DNA Extraction Efficiency Research Articles

Comparing sediment preservation methods for genomic biomonitoring of coastal marine ecosystems

To avoid loss of genetic information in environmental DNA (eDNA) field samples, the preservation of nucleic acids during field sampling is a critical step. In the development of standard operating procedures (SOPs) for eDNA-based compliance monitoring, the effect of different routinely used sediment preservations on biological community structures serving as bioindicators has gone untested. We compared eDNA metabarcoding results of marine bacterial communities from sample aliquots that were treated with a nucleic acid preservation solution (treated samples) and aliquots that were frozen without further treatment (non-treated samples). Sediment samples were obtained from coastal locations subjected to different stressors (aquaculture, urbanization, industry). DNA extraction efficiency, bacterial community profiles, and measures of alpha- and beta-diversity were highly congruent between treated and non-treated samples. As both preservation methods provide the same relevant information to environmental managers and regulators, we recommend the inclusion of both methods into SOPs for biomonitoring in marine coastal environments.

Postsynthetic Modification of the Magnetic Zirconium-Organic Framework for Efficient and Rapid Solid-Phase Extraction of DNA.

In recent years, several approaches have been applied to modify metal-organic frameworks (MOFs) owing to their excellent structural tunability such as higher extraction efficiency than that of primitive crystals. Herein, Zr-based MOFs (UiO-66-NH2) with a suitable size modulated by acetic acid were successfully synthesized for effective DNA extraction. The bonding conformations and adsorption mechanism indicated a high affinity between UiO-66-NH2 and the DNA molecules. Furthermore, Fe3O4 nanoparticles were immobilized on the UiO-66-NH2 surface to allow MOFs with magnetism. The magnetic zirconium-organic framework (MZMOF) retained the intact structure of MOFs and simplified subsequent extraction operations. In the DNA recovery investigation, MZMOF showed high recovery efficiency for both short-stranded DNA (90.4%) and pseudovirus DNA (95.1%). In addition, it showed superior DNA extraction efficiency from plasma (57.6%) and swab preservation solution (86.5%). The prepared MZMOF was employed for highly specific extraction of viral DNA and cfDNA from samples. To further simplify the extraction process, MZMOF was applied to immiscible phase filtration assisted by a surface tension (IFAST) chip for facilitating rapid DNA extraction with sensitive point-of-care testing. The developed MZMOF-based extraction method has significant potential for increasing the demand for rapid and efficient nucleic acid extraction.

Evaluation of Some Potential Protocols to Extract DNA from Paddy Soil

Isolation of DNA from environmental samples is a crucial step in microbial community analyses through molecular methods. The present study was conducted to evaluate a DNA extraction protocol from paddy soil with a comparison on quality, quantity and integrity of the isolated DNA and to determine the suitability of extracted DNA for downstream applications in microbial community analyses. Three protocols (i.e. PEG/NaCl, Mannitol/C TAB and Sodium Phosphate Buffer) used for the extraction of DNA from different types of soil were attempted on paddy soil. The quality and quantity of the extracted genomic DNA was quantified spectrophotometrically and integrity was checked by gel electrophoresis. The efficiency of DNA extraction by the three protocols was compared with a commercia l soil DNA extraction kit (Norgen’s Soil DNA Isolation Plus Kit). Further, quality of the extracted DNA for PCR amplification was assessed using universal primer pairs for bacteria and fungi. DNA extracted using PEG/NaCl method resulted in the highest DNA concentration, while the highest purity was recorded by the DNA extracted by Mannitol/CTAB method (A 260/A280 = 1.61 and A260/A230 = 1.15). Expected PCR products targeting 16s rDNA and ITS regions were obtained from the DNA samples extracted by Mannitol/CTAB method. Therefore, Mannitol/CTAB method used in the present study is suitable to extract high-quality DNA from paddy soil for molecular microbial studies.

A workflow for the relative quantification of multiple fish species from oceanic water samples using environmental DNA (eDNA) to support large-scale fishery surveys.

