Isolation Of Mitochondrial DNA Research Articles

Mitochondrial DNA Isolation from Plants.

For most eukaryotes, sequencing and assembly of the mitochondrial DNA (mtDNA) is possible by starting the analysis from total cellular DNA, but the exploration of the mtDNA of plants is more challenging because of the low copy number, limited sequence conservation, and complex structure of the mtDNA. The very large size of the nuclear genome of many plant species and the very high ploidy of the plastidial genome further complicate the analysis, sequencing, and assembly of plant mitochondrial genomes. An enrichment of mtDNA is therefore necessary. For this, plant mitochondria are purified prior to mtDNA extraction and purification. The relative enrichment in mtDNA can be assessed by qPCR, while the absolute enrichment can be deduced from the proportion of NGS reads mapping to each of the three genomes of the plant cell. Here we present methods for mitochondrial purification and mtDNA extraction applied to different plant species and tissues, and compare the mtDNA enrichment obtained with the different procedures.

A new D-Loop mitochondrial nucleotide variation from individual at Naga and Kuta traditional village

The D-Loop region of human mitochondrial DNA (mtDNA) has a higher level of polymorphism compared to nuclear DNA. Therefore, the mtDNA D-Loop area can determine a particular individual or ethnic identity. This study aims to determine the variation of the D-Loop region nucleotide sequence, namely the Hipervariable I (HV1) region in normal tribal individuals in Kampung Naga and Kampung Kuta. The sample used is the hair root. In determining the variation of nucleotides has been carried out a series of stages of research, namely the isolation of mitochondrial DNA from hair root samples using buffer lysis, mtDNA sample amplification using PCR techniques with M1 primers and HV2R primers, detection of D-Loop region mtDNA fragments by agarose gel electrophoresis, and sequencing of PCR products by the Sanger Dideoxy method and nucleotide sequence analysis of the sequencing results. The results of DNA amplification by PCR showed a band in the area of 1.0 kb. Homology analysis was carried out by comparing the nucleotide sequences of samples with the Cambridge nucleotide sequence, which shows that there are 10 variations. The new variation (morph) is determined by comparing the results of the variations of the two samples with variations from Homo Sapiens Indonesia including Baduy, Sundanese, Javanese (Sangiran), and Madura. Homology results showed 4 new variations, namely c(16184)A, t(16209)C, a(16272)G, t(16519)C. Sapiens Kuta village and Sundanese (general) have a close genetic relationship. The ancestors of the two Homo Sapiens were closely related to Homo Sapiens Naga, Homo Sapiens Naga are closely related to the Homo Sapiens of the Baduy. The ancestors of Homo sapiens, Kuta, Sundanese, Naga and Baduy, are closely related to Homo sapiens Sangiran (Java). Homo Sapiens Madura is most likely the oldest Homo sapiens seen from phylogenetic trees. The results of this study are expected to be able to add Indonesia’s normal human database.

Methods for simultaneous and quantitative isolation of mitochondrial DNA, nuclear DNA and RNA from mammalian cells.

The aim of this study was to assess two protocols for their capacities to simultaneously isolate RNA, mtDNA and ncDNA from mammalian cells. We compared the Invitrogen TRIzol-based method and Qiagen DNeasy columns, using the HepG2 cell line and human primary glioblastoma stem cells. Both methods allowed the isolation of all three types of nucleic acids and provided similar yields in mtDNA. However, the yield in ncDNA was more than tenfold higher on columns, as observed for both cell types. Conversely, the TRIzol method proved more reproducible and was the method of choice for isolating RNA from glioblastoma cells, as demonstrated for the housekeeping genes RPLP0 and RPS9.

