Amplification and cloning of NR3 region of NMDA receptor subunit / Nurul Buda Ahmad Shahrir

This thesis focused on the amplification and verification of NR3 subunits of the excitatory glutamate receptor, ionotropic N-methyl-0-aspartate. The functional unit of cation channel gated is believed to be a tetrametric complex structure which consists of NR1, NR2 and NR3 subunits. The glycine binding to NR3 subunit is required for the opening of the ion channel. We cloned the mice NR3A and NR3B which has only minor differences in homology to rat NR3 subunit. The RNA was extracted from adult albino mice brain tissue and converted into cDNA by using two steps Reverse-Transcriptase Polymerase Chain Reaction. The NR3A and NR3B were amplified by using specific primer which has been synthesized based on previous studies. The NR3A and NR3B were constructed into pcDNA and pTARGET respectively and cloned into E.coli. Both plasmid then were extracted from E.coli and purified before they were sent for sequencing and verification by using NCBI. Only NR3B was successfully constructed into pT AR GET showing specific region from residue TM1 until S2. Eventhough NR3A was not successfully constructed into pCDNA but the NR3A PCR product was blasted and attributed to specific region C-terminal phosphorylation site.