Polymerase chain reaction (PCR) is a widely used molecular biology technique for amplifying specific DNA sequences. However, amplifying templates with a high GC content (>60 %) poses challenges owing to strong hydrogen bonds and secondary structure formation, hindering DNA polymerase activity and primer annealing. Given these challenges, our study focuses on refining PCR protocols for the nicotinic acetylcholine receptor subunits, pivotal for understanding signal transduction in various organisms and potential important drug targets. We optimized the PCR protocol to efficiently amplify the beta1 and alpha1 subunits of the nicotinic acetylcholine receptor from Ixodes ricinus (Ir-nAChRb1) and Apis mellifera (Ame-nAChRa1). Ir-nAChRb1 and Ame-nAChRa1 have open reading frames of 1743 and 1884 bp, respectively, with overall GC contents of 65 % and 58 %. Various DNA polymerases and organic additives have been evaluated at different annealing temperatures. The tailored protocol incorporated organic additives, such as DMSO and betaine, increased enzyme concentration, and adjusted annealing temperatures. This study demonstrates the importance of a multipronged approach involving various organic molecules, DNA polymerases, PCR conditions, and primer adjustments to overcome the challenges of amplifying GC-rich sequences.