Abstract:
BACKGROUND:Sequencing based mutation screening assays of genes encompassing large numbers of exons could be substantially optimized by multiplex PCR, which enables simultaneous amplification of many targets in one reaction. In the present study, a multiplex PCR protocol originally developed for fragment analysis was evaluated for sequencing based mutation screening of the ornithine transcarbamylase (OTC) and the medium-chain acyl-CoA dehydrogenase (MCAD) genes. METHODS:Single exon and multiplex PCR protocols were applied to generate PCR templates for subsequent DNA sequencing of all exons of the OTC and the MCAD genes. For each PCR protocol and using the same DNA samples, 66 OTC and 98 MCAD sequence reads were generated. The sequences derived from the two different PCR methods were compared at the level of individual signal-to-noise ratios of the four bases and the proportion of high-quality base-signals. RESULTS:The single exon and the multiplex PCR protocol gave qualitatively comparable results for the two genes. CONCLUSIONS:Many existing sequencing based mutation analysis protocols may be easily optimized with the proposed method, since the multiplex PCR protocol was successfully applied without any re-design of the PCR primers and other optimization steps for generating sequencing templates for the OTC and MCAD genes, respectively.
journal_name
Clin Chem Lab Medjournal_title
Clinical chemistry and laboratory medicineauthors
Horn MP,Mäder-Heinemann G,Andrey G,Largiadèr CRdoi
10.1515/CCLM.2009.012subject
Has Abstractpub_date
2009-01-01 00:00:00pages
56-9issue
1eissn
1434-6621issn
1437-4331pii
10.1515/CCLM.2009.012journal_volume
47pub_type
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