title: Mutation screening of the medium-chain acyl-CoA dehydrogenase (MCAD) and the ornithine transcarbamylase (OTC) genes by multiplex PCR amplification and sequencing.
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.
authors.affiliation: Molecular Diagnostics DOLS, Inselspital, Bern University Hospital and University of Bern, Bern, Switzerland.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Horn
authors.forename: Gabriela
authors.initials: G
authors.lastname: Mäder-Heinemann
authors.forename: Gisela
authors.initials: G
authors.lastname: Andrey
authors.forename: Carlo R
authors.initials: CR
authors.lastname: Largiadèr
mesh_terms: D042964:Acyl-CoA Dehydrogenase / Q000235:genetics*; D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D009154:Mutation; D009954:Ornithine Carbamoyltransferase / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D042964:Acyl-CoA Dehydrogenase; D009954:Ornithine Carbamoyltransferase