While the number of published marine studies using environmental DNA (eDNA) has increased substantially in recent years, marine fish surveys are still scarce. To examine the potential for eDNA to support marine fisheries monitoring surveys, we optimized an eDNA isolation method, developed a multispecies assay and tested it on eDNA samples collected along the Pacific coast of the United States. Four commercial DNA extraction kits that exploit the capability of the nucleic acids binding a solid phase (two using a silica matrix and two magnetic beads) as well an organic separation method were tested. A species-specific multiplex qPCR assay was developed and tested to simultaneously target Pacific hake (Merluccius productus), Pacific lamprey (Entosphenus tridentatus) and eulachon (Thaleichthys pacificus). The specificity of the assay was tested in silico, in vitro and in natura. Environmental DNA isolation using phenol:chloroform:isoamyl purification with a phase lock was optimized and yielded the highest amount of total and target DNA and was used to extract 46 marine water samples for the detection of the three species of interest. The multiplex qPCR assay used in the quantification process was also optimized to provide convenience and accuracy. Pacific hake was present in 44% of the eDNA samples while the other two species were absent. Here, we present a complete workflow for the simultaneous detection and quantification of multiple marine fish species using eDNA. This workflow supports large-scale at-sea sampling efforts with preservation at ambient temperatures and has demonstrated DNA extraction efficiency and reliability. The multiplex qPCR assay is shown to be sensitive and specific for the purposes of simultaneously monitoring the relative abundance of multiple targeted fish species.

New Estimation of Antibiotic Resistance Genes in Sediment Along the Haihe River and Bohai Bay in China: A Comparison Between Single and Successive DNA Extraction Methods.

Sediment is thought to be a vital reservoir for antibiotic resistance genes (ARGs). Often, studies describing and comparing ARGs and their potential hosts in sediment are based on single DNA extractions. To date, however, no study has been conducted to assess the influence of DNA extraction efficiency on ARGs in sediment. To determine whether the abundance of ARGs is underestimated, we performed five successive extraction cycles with a widely used commercial kit in 10 sediment samples collected from the Haihe River and Bohai Bay. Our results showed that accumulated DNA yields after five extractions were 1.8–3.1 times higher than that by single DNA extractions. High-throughput sequencing showed that insufficient DNA extraction could generate PCR bias and skew community structure characterization in sediment. The relative abundances of some pathogenic bacteria, such as Enterobacteriales, Lactobacillales, and Streptomycetales, were significantly different between single and successive DNA extraction samples. In addition, real-time fluorescent quantitative PCR (qPCR) showed that ARGs, intI1, and 16S rRNA gene abundance strongly increased with increasing extraction cycles. Among the measured ARGs, sulfonamide resistance genes and multidrug resistance genes were dominant subtypes in the study region. Nevertheless, different subtypes of ARGs did not respond equally to the additional extraction cycles; some continued to have linear growth trends, and some tended to level off. Additionally, more correlations between ARGs and bacterial communities were observed in the successive DNA extraction samples than in the single DNA extraction samples. It is suggested that 3–4 additional extraction cycles are required in future studies when extracting DNA from sediment samples. Taken together, our results highlight that performing successive DNA extractions on sediment samples optimizes the extractable DNA yield and can lead to a better picture of the abundance of ARGs and their potential hosts in sediments.

Technical note: Polyvinylpyrrolidone (PVP) and proteinase-K improve the efficiency of DNA extraction from Japanese larch wood and PCR success rate

