Characterization and Optimization Of pH PCR In Tube Using Seminaphtharhodafluor (SNARF) Indicator Dye

This Semester-in-Residence Project was conducted at DNA Electronics, Inc. in the R&D Molecular Biology Department located in Carlsbad, California. DNA Electronics is a molecular diagnostics company seeking to develop real-time diagnostics products. The objective of this project was to optimize the current pH PCR formulation using Seminaphtharhodafluor (SNARF) pH dye as an indicator for PCR amplification. This will be used as a screening assay development tool for optimizing and characterizing factors leading to the greatest pH change during PCR. Specific aims were to develop a written protocol using SNARF dye as an indicator in pH PCR, looking specifically at the effects of buffering capacity, reagent concentration, and amplicon size on the delta pH and Ct signal. This technology shows amplification based on the release of hydrogen ions during PCR. It was necessary to design a solution for transforming raw data from a real-time PCR machine and applying an algorithm to graph the change in pH during the amplification step of PCR and create Ct values. Although using the original formulation produced the largest signal noise delta pH, new primers were designed for the amplification of a longer amplicon which generated an even larger delta pH signal. This final formulation will be transferred to Product Development for further testing.