Cloning and site-directed mutagenesis in NALP3 gene

Cloning and site-directed mutagenesis in NALP3 gene has been performed mediated by ligation independent cloning vector. Accurate overhang was created in plasmid carrying the NALP3 gene by using NALP3 T436P plasmid DNA in proof reading PCR and the mutated NALP3 gene was successfully inserted to ligation independent vector, pTriEx-6 3C/LIC. Specific band was found in agarose gel electrophoresis followed by colony PCR. In addition to it, mutation was created at the 779 nucleotide position of the NALP3 gene (c.779G>T) which was initiated by mutagenic primers and QuikChange site-directed mutagenesis kit. The mutated sequence was blasted to find out any homology within genome database which showed 92 sequence similarity with several Homo sapiens NLR family. Altogether, these results suggest that our methodological strategies could be used as a potential protocol for studying genetic disorders related to NALP3.