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

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.

Detection of RTX toxin genes in gram-negative bacteria with a set of specific probes

The family of RTX (RTX representing repeats in the structural toxin) toxins is composed of several protein toxins with a characteristic nonapeptide glycine-rich repeat motif. Most of its members were shown to have cytolytic activity. By comparing the genetic relationships of the RTX toxin genes we established a set of 10 gene probes to be used for screening as-yet-unknown RTX toxin genes in bacterial species. The probes include parts of apxIA, apxIIA, and apxIIIA from Actinobacillus pleuropneumoniae, cyaA from Bordetella pertusis, frpA from Neisseria meningitidis, prtC from Erwinia chrysanthemi, hlyA and elyA from Escherichia coli, aaltA from Actinobacillus actinomycetemcomitans and lktA from Pasteurella haemolytica. A panel of pathogenic and nonpathogenic gram-negative bacteria were investigated for the presence of RTX toxin genes. The probes detected all known genes for RTX toxins. Moreover, we found potential RTX toxin genes in several pathogenic bacterial species for which no such toxins are known yet. This indicates that RTX or RTX-like toxins are widely distributed among pathogenic gram-negative bacteria. The probes generated by PCR and the hybridization method were optimized to allow broad-range screening for RTX toxin genes in one step. This included the binding of unlabelled probes to a nylon filter and subsequent hybridization of the filter with labelled genomic DNA of the strain to be tested. The method constitutes a powerful tool for the assessment of the potential pathogenicity of poorly characterized strains intended to be used in biotechnological applications. Moreover, it is useful for the detection of already-known or new RTX toxin genes in bacteria of medical importance.