Comparison of small scale methods for the rapid and efficient extraction of mitochondrial DNA from wheat crop suitable for down-stream processes

Ejaz, Muhammad; Zhang, Gaisheng; Na, N; Zhao, Huiyan; Zhu, Qidi; Qunzhu, W

doi:10.4238/2014.december.4.27

Cited by 2 publications

(3 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It is worth noting that if qPCR is not available, end-point PCR (using the same primers as for qPCR) provides the qualitative assessment of organellar purity. In this case, pure organellar DNA samples will show amplification for the mitochondrial and plastid amplicons, but no detectable amplification of the nuclear amplicon on the agarose gel, whereas total genomic DNA shows amplification for all three primer sets, as demonstrated in previous studies 11,12 .…”

Section: Both Methods Enrich For Organellar Dna Albeit With Differing Proportions Of Mitochondria and Plastid Sequencessupporting

confidence: 60%

See 1 more Smart Citation

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Miller

Liberatore

Kianian

2017

JoVE

View full text Add to dashboard Cite

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

show abstract

Section: Both Methods Enrich For Organellar Dna Albeit With Differing Proportions Of Mitochondria and Plastid Sequencessupporting

confidence: 60%

“…To assess sample purity following organelle isolation, most studies to date only use end-point PCR and gel electrophoresis 11,12 . This gives a fair qualitative measure of sample purity.…”

Section: Namementioning

confidence: 99%

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Miller

Liberatore

Kianian

2017

JoVE

View full text Add to dashboard Cite

show abstract

“…Different protocols for isolating functional mitochondria have been reported in different crop species and plant cell culture: Arabidopsis [ 6 – 10 ], pea [ 11 , 12 ], wheat [ 13 ] or plant cell culture [ 14 ]. Protocols for extracting proteins [ 15 – 17 ] and mtDNAs [ 18 – 21 ] form different crop species also exist for non-functional analysis of mitochondria. However, issues associated with these protocols are not lacking and the impurity of isolated mitochondria is the major concern, particularly for a non-functional analysis [ 22 – 24 ].…”

Section: Introductionmentioning

confidence: 99%

An improved method with a wider applicability to isolate plant mitochondria for mtDNA extraction

Ahmad

2015

Plant Methods

View full text Add to dashboard Cite

BackgroundMitochondria perform a principal role in eukaryotic cells. Mutations in mtDNA can cause mitochondrial dysfunction and are frequently associated with various abnormalities during plant development. Extraction of plant mitochondria and mtDNA is the basic requirement for the characterization of mtDNA mutations and other molecular studies. However, currently available methods for mitochondria isolation are either tissue specific or species specific. Extracted mtDNA may contain substantial chloroplast DNA (cpDNA) and nuclear DNA (nDNA) and its end use efficiency can be reduced. Clearly, an effective mitochondria isolation method is warranted with wider applicability and with minimum contamination from cpDNA and nDNA.ResultsHere we reported an improved method for isolating mitochondria from dry wheat seeds and its extension to dead seeds, viable seeds, etiolated leaf tissue and several other plant species: oat, Arabidopsis, flax, and yellow mustard. The isolated mitochondria were successfully used to extract mtDNA with QIAamp DNA mini kit (Qiagen). The extracted mtDNA from the assayed samples of these species was intact in large quantity and showed little contamination from nDNA, cpDNA, RNA, and proteins. The mtDNA extracted from dead wheat seeds was also substantial, but more degraded and less intact when compared to those from viable seeds and other tissues.ConclusionThe improved method was successfully applied to isolate mitochondria and extract mtDNA from several different tissues and plant species. The major advance in the improvement lies in its wider application with the same mitochondria extraction medium to different tissues and species. The improvement is significant, as it helps to widen the scope of future plant mitochondria research.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-015-0099-x) contains supplementary material, which is available to authorized users.

show abstract

Comparison of small scale methods for the rapid and efficient extraction of mitochondrial DNA from wheat crop suitable for down-stream processes

Cited by 2 publications

References 21 publications

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

An improved method with a wider applicability to isolate plant mitochondria for mtDNA extraction

Contact Info

Product

Resources

About