Illegal distribution of timber disrupts the timber market and depletes forest resources. DNA markers are used to verify the legal distribution of wood. However, it is difficult to obtain the quantity and quality of DNA suitable for genetic analysis because of the physicochemical properties of wood; therefore, an efficient wood DNA extraction method is required. In this study, to prepare an efficient DNA extraction method from Japanese larch (Larix kaempferi) wood, we investigated the ability of polyvinylpyrrolidone (PVP) and proteinase-K to improve DNA extraction efficiency and PCR success rate. It was found that the addition of PVP resulted in a significant increase in the DNA concentration of the treatment group compared to that of the control group, while the purity (A260/A280) showed no difference. Moreover, the addition of proteinase-K significantly increased both the DNA concentration and purity of the treatment group compared to those of the control group. Further analysis showed that the PCR success rate of psbC (approximately 350 bp) was higher than 90% in the control, PVP treatment, and proteinase-K treatment groups. However, in the PCR success rate of rbcL (approximately 1.3 kb) was higher in the proteinase-K and PVP treatment groups than in the control group. The addition of PVP and proteinase-K increased the success rate of PCR amplification for long regions by preventing DNA damage caused by phenolic compounds and proteins in the wood. The results of this study can thus develop DNA extraction methods to identify the species and origin of woods.

Evaluation of eight protocols for genomic DNA extraction of Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes).

The principle and the techniques applied in DNA extraction play a pivotal role in the obtention of a purified genetic material. The present study investigates the efficiency of eight protocols in the DNA extraction of Hypostomus commersoni, an essential component of South American freshwater ichthyofauna. The quality of samples was assessed through spectrophotometry, gel electrophoresis, and PCR-RAPD markers amplification. The efficiency of DNA extraction was influenced both by the method applied and the target-tissue of choice. Higher concentrations and yield of DNA were obtained from ocular tissue, with a positive spectrum of incubation in lysis buffer for up to 36 hours after sample collection, using fresh tissues and in the presence of a high concentration of Proteinase K (20 mg.ml-1). In these conditions, samples were successfully amplified. To date, there is no record of description for the parameters analyzed in this work, neither the description of RAPD markers for the species H. commersoni.

Quantitative detection of human- and canine-associated Bacteroides genetic markers from an urban coastal lagoon.

The contamination of water catchments by nonpoint source faecal pollution is a major issue affecting the microbial quality of receiving waters and is associated with the occurrence of a range of enteric illnesses in humans. The potential sources of faecal pollution in surface waters are diverse, including urban sewage leaks, surface runoff and wildlife contamination originating from a range of hosts. The major contributing hosts require identification to allow targeted management of this public health concern. In this study, two high-performing Microbial Source Tracking (MST) assays, HF183/Bac242 and BacCan-UCDmodif, were used for their ability to detect host-specific Bacteroides 16Sr RNA markers for faecal pollution in a 12-month study on an urban coastal lagoon in Sydney, Australia. The lagoon was found to contain year-round high numbers of human and canine faecal markers, as well as faecal indicator bacteria counts, suggesting considerable human and animal faecal pollution. The high sensitivity and specificity of the HF183/Bac242 and BacCan-UCDmodif assays, together with the manageable levels of PCR inhibition and high level DNA extraction efficiency obtained from lagoon water samples make these markers candidates for inclusion in an MST 'toolbox' for investigating host origins of faecal pollution in urban surface waters.

DNA Isolation: A Method for Improving the Efficiency of DNA Extraction From Clotted Blood Samples

In generally, the genome research of high DNA extraction from clotted blood produced a low quality. The aim of this study is to develop a simple and safe technique to dispering the blood clots by the ball bearing metal shots. The yield and purity of DNA obtained by three steps were significantly different (P < 0.0001), with a higher yield in the modified salting-out method. The salting-out method is simple, efficient and economical for DNA isolation from old clotted blood samples.

Metal-Doped Polydopamine Nanoparticles for Highly Robust and Efficient DNA Adsorption and Sensing.

