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 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 Performance comparison of different microbial DNA extraction methods on bird feces

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xian Hou ◽  
Shengkai Pan ◽  
Zhenzhen Lin ◽  
Jiliang Xu ◽  
Xiangjiang Zhan
Keyword(s):  
Dna Extraction ◽  
Relative Abundance ◽  
Alpha Diversity ◽  
Cell Lysis ◽  
Extraction Methods ◽  
Dna Integrity ◽  
Microbial Composition ◽  
Dna Yield ◽  
Dna Extraction Methods ◽  
Microbial Dna

Abstract Background As an important player during food digestion, gut microbiota has attracted much attention in diet adaptation studies in birds. Microbiota extracted from feces has been widely used as a proxy for gut microbiota. Although several methods have been developed for microbial DNA extraction, their performances in the bird feces have not been systematacially evaluated yet. Methods In this study, we applied three DNA extraction methods (Qiagen, MoBio and Bead) to extract DNA from feces of three avian dietary guilds (granivore, omnivore and carnivore), sequenced V4 region of 16S rRNA gene for each extract and evaluated the performances of DNA yield, DNA integrity, microbial composition, cell lysis capacity and alpha diversity for the three methods on each dietary guild. Results Bead method was the best on the performance of both DNA yield and DNA integrity regardless of dietary guild. In granivore, microbial relative abundance at both species and phylum levels, alpha diversity and cell lysis capacity were comparable among all methods. In omnivore, Qiagen had the best performance on alpha diversity, followed by Bead and MoBio. There were small variations on microbial relative abundance at both species and phylum levels among different extraction methods. MoBio exhibited the best performance on cell lysis capacity. In carnivore, considerable variations were found on microbial relative abundance at both species and phylum levels. Qiagen had the best performance on alpha diversity, followed by MoBio and Bead. MoBio had the highest cell lysis capacity. Conclusions DNA yield and integrity have no obvious impact on microbial composition, alpha diversity or cell lysis capacity. The microbiota results (e.g., microbial composition, cell lysis capacity, alpha diversity) obtained from different methods are comparable in granivorous avian species but not in omnivorous or carnivorous birds. Either method could be used in granivore microbiota studies. For omnivores and carnivores, we recommend Qiagen method when the research purpose is microbial diversity and MoBio when gram-positive bacteria is the research target.

scholarly journals Impact of bead-beating intensity on microbiome recovery in mouse and human stool: Optimization of DNA extraction

2020 ◽  
Author(s):  
Bo Zhang ◽  
Matthew Brock ◽  
Carlos Arana ◽  
Chaitanya Dende ◽  
Lora Hooper ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Extraction Methods ◽  
Dna Integrity ◽  
Sequencing Data ◽  
16S Sequencing ◽  
Bead Beating ◽  
Dna Yield ◽  
Dna Extraction Methods ◽  
The Impact ◽  
Human Stool

AbstractDNA extraction methods play an important role in the acquisition of accurate and reproducible 16S sequencing data in microbiome studies. In this study, we assessed the impact of bead-beating intensity during DNA extraction on microbiome recovery in mouse and human stool. We observed a higher DNA yield, better DNA integrity, higher Shannon’s entropy and Simpson’s index in samples beaten for 4 and 9 minutes as compared to unbeaten samples. 16S sequencing data showed that bead beating has a statistically-significant (p<0.05) impact on the recovery of many clinically relevant microbes that live in the mouse and human gut, including Bifidobacterium, Sutterella and Veillonella. It was observed that 4 minutes of bead beating promotes recovery of about 70% of OTUs in mouse and human stool, while the remaining 30% requires longer bead beating. In conclusion, our study indicates adjustments in bead beating treatment based on the composition of the specimen and the targeted bacteria.

scholarly journals Analysis of automatically extracted white blood cell DNA yield

2020 ◽  
pp. 40-50
Author(s):  
М.В. Олькова ◽  
Е.В. Балановская ◽  
Л.С. Бычковская ◽  
О.П. Балановский
Keyword(s):  
Blood Cell ◽  
Dna Extraction ◽  
White Blood Cell ◽  
Regression Models ◽  
Extraction Methods ◽  
Reference Points ◽  
Linear Regression Models ◽  
Dna Concentration ◽  
Dna Yield ◽  
Dna Extraction Methods

Все более широкое применение поточных методов экстракции ДНК влечет за собой необходимость стандартизации и проверки качества ее выделения. Мы провели детальное количественное изучение процесса выделения и определили стандартизирующие опорные точки для одного из наиболее производительных методов - выделения на магнитных частицах с использованием автоматической станции QIAsymphony SP. Показано, что концентрация ДНК в индивидуальном образце в основном определяется содержанием лейкоцитов в исходном образце крови (корреляция около 0,9). Концентрация ДНК также зависит от метода измерения. Это позволило нам построить линейные регрессионные модели и вывести формулы, точно прогнозирующие концентрацию ДНК в образце для случаев применения двух широко распространенных методов - спектрофотометрического (Nanodrop) и флуоресцентного (Qubit). Обнаружено, что последняя модель Nanodrop OneC, благодаря встроенному алгоритму идентификации примесей и корректировки концентрации, дает более точную оценку концентрации, чем Qubit 4.0. Для быстрого, но приблизительного прогноза концентрации ДНК вместо регрессионных моделей могут применяться стандарты, рассчитанные нами для референсных значений содержания лейкоцитов. The continuing development of mass DNA extraction methods entails the need to set standardisation and quality verification reference points. A study has been conducted and standards set for one of the many methods of DNA extraction - the automated мagnetic beads-based extraction using QIAsymphony SP station. It was shown that the concentration of DNA in an individual sample is mainly determined by the leukocyte content in the initial blood sample (correlation of about 0.9). DNA concentration also depends on the measurement method. It allowed us to build linear regression models and derive formulae that accurately predict the concentration of DNA in the sample for the use of two widely used methods - spectrophotometric (Nanodrop) and fluorescence (Qubit). It was found that the latest Nanodrop OneC model, thanks to a built-in algorithm for identifying impurities and adjusting the concentration, provides an even more accurate concentration estimate than Qubit 4.0. For a quick but rough forecast of DNA concentration, instead of regression models, standards calculated by us for reference values of the white blood cell count can be used.

