Pustaka Ilmiah Universitas Padjadjaran

Abstrak

Making of the A3243g Mutant Template Through Site Directed Mutagenesis as Positive Control in PASA-Mismatch Three Bases

Nucleotide mutation at position 3243 that changes adenine to guanine nucleotide (A3243G) on tRNA Leu(UUR) gene in mtDNA is the most discovered mutation and is expressed in phenotype MIDD (Maternally Inherited Diabetes and Deafness) and MELAS (Mitochondrial myopathy, Encephalopathy, Lactic Acidosis, and Stroke-like episodes) . The A3243G mutation was heteroplasmy, so it was difficult to analyze with low levels, especially in the blood. Some of methods have limited sensitivity and potentially cause mistake. In previous research has been used PASA-mismatch three bases, but it was still less validity, because it didn’t have positive control of the A3243G mutant. One of the method commonly used to make mutant template is site directed mutagenesis (SDM).This research aims to make the A3243G mutant template by SDM which will be used as positive control in PASA-mismatch three bases and to determine optimization condition of SDM. Normal mtDNA was isolated from lymphocytes, then adenine base in position 3243 of tRNALeu(UUR) gene was mutated into guanine base and amplified using mutant forward primer Mt3243 F and reverse primer Mt3243 R. Determination of PCR optimum condition was done including varied annealing temperature and mutant primer concentration. Then, to optimize mutant template concentration which can be detected by PASA-mismatch three bases. The presence of the A3243G mutant template was confirmed by direct sequencing. The result of SDM showed the A3243G mutant template with size of 489 bp in the optimize of annealing temperature and ratio of mutant primer to normal primer were 74 OC and 3:1, respectively. Mutant template with 100× optimum dilution can be used as a PASA-mismatch three bases positive control in A3243G mtDNA mutation analysis.