From your eyes only: Efficiency of nuclear and mitochondrial DNA isolation from contact lenses at crime scenes

AbstractRecovering appropriate evidence from crime scenes to identify the perpetrator, or in certain cases, the victim, is one of the main goals of forensic investigation. The current practice includes collection of objects from which “trace” or “touch” DNA may be retrieved. Contact lenses, worn by the perpetrator/victim, represent a potentially good source of DNA. This is based on their exposure to touch DNA and the release and regeneration of epithelial cells from the eye. This study takes these premises into account with the objective of analyzing the efficiency of human nuclear and mitochondrial DNA isolation from contact lenses. DNA was collected using a non‐destructive technique (double‐swab) and subsequently isolated using a silica‐based methodology. Although DNA quantification was variable among subjects and contact lenses, it was sufficient to amplify some STRs and successfully sequence mtDNA. The findings from this research provide a proof‐of‐concept that it is possible to recover, isolate, amplify human nuclear DNA, and characterize mitochondrial DNA from contact lenses left at crime scenes. These results show the potential importance of recovering unusual items from crime scenes as a potential source of DNA to identify the victim and/or suspect.

An Efficient Method of Mitochondrial DNA Isolation from Vigna radiata for Genomic Studies.

Isolation of mitochondrial DNA from root tissues of mung bean (Vigna radiata) is quite tedious, complex, and often results in low yield. Hence here we show a simple, rapid, and improved protocol for isolation of mitochondrial DNA from root tissues of hydroponically grown mung bean plants. This method involves purification of mitochondria and subsequent isolation of DNA from obtained purified mitochondria. For this purpose, mitochondria were isolated using a discontinuous Percoll gradient centrifugation followed by RNase I treatment to the isolated DNA to remove any traces of RNA contamination. The mitochondrial DNA was isolated from mitochondrial samples by commonly used CTAB method. The specificity of isolated mitochondrial DNA was confirmed using mtDNA-specific genes (NAD1 and COX3). β-Actin primer was used to check the nuclear DNA contamination. PCR amplification was observed in mtDNA specific genes NAD1 and COX3 except nuclear encoded β-actin gene suggesting that mitochondrial DNA was not contaminated by nuclear DNA.

A rapid and efficient method for enriching mitochondrial DNA from plants

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.

Isolation of Mitochondrial DNA from Single, Short Hairs without Roots Using Pressure Cycling Technology.

Hairs are commonly submitted as evidence to forensic laboratories, but standard nuclear DNA analysis is not always possible. Mitochondria (mt) provide another source of genetic material; however, manual isolation is laborious. In a proof-of-concept study, we assessed pressure cycling technology (PCT; an automated approach that subjects samples to varying cycles of high and low pressure) for extracting mtDNA from single, short hairs without roots. Using three microscopically similar donors, we determined the ideal PCT conditions and compared those yields to those obtained using the traditional manual micro-tissue grinder method. Higher yields were recovered from grinder extracts, but yields from PCT extracts exceeded the requirements for forensic analysis, with the DNA quality confirmed through sequencing. Automated extraction of mtDNA from hairs without roots using PCT could be useful for forensic laboratories processing numerous samples.

Development of a rapid mitochondrial DNA extraction method for species identification in milk and milk products

Isolation of mitochondrial DNA (mtDNA) from milk offers an effective way to monitor aspects of quality control and traceability to ensure food safety. A few methods of DNA isolation from milk have been reported, but many of them are time consuming and expensive. Here, we report a rapid, simple, and efficient method of mtDNA extraction from raw and processed milk (pasteurized, retorted, and UHT milk) to generate substrate for analysis using any PCR analysis platform. Various techniques used for the separation of mitochondria were explored and combined with a sodium dodecyl sulfate method for mtDNA extraction from raw and processed milk. The optimized protocol supports the efficient amplification of mtDNA independent of sample origin and is sufficiently straightforward to allow its widespread adoption by industry.