Controlling DNA adsorption on nanomaterials is crucial for a wide range of applications in analytical and biomedical sciences. Polydopamine (PDA) is a versatile material that can be coated on nearly any surface, and thus adsorbing DNA onto PDA can be a general method for indirect DNA functionalization of surfaces. Polyvalent metal ions were reported to promote DNA adsorption on PDA nanoparticles (NPs), but previous works added the metal ions after the formation of PDA. Herein, we compared the effect of polyvalent metal ions added during the synthesis of PDA NPs (called metal-doped) with the effect of polyvalent metal ions added after the synthesis (metal-adsorbed). A series of metal ions including Ca2+, Zn2+, Ni2+, Fe3+, and Gd3+ were tested, and Zn2+ was studied in detail due to its excellent ability for promoting DNA adsorption. With 100 μM Zn2+, metal-doped NPs were ∼30% more efficient than metal-adsorbed NPs for DNA adsorption in buffer attributable to a higher metal loading on the surface of the metal-doped NPs. Metal leaching was negligible from the metal-doped NPs, and they showed a remarkably higher robustness than the metal-adsorbed NPs, resulting in a 20-fold higher DNA extraction efficiency from serum. Based on the desorption studies, a higher adsorption affinity for the metal-doped NPs was confirmed. Finally, the Zn2+-doped PDA NPs were used for sensitive DNA detection with a limit of detection of 0.45 nM, and the sensor was highly resistant to nonspecific protein and phosphate displacement.

A Synthetic DNA Construct to Evaluate the Recovery Efficiency of Cell-Free DNA Extraction and Bisulfite Modification.

Despite improvements in the genetic and epigenetic analysis of cell-free DNA (cfDNA), there has been limited focus on assessing the preanalytical variables of recovery efficiency following cfDNA extraction and bisulfite modification. Quantification of recovery efficiency after these steps can facilitate quality assurance and improve reliability when comparing serial samples. We developed an exogenous DNA Construct to Evaluate the Recovery Efficiency of cfDNA extraction and BISulfite modification (CEREBIS) after cfDNA extraction and/or subsequent bisulfite modification from plasma. The strategic placement of cytosine bases in the 180 bp CEREBIS enabled PCR amplification of the construct by a single primer set both after plasma DNA extraction and following subsequent bisulfite modification. Plasma samples derived from 8 organ transplant donors and 6 serial plasma samples derived from a liver transplant recipient were spiked with a known number of copies of CEREBIS. Recovery of CEREBIS after cfDNA extraction and bisulfite modification was quantified with high analytical accuracy by droplet digital PCR. The use of CEREBIS and quantification of its recovery was useful in identifying problematic extractions. Furthermore, its use was shown to be invaluable towards improving the reliability of the analysis of serial samples. CEREBIS can be used as a spike-in control to address the preanalytical variable of recovery efficiency both after cfDNA extraction from plasma and following bisulfite modification. Our approach can be readily implemented and its application may have significant benefits, especially in settings where longitudinal quantification of cfDNA for disease monitoring is necessary.

Influence of ink and smoke ATM security systems on dactyloscopy and subsequent DNA analysis after detonation

Automated Teller Machine bombings are an increasing societal problem that are often committed using Improvised Explosive Devices. The evolution in IEDs and the negative consequences for society require new security measures to prevent these crimes. Ink staining and security smoke devices are added to cash cassettes, in order to protect ATMs and prevent ATM bombings. Traces found at crime scenes, such as fingerprints and DNA, can contribute to the identification of perpetrators. However, the effect of ink staining and security smoke devices on dactyloscopy and DNA profiling is still unknown. In the current study, we demonstrate that procedures using Citrus Cleaner or sulfosalicylic acid were successful in removing ink and security smoke deposited on plastic plates but did result in the massive loss of fingerprint information as only a low number (4%) of good quality fingerprints were recovered after smoke contamination. Secondly, security ink Sun Blue ES2, but not SICPA Green and Sun Blue ES1, had a significant impact on DNA profiling success. DNA concentrations obtained from blood spiked swabs decreased with increasing ink concentration resulting in a complete loss of genotype information with the addition of ≥10 μl Sun Blue ES2. No noticeable PCR inhibition or DNA degradation was detected during quantification, but a decreased efficiency of DNA extraction could not be excluded. Security smoke, on the other hand, does not seem to have a significant influence on DNA analysis. Precautions must therefore be taken in order to avoid contaminating DNA swabs with ink during sampling. Thirdly, only a single negative impression of a glove in ink and a single glove print were able to be visualized with white fingerprint powder on detonated cash cassettes. In conclusion, the detection of glove prints and fingerprints is limited and security ink, contrary to smoke, after detonation can have a negative influence on downstream DNA analysis procedures.

