Andrella Zuelke


Amplifying a Rare Edited mRNA of Trypanosoma cruzi

Trypanosoma cruzi is a vector-borne protozoan parasite that causes Chagas disease in Central and South America. Basic biological processes in the organism are not well understood. Establishing fully edited translatable sequences through high-throughput RNA sequencing is difficult due to the unique, and processive yet stochastic editing of its mitochondrial RNA. I have worked to develop a new methodology of generating higher levels of the target translatable mRNAs for sequencing. In order to obtain sufficient amounts of these edited sequences, I first had to generate mitochondrial cDNA of two edited genes from the total RNA population in the organism. This single stranded cDNA was then circularized through an optimized 5’ adenylation and ligation procedure. Finally, rolling circle amplification using REPLI-g amplified the circularized cDNA products. PCR amplification using primers specific for circularized products allowed for identification of any successful products. After visualization on an agarose gel, it was found that the fully edited version of RPS12 was successfully converted to cDNA, circularized, and amplified to a concentration suitable for sequencing. Further optimization of primers and rolling circle amplification should result in a reliable system for generating T. cruzi translatable mRNA for sequencing. 

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