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A novel amplification strategy for genotyping with liquid chromatography-electrospray ionization mass spectrometry.
MedLine Citation:
PMID:  23034565     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
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.
Authors:
Beate Beer; Kaarel Krjutškov; Robert Erb; Triin Viltrop; Herbert Oberacher
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-10-4
Journal Detail:
Title:  The Analyst     Volume:  -     ISSN:  1364-5528     ISO Abbreviation:  Analyst     Publication Date:  2012 Oct 
Date Detail:
Created Date:  2012-10-4     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0372652     Medline TA:  Analyst     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Affiliation:
Institute of Legal Medicine, Innsbruck Medical University, Muellerstrasse 44, 6020 Innsbruck, Austria. herbert.oberacher@i-med.ac.at.
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