External and semi-internal controls for PCR amplification of homologous sequences in mixed templates.
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.
Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Allmas alle 5, 75007 Uppsala, Sweden. Electronic address: Elena.Kalle@terrapretation.com.
Elena
E
Kalle
Alexander
A
Gulevich
Christopher
C
Rensing
D000180:Acrylic Resins / Q000737:chemistry; D004269:DNA, Bacterial / Q000302:isolation & purification*; D018744:DNA, Plant / Q000302:isolation & purification*; D058645:Denaturing Gradient Gel Electrophoresis; D006109:Poaceae / Q000235:genetics / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012015:Reference Standards; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
D000180:Acrylic Resins; D004269:DNA, Bacterial; D018744:DNA, Plant; C016680:polyacrylamide gels
BSA; CTAB; DG-DGG; DG-DGGE; DGGE; DHPLC; DMSO; Denaturing Gradient Gel Electrophoresis; Double-Gradient Denaturing Gradient Gel Electrophoresis; Guidelines; Microbial diversity; OTU; PAAG; PCR; PCR bias; PCR protocol; Polymerase; Quality assurance; TMA; bovine serum albumin; cetyltrimethylammonium bromide; denaturing high-performance liquid chromatography; dimethyl sulfoxide; double-gradient denaturing gradient gel; operational taxonomic unit; polyacrylamide gel; polymerase chain reaction; tetramethylammonium chloride