Technical Application:
2-Step PCR Pathogen DNA Detection


​​Background
Pathogen detection by PCR analysis has become an area of significant interest and product development. Many commercial systems can detect large panels of pathogens but utilize expensive closed systems, allowing the user little opportunity to use their existing equipment or modify the biochemistry to their needs. Reagent kits exist for open formats, but typically require significant upfront sample preparation steps. In this study we utilized KlenTaq (DNA Polymerase Technologies, Inc.) to amplify DNA in a mixture of 20% human blood to model crude sample preparation techniques. We then demonstrate it is possible to achieve much clearer gel electrophoresis results and overcome qPCR signal inhibition by performing a second step of nested PCR...

Technical Application:
2-Step qPCR using the Alluvia System


Background
The Alluvia system is intended to enable 2-step qPCR and subsequent loading on an electrophoresis gel while keeping the PCR products continuously contained. 2-step PCR is defined as performing two rounds of PCR where the products of the first round of PCR (typically purified or just diluted) become the template for the second round of PCR. More often, the primers for the second round are positioned internally ("nested") relative to the primers used in the first round to improve PCR specificity. Specificity is improved because it is unlikely that the second set of primers will amplify the same unwanted region as the first set of primers. Nesting is particularly useful when the first round of PCR is prone to generating unwanted products, for example, multiplex PCR reaction...  

Technical Application:
2-Step PCR DNA Methylation Detection

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​​Background
DNA methylation is a form of epigenetics that is widely studied in development and cancer research...The most common methods used to determine the DNA methylation pattern involve treating a DNA sample with bisulfite, which converts all non-methylated C bases to U(T) residues, while leaving methylated C bases intact. Differentiation of C or T bases can subsequently be determined by biochemical methods such as PCR, sequencing, or DNA melting analysis...Draht et al. (Clinical Epigenetics 8:44, 2016) report that two steps of PCR utilizing a preamplification with non-MSP primers followed by MSP can be more robust than a single MSP step, particularly when the samples derive from FFPE tissue samples. In this study we compared the sensitivity of single-step MSP vs. two-step MSP...