scholarly journals Assessment of sampling and DNA extraction methods for identification of grapevine trunk microorganisms using metabarcoding

2018 ◽  
Vol 71 ◽  
pp. 10-18 ◽  
Author(s):  
Dion C. Mundy ◽  
Bhanupratap R. Vanga ◽  
Sarah Thompson ◽  
Simon Bulman
Keyword(s):  
New Zealand ◽  
Dna Extraction ◽  
Dna Sequences ◽  
Low Cost ◽  
Pcr Amplification ◽  
Extraction Methods ◽  
Subsequent Work ◽  
Dna Metabarcoding ◽  
Ctab Method ◽  
Dna Extraction Methods

For a deeper understanding of grapevine trunk disease (GTD) in New Zealand, a cheap, rapid, sensitive method for identifying within-vine microbial communities is required. Wood tissue from grapevine trunks was collected and three different DNA extraction methods were compared: a cetyltrimethylammonium bromide (CTAB) method, the Geneaid Plant Genomic DNA Mini Kit and the Qiagen DNeasy Plant Mini Kit. DNA samples from the CTAB and Geneaid methods were used for MiSeq DNA metabarcoding targeting the ribosomal internal transcribed spacer 1 (ITS1) region. DNA produced by the CTAB method was of a greater quantity and quality than for the other two methods, although the majority of the DNA samples provided polymerase chain reaction (PCR) amplification of fungal DNA sequences. Fungal metabarcoding profiles from the CTAB and Geneaid samples indicated the presence of fungi normally associated with GTD in New Zealand. The CTAB method was chosen for subsequent work due to its low-cost, simplicity and effective detection of typical GTD fungi. The complete process of sampling through to metabarcoding is now used annually as part of a wider ecological study, screening more than 600 vines at 12 Marlborough vineyards.

scholarly journals Determining the optimal method for DNA isolation from fruit jams

2018 ◽  
Vol 36 (No. 2) ◽  
pp. 126-132
Author(s):  
Sovová Tereza ◽  
Křížová Barbora ◽  
Ovesná Jaroslava
Keyword(s):  
Dna Extraction ◽  
Dna Isolation ◽  
Extraction Methods ◽  
Food Samples ◽  
Pcr Analysis ◽  
Uv Spectrophotometry ◽  
Pcr Assay ◽  
Optimal Method ◽  
Ctab Method ◽  
Dna Extraction Methods

DNA extraction is a crucial step in PCR analysis especially when analysing food samples that can be degraded and can potentially contain PCR-inhibiting substances. In this study, we compared the suitability of three DNA extraction methods – two kits: DNeasy<sup>®</sup> Plant Mini Kit and NucleoSpin<sup>®</sup> Food, and the CTAB method – for DNA extraction from commercial fruit jams. Fourteen jams with different contents of fruit, sugar and other additives were extracted in triplicate using the above-mentioned methods directly and after a washing step. The concentration and optical density were analysed using UV spectrophotometry and the amplifiability of the obtained DNA was evaluated using a PCR assay targeting a sequence coding for chloroplast tRNA-Leu. Samples isolated using the NucleoSpin<sup>®</sup> Food kit contained non-amplifiable DNA in eight cases, and samples isolated using the CTAB method could not be quantified. The DNeasy<sup>®</sup> Plant Mini Kit thus proved to be the most suitable method, since well-amplifiable DNA was obtained for all the analysed samples.

scholarly journals ALKALINE LYSIS AS AN EFFICIENT AND ECONOMICAL ALTERNATIVE FOR DNA EXTRACTION FROM HORSEHAIR HAIR SAMPLES: COMPARISON BETWEEN DIFFERENT METHODS

2021 ◽  
Vol 9 (02) ◽  
pp. 836-841
Author(s):  
Myriam Janeth Ortega Torres ◽  
◽  
Jessica Almeida Braga ◽  
Camilo Torres ◽  
Ahmed Sami Shaker ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Hypervariable Region ◽  
Pcr Amplification ◽  
Extraction Methods ◽  
Forensic Population ◽  
Alkaline Lysis ◽  
Hair Samples ◽  
Genomic Studies ◽  
Dna Extraction Methods ◽  
Short Time

