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Despite the raising preoccupation, the critical question of how the plant community is composed belowground still remains unresolved, particularly for the conservation priority types of vegetation. The usefulness of metabarcoding analysis of the belowground parts of the plant community is subjected to a considerable bias, that often impedes detection of all species in a sample due to insufficient DNA quality or quantity. In the presented study we have attempted to find environmental factors that determine the amount and quality of DNA extracted from total plant tissue from above- and belowground samples (1,000 and 10,000 cm2). We analyzed the influence of land use intensity, soil properties, species composition, and season on DNA extraction. The most important factors for DNA quality were vegetation type, soil conductometry (EC), and soil pH for the belowground samples. The species that significantly decreased the DNA quality were Calamagrostis epigejos, Coronilla varia, and Holcus lanatus. For the aboveground part of the vegetation, the season, management intensity, and certain species — with the most prominent being Centaurea rhenana and Cirsium canum — have the highest influence. Additionally, we found that sample size, soil granulation, MgO, organic C, K2O, and total soil N content are important for DNA extraction effectiveness. Both low EC and pH reduce significantly the yield and quality of DNA. Identifying the potential inhibitors of DNA isolation and predicting difficulties of sampling the vegetation plots for metabarcoding analysis will help to optimize the universal, low-cost multi-stage DNA extraction procedure in molecular ecology studies.

Los estudios sobre salud pública y biodiversidad de mamíferos silvestres incluyen un componente genético. Para las muestras de sangre, se debe tener condiciones óptimas de colección, ya que pueden afectar la preservación y la extracción del ADN. Este estudio evaluó el uso de métodos de preservación de ADN líquido y seco y métodos de extracción de ADN comerciales y no comerciales, en muestras de sangre, recolectadas en campo. Para ello, se recogieron 264 muestras de sangre totales de mamíferos salvajes. Se preservó un primer grupo de muestras en clorhidrato de guanidina (GuHCl) y se extrajo el ADN, utilizando seis kits comerciales: Bioline, Norgen, Invitrogen, Promega y Qiagen, además de dos protocolos no comerciales: fenol-cloroformo isoamil alcohol (PC) y guanidina tiocianato (GIT). Otro grupo de muestras, se preservó en tarjetas Whatman® FTA® y se extrajo el ADN, con PC y GIT. Las extracciones con GIT y PC mostraron los valores y variaciones más altas en la concentración de ADN (ng/µL), mientras que el kit comercial mostró una baja variación. La preservación de la muestra en tarjetas Whatman® FTA® proporcionó una baja variación y cantidad de ADN extraído, en comparación con el uso de GuHCl. En cuanto a la calidad del ADN, los kits comerciales produjeron una mayor pureza (A260/280), mientras que los protocolos basados en GIT y PC proporcionaron resultados muy variables. Además, el uso de GIT y PC originó una mayor cantidad de ADN, pero de calidad variable. En general, la extracción basada en kits comerciales y la conservación Whatman® FTA® permitió obtener calidades y cantidades de ADN más estandarizadas. Studies on public health and wild mammal biodiversity include a genetic component. For blood samples, there must be optimal sample collection conditions since these can affect DNA preservation and extraction. This study evaluated the use of liquid and dry DNA preservation methods and commercial and non-commercial DNA extraction methods on field-collected blood samples. For this, 264 total blood samples were collected from wild mammals. A first group of samples was preserved in guanidine hydrochloride (GuHCl) and DNA was extracted using six commercial kits: Bioline, Norgen, Invitrogen, Promega, and Qiagen, in addition to phenol-chloroform isoamyl alcohol (PC) and guanidine thiocyanate (GIT). Another group of samples was preserved in Whatman® FTA® cards and DNA was extracted with PC and GIT. The extractions with GIT and PC showed the highest values (ng/µL) and variation in DNA concentration, while the commercial kit showed low variation. Sample preservation in Whatman® FTA® cards provided low variation and quantity of the extracted DNA compared with the use of GuHCl. Concerning DNA quality, the commercial kits yielded higher purity, while GIT and PC-based protocols provided highly variable results. Furthermore, the use of GIT and PC yielded a higher amount of DNA, yet, of variable quality. Overall, extraction based on commercial kits and Whatman® FTA® preservation allowed obtaining more standardized DNA qualities and quantities Incluye referencias bibliográficas

