External and semi-internal controls for PCR amplification of homologous sequences in mixed templates

Abstract

In a mixed template, the presence of homologous target DNA sequences creates environments that almost inevitably give rise to artifacts and biases during PCR. Heteroduplexes, chimeras, and skewed template-to-product ratios are the exclusive attributes of mixed template PCR and never occur in a single template assay. Yet, multi-template PCR has been used without appropriate attention to quality control and assay validation, in spite of the fact that such practice diminishes the reliability of results. External and internal amplification controls became obligatory elements of good laboratory practice in different PCR assays. We propose the inclusion of an analogous approach as a quality control system for multi-template PCR applications. The amplification controls must take into account the characteristics of multi-template PCR and be able to effectively monitor particular assay performance. This study demonstrated the efficiency of a model mixed template as an adequate external amplification control for a particular PCR application. The conditions of multi-template PCR do not allow implementation of a classic internal control: therefore we developed a convenient semi-internal control as an acceptable alternative. In order to evaluate the effects of inhibitors, a model multi-template mix was amplified in a mixture with DNAse-treated sample. Semi-internal control allowed establishment of intervals for robust PCR performance for different samples, thus enabling correct comparison of the samples. The complexity of the external and semi-internal amplification controls must be comparable with the assumed complexity of the samples. We also emphasize that amplification controls should be applied in multi-template PCR regardless of the post-assay method used to analyze products. (C) 2013 Elsevier B.V. All rights reserved.

Keywords

Microbial diversity; Polymerase; PCR bias; PCR protocol; Quality assurance; Guidelines

Published in

Journal of Microbiological Methods
2013, volume: 95, number: 2, pages: 285-294
Publisher: ELSEVIER SCIENCE BV

SLU Authors

• Kalle Kossio, Elena

  • Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences
    

UKÄ Subject classification

Microbiology
Biochemistry
Molecular Biology

Publication identifier

• DOI: https://doi.org/10.1016/j.mimet.2013.09.014

Permanent link to this page (URI)

https://res.slu.se/id/publ/55143