Studies related to DNA extraction are becoming more ambitious in the sense that large studies are intended to be carried out with minimum DNA sources. The DNA extracted must be of quality for genetic, forensic, population and genomic studies, these samples must be easy to obtain and product of efficient manipulation.Samples obtained from horsehair are an important technical challenge since they constitute the preferred non-invasive sample for genetic studies in horses, which has been shown to obtain reliable results in a short time. In this sense, working into effective techniques to optimizeDNA extraction of scarse samples is a pertinent task. In this study, different DNA extraction methods were evaluated from mane samples obtained from a population of wild horses from the Region of Arauca in eastern Colombia.Three DNA extraction methods were evaluated (phenol chloroform, alkaline lysis and twocommercial DNA extraction kit), DNA concentration, purity and qualityweredeterminate and PCR amplification product were obtain using primers for a hypervariable region of DNA mitochondrial. DNA preparation from hair roots using alkaline lysis was the most economical and efficient method with which it was possible to obtain high quality and quantity DNA.

scholarly journals Extracting the invisible: obtaining high quality DNA is a challenging task in small arthropods

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6753 ◽  
Author(s):  
Andrea Lienhard ◽  
Sylvia Schäffer
Keyword(s):  
Dna Extraction ◽  
Low Cost ◽  
Extraction Methods ◽  
Chelating Resin ◽  
Precipitation Method ◽  
Ammonium Bromide ◽  
User Friendliness ◽  
Silica Membrane ◽  
High Quality ◽  
Dna Extraction Methods

BackgroundThe application of an appropriate extraction method is a relevant factor for the success of all molecular studies.MethodsSeven different DNA extraction methods suitable for high-throughput DNA sequencing with very small arthropods were compared by applying nine different protocols: three silica gel based spin methods, two cetyltrimethyl ammonium bromide (CTAB) based ones (one with an additional silica membrane), a protein precipitation method and a method based on a chelating resin (applying different protocols). The quantity (concentration) and quality (degradation, contamination, polymerase chain reaction (PCR) and sequencing success) of the extracted DNA as well as the costs, preparation times, user friendliness, and required supplies were compared across these methods. To assess the DNA quantity, two different DNA concentration measurements were applied. Additionally, the effect of varying amounts of starting material (different body sizes), variable lysis temperatures and mixing during DNA extraction was evaluated.ResultsAlthough low DNA concentrations were measured for all methods, the results showed that—with the exception of two methods—the PCR success was 100%. However, other parameters show vast differences. The time taken to perform DNA extraction varied from 20 min to 2.5 h (Chelex vs. CTAB) and the costs from 0.02 to 3.46 € (Chelex vs. QIAamp kit) per sample. High quality genomic DNA was only gained from four methods. Results of DNA quantity measurements further indicated that some devices cannot deal with small amounts of DNA and show variant results.DiscussionIn conclusion, using Chelex (chelating resin) turned out as a rapid, low-cost method which can provide high quality DNA for different kinds of molecular investigations.

Effect of Source of DNA on the Quantitative Analysis of Genetically Engineered Traits Using Digital PCR and Real-Time PCR

2017 ◽  
Vol 100 (2) ◽  
pp. 492-498 ◽  
Author(s):  
Tigst Demeke ◽  
Jemima Malabanan ◽  
Michelle Holigroski ◽  
Monika Eng
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Extraction Methods ◽  
Absolute Quantification ◽  
Genetically Engineered ◽  
Rt Pcr ◽  
Ctab Method ◽  
Dna Extraction Methods

Abstract Seven commercially available DNA extraction kits were compared with a cetyltrimethylammonium bromide (CTAB)method to determine the suitability of the extracted DNA for RainDrop digital PCR (dPCR) and real-time PCR (RT-PCR) quantification of OXY235 canola, FP967 flax, and DP305423 soybean (spiked at the 0.1% level). For the kits, the highest amount of DNA extracted from a 0.2 g sample was obtained using OmniPrep for Plant for flax andDNeasy mericon Food for canola and soybean. For canola, DNA extracted with the Fast ID Genomic DNA Extraction Kit, FastDNA Spin Kit, GM Quicker 2, NucleoSpin Food, and DNeasy mericon Food was suitable for dPCR and RT-PCR. For flax, DNA extracted with Fast ID, FastDNA Spin Kit, OmniPrep for Plant, and NucleoSpin Food was suitable for RT-PCR. However, only Fast ID yielded DNA suitable for dPCR. For soybean, DNA extracted with five and six of the seven DNA extraction kits was suitable for dPCR and RT-PCR, respectively. Overall, Fast ID provided reliable results regardless of species or analysis method used. Canola, flax, and soybean DNA extracted with the CTAB method and then purified were suitable for both dPCR and RT-PCR. This is the first report showing the effect of different DNA extraction methods on the absolute quantification of genetically engineered traits using dPCR.

scholarly journals At the crossroads of botanical collections and molecular genetics laboratory: a preliminary study of obtaining amplifiable DNA from moss herbarium material

