scholarly journals Rapid Extraction of Genomic DNA from Medically Important Yeasts and Filamentous Fungi by High-Speed Cell Disruption

1998 ◽  
Vol 36 (6) ◽  
pp. 1625-1629 ◽  
Author(s):  
Frank-Michael C. Müller ◽  
Katherine E. Werner ◽  
Miki Kasai ◽  
Andrea Francesconi ◽  
Stephen J. Chanock ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Filamentous Fungi ◽  
Genomic Dna ◽  
High Speed ◽  
Dna Isolation ◽  
Random Amplified Polymorphic Dna ◽  
Extraction Procedure ◽  
Cell Disruption ◽  
Pseudallescheria Boydii ◽  
Rapid Extraction

Current methods of DNA extraction from different fungal pathogens are often time-consuming and require the use of toxic chemicals. DNA isolation from some fungal organisms is difficult due to cell walls or capsules that are not readily susceptible to lysis. We therefore investigated a new and rapid DNA isolation method using high-speed cell disruption (HSCD) incorporating chaotropic reagents and lysing matrices in comparison to standard phenol-chloroform (PC) extraction protocols for isolation of DNA from three medically important yeasts (Candida albicans, Cryptococcus neoformans, andTrichosporon beigelii) and two filamentous fungi (Aspergillus fumigatus and Fusarium solani). Additional extractions by HSCD were performed on Saccharomyces cerevisiae, Pseudallescheria boydii, andRhizopus arrhizus. Two different inocula (108and 107 CFU) were compared for optimization of obtained yields. The entire extraction procedure was performed on as many as 12 samples within 1 h compared to 6 h for PC extraction. In comparison to the PC procedure, HSCD DNA extraction demonstrated significantly greater yields for 108 CFU of C. albicans, T. beigelii, A. fumigatus, andF. solani (P ≤ 0.005), 107CFU of C. neoformans (P ≤ 0.05), and 107 CFU of A. fumigatus (P ≤ 0.01). Yields were within the same range for 108 CFU ofC. neoformans and 107 CFU of C. albicans for both HSCD extraction and PC extraction. For 107 CFU of T. beigelii, PC extraction resulted in a greater yield than did HSCD (P ≤ 0.05). Yields obtained from 108 and 107 CFU were significantly greater for filamentous fungi than for yeasts by the HSCD extraction procedure (P < 0.0001). By the PC extraction procedure, differences were not significant. For all eight organisms, the rapid extraction procedure resulted in good yield, integrity, and quality of DNA as demonstrated by restriction fragment length polymorphism, PCR, and random amplified polymorphic DNA. We conclude that mechanical disruption of fungal cells by HSCD is a safe, rapid, and efficient procedure for extracting genomic DNA from medically important yeasts and especially from filamentous fungi.

scholarly journals OPTIMIZING RAPD MARKERS FOR ONION GENOMIC DNA ANALYSIS

HortScience ◽  
1995 ◽  
Vol 30 (3) ◽  
pp. 435a-435
Author(s):  
Ruwanthi C. Wettashinghe ◽  
Ellen B. Peffley
Keyword(s):  
Cost Effectiveness ◽  
Plant Breeding ◽  
Dna Extraction ◽  
Genetic Markers ◽  
Plant Tissue ◽  
Genomic Dna ◽  
Rapd Markers ◽  
Dna Analysis ◽  
Dna Isolation ◽  
Random Amplified Polymorphic Dna

Random amplified polymorphic DNA (RAPD) are genetic markers that facilitate selection in plant breeding. To obtain clear reproducible, and repeatable RAPD bands, four DNA extraction protocols and two Taq polymerases were compared using thirteen TG1015Y (Allium cepa) genotypes. Protocols for DNA extraction followed those of a modified Tai and Tanksley, 1989 (PMBR); a modified Dellaporta et al., 1983 (PMBR); a modified Guillemunt et al., 1992 (PMBR); and extracted with a plant tissue DNA isolation kit from Gentra System (Minneapolis). The modified Guillemunt protocol was selected due to ease of extraction and cost effectiveness. Polymerases compared were Taq and Taq Stoffel fragment. Results are based on three separate amplifications and electrophoretic assays. PCR amplifications of Stoffel fragment produced more scorable and reproducible RAPD bands compared to bands produced using Taq polymerase.

