Mutation screening of the medium-chain acyl-CoA dehydrogenase (MCAD) and the ornithine transcarbamylase (OTC) genes by multiplex PCR amplification and sequencing

Horn, MP; Mäder-Heinemann, G; Andrey, G; Largiadèr, CR (2009). Mutation screening of the medium-chain acyl-CoA dehydrogenase (MCAD) and the ornithine transcarbamylase (OTC) genes by multiplex PCR amplification and sequencing. Clinical chemistry and laboratory medicine, 47(1), pp. 56-9. Berlin: Walter de Gruyter 10.1515/CCLM.2009.012

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Department of Haematology, Oncology, Infectious Diseases, Laboratory Medicine and Hospital Pharmacy (DOLS) > Institute for Immunology (discontinued)
04 Faculty of Medicine > Department of Haematology, Oncology, Infectious Diseases, Laboratory Medicine and Hospital Pharmacy (DOLS) > Institute of Clinical Chemistry

UniBE Contributor:

Horn, Michael Peter and Largiadèr, Carlo Rodolfo

ISSN:

1434-6621

Publisher:

Walter de Gruyter

Submitter:

Factscience Import

Date Deposited:

04 Oct 2013 15:13

Last Modified:

25 Oct 2019 18:41

Publisher DOI:

10.1515/CCLM.2009.012

Web of Science ID:

000263224200010

BORIS DOI:

10.7892/boris.32210

URI:

https://boris.unibe.ch/id/eprint/32210 (FactScience: 197203)

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