Technical Application:
2-Step PCR Pathogen DNA Detection


​​Background
Pathogen detection by PCR analysis has become an area of significant interest and product development.  Commercial systems exist that can detect panels of pathogens using both one-step and two-step PCR approaches but utilize enclosed systems (a sealed consumable). While enclosed systems are ideal for point-of-care applications, they are typically expensive, inflexible, and do not utilize existing laboratory equipment. Reagent kits exist that are compatible with standard laboratory equipment, but all use single-step PCR to avoid the added steps and contamination risks associated with two-step PCR.  Since the Alluvia system can enable the development of two-step PCR pathogen detection reagent kits for standard laboratory equipment, we investigated if the added PCR step will significantly improve PCR results...

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 when PCR products (typically purified or just diluted) become templates for a second round of PCR. More often, the primers for the second PCR round are positioned internally ("nested") relative to the primers used in the first round in order to improve PCR specificity 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 reactions....  

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...