Obtaining high-quality plant materials for experiments is challenging for many research projects. Therefore, it is of special importance to determine the best method for preserving biological macromolecules like DNA, which degrade over time. Although some research has demonstrated that DNA degradation has little effect on traditional molecular markers, the effects of DNA degradation on ddRAD-seq, a popular reduced-representation sequencing technology, have not been adequately investigated. In this study, we first chose six woody bamboo species (Bambusoideae, Poaceae) to explore appropriate methods for preserving molecular materials with two DNA extraction approaches. Then we sequenced twenty-one bamboos and examined the effects of DNA quality on data generation using the ddRAD-seq technique (MiddRAD-seq). Finally, we reconstructed phylogenies of twenty woody bamboo species. We found that the integrity of dry-powdered DNA was preserved longer than that of TE-dissolved DNA, regardless of whether the DNA was extracted by a modified CTAB protocol or DNAsecure plant kit. The ddRAD-seq data were robust, except when DNA was severely degraded. In addition, we resolved the phylogenetic positions of the sampled Phyllostachys spp. Our results suggest that dry-powdered DNA is the most appropriate preservation method for plant molecular materials. Furthermore, a moderate level of DNA degradation has little effect on reduced representation sequencing techniques represented by ddRAD-seq. Keywords: Molecular materials, DNA extraction and preservation, DNA quality, ddRAD-seq, Bamboo, Phylogeny

Günümüzde moleküler biyoloji ve biyoinformatik alanındaki gelişmelere paralel olarak metagenomik, biyoteknoloji, tüm genom dizilemesi, genom evrimi gibi alanlarda çok sayıda araştırma yapılmaktadır. Böcek türleri ile yapılan çalışmalarda ilk ve en önemli adım etkili bir DNA izolasyonu yönteminin kullanılmasıdır. Kitin yapılı bir dış iskelete sahip olan böceklerin çoğunun küçük canlılar olması DNA izolasyonunu zorlaştırmaktadır. Yapılan bu çalışma ile Cephus pygmeus (Linnaeus, 1767) türüne ait bireylerden dokuz farklı DNA izolasyon yöntemi ile total DNA izolasyonu gerçekleştirilmiştir. Elde edilen DNA örnekleri verim, saflık, maliyet, zaman ve PCR başarısı kriterleri altında değerlendirilmiştir. Maliyet ve kullanılan kimyasalların toksisitesi göz önüne alındığında ise tuzla çöktürme yönteminin böcek dokularından DNA izolasyonu için en uygun yöntem olduğu yorumlanmıştır., Today, there are many studies conducted in the areas such as metagenomics, biotechnology, whole genome sequencing, genome evolution, and so on parallel to the developments in molecular biology and bioinformatics in molecular biology and bioinformatics. The first and most important step in studies with insect species is the use of an effective DNA isolation method. Small body and external chitin skeleton of the insects cause difficulties in isolating the insect DNAs. In this study, nine different DNA isolation methods were applied to carry out total DNA isolation from the individuals belonging to the species Cephus pygmeus (Linnaeus, 1767). The results were interpreted in terms of DNA yield, purity, cost, time and PCR performance criteria. When the cost and the toxicity of the used chemicals are taken into consideration, it is interpreted that the salting out method is the most suitable method for DNA isolation from insect tissues.

Background Genomic DNA extracted from species of Cactaceae is often contaminated with significant amounts of mucilage and pectin. Pectin is one of the main components of cellular walls, whereas mucilage is a complex polysaccharide with a ramified structure. Thus, pectin- and mucilage-free extraction of DNA is a key step for further downstream PCR-based analyses. Results We tested our DNA extraction method on cladode tissue (juvenile, adult, and herbaria exemplars) of 17 species of Opuntia Mill., which are characterized by a large quantity of pectin and mucilage. Conclusion We developed a method for the extraction of gDNA free of inhibitory compounds common in species of Opuntia Mill., such as pectin and mucilage. Compared to previously extraction protocols, our method produced higher yields of high-quality genomic DNA.