Human mitochondrial DNA and nuclear DNA isolation from food bite marks

ObjectiveBite mark analysis is used for comparison between bite marks on a bitten object and the suspects’ teeth. However, if it is not possible to obtain a correct match, it is important to recover salivary DNA. Previous studies have tried to isolate human nuclear DNA from bitten foods but were not completely successful. In the present work, we studied the efficiency of human nuclear and mitochondrial DNA isolation from bite marks in cheese, a donut and an apple. DesignUsing a double swab technique and silica-based DNA extraction kit, nuclear and mitochondrial DNA were isolated. Human housekeeping genes were amplified to analyse the efficiency of nuclear DNA profiling. mtDNA was sequencing and haplogroup assign. ResultsAlthough cheese and apple samples showed the highest concentration of DNA, the purity of DNA on the apple was low. Moreover, apple samples failed to amplify the two human housekeeping genes, GAPDH and RPL22. In contrast, cheese samples have high purity and amplification efficiency. Donut samples showed an intermediate value and low amplification efficiency. In spite of these results, isolation and characterization/sequencing of human mitochondrial DNA was completely successful in the three samples, which pointed out the possibility of identification through this type of DNA. ConclusionsThis research indicated that it is possible to recover and isolate human nuclear and mitochondrial DNA from bitten foods, although the quantity and purity of nuclear DNA depends on the type of food. That is of significance important in forensic sciences for the correct identification of a suspect.

Real-Time Polymerase Chain Reaction for Halal Authentication of Gelatin in Soft Candy

Currently, along with the development of science and technology, the diversification of food products occurs in the market. Food products can contain non-halal components like porcine gelatine. One of food suspected to use gelatine is soft candy. Gelatin can be made from pork or beef or other animal. The presence of porcine gelatine in any food products is not allowed for Moslem community, therefore an analytical method offering reliable results must be developed. This study is intended to use Real-Time Polymerase Chain Reaction (RT-PCR) for analysis of porcine gelatine in soft candy. Isolation of DNA was performed with mitochondrial DNA Isolation Kit K280-50 (Bio-Vision). The DNA was analyzed by RT-PCR using primer D-Loop 318. Analysis for the primer specificity was performed on fresh tissue (pig, cows, chickens, goats and rats) and gelatin sources (beef, pigs and catfish). Primer D-loop318 can amplify porcine DNA at the optimum temperature 61.4°C. Repeatability test demonstrated amplification of all positive response samples containing porcine DNA in serial dilution of 10000-1 pg). The Coefficient of Variation (CV) is 6.32%. The repeatability test was also performed on soft candy 100% having CV of 1.06%. The commercial soft candy samples evaluated do not contain porcine DNA.

A comparison of mitochondrial DNA isolation methods in frozen post-mortem human brain tissue--applications for studies of mitochondrial genetics in brain disorders.

Given that many brain disorders are characterized by mitochondrial dysfunction, there is a growing interest in investigating genetic and epigenetic variation in mitochondrial DNA (mtDNA). One major caveat for such studies is the presence of nuclear-mitochondrial pseudogenes (NUMTs), which are regions of the mitochondrial genome that have been inserted into the nuclear genome over evolution and, if not accounted for, can confound genetic studies of mtDNA. Here we provide the first systematic comparison of methods for isolating mtDNA from frozen post-mortem human brain tissue. Our data show that a commercial method from Miltenyi Biotec, which magnetically isolates mitochondria using antibodies raised against the mitochondrial import receptor subunit TOM22, gives significant mtDNA enrichment and should be considered the method of choice for mtDNA studies in frozen brain tissue.

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

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.

TGF-β1-mediated differentiation of fibroblasts is associated with increased mitochondrial content and cellular respiration.

