Bylo provedeno srovnání devíti metod izolace specifické DNA etiologického agens humánní granulocytárníehrlichiózy z lidské krve pro účely vyšetření PCR. Do plné krve zdravého dárce byladefinovaně přimíšena laboratorní kultura agens a testována účinnost izolace a stabilita templátuza podmínek, kdy byla krev čerstvá nebo zmražená. K univerzálnímu použití byl nejvhodnějšíQIAamp® DNA Mini Kit (QIAGEN), zmražená krev byla nejlépe extrahována pomocí Nucleo SpinTissue (Macherey-Nagel).
The author compared nine methods for isolation of specific DNA of the etiological agent of humangranulocytic ehrlichiosis from human blood for examination of the PCR. To full blood of healthydonors a laboratory culture of the agent was added and the effectiveness of isolation an stability ofthe template was tested under conditions when the blood was fresh or frozen. For universal useQIAamp® (QIAGEN) was most suitable, frozen blood was extracted best using NucleoSpin® Tissue(Macherey-Nagel).
- MeSH
- Anaplasma pathogenicity MeSH
- DNA MeSH
- Ehrlichiosis diagnosis etiology MeSH
- Research Support as Topic MeSH
- Clinical Laboratory Techniques methods MeSH
- Humans MeSH
- Polymerase Chain Reaction MeSH
- Serial Extraction methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Review MeSH
- Comparative Study MeSH
The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT, -80 °C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using high-throughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (P < 0.01) in eDNA samples. The qPCR indicated significantly higher amount of Firmicutes in iDNA sample frozen with glycerol (P < 0.01). Deep sequencing analysis of iDNA samples revealed the prevalence of Bacteroidetes and similarity of samples frozen with and without cryoprotectants, which differed from sample stored with ethanol at room temperature. Centrifugation and consequent filtration of rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored at -80 °C was found as the optimal method to study ruminal bacterial profile.
- MeSH
- Rumen microbiology MeSH
- Bacteroidetes classification genetics isolation & purification MeSH
- Denaturing Gradient Gel Electrophoresis MeSH
- DNA, Bacterial isolation & purification MeSH
- Phylogeny MeSH
- Gram-Positive Bacteria classification genetics isolation & purification MeSH
- Cryopreservation * MeSH
- Real-Time Polymerase Chain Reaction MeSH
- Specimen Handling methods MeSH
- Cattle MeSH
- DNA Barcoding, Taxonomic * MeSH
- High-Throughput Nucleotide Sequencing MeSH
- Animals MeSH
- Check Tag
- Cattle MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
With the expansion of molecular techniques, the historical collections have become widely used. Studying plant DNA using modern molecular techniques such as DNA sequencing plays an important role in understanding evolutionary relationships, identification through DNA barcoding, conservation status, and many other aspects of plant biology. Enormous herbarium collections are an important source of material especially for specimens from areas difficult to access or from taxa that are now extinct. The ability to utilize these specimens greatly enhances the research. However, the process of extracting DNA from herbarium specimens is often fraught with difficulty related to such variables as plant chemistry, drying method of the specimen, and chemical treatment of the specimen. Although many methods have been developed for extraction of DNA from herbarium specimens, the most frequently used are modified CTAB and DNeasy Plant Mini Kit protocols. Nine selected protocols in this chapter have been successfully used for high-quality DNA extraction from different kinds of plant herbarium tissues. These methods differ primarily with respect to their requirements for input material (from algae to vascular plants), type of the plant tissue (leaves with incrustations, sclerenchyma strands, mucilaginous tissues, needles, seeds), and further possible applications (PCR-based methods or microsatellites, AFLP).
- MeSH
- Cetrimonium Compounds chemistry MeSH
- Chemical Fractionation methods MeSH
- DNA, Plant chemistry genetics isolation & purification MeSH
- Species Specificity MeSH
- Filtration MeSH
- Nitric Acid chemistry MeSH
- Plant Leaves chemistry MeSH
- Microsatellite Repeats MeSH
- Plants chemistry MeSH
- Seeds chemistry MeSH
- Tissue Preservation * MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Several methods of DNA extraction, coupled with 'DNA barcoding' species identification, were compared using specimens from early developmental stages of forensically important flies from the Calliphoridae and Sarcophagidae families. DNA was extracted at three immature stages - eggs, the first instar larvae, and empty pupal cases (puparia) - using four different extraction methods, namely, one simple 'homemade' extraction buffer protocol and three commercial kits. The extraction conditions, including the amount of proteinase K and incubation times, were optimized. The simple extraction buffer method was successful for half of the eggs and for the first instar larval samples. The DNA Lego Kit and DEP-25 DNA Extraction Kit were useful for DNA extractions from the first instar larvae samples, and the DNA Lego Kit was also successful regarding the extraction from eggs. The QIAamp DNA mini kit was the most effective; the extraction was successful with regard to all sample types - eggs, larvae, and pupari.
