کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2090051 1081467 2013 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
External and semi-internal controls for PCR amplification of homologous sequences in mixed templates
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی بیوتکنولوژی یا زیست‌فناوری
پیش نمایش صفحه اول مقاله
External and semi-internal controls for PCR amplification of homologous sequences in mixed templates
چکیده انگلیسی


• Multi-template PCR needs specific amplification controls.
• The characteristics of amplification controls for multi-template PCR are described.
• External control consists of several homologous templates amplified together.
• External control monitors reaction failure and PCR-induced artifacts and biases.
• Semi-internal control monitors the level of inhibition.

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.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Microbiological Methods - Volume 95, Issue 2, November 2013, Pages 285–294
نویسندگان
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