Objectives The aim of this study was to explore the variability in DNA quality and quantity along a gradient of industrial processing of botanical ingredients from raw materials to extracts. Methods A data matrix was assembled for 1242 botanical ingredient samples along a gradient of industrial processing commonly used in the Natural Health Product (NHP) industry. Multivariate statistics was used to explore dependant variables for quality and quantity. The success of attaining a positive DNA test result along a gradient of industrial processing was compared among four biotechnologies: DNA barcoding, NGS, Sanger sequencing and qPCR. Results There was considerable variance in DNA quality and quantity among the samples, which could be interpreted along a gradient from raw materials with greater quantities (50–120 ng/μL) of DNA and longer DNA (400-500bp) sequences to extracts, which were characterized by lower quantities (0.1–10.0 ng/μL) and short fragments (50-150bp). Conclusions Targeted molecular diagnostic tests for species identity can be used in the NHP industry for raw and processed samples. Non-targeted tests or the use of NGS for any identity test needs considerable research and development and must be validated before it can be used in commercial operations as these methods are subject to considerable risk of false negative and positive results. Proper use of these tools can be used to ensure ingredient authenticity, and to avert adulteration, and contamination with plants that are a health concern. Lastly these tools can be used to prevent the exploitation of rare herbal species and the harvesting of native biodiversity for commercial purposes.

A global decline in pollinator abundance and diversity has demanded increased research attention to the ecology and genetics of bumblebees. However, as progressively more restrictions are placed on sampling for insects, researchers are increasingly obliged to use archival specimens collected for purposes other than genetic analyses. In this study we assessed the suitability, for population genetic studies, of popular, low-cost methods for preservation and storage of bumblebee specimens. Specimens of Bombus terrestris L. were held under six storage regimes for up to two years. DNA was extracted from the samples using three extraction protocols and the quality of the DNA was examined using PCR amplification of a mitochondrial and a nuclear gene. All extraction and storage methods provided sufficient DNA for successful PCR amplification. However, samples preserved in acetone or at freezing temperatures yielded the highest DNA concentrations. DNA yields from pinned specimens at room temperature declined over time, particularly when using standard extraction techniques. DNA concentrations were significantly lower from speci- mens preserved in 70% ethanol compared to all other extraction techniques and declined linearly over the two years of storage. These results indicate that two of the most popular insect storage methods (pinning and storage in ethanol) should be avoided for the long-term preservation of genetic material for future studies. We suggest that optimal insect preservation methods should be incorpo- rated into research protocols in order to best capitalise on limited collection opportunities.

Extraction of genomic DNA is the very first experiment in the study cascade of human inherited diseases. Blood is preferred to be the material for DNA extraction. In the case of limitation of the material source, for example, new born screening program, blood is saved as dried spot in filter paper. In practical, long term storing blood sample as the dried spots on filter paper leads to more advantages for DNA extraction essay which need to be performed several times. This study provides an efficient method with 2 step lyses allow high concentration of DNA is extracted from samples. Comparing to the commercial QIAamp® DNA Mini Kit DNA concentration extracted from this method is higher, the quality of DNA is equal and the cost is lower. The optimal DNA extraction method based on phenol in this study is useful tool which may apply for further molecular analysis studies using dried blood spot samples.

International audience; Many methods have been developed for DNA integrity assessment including electrophoresis-based procedures, quantitative PCR, and, more recently, microfluidics-based procedures. DNA integrity evaluation can be employed for characterizing biological samples quality before extensive genomic analysis and also finds applications in reproductive medicine, prenatal diagnostics, or cancer research. In this chapter, we will focus on the assessment of DNA integrity in cancer research. In particular, we will present the application of the determination of DNA integrity for tracking of circulating tumor DNA. Finally, we will conclude by illustrating the potential innovative application of DNA integrity as a biomarker in clinical research, especially for prognostic purposes, patient follow-up, or early diagnosis.

In the context of population genetic research, a faster and less invasive method of DNA sampling would allow large-scale assessments of genetic diversity and genetic differentiation with the help of volunteer observers. The aim of this study was to investigate the usefulness of eggshell membranes as a DNA source for population genetic research, by addressing eggshell membrane DNA quality, degeneration and cross-contamination. To this end, a comparison was made with blood-derived DNA samples. We have demonstrated 100% successful DNA extraction from post-hatched Black-tailed Godwit (Limosa limosa) eggshell membranes as well as from blood samples. Using 11 microsatellite loci, DNA amplification success was 99.1% for eggshell membranes and 97.7% for blood samples. Genetic information within eggshell membrane DNA in comparison to blood DNA was not affected (F(ST) = -0.01735, P = 0.999) by degeneration or possible cross-contamination. Furthermore, neither degeneration nor cross-contamination was apparent in total genotypic comparison of eggshell membrane DNA and blood sample DNA. Our research clearly illustrates that eggshell membranes can be used for population genetic research.