- MeSH
- Diptera classification genetics growth & development MeSH
- DNA isolation & purification MeSH
- Insect Proteins genetics MeSH
- Reagent Kits, Diagnostic MeSH
- Electron Transport Complex IV genetics MeSH
- Sequence Analysis, DNA MeSH
- Forensic Genetics methods MeSH
- DNA Barcoding, Taxonomic methods MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
- Geographicals
- Czech Republic MeSH
Microbial community profiling using high-throughput sequencing relies in part on the preservation of the DNA and the effectiveness of the DNA extraction method. This study aimed at understanding to what extent these parameters affect the profiling. We obtained samples treated with and without a preservation solution. Also, we compared DNA extraction kits from Qiagen and Zymo-Research. The types of samples were defined strains, both as single species and mixtures, as well as undefined indigenous microbial communities from soil. We show that the use of a preservation solution resulted in substantial changes in the 16S rRNA gene profiles either due to an overrepresentation of Gram-positive bacteria or to an underrepresentation of Gram-negative bacteria. In addition, 16S rRNA gene profiles were substantially different depending on the type of kit that was used for extraction. The kit from Zymo extracted DNA from different types of bacteria in roughly equal amounts. In contrast, the kit from Qiagen preferentially extracted DNA from Gram-negative bacteria while DNA from Gram-positive bacteria was extracted less effectively. These differences in kit performance strongly influenced the interpretation of our microbial ecology studies.
Cíl studie : Stanovit trendy a potencionální prognostický význam změn hladin apoptotické (aDNA) a genomické DNA (gDNA) u pacientů, u nichž inzult různého charakteru vedl k selhávání vitálních funkcí. Typ studie: Prospektivní otevřená studie. Název a sídlo pracoviště:Klinika anesteziologie a resuscitace a Chirurgická klinika 3. LF UK a FNKV, Praha; Ústav farmakologie 3. LF UK, Praha. Materiál a metody:Do studie byla zařazeno 94 kriticky nemocných. Vzorky krve k analýze byly odebírány v den přijetí a potom 3. a 5. den hospitalizace. U každého pacienta byla sledována základní diagnóza, věk a pohlaví, pří- jmové APACHE II skóre a orgánová dysfunkce; 3. a 5. den byla hodnocena metodikou SOFA délka hospitalizace ve dnech, přežití či úmrtí, z ukazatelů zánětu C-reaktivní protein a počet leukocytů. Analýza byla provedena na sekvenátoru (ABI Prism 377) s užitím originální fenolové extrakční metody. Získané hodnoty plazmy pacientů byly porovnávány s normou získanou z plazmy 86 zdravých dobrovolníků (norma aDNA a gDNA = 100%). Výsledky:Hladina volné aDNA po zátěži ovlivňuje celkovou hladinu volné DNA zásadním způsobem. Její navýše- ní v hodnotě mediánu je 16,3násobně vyšší než u gDNA. V trendu dalších 4 dnů vykazovala v celé hodnocené skupině snížení hladin, gDNA naopak zvýšení. Rozdíl v průběhu trendů i obě kvality DNA hodnocené proti normě byly na hladině statistické významnosti p < 0,001. Byly zjištěny statisticky významná korelace mezi hladinou aDNA v době přijetí a mortalitou nemocných. Závěr:U kriticky nemocných dochází bezprostředně po inzultu k výraznému zvýšení volné aDNA v plazmě, která je v dalším průběhu i ukazatelem přežití nemocných (p < 0,05).
Objective: To establish the trends and prognostic value of plasma levels of the free apoptotic (aDNA) and geno- mic DNA (gDNA) in patients with failure of vital functions. Type of study: Prospective observational study. Setting: University Dept. of Anaesthesiology/CCM and Dept. of Surgery, Charles University, 3rd School of Medici- ne, Prague. Material and Methods : 94 critically ill patientts were evaluated. The blood samples for DNA analysis were taken on the day of admission, the third and fifth day of hospital stay. The following data were collected: ICU admission diagnosis, age and gender, APACHE II, SOFA, ICU stay in days, ICU survival, CRP and WBC count. The analy- sis was done on a sequencer (ABI PRISM 377) using of the original phenol extraction method. The results were correlated to the plasma free DNA levels of 86 healthy volunteers (normal value = 100%). Results:The contribution of aDNA to the total plasma DNA in the critically ill was ~16fold greater than the contri- bution of gDNA. Apoptotic DNA levels were highest on the day of admission and declined thereafter (P < 0.001), whilst the opposite was true for gDNA (P < 0.001) The difference in trends and the aDNA and gDNA levels in cor- relation to normal levels was found statistically significant (P < 0.001). Apoptotic DNA on the day of admission sig- nificantly differs in survivors and non-survivors (P < 0.05). Conclusion:The free apoptotic DNA plasma level on admission could be a predictor of mortality in critical illness.
