). A total of 399 SNPs were retyped by Sanger sequencing (concordance rate 99.8%). The PCR-ICEMS assay turned out to be an accurate, reliable, cost-effective and a ready-to-use tool for pharmacogenetic genotyping." @default.
• W2153123986 created "2016-06-24" @default.
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• W2153123986 date "2012-01-01" @default.
• W2153123986 modified "2023-09-23" @default.
• W2153123986 title "A novel amplification strategy for genotyping with liquid chromatography-electrospray ionization mass spectrometry" @default.
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• W2153123986 doi "https://doi.org/10.1039/c2an35440c" @default.
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