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.

An improved method to extract DNA from mango Mangifera indica

Biologia ◽  
2014 ◽  
Vol 69 (2) ◽  
Author(s):  
Mohammad Uddin ◽  
Wenli Sun ◽  
Xinhua He ◽  
Jaime Teixeira da Silva ◽  
Qi Cheng
Keyword(s):  
Secondary Metabolites ◽  
Dna Extraction ◽  
Genomic Dna ◽  
Mangifera Indica ◽  
Leaf Tissue ◽  
Poor Quality ◽  
Ammonium Bromide ◽  
High Quality ◽  
Woody Perennials ◽  
Ctab Method

AbstractHigh quality genomic DNA is the first step in the development of DNA-based markers for fingerprinting and genetic diversity of crops, including mango (Mangifera indica L.), a woody perennial. Poor quality genomic DNA hinders the successful application of analytical DNA-based tools. Standard protocols for DNA extraction are not suitable for mango since the extracted genomic DNA often contains secondary metabolites that interfere with analytical applications. In this study, we employed an additional step to remove polysaccharides, polyphenols and secondary metabolites from genomic DNA extracted from young or mature leaf tissue; then a modified traditional cetyl trimethyl ammonium bromide (CTAB) method was applied. The use of 0.4 M glucose improved DNA quality and avoided contamination and browning by polyphenolics, relative to the traditional CTAB method. This is an easy and efficient method for genomic DNA extraction from both young and mature leaves of mango. The isolated DNA was free of polysaccharides, polyphenols, RNA and other major contaminants, as judged by its clear colour, its viscosity, A260/A280 ratio and suitability for PCR-based reactions. This modified protocol was also used to extract high quality genomic DNA from other woody perennials, including walnut, guava, lychee, pear, grape and sugarcane.

scholarly journals Optimization of high molecular weight DNA extraction methods in shrimp for a long-read sequencing platform

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10340
Author(s):  
Pacharaporn Angthong ◽  
Tanaporn Uengwetwanit ◽  
Wirulda Pootakham ◽  
Kanchana Sittikankaew ◽  
Chutima Sonthirod ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Genomic Dna ◽  
Extraction Methods ◽  
Marine Organisms ◽  
High Quality ◽  
Sequencing Platform ◽  
Global Food Security ◽  
Long Read ◽  
Ctab Method ◽  
Global Food

Marine organisms are important to global food security as they are the largest source of animal proteins feeding mankind. Genomics-assisted aquaculture can increase yield while preserving the environment to ensure sufficient and sustainable production for global food security. However, only few high-quality genome sequences of marine organisms, especially shellfish, are available to the public partly because of the difficulty in the sequence assembly due to the complex nature of their genomes. A key step for a successful genome sequencing is the preparation of high-quality high molecular weight (HMW) genomic DNA. This study evaluated the effectiveness of five DNA extraction protocols (CTAB, Genomic-tip, Mollusc DNA, TIANamp Marine Animals DNA, and Sbeadex livestock kits) in obtaining shrimp HMW DNA for a long-read sequencing platform. DNA samples were assessed for quality and quantity using a Qubit fluorometer, NanoDrop spectrophotometer and pulsed-field gel electrophoresis. Among the five extraction methods examined without further optimization, the Genomic-tip kit yielded genomic DNA with the highest quality. However, further modifications of these established protocols might yield even better DNA quality and quantity. To further investigate whether the obtained genomic DNA could be used in a long-read sequencing application, DNA samples from the top three extraction methods (CTAB method, Genomic-tip and Mollusc DNA kits) were used for Pacific Biosciences (PacBio) library construction and sequencing. Genomic DNA obtained from Genomic-tip and Mollusc DNA kits allowed successful library construction, while the DNA obtained from the CTAB method did not. Genomic DNA isolated using the Genomic-tip kit yielded a higher number of long reads (N50 of 14.57 Kb) than those obtained from Mollusc DNA kits (N50 of 9.74 Kb). Thus, this study identified an effective extraction method for high-quality HMW genomic DNA of shrimp that can be applied to other marine organisms for a long-read sequencing platform.

scholarly journals Comparison of genomic DNA extraction methods to obtain high DNA quality from barnyard grass (Echinochloa colona)

2020 ◽  
Author(s):  
Romesh Kumar Salgotra ◽  
Bhagirath Singh Chauhan
Keyword(s):  
Essential Oils ◽  
Dna Extraction ◽  
Leaf Tissue ◽  
Extraction Methods ◽  
High Quality ◽  
Time Saving ◽  
Plant Leaf ◽  
Plant Parts ◽  
Molecular Studies ◽  
Leaf Tissues

Abstract Background: The study of weed genomics is important for the effective management of weeds to enhance crop yield. A rapid, inexpensive and high quality DNA extraction is needed for genomic and other molecular studies. Here, we describe the protocols for DNA extraction from two different parts of the Echinochloa colona plant using modified cetyltrimethylammonium bromide (CTAB) and a commercial kit.Results: In the study, it was observed that the DNA extracted from plant leaf tissues and dry seeds with a modified CTAB protocol was of good quality, with no contaminations of polysaccharides and essential oils. Quality of DNA extracted from dry seeds was comparable with that of plant leaves under both protocols. The extracted DNA from both plant parts was successfully amplified by PCR using the EPSPS microsatellite marker. Compared to the protocol of DNA extracted from leaf tissue, dry seeds will save time and other valuable resources. Moreover, the same protocols can be implemented for the extraction of high-quality DNA for molecular studies in other plant species where a large amount of polysaccharides, secondary metabolites and essential oils are present.Conclusions: Modified methods of DNA extraction from dry seeds are efficient and time-saving which can be used in genotypic and other molecular approaches. High-quality DNA can be isolated from plant leaf tissues using modified CTAB and commercial kits, however, DNA extracted from dry seeds will save time and other valuable resources.

