Synthesis and molecular cloning of antimicrobial peptide chromogranin A N-46 gene using conventional PCR

Background: Chromogranin A N-46 (CGA-N46) is an antimicrobial peptide (AMP) consisting of partial N terminus of human chromogranin A. Objective: Synthesis and molecular cloning the gene encoding CGA-N46. Materials and methods: The coding sequence of CGA-N46 gene was synthesized using free bioinformatics tool (Gene2Oligo) that allows the development of in vitro gene synthesis. In this study two methods have been established, stepwise PCR and One Step Simplified Gene Synthesis (SGS) methods. The gene was obtained in stepwise PCR by three steps: 1st PCR to generate short DNA fragments using specific oligonucleotides, 2nd step was the overlap extension PCR (OE-PCR) to bond the short DNA fragments and the final step was the whole gene assembly using amplification primers. Nevertheless, the SGS method was achieved by only one step PCR with various concentrations of oligonucleotides (1000, 500, 250, 100, 50 and 25 nM) and amplification primers (400 nM). In order to achieve a DNA library for future studies, the obtained CGA-N46 gene was cloned into pCRII TOPO TA cloning vector to create a recombinant vector which was preserved in Escherichia soli (E. coli) TOP10 bacterial stock using transformation process. Results: The stepwise PCR method generated a sharp band at expected MW (150 bp). In addition, the SGS method gave one faint band at 1 mu M of oligonucleotides. Digestion and PCR analysis revealed that the CGA-N46 gene was successfully cloned in pCR-II TOPO TA cloning vector.