- MeSH
- Apoptosis MeSH
- Blood Chemical Analysis methods instrumentation statistics & numerical data MeSH
- C-Reactive Protein MeSH
- DNA MeSH
- Research Support as Topic MeSH
- Genome MeSH
- Critical Illness nursing therapy MeSH
- Leukocytes MeSH
- Critical Care methods MeSH
- Review Literature as Topic MeSH
- Prognosis MeSH
- Publication type
- Comparative Study MeSH
DNA extraction from soil samples is a critical step for molecular biology analyses. The present study compared the efficiency of two DNA isolation methods from non-polluted and polluted soils with or without the presence of a plant. Both applied methods used chemical and physical lyses, but method 1 had an additional physical disruption. The main difference between these two methods was the humic acid purification technique as it was carried out during cell lysis for method 1 and after cell lysis for method 2. Samples were assessed on the basis of their yield and DNA purity as well as their bacterial quantity and diversity. Based on our results, method 1 proved to be more effective at removing protein and RNA, whereas method 2 proved to be more effective at removing humic acids. Although no differences were obtained in terms of the DNA yield, both the bacterial quantity and community structure were affected by the method used. Method 1 allowed for the recovery of more information than method 2, and polluted soil was more sensitive to the DNA extraction procedure. We recommend carefully selecting the DNA extraction method, especially when soil is disturbed.
- MeSH
- Bacteria classification genetics isolation & purification metabolism MeSH
- Chemistry Techniques, Analytical methods MeSH
- DNA, Bacterial genetics isolation & purification MeSH
- Humic Substances MeSH
- Soil Pollutants analysis metabolism MeSH
- Polymerase Chain Reaction MeSH
- Soil chemistry MeSH
- Soil Microbiology MeSH
- Environmental Pollution MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
The efficiency of solid phase extraction (SPE) of DNA on polymer particles is limited by the features of the applied solid support, such as size, hydrophilicity, and functionality and their application in SPE also requires additional steps and compounds to finally obtain sufficient amount of high-quality DNA. The present study describes a preparation of sub-micrometer monodisperse poly(methacrylic acid-co-ethylene dimethacrylate) (PME) particles by precipitation polymerization. The effect of the ethylene dimethacrylate (EDMA) crosslinker concentration on morphology and particle size, which varied from 730 to 900 nm, was investigated. The particles with 5 and 15 wt% EDMA were selected for a study of SPE of plasmid DNA under various adsorption and elution conditions, followed by the enzymatic restriction of isolated DNA to verify a quality the nucleic acid. The particles with 15 wt% EDMA were suitable for the SPE because they retained better colloidal stability during the adsorption without additional induction of DNA conformational change. The quality of isolated DNA was finally verified by enzymatic restriction by restriction endonuclease EcoRI. Moreover, the developed method using PME particles was successfully utilized for DNA isolation from Escherichia coli lysate.
- MeSH
- DNA, Bacterial chemistry isolation & purification MeSH
- DNA chemistry isolation & purification MeSH
- Solid Phase Extraction * methods MeSH
- Hydrophobic and Hydrophilic Interactions MeSH
- Hydrogen-Ion Concentration MeSH
- Polymers chemistry MeSH
- Polymethyl Methacrylate chemistry MeSH
- Particle Size MeSH
- Publication type
- Journal Article MeSH
Epitachophoresis is a novel next generation extraction system capable of isolating DNA and RNA simultaneously from clinically relevant samples. Here we build on the versatility of Epitachophoresis by extracting diverse nucleic acids ranging in lengths (20 nt-290 Kbp). The quality of extracted miRNA, mRNA and gDNA was assessed by downstream Next-Generation Sequencing.
- MeSH
- DNA, Neoplasm analysis chemistry isolation & purification MeSH
- Tissue Fixation MeSH
- Colorectal Neoplasms genetics pathology MeSH
- Humans MeSH
- Tumor Cells, Cultured MeSH
- Lung Neoplasms genetics pathology MeSH
- RNA, Neoplasm analysis chemistry isolation & purification MeSH
- High-Throughput Nucleotide Sequencing methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