scholarly journals Optimizing RAPD Markers for Onion Genomic DNA Analysis

HortScience ◽  
1995 ◽  
Vol 30 (4) ◽  
pp. 877E-877
Author(s):  
Ruwanthi C. Wettasinghe ◽  
Ellen B. Peffley
Keyword(s):  
Cost Effectiveness ◽  
Dna Extraction ◽  
Genetic Markers ◽  
Allium Cepa ◽  
Plant Tissue ◽  
Genomic Dna ◽  
Rapd Markers ◽  
Dna Analysis ◽  
Dna Isolation ◽  
Random Amplified Polymorphic Dna

Random amplified polymorphic DNA (RAPD) have potential as genetic markers that may facilitate selection in plant improvement. To obtain clear, reproducible, and repeatable RAPD bands, four DNA extraction protocols and two Taq polymerases were compared. DNA extraction followed modified Tai and Tanksley (PMBR), Dellaporta et al. (PMBR), and Guilllemant et al. (PMBR) protocols, and a plant tissue DNA isolation kit from Gentra Systems was used. The modified Guillemant protocol was selected because of ease of extraction and cost effectiveness. Genotypes studied were TG1015Y (Allium cepa). Polymerases compared were Taq and Taq Stoffel fragment. Results are based on separate amplifications and electrophoretic assays. PCR amplifications of Stoffel fragment produced more scorable and reproducible RAPD bands compared to bands produced using Taq polymerase.

scholarly journals The Applicability of Three DNA Isolation Methods in SSR Analysis of Hexaploid Plum (Prunus domestica L.) Cultivars

2018 ◽  
Vol 2 (1) ◽  
pp. 1
Author(s):  
Jasna Hanjalić ◽  
Lejla Lasić ◽  
Fuad Gaši ◽  
Mekjell Meland ◽  
Jasmin Grahić ◽  
...  
Keyword(s):  
Medicinal Plants ◽  
Dna Extraction ◽  
Genomic Dna ◽  
Dna Isolation ◽  
Extraction Procedure ◽  
Plant Tissues ◽  
Prunus Domestica ◽  
Ssr Analysis ◽  
Isolation Methods ◽  
Downstream Analysis

The main goal of any DNA extraction procedure is to ensure reliable and reproducible results in a simple, fast and inexpensive manner. When it comes to plant tissues, this goal is challenging to achieve due to the presence of a variety of metabolites that interfere with DNA during isolation and downstream analysis. In this study, we compared the efficiency of three methods for DNA extraction from plum kernels: 1) the standard CTAB Soltis method which is the most common protocol for DNA extraction from various plant tissues (seeds, young leafs, mature leafs, root); 2) CTAB-based method originally described for DNA isolation from medicinal plants with high levels of secondary metabolites; 3) and one of various commercially available kits. The usefulness of the obtained DNA was evaluated by SSR analysis with seven microsatellite markers. Although the latter two extraction protocols retrieved genomic DNA that gave positive PCR results, only DNA isolated by kit produced full SSR profile

scholarly journals Comparing Three DNA Extraction Procedures for Cost, Efficiency, and DNA Yield

HortScience ◽  
2002 ◽  
Vol 37 (5) ◽  
pp. 822-825 ◽  
Author(s):  
D.W. Lickfeldt ◽  
N.E. Hofmann ◽  
J.D. Jones ◽  
A.M. Hamblin ◽  
T.B. Voigt
Keyword(s):  
Dna Extraction ◽  
Poa Pratensis ◽  
Random Amplified Polymorphic Dna ◽  
Extraction Procedure ◽  
Kentucky Bluegrass ◽  
Ammonium Bromide ◽  
Individual Plant ◽  
Extraction Procedures ◽  
Dna Yield ◽  
Ctab Method

