A Single-Step PCR For the Amplification of Highly Repetitive PolyQ Stretches

Author(s): Mukul Jain, Pooja Bhadoriya , Bano Saidullah, Geeta Kaicker

Abstract: The expression, misfolding and aggregation of long-repetitive polyglutamine (polyQ) residues contribute to a number of neurodegenerative diseases where, protein-aggregates are formed, their degradation process is still not understood. Thus, to understand these diseases, new model systems are required. We exploited Dictyostelium discoideum, a non-mammalian system and successfully expressed varying polyQ repeat lengths, which could help delineate the pathways. Traditionally, the (CAG)/(CAA) repeats (coding for polyQ) used in different organism are PCR amplified from disease-related genes like, in case of E. coli, human DRPLA (containing the different number of (CAG)/(CAA) repeats) cDNA was used as a template. To avoid the use of different templates for the synthesis of varying (CAG)/(CAA) repeats, we have made use of a single step PCR, using only one template for the synthesis of varying lengths of (CAG)/(CAA) repeats. The advantages of this technique are as below: • A single step PCR amplification enables generation of variable polyQ repeats. • This protocol reduces the time and consumption of raw materials for preparing varying lengths of (CAG)/(CAA) repeats. • This may help identify the molecular mechanism(s) of pathogenesis of polyQ-repeat diseases and be a useful screen to identify potential therapeutic compounds in a non-mammalian model.

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