An Improved Method for Prioritizing Polymerase Chain Reaction (PCR) Primer Design in Sanger Sequencing

Designing primers is a challenge for researchers to design primers that can amplify sequence genes. Primer design has several parameters considered, namely primer length, thermal melting temperature (TM), and guanine-cytosine content (GC-content). Therefore, bioinformatics is needed to help design primers more informatively by showing the parameter values of the primer design. One of the programs used to design primers is Primer3Plus. Primer3Plus can create several primer designs. Thus, Primer3Plus requires an expert to choose a primer pair that fits with the primer length, TM, and GC content parameters. This research proposed an improvised method for prioritizing design primer pairs from the Primer3Plus results to obtain the most suitable primer pairs according to the specified parameters. Before being processed into Primer3Plus, the gene sequence is split into several sequences with a length of around 1000bp to suit Sanger Sequencing (SGS). Therefore, the primers designed from each sequence can amplify all parts of the gene sequence. The experiment used the sequence gene Sorghum bicolor phytochrome A (PHYA) gene from the National Center for Biotechnology Information (NCBI) and implemented using Python with the Biopython library. The results get the best pair primers from each sequence based on primer length, TM, and GC content parameters with a product size of 900-1000bp for PCR and sequencing using SGS.