Abstract
Current mitochondrial purification techniques are tedious and protracted due to their emphasis on recovering physiologically active mitochondria. However, for studies that are exclusively interested in isolating mitochondrial DNA (mtDNA) for applications such as PCR and sequencing, respiring mitochondria − and the complex procedures that stem from the need to retain their function − are unnecessary. Still, global DNA extraction methods have proven insufficient for mitochondrial DNA isolation because nuclear mitochondrial DNA segments (NUMTs) pose unique challenges to accurate mtDNA quantification and characterization. We present a rapid and simple extraction technique that maximizes recovery of mitochondrial DNA from plant cells, while minimizing the presence of nuclear DNA. Through real-time PCR, we show that this method provides a significant increase in the enrichment of mitochondrial DNA compared to that of nuclear DNA in both Arabidopsis thaliana and Brassica rapa. This method has important implications for future mitochondrial DNA analyses as it possesses few procedural limitations and minimizes the analytical problems typically associated with mtDNA purification by other techniques.
Highlights
For applications involving polymerase chain reactions (PCR) and sequencing, the presence of the entire mitochondrial genome or fragments of it (Lin et al 1999; Stupar et al 2001; Richly and Leister 2004; Hazkani-Covo et al 2010) in the nuclear chromosomes can confound results that depend on strict amplification of the mitochondrial copies of DNA
Without purification or enrichment of mitochondrial DNA (mtDNA), nuclear DNA will comprise the majority of NextGen DNA sequencing reads, increasing the total amount of sequence data required and the overall cost to sequence the mitochondrial genome
Treating purified mitochondria with DNAses in order to degrade contaminating nuclear DNA is problematic because this often results in simultaneous degradation of the mitochondrial DNA (Li et al 2006)
Summary
For applications involving PCR and sequencing, the presence of the entire mitochondrial genome or fragments of it (Lin et al 1999; Stupar et al 2001; Richly and Leister 2004; Hazkani-Covo et al 2010) in the nuclear chromosomes can confound results that depend on strict amplification of the mitochondrial copies of DNA. Treating purified mitochondria with DNAses in order to degrade contaminating nuclear DNA is problematic because this often results in simultaneous degradation of the mitochondrial DNA (Li et al 2006) Due to their emphasis on retaining physiologically active mitochondria for use in metabolic or respiration experiments, existing procedures (Hayes et al 1991) may not provide good yields of mtDNA suitable for molecular biology applications and they may retain nuclear DNA. These protocols tend to be lengthy and tedious compared with standard DNA purification procedures
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
