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.

• Klíčová slova
• Bacterial diversity, Bacteroidetes, Extracellular DNA, Firmicutes, Intracellular DNA, Metabarcoding, PCR-DGGE, Q-PCR, Rumen fluid, Storage conditions,
• MeSH
• bachor mikrobiologie   MeSH
• Bacteroidetes klasifikace   genetika   izolace a purifikace   MeSH
• denaturační gradientová gelová elektroforéza MeSH
• DNA bakterií izolace a purifikace   MeSH
• fylogeneze MeSH
• grampozitivní bakterie klasifikace   genetika   izolace a purifikace   MeSH
• kryoprezervace * MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• odběr biologického vzorku metody   MeSH
• skot MeSH
• taxonomické DNA čárové kódování * MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH

With the expansion of molecular techniques, the historical collections have become widely used. The last boom started with using next- and second-generation sequencing in which massive parallel sequencing replaced targeted sequencing and third-generation technology involves single molecule technology. Studying plant DNA using these modern molecular techniques 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 taxonomic long-standing issues, 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. The result of these applications is often fragmented DNA. The reason new sequencing approaches have been so successful is that the template DNA needs to be fragmented for proper library building, and herbarium DNA is exactly that. 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, microsatellites, AFLP or next-generation sequencing).

• Klíčová slova
• AFLP, DNA extraction, Difficult plant tissues, Herbarium specimens, Microsatellites, Next-generation sequencing, PCR,
• MeSH
• analýza polymorfismu délky amplifikovaných restrikčních fragmentů MeSH
• chemická frakcionace metody   MeSH
• DNA rostlinná genetika   izolace a purifikace   MeSH
• listy rostlin genetika   MeSH
• mikrosatelitní repetice MeSH
• orgánová specificita MeSH
• polymerázová řetězová reakce MeSH
• reagenční diagnostické soupravy MeSH
• rostliny klasifikace   genetika   MeSH
• sekvenční analýza DNA MeSH
• taxonomické DNA čárové kódování metody   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH
• reagenční diagnostické soupravy MeSH

A fast, very simple and efficient method of DNA extraction is described which takes advantage of DNA sedimentation induced by millimolar concentrations of ZnCl2. The zinc-induced sedimentation is furthermore strongly promoted by submillimolar phosphate anion concentrations. Within <30 min, the method recovers >90% of DNA irrespective of whether a plasmid DNA or short oligonucleotides are the extracted material. The method works with plasmid DNA and oligonucleotide concentrations as low as 100 ng/ml and 10 microg/ml, respectively, without using any expensive facilities or toxic chemicals.

• MeSH
• chloridy chemie   MeSH
• DNA chemie   izolace a purifikace   MeSH
• indikátory a reagencie MeSH
• pufry MeSH
• sloučeniny zinku chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chloridy MeSH
• DNA MeSH
• indikátory a reagencie MeSH
• pufry MeSH
• sloučeniny zinku MeSH
• zinc chloride MeSH Prohlížeč

Environmental DNA (eDNA) metabarcoding (parallel sequencing of DNA/RNA for identification of whole communities within a targeted group) is revolutionizing the field of aquatic biomonitoring. To date, most metabarcoding studies aiming to assess the ecological status of aquatic ecosystems have focused on water eDNA and macroinvertebrate bulk samples. However, the eDNA metabarcoding has also been applied to soft sediment samples, mainly for assessing microbial or meiofaunal biota. Compared to classical methodologies based on manual sorting and morphological identification of benthic taxa, eDNA metabarcoding offers potentially important advantages for assessing the environmental quality of sediments. The methods and protocols utilized for sediment eDNA metabarcoding can vary considerably among studies, and standardization efforts are needed to improve their robustness, comparability and use within regulatory frameworks. Here, we review the available information on eDNA metabarcoding applied to sediment samples, with a focus on sampling, preservation, and DNA extraction steps. We discuss challenges specific to sediment eDNA analysis, including the variety of different sources and states of eDNA and its persistence in the sediment. This paper aims to identify good-practice strategies and facilitate method harmonization for routine use of sediment eDNA in future benthic monitoring.

• Klíčová slova
• Aquatic ecosystems, Environmental DNA, Metabarcoding, Monitoring, Sediments,
• MeSH
• biodiverzita MeSH
• DNA genetika   MeSH
• ekosystém MeSH
• environmentální DNA * MeSH
• monitorování životního prostředí metody   MeSH
• taxonomické DNA čárové kódování MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• DNA MeSH
• environmentální DNA * 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.