DNA extraction methods were evaluated for the yield and purity of DNA recovered from mycorrhized roots and whether the recovered DNA is suitable for amplification of arbuscular mycorrhizal (AM) fungal SSU rDNA. The DNeasy Plant Mini Kit and three extraction buffers were used alone or in combination with either polyvinylpyrrolidone (PVP), polyvinylpolypyrrolidone (PVPP) and/or activated charcoal (AC). Among the extraction methods tested, those based on the CTAB buffers yielded more DNA than those based on the TE buffer and the DNeasy Plant Mini Kit. Moreover, the use of AC alone or in combination with PVPP reduced DNA yield, while it significantly improved the purity of recovered DNA, whatever the extraction buffer. On the other hand, the success of nested-PCR amplification was negatively correlated with the amount of template DNA and positively correlated with the purity of recovered DNA. Three methods based on the TE buffer, two on the CTAB-βM buffer and one on the DNeasy Plant Mini Kit produced high-quality DNA in terms of purity and PCR performance. However, the TE buffer-based methods are less time consuming than the CTAB-βM buffer-based methods, and cheaper than the method based on the DNeasy Plant Mini Kit.

Nowadays, molecular analyses play an important role in studies of soil dwelling animals, for example in taxonomy, phylogeography or food web analyses. The quality of the DNA, used for later molecular analyses, is an important factor and depends on collection and preservation of samples prior to DNA extraction. Ideally, DNA samples are frozen immediately upon collection, but if samples are collected in the field, suitable preservation methods might be limited due to unavailability of resources or remote field sites. Moreover, shipping samples over long distances can cause loss of DNA quality e.g. by thawing or leaking of preservation liquid. In this study we use earthworms, a key organism in soil research, to compare three different DNA preservation methods – freezing at −20 °C, storing in 75% ethanol, and freeze drying. Samples were shipped from the United States of America to Austria. The DNA of the samples was extracted using two different extraction methods, peqGOLD™ and Chelex® 100. The DNA amplification success was determined by amplifying four DNA fragments of different length. The PCR amplification success is significantly influenced by preservation method and extraction method and differed significantly depending on the length of the DNA fragment. Freeze drying samples was the best preservation method when samples were extracted using the silica based extraction method peqGOLD™. For samples that were extracted with Chelex® 100, storage in ethanol was the best preservation method. However, the overall amplification success was significantly lower for the extraction procedure based on Chelex® 100. The detection of the small DNA fragments was higher and independent from the extraction method, while the amplification success was significantly reduced for the longer DNA fragments. We recommend freeze drying of DNA samples, especially when they have to be shipped for longer distances. No special packaging or declaration is needed for freeze dried samples, and the risk of thawing is excluded. Storage of freeze dried samples also reduces costs because samples can be kept at room temperature in a desiccator. It should be noted, that the extraction methods showed significant differences in DNA amplification success. Thus, the extraction method should be taken into account when choosing the preservation method., Highlights • How to best store and ship DNA samples generated in the USA for analyses in Austria? • Different preservation and extraction methods are compared by analysing tissue samples. • Freeze drying is the recommended preservation method, especially when samples have to be shipped. • The DNA amplification success is influenced by preservation and extraction method.

Background: Array Comparative Genomic Hybridization (array CGH) is increasingly applied on DNA obtained from formalin-fixed paraffin-embedded (FFPE) tissue, but in a proportion of cases this type of DNA is unsuitable. Due to the high experimental costs of array CGH and unreliable methods for DNA quality testing, better prediction methods are needed. The aim of this study was to accurately determine the quality of FFPE DNA input in order to predict quality of array CGH outcome. Material and Methods: DNA quality was assessed by isothermal amplification and compared to array CGH quality on 59 FFPE gastric cancer samples, one FFPE colorectal cancer sample, two FFPE normal uvula samples, one fresh frozen and six FFPE HNSCC samples. Gastric cancer DNA was also quality tested by β-globin PCR. Results: Accurate prediction of DNA quality using the isothermal amplification was observed in the colorectal carcinoma, HNSCC and uvula samples. In gastric cancer samples, the isothermal amplification was a more accurate method for selecting good quality DNA for array CGH compared to using PCR product lengths. The isothermal amplification product was used for array CGH and compared to the results achieved using non-amplified DNA in four of the samples. DNAs before and after amplification yielded the same segmentation patterns of chromosomal copy number changes for both the fresh DNA sample and the FFPE samples. Conclusion: The efficiency of isothermal DNA amplification is a reliable predictor for array CGH quality. The amplification product itself can be used for array CGH, even starting with FFPE derived DNA samples.