A New Method for Improving Extraction Efficiency and Purity of Urine and Plasma Cell-Free DNA.

This study assessed three commercially available cell-free DNA (cfDNA) extraction kits and the impact of a PEG-based DNA cleanup procedure (DNApure) on cfDNA quality and yield. Six normal donor urine and plasma samples and specimens from four pregnant (PG) women carrying male fetuses underwent extractions with the JBS cfDNA extraction kit (kit J), MagMAX Cell-Free DNA Extraction kit (kit M), and QIAamp Circulating Nucleic Acid Kit (kit Q). Recovery of a PCR product spike-in, endogenous TP53, and Y-chromosome DNA was used to assess kit performance. Nucleosomal-sized DNA profiles varied among the kits, with prominent multi-nucleosomal-sized peaks present in urine and plasma DNA isolated by kits J and M only. Kit J recovered significantly more spike-in DNA than did kits M or Q (p < 0.001) from urine, and similar amounts from plasma (p = 0.12). Applying DNApure to kit M- and Q-isolated DNA significantly improved the amplification efficiency of spike-in DNA from urine (p < 0.001) and plasma (p ≤ 0.013). Furthermore, kit J isolated significantly more Y-chromosome DNA from PG urine compared to kit Q (p = 0.05). We demonstrate that DNApure can provide an efficient means of improving the yield and purity of cfDNA and minimize the effects of pre-analytical biospecimen variability on liquid biopsy assay performance.

Magnetic Bead Chain-Based Continuous-Flow DNA Extraction for Microfluidic PCR Detection of Salmonella.

Nucleic acid extraction is crucial for PCR detection of pathogenic bacteria to ensure food safety. In this study, a new magnetic extraction method was developed using 3D printing and magnetic silica beads (MSBs) to extract the target DNA from a large volume of bacterial sample and combined with microfluidic PCR to determine the bacteria. After proteinase K was added into a bacterial sample to lyse the bacteria and release the DNA, it was continuous-flow injected into the serpentine channel of the extraction chip, where magnetic silica bead chains had been formed in advance using a homogeneous magnetic field generated by two concentric semicircle magnets to capture the MSBs. Then, the flowing DNA was captured by the MSB chains, washed with alcohol, dried with gas, and eluted with deionized water to obtain the purified and concentrated DNA. Finally, the extracted DNA templates were injected into a microfluidic PCR chip with lyophilized amplification reagents and determined using a commercial qPCR device. The experimental results showed that the DNA extraction efficiency was more than 90%, and the lower detection limit of Salmonella was 102 CFU/mL. This new Salmonella detection method is promising to provide the rapid, sensitive, and simultaneous detection of multiple foodborne pathogens.

Evaluation of 11 DNA Automated Extraction Protocols for the Detection of the 5 Mains Candida Species from Artificially Spiked Blood.

The molecular detection of Candida plays an important role in the diagnosis of candidaemia, a major cause of morbidity and mortality. The sensitivity of this diagnosis is partly related to the efficiency of yeast DNA extraction. In this monocentric study, we investigated the suitability of 11 recent automated procedures for the extraction of low and high amounts of Candida DNA from spiked blood. The efficacy of the DNA extraction procedures to detect Candida spp. in blood samples ranged from 31.4% to 80.6%. The NucliSENSTM easyMAGTM procedure was the most efficient, for each species and each inoculum. It significantly outperformed the other procedures at the lower Candida inocula mimicking the clinical setting. This study highlighted a heterogeneity in DNA extraction efficacy between the five main Candida species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei). Up to five automated procedures were appropriate for C. krusei DNA extraction, whereas only one method yielded an appropriate detection of low amount of C. tropicalis. In the era of the syndromic approach to bloodstream infection diagnosis, this evaluation of 11 automated DNA extraction methods for the PCR diagnosis of candidaemia, puts the choice of an appropriate method in routine diagnosis within the reach of laboratories.

