Dengue Virus (DENV) is a positive single stranded RNA virus presented in four serotypes, DEN1V, DEN2V, DEN3V and DEN4V. Its structure is composed of three structural proteins, capsid, precursor membrane (prM) and envelope, and seven non structural (NS) proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. NS5 has a vital role in the replication of flavivirus. In this project, four different experimental procedures based on standard protocols, were used to create mutations in NS5 of DEN2V. There was no growth in original plates after overnight observation in experiment 1 and in experiment 2, competent cells were streaked on LB ampicillin plates but overgrowth reported in the result after overnight observation. Experience.3 the result of sequencing demonstrated that there was no change in the sequence of NS5 (DEN2V) as compared to the original sequence. Out of all the experimental procedures, optimized mutational results in experiment. 4 i-e., insertion of stop codon TAA and TGA were achieved in NS5 of DEN2V Replicon. It is clear from the sequencing analysis of NS5 of DEN2V Replicon after site directed mutagenesis that two stop codons (TAA & TGA) are inserted instead of AAA & AGA in the original SN5 sequence of DEN2V.