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9109
Author(s):  
Marta Saługa
Keyword(s):  
Dna Extraction ◽  
Extreme Environments ◽  
Pcr Amplification ◽  
Extraction Methods ◽  
Herbarium Specimens ◽  
Polar Regions ◽  
Moss Species ◽  
Dna Yield ◽  
Herbarium Collections ◽  
Dna Extraction Methods

Background Research focused on extreme environments is often associated with difficulties in obtaining fresh plant material. Herbaria may provide great support as they house large collections of specimens from different parts of the world. Accordingly, there is also a growing interest in methods using herbarium specimens in molecular studies. Much of the literature on herbarium DNA is aimed to improve extraction and PCR amplification and is focused mostly on vascular plants. Here, I provide a brief study of DNA extraction efficiency from moss herbarium specimens, emphasizing the importance of herbaria as an invaluable source of material from hard-to-access geographical areas, such as the Antarctic region. Methods The presented study is based on herbarium collections of 25 moss species collected in the austral polar regions between 1979 and 2013. The majority of samples were obtained using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The remaining, smaller part was extracted using an adapted CTAB-based approach. The performance of DNA extraction methods in terms of PCR amplification success was measured by testing several DNA fragments of various size. Furthermore, in order to estimate of DNA fragmentation level, an automated on-chip electrophoresis system was used. Results Results reveal that DNA purity and the length of the target genetic region are the fundamental agents which drive the successful PCR reaction. Conversely, the DNA yield and specimen age seem to be less relevant. With this study, I present also an optimized CTAB-based approach which may effectively suppress inhibitors in the herbarium DNA. This method can be considered a cheaper alternative to column-based technology, particularly useful for dealing with a large number of samples. Results of this study confirmed previous reports and contribute to filling the existing gap in molecular analyses which involve the use of herbarium collections of mosses.

scholarly journals Comparative investigation of DNA extraction methods in black gram (Vigna mungo (L.)

2019 ◽  
Author(s):  
M. . Prakash ◽  
B. . Priyadharshini ◽  
M. . Vignesh ◽  
R. . Anandan
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Large Scale ◽  
Dna Isolation ◽  
Extraction Methods ◽  
Comparative Investigation ◽  
Black Gram ◽  
Dna Extraction Method ◽  
Ctab Method ◽  
Dna Extraction Methods

Isolation of intact, double stranded, pure and non- contaminated genomic DNA is prerequisite for large scale genotyping analysis including DNA-banks. Three methods of DNA isolation (Dellaporta, CTAB and Hi-PurAg DNA isolation kits) from 25 black gram genotypes were compared in terms of the yield, purity, integrity, and stability of extracted DNA. Purity and quantification of isolated DNA samples was confirmed by using the UV nano-spectrophotometer at OD260/280 and the same is confirmed based by agarose gel electrophoresis. The CTAB method showed the best results followed by Hi-PurAg and Dellaporta method. The CTAB DNA extraction method was found to be the most efficient DNA extraction method, capable of providing high quality, pure and stable DNA and could be used for various molecular related works. All the 25 black gram genotypes for this research gave good yield of DNA from the established modified CTAB protocol.

scholarly journals Comparison of DNA extraction methods for non-marine molluscs: Is modified CTAB DNA extraction method more efficient than DNA extraction kits?

2019 ◽  
Author(s):  
Sudeshna Chakraborty ◽  
Anwesha Saha ◽  
N.A. Aravind
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Pcr Amplification ◽  
Extraction Methods ◽  
High Molecular Weight Dna ◽  
Long Term Storage ◽  
Dna Extraction Method ◽  
Successful Amplification ◽  
Dna Extraction Methods ◽  
Sequence Quality

AbstractIsolation of high molecular weight DNA from gastropod molluscs and its subsequent PCR amplification is considered difficult due to excessive mucopolysaccharides secretion which co-precipitate with DNA and obstruct successful amplification. In an attempt to address this issue, we describe a modified CTAB DNA extraction method that proved to work significantly better with a number of freshwater and terrestrial gastropod taxa. We compared the performance of this method with Qiagen® DNeasy Blood and Tissue Kit. Reproducibility of amplification was verified using a set of taxon-specific primers wherein, modified CTAB extracted DNA could be replicated at least four out of five times but kit extracted DNA could not be replicated. Additionally, sequence quality was significantly better with CTAB extracted DNA. This could be attributed to the removal of polyphenolic compounds by polyvinyl pyrrolidone (PVP) which is the only difference between conventional and modified CTAB DNA extraction methods for animals. The genomic DNA isolated using modified CTAB protocol was of high quality (A260/280 ≥ 1.80) and could be used for downstream reactions even after long term storage (more than two years).

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