scholarly journals Optimized protocol to isolate high quality genomic DNA from different tissues of a palm species

Hoehnea ◽  
2019 ◽  
Vol 46 (2) ◽  
Author(s):  
Marília Souza Lucas ◽  
Carolina da Silva Carvalho ◽  
Giovane Böerner Hypolito ◽  
Marina Corrêa Côrtes
Keyword(s):  
Dna Extraction ◽  
Genomic Dna ◽  
Dna Isolation ◽  
Molecular Techniques ◽  
Tissue Type ◽  
High Quality ◽  
Optimized Protocol ◽  
Palm Species ◽  
Different Tissues

ABSTRACT The application of molecular techniques to tackle ecological and evolutionary questions requires genomic DNA in good quality and quantity. The quality of the isolated DNA, however, can be influenced by the tissue type and the way the sample was conserved and manipulated. Therefore, customizing protocols to improve the DNA isolation and locus amplification is crucial. We optimized a cheap and manual protocol of DNA extraction and microsatellites amplification using five different tissues of a palm species of the brazilian Atlantic Forest. We successfully extracted DNA from all five tissue types. Leaf, stem, and endocarp of non-dispersed seeds presented the highest rates of successful DNA extraction and microsatellite amplification; whereas root, endocarp of dispersed seeds, and embryo showed the lowest quality and quantity. Based on these results, we discussed the implications of using different tissues for studies about seed dispersal, pollination, and population genetics.

Efficient DNA isolation from moroccan arar tree [Tetraclinis articulata (Vahl) Masters] leaves and optimization of the rapd-pcr molecular technique. Extraction efficace de l’ADN des feuilles du thuya de berberie (Tetraclinis articulata (Vahl) masters) et optimisation de la technique moléculaire rapd-pcr

2010 ◽  
Vol 35 ◽  
pp. 97-106
Author(s):  
Salaheddine Bakkali Yakhlef ◽  
Imane Guenoun ◽  
Benaîssa Kerdouh ◽  
Noureddine Hamamouch ◽  
Mohamed Abourouh
Keyword(s):  
Dna Extraction ◽  
Genomic Dna ◽  
Dna Isolation ◽  
Molecular Genetic Analysis ◽  
Molecular Technique ◽  
High Quality ◽  
Fresh Tissue ◽  
Dna Yield ◽  
Tetraclinis Articulata ◽  
Rapd Pcr

 English.  Molecular genetic analysis of Arar tree [Tetraclinis articulata (Vahl) Masters] is often limited by the availability of fresh tissue and an efficient and reliable protocol for high quality genomic DNA extraction. In this study, two DNA extraction protocols were specifically developed for extracting high quality genomic DNA from Arar tree leaves: modified QIAgen DNA Kit and protocol developed by Ouenzar et al. (1998). DNA yield and purity were monitored by gel electrophoresis and by determining absorbance at UV (A260/A280 and A260/A230). Both ratios were between 1.7 and 2.0, indicating that the presence of contaminating metabolites was minimal. The DNA yield obtained ranged between 20 to 40 µg/g of plant materiel. The Ouenzar and collaborators protocol gave higher yield but was more time consuming compared to QIAgen Kit. However, both techniques gave DNA of good quality that is amenable to RAPD-PCR reactions.Additionally, restriction digestion and PCR analyses of the obtained DNA showed its compatibility with downstream applications. Randomly Amplified Polymorphic DNA profiling from the isolated DNA was optimized to produce scorable and clear amplicons. The presented protocols allow easy and high quality DNA isolation for genetic diversity studies within Arar tree.Français.  Les analyses en génétique moléculaire chez le thuya de Berberie [Tetraclinis articulata (Vahl) Masters] sont souvent limitées par la disponibilité du matériel végétal frais et le temps nécessaire pour l’extraction l’ADN ainsi que par sa qualité. Dans cette étude, deux protocoles d’extraction, à partir des feuilles du thuya, de l’ADN génomique de haute qualité, ont été développés : le Kit Qiagen et le protocole mis au point par Ouenzar et al. (1998) modifiés. La qualité et la quantité de l’ADN sont évaluées par électrophorèse sur gel d’agarose et par la mesure de l’absorbance en UV à (A260/A280) et (A260/A230). Ces deux rapports varient entre 1,7 et 2,0 indiquant la faible fréquence des métabolites contaminants. Le rendement d’ADN varie entre 20 et 40 µg/g du matériel végétal. Le protocole de Ouenzar et collaborateurs donne le meilleur rendement d’ADN mais nécessite plus de temps. Néanmoins, les deux protocoles donnent un ADN de bonne qualité utilisable dans les réactions RAPD-PCR. En outre, la restriction enzymatique et l’analyse PCR de l’ADN obtenu ont montré sa compatibilité avec les applications moléculaires ultérieures. Les paramètres intervenant dans les réactions RAPD ont été optimisés. Les protocoles présentés permettent l’extraction facile de l’ADN de haute qualité nécessaire pour des études de la diversité génétique au sein du thuya.

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