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.

journal_name

Clin Chem Lab Med

authors

Horn MP,Mäder-Heinemann G,Andrey G,Largiadèr CR

doi

10.1515/CCLM.2009.012

subject

Has Abstract

pub_date

2009-01-01 00:00:00

pages

56-9

issue

1

eissn

1434-6621

issn

1437-4331

pii

10.1515/CCLM.2009.012

journal_volume

47

pub_type

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