scholarly journals Preservation and maceration of amazon açai leaflet tissue to obtain genomic DNA

2019 ◽  
Vol 35 (4) ◽  
Author(s):  
Hellen Sandra Freires da Silva Azêvedo ◽  
Polinar Bandeira Rufino ◽  
José Marlo Araújo de Azevedo ◽  
Luciélio Manoel da Silva ◽  
Lúcia Helena de Oliveira Wadt ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Silica Gel ◽  
Genomic Dna ◽  
Leaf Tissue ◽  
Dna Concentration ◽  
Randomized Design ◽  
Molecular Studies ◽  
Tissue Maceration ◽  
Genomic Regions ◽  
Transport Buffer

The objective of this study was to test the efficiency of preservation and maceration methods for Euterpe precatoria leaflet tissue to obtain genomic DNA for molecular studies. The leaflets of E. precatoria were collected in an experimental field at Embrapa Acre, Brazil. The study was conducted in a completely randomized design with 10 replicates, in a 12 × 2 factorial structure, with 12 storage treatments (fresh; lyophiliser 3 days; refrigerator 3, 5, and 7 days; silica gel 7, 10, 20, and 30 days; and transport buffer 3, 5, and 7 days) and two leaf tissue maceration methods (liquid nitrogen and the TissueLyser®). Statistically significant differences in the obtained DNA concentration were found between the maceration and storage treatments. The TissueLyser® macerator produced higher DNA concentrations when compared to liquid nitrogen. For the storage treatments, five groups were formed based on DNA concentration when macerated with the TissueLyser® and two groups when macerated with liquid nitrogen. The DNA concentrations ranged from 285.00 ng/µL (7 days in transport buffer) to 702.00 ng/µL (30 days in silica gel) when the leaflets were macerated with liquid nitrogen, and from 572.73 ng/µL (30 days in silica gel) to 2,850.00 ng/µL (3 days in lyophiliser) using the TissueLyser® macerator. The DNA purity (A260/A280 nm) varied from 1.30 to 1.70 when the leaflets were macerated with liquid nitrogen and from 1.30 to 1.90 with the TissueLyser® macerator. Despite the variations in leaf tissue preservation and DNA concentration, all treatments were effective for DNA isolation and it was possible to amplify genomic regions of microsatellite markers by PCR. It was concluded that leaflets of E. precatoria stored in a lyophiliser and processed with an automatic macerator resulted in satisfactory DNA for molecular studies.

scholarly journals AVALIAÇÃO DE DOIS MÉTODOS DE TRITURAÇÃO FOLIAR DE Acianthera ciliata (Orchidaceae) PARA EXTRAÇÃO DE DNA

Nativa ◽  
2020 ◽  
Vol 8 (1) ◽  
pp. 97
Author(s):  
Eliane Cristina Moreno de Pedri ◽  
Catiane Dos Santos Braga ◽  
Carlos Alberto Da Cunha Oliveira ◽  
Auana Vicente Tiago ◽  
Ana Aparecida Bandini Rossi
Keyword(s):  
Genetic Diversity ◽  
Molecular Markers ◽  
Liquid Nitrogen ◽  
Dna Extraction ◽  
Plant Species ◽  
Genomic Dna ◽  
Extraction Buffer ◽  
Foliar Tissue ◽  
Genomic Regions ◽  
Issr Primers

