A Rapid, low cost, and efficient method for isolation of high quality mitochondrial DNA from Oryza sativa
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J. Crop Sci. Biotech. 2015 (September) 18 (3) : 155 ~ 160 DOI No. 10.1007/s12892-014-0095-0 RESEARCH ARTICLE
A Rapid, Low Cost, and Efficient Method for Isolation of High Quality Mitochondrial DNA from Oryza sativa Abhinav Chaudhary*, Spandan Chaudhary, Arpita Ghosh, Srinivas Vuduthala, K. M. Singh, Surendra K Chikara Xcelris Labs Limited, Old Premchand Nagar Road, Opp, Satyagrah Chhavani, Bodakdev, Ahmedabad, Gujarat, India Received: September 10, 2014 / Revised: October 21, 2014 / Accepted: February 25, 2015 Ⓒ Korean Society of Crop Science and Springer 2015
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. Key words : β-actin, DNA extraction, Mitochondria, Oryza sativa, sequencing, sucrose gradient.
Introduction In recent years, there has been an increase in the number of researchers focusing on mitochondria. The availability of mitochondrial genomes of many plant species such as carrot, rice, wheat, cucumber, bamboo, and tobacco has increased our understanding of mitochondrial genome organization and its functional roles in development and productivity of plants. Plant mitochondria are the main site of the ATP synthesis and play an important role in nitrogen assimilation, amino acids biosynthesis, plant development, productivity, fertility, and disease resistance (Millar et al. 2007). Mitochondria have been studied for cytoplasmic male sterility (Zhang et al. 2012), abiotic stress tolerance (Amirsadeghi et al. 2007), alternative energy pathways in plants (Mackenzie and McIntosh 1999) etc. However, isolating pure mitochondrial DNA is often difficult for many studies due to mitochondrial Abhinav Chaudhary (
) E-mail: [email protected] Address: Xcelris Labs Limited, Old Premchand Nagar Road, Opp, Satyagrah Chhavani, Bodakdev, Ahmedabad, Gujarat, India
The Korean Society of Crop Science
physiology. (Isolation of Mitochondrial DNA, 2001) Plant mitochondria is about 1% of the total cellular content of the cell (Day 1997) and about 150 - 500 mitochondria are present i
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