A Rapid, low cost, and efficient method for isolation of high quality mitochondrial DNA from Oryza sativa

Abstract

A rapid and inexpensive protocol for isolation of mitochondrial DNA from Oryza sativa with negligible genomic DNA contamination is developed without use of density-gradients materials. Mitochondria were isolated from rice seedlings in an in-house lysis buffer containing sucrose followed by DNase I treatment to remove nuclear DNA. Modified CTAB method was used to isolate mitochondrial DNA from isolated mitochondria. The presence of mitochondrial DNA was confirmed by using selective amplification of mtDNA specific genes. PCR amplification was observed in all genes except β-actin gene. In addition, Sanger sequencing and gene mapping to reference gene sequences in public database was performed to confirm the presence of mitochondrial DNA. The mapping analysis showed 99.71% similarity with mitochondrial DNA. The protocol demonstrated high specificity and yielded high purity mitochondrial DNA. It is concluded that the protocol described here will be beneficial for scientific communities by providing a cheap and robust mitochondrial DNA isolation protocol for potential applications.