O estabelecimento de protocolo de extração de DNA de espécies vegetais é uma técnica empregada para a obtenção de um DNA puro e de qualidade. Diante disso, objetivou-se neste estudo padronizar um protocolo para a extração de DNA da espécie Acianthera ciliata, visando posteriores estudos de diversidade genética. Foram testados dois métodos de trituração do tecido foliar, sendo eles: Tampão STE e nitrogênio líquido. Para cada método de trituração foram testadas duas concentrações de β-mercaptoetanol (0% e 2%). Os dois métodos utilizados, foram eficientes na extração do DNA genômico de A. ciliata. As amostras extraídas com 0% de β-mercaptoetanol, para os dois métodos, STE e nitrogênio líquido, apresentaram menor quantidade de DNA quando comparado com as amostras extraídas com 2% de β-mercaptoetanol. Os dois primers testados amplificaram regiões do genoma de A. ciliata. Para a extração de DNA de A. ciliata indica-se a utilização de CTAB 5% no tampão de extração e β-mercaptoetanol a 2%. Os iniciadores ISSR foram eficientes na amplificação e são recomendados para estudos de diversidade genética de A. ciliata.Palavras-chave: diversidade genética; CTAB; marcadores moleculares; orquídeas. EVALUATION OF TWO MACERATION METHODS IN Acianthera ciliata (Orchidaceae) LEAVES FOR DNA EXTRACTION ABSTRACT: The establishment of DNA extraction protocol for plant species is a technique employed to obtain pure and good quality DNA. In this study, we standardized a protocol for the extraction of DNA of the species Acianthera ciliata, aiming studies of genetic diversity subsequently. Two maceration methods for foliar tissue were tested, and they were STE buffer and liquid nitrogen. Two concentrations of β-mercaptoethanol (0% and 2%) were tested for each method. The two methods used were efficient for genomic DNA extraction of A. ciliata. In both methods the samples extracted using 0% of β-mercaptoethanol, they presented lesser amount of DNA than the samples extracted using 2% of β-mercaptoethanol. The two tested primers amplified genomic regions of A. ciliata. For the DNA extraction of A. ciliata, we indicated the use of CTAB 5% in the extraction buffer as well as β-mercaptoethanol to 2%. The ISSR primers were efficient in amplification and thus they are indicated for studies of genetic diversity of A. ciliata.Keywords: genetic diversity; CTAB; molecular markers; orchids.

scholarly journals Comparison of Different Leaf Preservation Methods To Obtain High Quality DNA From Enset (Ensete Ventricosum (Welw.) Cheesman), A Native And Orphan Food Security Crop In Ethiopia

2021 ◽  
Author(s):  
Alye Tefera Haile ◽  
Sylvia Sagen Johnsen ◽  
Mallikarjuna Rao Kovi ◽  
Trine Hvoslef-Eide ◽  
Bizuayehu Tesfaye ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Silica Gel ◽  
Genomic Dna ◽  
Leaf Tissue ◽  
Endemic Plant ◽  
High Quality ◽  
Crop Species ◽  
Ensete Ventricosum ◽  
Ctab Solution ◽  
Ctab Method

Abstract Background: Enset (Ensete ventricosum (Welw.) Cheesman) is a staple food for more than 20 million Ethiopians and only cultivated in the native indigenous farming systems of Ethiopia. In contrast to other cultivated species in the Musaceae family, enset has been relatively little studied at the molecular level. Application of advanced molecular genetic techniques requires rapid extraction of DNA of high quality and quantity. Fresh, lyophilized tissues, as well as tissues stored in liquid nitrogen are mainly preferred to avoid DNA degradation, thus most of the DNA extraction protocols recommend these types of tissues as starting material. However, such sample processing techniques are difficult to utilize in many developing countries and at collection sites of many endemic plant species, underutilized or orphan crop species like enset. These situations necessitate the development of alternative protocols for leaf preservation and optimized methods for isolating high-quality DNA from dried or preserved leaf samples. Results: In this study, three different leaf preservation and two DNA extraction methods were compared. Fresh young leaf tissue was preserved using the minor modified saturated NaCl-CTAB solution, silica gel or 96% ethanol at ambient temperature for more than 35 days. Subsequently, DNA was extracted using either the DNeasy Plant Mini Kit or the CTAB method. As compared to silica gel and 96% ethanol, the minor modified saturated NaCl-CTAB solution preserved the quality, quantity, and integrity of enset genomic DNA. This method consistently produced genomic DNA of high-quality and quantity at affordable cost. The DNeasy Plant Mini Kit method was found to be more efficient than the standard CTAB method, being faster and producing genomic DNA of higher quality. Conclusions: Using saturated NaCl-CTAB solution is an accessible, efficient, scalable, and inexpensive way to preserve enset leaves during collection and transportation. The preservation protocol was validated for leaf tissues of all cultivated and wild enset, and Entada landraces. Genomic DNA of high quality and quantity was obtained from preserved enset leaves, which can be used for further downstream applications including PCR and sequencing.