• Klíčová slova
• DNA, EcoRI, Enzymatic restriction, Microparticles, Poly(methacrylic acid-co-ethylene dimethacrylate), Solid phase extraction,
• MeSH
• DNA bakterií chemie   izolace a purifikace   MeSH
• DNA chemie   izolace a purifikace   MeSH
• extrakce na pevné fázi * metody   MeSH
• hydrofobní a hydrofilní interakce MeSH
• koncentrace vodíkových iontů MeSH
• polymery chemie   MeSH
• polymethylmethakrylát chemie   MeSH
• velikost částic MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA bakterií MeSH
• DNA MeSH
• poly(MAA-co-EDMA) MeSH Prohlížeč
• polymery MeSH
• polymethylmethakrylát MeSH

Approaches to DNA extraction play a crucial role in determining the variability of results obtained through 16S rRNA amplicon sequencing. Particularly, clay-rich samples can impede the efficiency of various standard cultivation-independent techniques. We conducted an inter-laboratory comparison study to thoroughly assess the efficacy of two published DNA extraction methods (kit-based and phenol-chloroform-based) specifically designed for bentonite samples. To this end, we spiked Wyoming MX 80 bentonite with two different mock communities and compared the obtained DNA yield and purity, the presence of contaminants and the community profile. Our findings suggest that both methods are equally viable, with the best choice depending on the specific requirements of the downstream analysis. However, it is crucial to maintain consistency in the chosen method, as comparing results becomes challenging, particularly in the presence of bentonite. In summary, our study emphasizes the significance of standardized DNA extraction methods and underscores the importance of validating these methods using appropriate controls when studying microbial communities with 16S rRNA amplicon sequencing, particularly in environments characterized by low biomass and clay-rich compositions. Additionally, slight modifications to one of the extraction methods can substantially enhance its efficiency.

• MeSH
• Bacteria genetika   klasifikace   izolace a purifikace   MeSH
• bentonit * chemie   MeSH
• DNA bakterií * genetika   izolace a purifikace   MeSH
• mikrobiota genetika   MeSH
• půdní mikrobiologie MeSH
• RNA ribozomální 16S * genetika   MeSH
• sekvenční analýza DNA MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• bentonit * MeSH
• DNA bakterií * MeSH
• RNA ribozomální 16S * MeSH

Our understanding of human gut microbiota in health and disease depends on accurate and reproducible microbial data acquisition. The critical step in this process is to apply an appropriate methodology to extract microbial DNA, since biases introduced during the DNA extraction process may result in inaccurate microbial representation. In this study, we attempted to find a DNA extraction protocol which could be effectively used to analyze both the bacterial and fungal community. We evaluated the effect of five DNA extraction methods (QIAamp DNA Stool Mini Kit, PureLinkTM Microbiome DNA Purification Kit, ZR Fecal DNA MiniPrepTM Kit, NucleoSpin® DNA Stool Kit, and IHMS protocol Q) on bacterial and fungal gut microbiome recovery using (i) a defined system of germ-free mice feces spiked with bacterial or fungal strains, and (ii) non-spiked human feces. In our experimental setup, we confirmed that the examined methods significantly differed in efficiency and quality, which affected the identified stool microbiome composition. In addition, our results indicated that fungal DNA extraction might be prone to be affected by reagent/kit contamination, and thus an appropriate blank control should be included in mycobiome research. Overall, standardized IHMS protocol Q, recommended by the International Human Microbiome Consortium, performed the best when considering all the parameters analyzed, and thus could be applied not only in bacterial, but also in fungal microbiome research.

• Klíčová slova
• 16S rDNA, DNA extraction method, ITS rDNA, fungal microbiota, gut microbiome, gut microbiota, gut mycobiome, gut mycobiota,
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• Forensic science, cytochrome oxidase subunit I, forensic entomology, molecular markers, species identification,
• MeSH
• Diptera klasifikace   genetika   růst a vývoj   MeSH
• DNA izolace a purifikace   MeSH
• hmyzí proteiny genetika   MeSH
• reagenční diagnostické soupravy MeSH
• respirační komplex IV genetika   MeSH
• sekvenční analýza DNA MeSH
• soudní genetika metody   MeSH
• taxonomické DNA čárové kódování metody   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• DNA MeSH
• hmyzí proteiny MeSH
• reagenční diagnostické soupravy MeSH
• respirační komplex IV 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
• cetrimoniové sloučeniny chemie   MeSH
• cetrimonium MeSH
• chemická frakcionace metody   MeSH
• DNA rostlinná chemie   genetika   izolace a purifikace   MeSH
• druhová specificita MeSH
• filtrace MeSH
• kyselina dusičná chemie   MeSH
• listy rostlin chemie   MeSH
• mikrosatelitní repetice MeSH
• rostliny chemie   MeSH
• semena rostlinná chemie   MeSH
• uchovávání tkání * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cetrimoniové sloučeniny MeSH
• cetrimonium MeSH
• DNA rostlinná MeSH
• kyselina dusičná 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.

• MeSH
• DNA bakterií * genetika   izolace a purifikace   MeSH
• genetické techniky * normy   MeSH
• mikrobiota * genetika   MeSH
• monitorování životního prostředí * metody   MeSH
• půdní mikrobiologie * MeSH
• RNA ribozomální 16S genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA bakterií * MeSH
• RNA ribozomální 16S MeSH