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).

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 Endocervical swabs transported in first void urine as combined specimens in the detection of Mycoplasma genitalium by real-time PCR

2009 ◽  
Vol 58 (1) ◽  
pp. 117-120 ◽  
Author(s):  
Andreas Edberg ◽  
Fredrik Aronsson ◽  
Eva Johansson ◽  
Elisabeth Wikander ◽  
Thomas Ahlqvist ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Mycoplasma Genitalium ◽  
Extraction Methods ◽  
Pcr Inhibition ◽  
Dna Yield ◽  
Extraction Study ◽  
Dna Extraction Methods ◽  
Swab Specimen

The aim of this study was to determine whether a patient's endocervical swab specimen can be transported in first void urine (FVU) as combined specimens for the detection of Mycoplasma genitalium by real-time PCR. The study also compared two different DNA extraction methods for observation of possible PCR inhibition. Three specimens, one endocervical swab specimen transported in 2-SP medium, one endocervical swab specimen transported in FVU and a FVU specimen, were collected from 329 women. All sample types underwent manual DNA extraction whereas in the DNA extraction study, 329 endocervical swab specimens transported in FVU were subjected to both manual Chelex and automated BioRobot M48 DNA extraction. A total of 100 endocervical swab specimens transported in FVU from patients PCR-negative for M. genitalium in the study were used in the PCR inhibition analysis. M. genitalium was detected in 25/329 (7.6 %) women. The endocervical swab specimens transported in 2-SP medium and transported in FVU were positive for M. genitalium in 17/25 (68 %) and 24/25 (96 %) women, respectively. The FVU specimens alone were positive for M. genitalium in 22/25 (88 %) women. In the DNA extraction study, M. genitalium DNA was detected in 24/329 (7.3 %) and 28/329 (8.5 %) of endocervical swab specimens transported in FVU subjected to manual Chelex extraction and automated BioRobot M48 extraction, respectively. Partial PCR inhibition was detected in 6 % of samples subjected to manual Chelex extraction whereas no inhibition was detected with the automated BioRobot M48 extraction. Thus endocervical swab specimens transported in FVU demonstrate higher sensitivity than FVU specimens only and have considerably increased sensitivity compared with endocervical swab specimens transported in 2-SP medium for detection of M. genitalium DNA. Moreover, automated BioRobot M48 extraction was shown to be superior to a crude manual Chelex extraction, leaving no PCR inhibition and giving a slightly higher DNA yield and/or better sensitivity.

scholarly journals A pilot study of DNA yield from bloodstains on various surfaces using Phenol chloroform isoamyl alcohol (PCIA) and Chelex DNA extraction methods

2020 ◽  
Vol 13 (2) ◽  
pp. 124-127
Author(s):  
Ishwar Prasad Dubey ◽  
R. K. Kumawat ◽  
I. P. Tripathi ◽  
Pankaj Shrivastava
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Short Tandem Repeats ◽  
Tandem Repeats ◽  
Isoamyl Alcohol ◽  
Extraction Methods ◽  
Forensic Dna ◽  
Dna Yield ◽  
Dna Extraction Methods ◽  
Short Tandem

Presently, Short Tandem Repeats (STRs) based forensic DNA typing technology is being globally used in solving a diverse range of forensic cases such as paternity, identification of unknown dead bodies/skeletal remains, or suspect in a case of rape or mass rape. The technology has invaded its tentacles in almost all areas of criminal investigation in the last few decades. The present forensic DNA technology is based on capillary electrophoresis and utilizes short tandem repeats(STRs).On one hand, the technology is extensively used in the investigation of crime in highly sensitive cases, but on the another hand, obtaining DNA profile from forensic samples are highly challenging many times. Advent of PCR has been a boon for handling the challenging samples in forensic DNA analysis. The quality DNA profiles from challenging samples rely on the yield and quality of DNA, which is mainly dependent upon the method used for DNA extraction. Any specific method can never be thought of to be useful for all variety of samples. Still, Phenol Chloroform Isoamyl Alcohol (PCIA) organic extraction method has been proven to be useful for a wide variety of samples from the simplest saliva/blood to complex teeth and bone samples. In the present study, we compared the yield of DNA from blood stains recovered from various surfaces using the PCIA extraction method and Chelex DNA extraction methods and their compatibility with present-day STR based capillary electrophoresis typing. The mean value of DNA yield was found 50.5 ng/ µl and 32.25 ng/ µl by PCIA and Chelex DNA extraction methods, respectively. Overall, the highest yield was observed from all the tested samples from the PCIA method.

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