scholarly journals Development of a loop-mediated isothermal amplification assay for the detection of Tilletia controversa based on genome comparison

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Somayyeh Sedaghatjoo ◽  
Monika K. Forster ◽  
Ludwig Niessen ◽  
Petr Karlovsky ◽  
Berta Killermann ◽  
...  
Keyword(s):  
Test Performance ◽  
Genomic Dna ◽  
Lamp Assay ◽  
Fungal Species ◽  
Isothermal Amplification ◽  
Genome Comparison ◽  
Performance Study ◽  
Loop Mediated Isothermal Amplification ◽  
Tilletia Controversa ◽  
Genomic Regions

AbstractTilletia controversa causing dwarf bunt of wheat is a quarantine pathogen in several countries. Therefore, its specific detection is of great phytosanitary importance. Genomic regions routinely used for phylogenetic inferences lack suitable polymorphisms for the development of species-specific markers. We therefore compared 21 genomes of six Tilletia species to identify DNA regions that were unique and conserved in all T. controversa isolates and had no or limited homology to other Tilletia species. A loop-mediated isothermal amplification (LAMP) assay for T. controversa was developed based on one of these DNA regions. The specificity of the assay was verified using 223 fungal samples comprising 43 fungal species including 11 Tilletia species, in particular 39 specimens of T. controversa, 92 of T. caries and 40 of T. laevis, respectively. The assay specifically amplified genomic DNA of T. controversa from pure cultures and teliospores. Only Tilletia trabutii generated false positive signals. The detection limit of the LAMP assay was 5 pg of genomic DNA per reaction. A test performance study that included five laboratories in Germany resulted in 100% sensitivity and 97.7% specificity of the assay. Genomic regions, specific to common bunt (Tilletia caries and Tilletia laevis together) are also provided.

scholarly journals Muscle biopsy essential diagnostic advice for pathologists

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Ana Cotta ◽  
Elmano Carvalho ◽  
Antonio Lopes da-Cunha-Júnior ◽  
Jaquelin Valicek ◽  
Monica M. Navarro ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Muscle Biopsy ◽  
Diagnostic Procedures ◽  
Mitochondrial Respiratory Chain ◽  
Muscle Disease ◽  
Diagnostic Workup ◽  
Main Body ◽  
Mitochondrial Myopathies ◽  
Muscle Enzymes ◽  
Molecular Studies

Abstract Background Muscle biopsies are important diagnostic procedures in neuromuscular practice. Recent advances in genetic analysis have profoundly modified Myopathology diagnosis. Main body The main goals of this review are: (1) to describe muscle biopsy techniques for non specialists; (2) to provide practical information for the team involved in the diagnosis of muscle diseases; (3) to report fundamental rules for muscle biopsy site choice and adequacy; (4) to highlight the importance of liquid nitrogen in diagnostic workup. Routine techniques include: (1) histochemical stains and reactions; (2) immunohistochemistry and immunofluorescence; (3) electron microscopy; (4) mitochondrial respiratory chain enzymatic studies; and (5) molecular studies. The diagnosis of muscle disease is a challenge, as it should integrate data from different techniques. Conclusion Formalin-fixed paraffin embedded muscle samples alone almost always lead to inconclusive or unspecific results. Liquid nitrogen frozen muscle sections are imperative for neuromuscular diagnosis. Muscle biopsy interpretation is possible in the context of detailed clinical, neurophysiological, and serum muscle enzymes data. Muscle imaging studies are strongly recommended in the diagnostic workup. Muscle biopsy is useful for the differential diagnosis of immune mediated myopathies, muscular dystrophies, congenital myopathies, and mitochondrial myopathies. Muscle biopsy may confirm the pathogenicity of new gene variants, guide cost-effective molecular studies, and provide phenotypic diagnosis in doubtful cases. For some patients with mitochondrial myopathies, a definite molecular diagnosis may be achieved only if performed in DNA extracted from muscle tissue due to organ specific mutation load.