An efficient deoxyribonucleic acid (DNA) extraction procedure that yields large quantities of DNA would provide adequate DNA for a large number of different analytical procedures. This study was conducted to compare three DNA extraction procedures for cost, time efficiency, and DNA content while extracting DNA from Kentucky bluegrass (Poa pratensis L.). Three students at the Univ. of Illinois with varying levels of DNA extraction experience conducted DNA extractions using Plant DNeasy™ Mini Kits, Plant DNAzol® Reagent, and a PEX/CTAB buffer. Costs varied significantly with cost (US$) per DNA sample of $3.04 for the DNeasy™ method, $0.99 for the DNAzol® method, and $0.39 for the PEX/CTAB extraction. The DNAzol® method was the fastest; although extracting 2.8 ng less DNA than the DNeasy™ method, it did not require the use of hazardous organic solvents, and random amplified polymorphic DNA (RAPD) markers were satisfactory for DNA fingerprinting of Kentucky bluegrass cultivars. The PEX/CTAB method, which did not include a tissue homogenization step, did not have reproducible banding patterns due to miniscule and inconsistent quantities of DNA extracted, or possibly due to inadequate purification. The investigator with the least DNA extraction experience was the slowest, while extracting 75% more DNA. All three methods are easily adapted to laboratories having personnel with different levels of experience. The DNAzol® Reagent method should save time and money, with reproducible results when many individual plant samples need to be identified. Chemical names used: potassium ethyl xanthogenate (PEX); cetyltrimethyl ammonium bromide (CTAB)

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.

scholarly journals Extraction of high-quality genomic DNA and identification of different DNA barcoding markers for chickpea (Cicer arietinum L.)

2020 ◽  
Vol 1 (1) ◽  
pp. 7
Author(s):  
Umavathi Saraswathi ◽  
Lakshmanan Mullainathan
Keyword(s):  
Dna Barcoding ◽  
Dna Extraction ◽  
Genomic Dna ◽  
Dna Isolation ◽  
Genomic Library ◽  
Pcr Amplification ◽  
Slight Modification ◽  
High Quality ◽  
Universal Primer ◽  
Genetic Studies

The genetic studies of individual plants, especially self-pollinated species like chickpea need to be evaluated at the DNA level with the help of molecular markers for identifying genetic variations among the plants. High-quality DNA extraction is a prerequisite for genetic studies. Extraction of intact genomic DNA with high – molecular mass is essential for the study of many molecular biology applications like Polymerase Chain Reaction, endonuclease restriction digestion, southern blot analysis, and also for the construction of a genomic library. Several plant DNA extraction methods are available, even though the DNA isolation methods that give good yield employing both quantity and quality is quite difficult especially for self-pollinated crops like a chickpea. This work was focused on developing a standard protocol for the extraction of genomic DNA and identifying different barcoding markers. The result revealed that the CTAB extraction method with slight modification in protocol had been optimized for DNA isolation. The purified DNA, which was isolated through the CTAB method, had excellent spectral qualities and is efficiently digested by a restriction endonuclease, and is found to be more suitable for long-fragment PCR amplification. DNA barcoding is considered as a promising tool because it provides a practical and standard identification of plants. The isolated DNA sample was processed with a classical DNA barcoding approach by amplifying and sequencing with a universal primer. According to the result, among the different barcoding markers studied, the RbcL and Mat K were found to given the best result for molecular species identification in chickpea.

scholarly journals Optimalisasi Teknik Ekstraksi dan Isolasi DNA Tanaman Suren (Toona Sureni Merr.) untuk Analisis Keragaman Genetik berdasarkan Random Amplified Polymorphic DNA (RAPD)

2012 ◽  
Vol 14 (1) ◽  
pp. 138 ◽  
Author(s):  
Muh Restu ◽  
Mukrimin Mukrimin ◽  
Gusmiaty Gusmiaty
Keyword(s):  
Genomic Dna ◽  
Extraction Method ◽  
Dna Isolation ◽  
Random Amplified Polymorphic Dna ◽  
Pcr Amplification ◽  
Secondary Compounds ◽  
Extraction Methods ◽  
Extraction Techniques ◽  
High Quality ◽  
Optimum Extraction

The species of trees have different secondary compounds that need optimum extraction techniques. Appropriate extraction techniquesdetermine the quality and quantity of DNA produced. This research aims to found optimal of extraction methods and DNA isolation, thento created genome DNA in high quality and quantity, so that it can be using for genetic variation analyses in Suren (Toona sureni Merr.) byRandom Amplified Polymorphic DNA (RAPD). This study shows that DNA concentrates were 763.3 μg/ml, 180.0 μg/ml, 383.3 μg/ml, and436.7 μg/ml. While based on the results of PCR amplification using the primers OPD 03 shows that the four extraction methods used, the extraction method of number 3 has been able to produce genomic DNA with better quality and more number of bands, although the quantityis lower.

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