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Using real-time PCR as strategy to evaluate performance of PCR and Sanger sequencing reactions

Abstract:

Sanger sequencing is an indispensable technique for mitochondrial ancestry studies. However, it is a time consuming process that requires several resources to obtain high quality electropherograms. To optimize the efficiency of DNA analysis, we used real-time PCR (qPCR) to evaluate Sanger sequencing reaction performance before capillary electrophoresis. qPCR was performed to amplify Mitochondrial Control Region (CR). We determined that final PCR product quantification can be done using qPCR final fluorescence instead of visualization in agarose gel. Real-time sequencing reactions were done using BigDye Terminator v3.1 and SYBR Safe (Invitrogen); in addition, a dissociation curve step from 65 °C to 95 °C was added at the end of the thermal cycler protocol. The sequencing qPCR products were purified and run in a 3130 Genetic Analyzer (Applied Biosystems). The results demonstrated that fluorescence at 65 °C during the dissociation curve was directly related with DNA quantity in the sequencing qPCR reaction. Furthermore, samples that presented higher fluorescence at 65 °C generated better quality electropherograms. We suggest that electropherogram quality pbkp_rediction before capillary electrophoresis is possible using sequencing qPCR. In this way, it is possible to increase workflow efficiency in laboratory procedures and identify errors during samples processing.