scholarly journals Optimization of DNA concentration to amplify short tandem repeats of human genomic DNA

2012 ◽  
Vol 12 (4) ◽  
pp. 236 ◽  
Author(s):  
Fahri Q. Gavazaj ◽  
Ilia I. Mikerezi ◽  
Valon H. Morina ◽  
Fatmir A. Cakaj ◽  
Ekrem B. Maloku ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Short Tandem Repeats ◽  
Tandem Repeats ◽  
Dna Concentration ◽  
Human Genomic ◽  
Human Genomic Dna ◽  
Short Tandem

scholarly journals Cryopreservation in liquid nitrogen of Brazilian orchid seeds Miltonia flavescens Lindl.

2020 ◽  
Vol 14 ◽  
Author(s):  
Jean Carlo Baudraz de Paula ◽  
Walter Aparecido Ribeiro Júnior ◽  
Gabriel Danilo Shimizu ◽  
Gabriel Barraca Men ◽  
Ricardo Tadeu De Faria
Keyword(s):  
Low Temperature ◽  
Water Content ◽  
Liquid Nitrogen ◽  
Biological Material ◽  
Control Treatment ◽  
Preservation Methods ◽  
Lower Performance ◽  
Randomized Design ◽  
Completely Randomized Design ◽  
Protocorm Formation

Miltonia flavescens is a species vulnerable to extinction, which justifies research on preservation methods. Cryopreservation in liquid nitrogen (LN) consists of maintaining biological material at a low-temperature (-196 °C). Thus, the aim of the experiment is to evaluate the influence of different cryoprotective solutions on cryopreservation in LN of the Brazilian orchid Miltonia flavescens seeds. The experiment was conducted in a completely randomized design (CRD), with eight treatments and six replications. The treatments were composed as follows: control; immersion in LN, no cryoprotectant adding; and immersion in LN, with the addition of cryoprotectants: sucrose 0,4 mol L-1; glycerol 2 mol L-1; protection by vitrification in solution (PVS)1; PVS2; PVS2 + 1% phloroglucinol, and PVS3. Except for the control treatment, which was kept in a freezer (10±2 °C), the others remained frozen for 15 days. After this period, the viability of the seeds was evaluated. These seeds were sown and, 30 days after germination, then the frequency of protocorm formation was verified. Before the cryopreservation, the seeds showed 75% viability and 9.5% water content. After cryopreservation, the seeds varied between 67 to 75% viability. However, treatment with glycerol 2 mol L-1 exhibited lower performance than the others (58%). The control treatment showed a higher percentage of protocorm formation (71%) followed by treatments PVS1 (63%), PVS2 (64%), and PVS2PHLO (66%). For the purpose of preserving Miltonia flavescens seeds in liquid nitrogen for a prolonged period, the treatments PVS1, PVS2, and PVS2PHLO proved to be viable and promising alternatives.

On the organisation of the regulatory region of the zebrafish deltaD gene

Development ◽  
2002 ◽  
Vol 129 (20) ◽  
pp. 4773-4784 ◽  
Author(s):  
Stefan Hans ◽  
José A. Campos-Ortega
Keyword(s):  
Genomic Dna ◽  
Transcriptional Control ◽  
Regulatory Region ◽  
Notch Pathway ◽  
Notch Receptor ◽  
Regulatory Sequences ◽  
Early Neurogenesis ◽  
Regulatory Feedback Loop ◽  
E Boxes ◽  
Genomic Regions