Centrifugation does not remove bacteria from the fat fraction of human milk

Analysis of the human milk microbiome is complicated by the presence of a variable quantity of fat. The fat fraction of human milk is typically discarded prior to analysis. It is assumed that all cells are pelleted out of human milk by high speed centrifugation; however, studies of bovine milk have reported that bacteria may remain trapped within the fat fraction. Here, the bacterial DNA profiles of the fat fraction and cell pellet of human milk (n = 10) were analysed. Human and bacterial DNA was consistently recovered from the fat fraction of human milk (average of 12.4% and 32.7%, respectively). Two low-abundance Staphylococcus species (< 0.5% relative abundance) was significantly more abundant in the cell pellet compared to the fat fraction (P < 0.04), and three low-abundance species (< 5% relative abundance) were recovered from one fraction only. However, inclusion of fat reduced the efficiency of DNA extraction by 39%. Culture-based methods were used to quantify the distribution of an exogenously added strain of Staphylococcus aureus in human milk fractions. S. aureus was consistently recovered from the fat fraction (average 28.9%). Bacterial DNA profiles generated from skim milk or cell pellets are not representative of the entire human milk microbiome. These data have critical implications for the design of future work in this field.

Impact of DNA extraction efficiency on the sensitivity of PCR-based plant disease diagnosis and pathogen quantification

PCR and qPCR are important methods for plant disease diagnosis and quantification of pathogen populations in host tissues or soil. For most plant diseases, DNA extracted from infected tissue or soil is a prerequisite for PCR or qPCR. The efficiency of DNA extraction should have a direct impact on the sensitivity of PCR and qPCR systems. In this study, plant pathogen DNA extraction efficiencies were evaluated based on three plant disease systems and three DNA extraction methods. DNA of bacterial (Pectobacterium atrosepticum), protist (Plasmodiophora brassicae) and fungal (Botrytis cinerea) pathogens was extracted and aliquots were prepared. Aliquots of the DNA were mixed with healthy tissues of the corresponding host plant and DNA was extracted again from the mixture. The resultant mixed DNA, as well as the original pathogen DNA, were analysed by qPCR to assess the quantities of the pathogen DNA. Extraction efficiency was calculated based on the qPCR Cq values of the mixed DNA and the original pathogen DNA for each extraction method on each pathogen. Our results indicated the pathogen DNA extraction efficiencies were generally less than, or near, 50%. This result called attention to the importance of: 1) including the DNA extraction efficiency in the evaluation and announcement of new qPCR diagnostic systems, and 2) developing and using high efficient DNA extraction methods in plant disease diagnosis and pathogen quantification.

Highly efficient and automated extraction of DNA from human remains using a modified EZ1 protocol

Bones and teeth often represent the only sources of DNA available for identifying human remains. DNA in bones and teeth is generally better preserved than that in soft tissues because of the presence of hard connective tissue with a high level of calcium. Because of the extensive mineralisation, the choice of an efficient DNA extraction procedure is important to minimise the sampling of a high level of minerals and to remove polymerase chain reaction (PCR) inhibitors. Some protocols are available for DNA extraction from bones and teeth as part of the Qiagen EZ1 DNA Investigator Kit using the EZ1 Advanced XL automated purification platform. To improve the efficiency of DNA extraction from skeletal remains, the present study focuses on a modification to these already available protocols. In this study, different bones and teeth collected between 1 and 50 years after death were subjected to DNA extraction using the standard EZ1 protocol, a supplementary protocol, and a modified protocol. The modified approach included a decalcification step, whereas the Qiagen protocols worked directly on non-decalcified powder. In all three procedures, 150 mg samples were used for DNA extraction. We evaluated the quantity of DNA recovered from samples, the presence of any PCR inhibitors co-extracted, the level of DNA degradation, the quality of short tandem repeat (STR) profiles, and the reproducibility of the modified procedure. When compared with the other protocols, the modified protocol resulted in the best recovery of DNA that was free of PCR inhibitors. Additionally, the STR profiles were reliable and of high quality. In our opinion, the decalcification step increases DNA recovery by softening tissues, which allows lysis solutions to act more effectively. Furthermore, the use of two lysis solutions and the variation added to the EZ1 purification step allow for DNA recovery with quality and quantity superior to those of the previously available Qiagen-based protocols. These findings may be helpful solutions to the problems commonly encountered when dealing with difficult samples, such as bones and teeth. Key points Bones and teeth often represent the only sources of DNA for identifying human remains. The choice of an efficient DNA extraction procedure is important for maximizing DNA recovery and removing PCR inhibitors. This study focuses on modifications to the previously available Qiagen-based protocols. The modified protocol enabled the best recovery of DNA, and both quality and quantity were superior to those of the previously available Qiagen-based protocols. The STR profiles obtained from samples extracted using the modified protocol were reliable and of high quality.