ObjectivsCytokine-dependent activation of fibroblasts to myofibroblasts, a key event in fibrosis, is accompanied by phenotypic changes with increased secretory and contractile properties dependent on increased energy utilization, yet changes in the energetic profile of these cells are not fully described. We hypothesize that the TGF-β1-mediated transformation of myofibroblasts is associated with an increase in mitochondrial content and function when compared to naive fibroblasts.MethodsCultured NIH/3T3 mouse fibroblasts treated with TGF-β1, a profibrotic cytokine, or vehicle were assessed for transformation to myofibroblasts (appearance of α-smooth muscle actin [α-SMA] stress fibers) and associated changes in mitochondrial content and functions using laser confocal microscopy, Seahorse respirometry, multi-well plate reader and biochemical protocols. Expression of mitochondrial-specific proteins was determined using western blotting, and the mitochondrial DNA quantified using Mitochondrial DNA isolation kit.ResultsTreatment with TGF-β1 (5 ng/mL) induced transformation of naive fibroblasts into myofibroblasts with a threefold increase in the expression of α-SMA (6.85 ± 0.27 RU) compared to cells not treated with TGF-β1 (2.52 ± 0.11 RU). TGF-β1 exposure increased the number of mitochondria in the cells, as monitored by membrane potential sensitive dye tetramethylrhodamine, and expression of mitochondria-specific proteins; voltage-dependent anion channels (0.54 ± 0.05 vs. 0.23 ± 0.05 RU) and adenine nucleotide transporter (0.61 ± 0.11 vs. 0.22 ± 0.05 RU), as well as mitochondrial DNA content (530 ± 12 μg DNA/106 cells vs. 307 ± 9 μg DNA/106 cells in control). TGF-β1 treatment was associated with an increase in mitochondrial function with a twofold increase in baseline oxygen consumption rate (2.25 ± 0.03 vs. 1.13 ± 0.1 nmol O2/min/106 cells) and FCCP-induced mitochondrial respiration (2.87 ± 0.03 vs. 1.46 ± 0.15 nmol O2/min/106 cells).ConclusionsTGF-β1 induced differentiation of fibroblasts is accompanied by energetic remodeling of myofibroblasts with an increase in mitochondrial respiration and mitochondrial content.

Analysis of pollutant-induced changes in mitochondrial DNA methylation.

There is increasing evidence that exposure to air pollutants is associated with human disease and may act through epigenetic modification of the nuclear genome, but there have been few publications describing their impact upon the mitochondrial genome. Mitochondrial DNA may be more susceptible to pollutant-induced changes via increased oxidative stress in the cell, and therefore this field of research is of growing interest. Many techniques employed to study DNA methylation of the nuclear genome are also applicable to mitochondrial epigenetic studies. In this chapter, we describe a protocol for the isolation of mitochondrial DNA from peripheral blood samples and the analysis of 5-methylcytosine content by bisulfite pyrosequencing.

Fast Mitochondrial DNA Isolation from Mammalian cells for Next-Generation Sequencing

Standard methods for mitochondrial DNA (mtDNA) extraction do not provide the level of enrichment for mtDNA sufficient for direct sequencing and must be followed by long-range-PCR amplification, which can bias the sequencing results. Here, we describe a fast, cost-effective, and reliable method for preparation of mtDNA enriched samples from eukaryotic cells ready for direct sequencing. Our protocol utilizes a conventional miniprep kit, paramagnetic bead-based purification, and an optional, limited PCR amplification of mtDNA. The first two steps alone provide more than 2000-fold enrichment for mtDNA when compared with total cellular DNA (~200-fold in comparison with current commercially available kits) as demonstrated by real-time PCR. The percentage of sequencing reads aligned to mtDNA was about 22% for non-amplified samples and greater than 99% for samples subjected to 10 cycles of long-range-PCR with mtDNA specific primers.

A Rapid and Efficient Method for the Isolation of Mitochondrial DNA From Wheat Crop

The mitochondrial DNA was isolated from wheat (Triticum aestivum) crop in combination of different centrifugation and density gradient ultracentrifugation, DNaseI enzyme treatment, sucrose sedimentation; lysis with SDS and potassium proteinase. Pure mitochondrial DNA was successfully obtained by phenol/chloroform/isoamyl alcohol extraction to remove protein, and digested with RNase phenol/chloroform extraction method. To detect mitochondrial DNA purity, specific primers were designed for nuclear (Beta-actin) and mitochondrial (COXII) gene. Isolated mitochondrial DNA is pure, suitable for PCR and genetic analysis.