deltaD is one of the four zebrafish Delta homologues presently known. Experimental evidence indicates that deltaD participates in a number of important processes during embryogenesis, including early neurogenesis and somitogenesis, whereby the protein it encodes acts as a ligand for members of the Notch receptor family. In accordance with its functional role, deltaD is transcribed in several domains of mesodermal and ectodermal origin during embryogenesis. We have analysed the organisation of the regulatory region of the deltaD gene using fusions to the reporter gene gfp and germline transgenesis. Cis-regulatory sequences are dispersed over a stretch of 12.5 kb of genomic DNA, and are organised in a similar manner to those in the regulatory region of the Delta-like 1 gene of mouse. Germline transformation using a minigene comprising 10.5 kb of this genomic DNA attached to the 3′ end of a full-length cDNA clone rescues the phenotype of embryos homozygous for the amorphic deltaD mutation after eightAR33. Several genomic regions that drive transcription in mesodermal and neuroectodermal domains have been identified. Transcription in all the neural expression domains, with one exception, is controlled by two relatively small genomic regions, which are regulated by the proneural proteins neurogenin 1 and zash1a/b acting as transcriptional activators that bind to so-called E-boxes. Transcriptional control of deltaD by proneural proteins therefore represents a molecular target for the regulatory feedback loop mediated by the Notch pathway in lateral inhibition.

scholarly journals Doses and forms of Azospirillum brasilense inoculation on maize crop

2018 ◽  
Vol 22 (6) ◽  
pp. 373-377 ◽  
Author(s):  
José M. K. Santini ◽  
Salatiér Buzetti ◽  
Marcelo C. M. Teixeira Filho ◽  
Fernando S. Galindo ◽  
Daniel N. Coaguila ◽  
...  
Keyword(s):  
Azospirillum Brasilense ◽  
Low Cost ◽  
Leaf Tissue ◽  
Physical Characteristics ◽  
Maize Crop ◽  
Nutrient Contents ◽  
Randomized Design ◽  
Zn Content ◽  
Completely Randomized Design ◽  
The Impact

ABSTRACT In search of a more sustainable agriculture, the use of beneficial microorganisms has been highlighted, because they are low-cost and can reduce the use of fertilizers and increase grain yield. The present study aimed to evaluate the efficiency of A. brasilense inoculation and the best form and dose of inoculation in maize, measuring the impact on some physical characteristics and on its nutrition. The experiment was conducted in a greenhouse, in Ilha Solteira, SP, Brazil, in a completely randomized design, with four replicates and eight treatments: 1) control; 2) Seed 1x; 3) Seed 2x; 4) Soil 1x; 5) Soil 2x; 6) Leaf 1x; 7) Leaf 2x; 8) Seed 1x + Leaf 1x, respectively representing in each treatment the site and dose of application (1x, dose recommended by the manufacturer; 2x, twice the dose recommended by the manufacturer). No differences were found in any physical characteristics evaluated between treatments; however, for nutrient contents in the leaf tissue, there was effect on Zn content. It was concluded that, regardless of the presence of A. brasilense inoculation, forms or dose (in hybrid DKB 350), in general, there were no improvements in the characteristics evaluated.

scholarly journals Genetic Diversity Analysis between Different Varieties of Chickpea (Cicer arietinum L.) Using SSR Markers

2019 ◽  
Vol 7 (2) ◽  
pp. 236-242
Author(s):  
Summy Yadav ◽  
Vaidehi Shah ◽  
Bhavya Mod
Keyword(s):  
Genetic Diversity ◽  
Ssr Markers ◽  
Genomic Dna ◽  
Physiological Data ◽  
Diversity Analysis ◽  
Hierarchical Tree ◽  
Cicer Arietinum L ◽  
Equal Distance ◽  
Genomic Regions ◽  
Simple Sequence

The paper aims at evaluating genetic diversity among genomes of chickpea comprising of 5 different varieties with the help of simple sequence repeats (SSR) molecular markers. Genomic DNA isolated from all varieties was checked with 15 different SSR markers specific for ENDPOINT PCR using PCR based techniques. Amplification bands with different markers enabled identification of the genomic regions responsible for Drought Tolerance in chickpea. All 15 SSR markers chosen gave monomorphic bands.  A hierarchical tree was also constructed using UPGMA Dendogram for figuring out the exact genetic distance of cultivars using band amplification data. It depicted GUJ-1 and GUJ-2 are closest of all cultivars. GUJ-5 is at the center having GUJ-3 and UJJAVAL at an almost equal distance but GUJ-5 and GUJ-3 are more related. Physiological data also supported this genetic evidence. Int. J. Appl. Sci. Biotechnol. Vol 7(2): 236-242

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