Оптимизация выделения мтДНК из клубней картофеля с использованием различных сорбционных носителей и разной степени очистки митохондрий

В настоящее время существует интерес к оценке повреждений мтДНК как к маркеру окисли-тельного стресса, при этом данный вопрос недостаточно изучен для мтДНК растений. Однако мито-хондриальный геном растений обладает рядом особенностей, которые могут существенно затруднять проведение ПЦР. При недостаточной очистке митохондрий пробы всё ещё могут быть контаминиро-ваны ядерной и пластидной ДНК, что может привести к неспецифичному отжигу праймеров. Целью данной работы является оптимизация процесса выделения мтДНК из картофеля (Solanum tube- rosum L.) при использовании различных сорбционных носителей. Применение методов сорбции на твердофазных носителях является наиболее эффективным для крупных по размерам молекул мтДНК. Нами было проведено сравнение эффективности трёх коммерческих наборов для выделения ДНК: diaGene (Диа-М, Россия), Проба-ГС (ДНК-Технология, Россия) и Plasmid miniPrep (Evrogen, Россия). Эти методы подразумевают связывание ДНК с поверхностью сорбционных колонок (diaGene, Plasmid miniPrep), либо на силикатном сорбенте (Проба-ГС). Для оценки эффективности рассмотренных ме-тодов с помощью количественной ПЦР был измерен уровень обогащения мтДНК. Также приведено сравнение эффективности выделения ДНК при разных степенях очистки исходного материала – при использовании гомогената картофеля, при очистке дифференциальным и центрифугированием в гра-диенте Перколла. Низкий уровень насыщенности мтДНК наблюдался при выделении из гомогената, высокий – при выделении митохондрий градиентным центрифугированием. При использовании гра-диентного центрифугирования высокий уровень обогащения мтДНК наблюдался только при выделе-нии ДНК набором Проба-ГС. Нами было обнаружено, что наибольшим насыщение материала мтДНК будет при использовании набора Plasmid miniPrep при предварительной очистке митохондрий гради-ентным центрифугированием. Только градиентное центрифугирование способно максимально очи-стить митохондрии от примесей. Использование набора Plasmid miniPrep, предназначенного для вы-деления плазмидной ДНК, показывает свою эффективность и для мтДНК, которые аналогично пла-стидным ДНК являются преимущественно кольцевыми. В результате удалось разработать оптимизи-рованный процесс выделения мтДНК картофеля, пригодного для проведения дальнейших молекуляр-но-генетических исследований. Этот метод может быть использован для любых научных изысканий, связанных с изучением митохондриального генома растений.

Application of megahertz ultrasound to isolate nucleic acids

The dependence of the yield of M. tuberculosis DNA from model solutions under the influence of megahertz ultrasound in the range from 1.2 to 3.0 W/cm2 on magnetic particles was studied. The dependences of the DNA yield upon sounding for 1, 5, and 10 minutes are obtained. Under ultrasound exposure, the maximum yield is 98%, which is 39% higher than the DNA yield in the absence of a similar effect. It was shown that the efficiency of DNA extraction on magnetic particles using ultrasound is not linearly dependent on the intensity of scoring. The result of the electrophoresis of the sample after ultrasonic treatment of different intensities in megahertz for 1, 5 and 10 minutes is also shown.