An efficient method for isolation of mitochondrial DNA in wheat

An efficient method for isolation of mitochondrial DNA (mtDNA) from etiolated tissues of wheat was developed. The protocol consists of mitochondria isolation with differential centrifugation, Dnase I treatment, lysis with SDS and proteinase K, removing protein by TE-saturated phenol/chloroform extraction and a final RNase A treatment for obtaining mtDNA. The mtDNA samples were tested using spectrophotometry and agarose gel electrophoresis. It was proved that the mtDNA isolated by this method not only have the high yield but also structural complete, and contains no impurities, such as nuclear DNA, RNA and protein. The result showed that this high quality mtDNA can be successfully used in PCR and other genetic studies. In addition, it was found that adjusting the lysis temperature has a noticeable effect on the mtDNA yield.

The relationships among mitochondrial uncoupling protein 2 and 3 expression, mitochondrial deoxyribonucleic acid single nucleotide polymorphisms, and residual feed intake in Angus steers1

The objective of this study was to determine the relationships of uncoupling protein 2 and 3 expression, SNP of mitochondrial DNA, and residual feed intake (RFI) in Angus steers selected to have high or low RFI. Individual feed intake was measured via the GrowSafe feed intake system over a 3-mo period and used to calculate RFI, a measure of efficiency. Based on these calculations, 6 low- (average RFI = -1.57 kg) and 6 high- (average RFI = 1.66 kg) RFI steers were selected for further study. Blood was collected via jugular venipuncture 1 wk before slaughter for the isolation of mitochondrial DNA. The steers were then killed to collect LM for the measurement of uncoupling protein 2 and 3 mRNA and protein expression. Protein and mRNA expression of uncoupling protein 2 and 3 were determined by Western blotting and quantitative PCR, respectively. To determine SNP of mitochondrial DNA, total DNA was isolated from blood via standard phenol/chloroform extraction; fragments were amplified with PCR and sequenced with an automated nucleotide sequencer. Average daily gain and carcass composition were not different (P > 0.13) between the high- and low-RFI steers; however, ADFI by the high-RFI animals was 3.77 kg greater (P < 0.001) than the low-RFI animals. No difference (P > 0.55) was observed between the high- and low-RFI animals in their expression of uncoupling protein 2 or 3 mRNA or protein. On average 9.8 and 8.9 polymorphisms were found per mitochondrial genome for the low- and high-RFI steers, respectively. None of these polymorphisms were related to RFI. It seems that the expression of uncoupling protein 2 and 3 and mitochondrial DNA sequence are not related to RFI status.

A rapid and efficient method for the isolation of mitochondrial DNA from angiosperm tree species. Application to the restriction fragment length polymorphism distinction between European and American ashes

AbstractA simplified method for the isolation of mitochondrial DNA (mtDNA) from several angiosperm tree species is described. The procedure does not require gradient ultracentrifugation or organic solvent extractions and can be performed in a single day. mtDNA was isolated from flower buds collected from adult trees just after the first pollen mitosis and purified by gravity flow through disposable columns containing an anion‐exchange resin. The method led to restrictable mtDNA from the tree species analysed. Restriction patterns enabled us to distinguish between European and American ashes.

Improved procedure for large‐scale isolation of mitochondrial DNA from mammalian tissues

Although there are several methods for the preparation of mitochondrial DNA (mtDNA) from mammalian tissues, most are relatively long ultracentri‐fugation or manipulations by a small‐scale method. We described a rapid method for large‐scale extraction of mtDNA from human placental and horse liver tissues. The method is based on the preparation and homogenization of tissues, purification of crude mitochondria by differential centrifugations, and isolation of mtDNA by alkaline lysis. It was improved from pre‐existing methods by replacing some steps with simpler ones and discarding many others. This method gives a high yield of pure mtDNA (approximately 1–5 mg from one placenta; ca. 400–600 g wet weight), depending on its sources (fresh tissue gave better results than frozen one). The resulting mtDNA indicated that this method can yield mtDNA in sufficient purity and quantity to identify the direct restriction analysis on agarose gel, random‐primed labeling as a probe, and end labeling. Therefore, the method ...