scholarly journals DNA Extraction Protocol for Plants with High Levels of Secondary Metabolites and Polysaccharides without Using Liquid Nitrogen and Phenol

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Sunil Kumar Sahu ◽  
Muthusamy Thangaraj ◽  
Kandasamy Kathiresan
Keyword(s):  
Salt Marsh ◽  
Secondary Metabolites ◽  
Liquid Nitrogen ◽  
Genomic Dna ◽  
Dna Isolation ◽  
Wide Spectrum ◽  
Pcr Amplification ◽  
Restriction Digestion ◽  
High Quality ◽  
Salt Marsh Species

Mangroves and salt marsh species are known to synthesize a wide spectrum of polysaccharides and polyphenols including flavonoids and other secondary metabolites which interfere with the extraction of pure genomic DNA. Although a plethora of plant DNA isolation protocols exist, extracting DNA from mangroves and salt marsh species is a challenging task. This study describes a rapid and reliable cetyl trimethylammonium bromide (CTAB) protocol suited specifically for extracting DNA from plants which are rich in polysaccharides and secondary metabolites, and the protocol also excludes the use of expensive liquid nitrogen and toxic phenols. Purity of extracted DNA was excellent as evident by A260/A280 ratio ranging from 1.78 to 1.84 and A260/A230 ratio was >2, which also suggested that the preparations were sufficiently free of proteins and polyphenolics/polysaccharide compounds. DNA concentration ranged from 8.8 to 9.9 μg μL−1. The extracted DNA was amenable to RAPD, restriction digestion, and PCR amplification of plant barcode genes (matK and rbcl). The optimized method is suitable for both dry and fresh leaves. The success of this method in obtaining high-quality genomic DNA demonstrated the broad applicability of this method.

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.

scholarly journals An alternative method for Staphylococcus aureus DNA isolation

2008 ◽  
Vol 60 (2) ◽  
pp. 299-306 ◽  
Author(s):  
L. Chapaval ◽  
D.H. Moon ◽  
J.E. Gomes ◽  
F.R. Duarte ◽  
S.M. Tsai
Keyword(s):  
Staphylococcus Aureus ◽  
Genomic Dna ◽  
Dna Isolation ◽  
Environmental Isolates ◽  
Reaction Volume ◽  
High Quality ◽  
Rapid Procedure ◽  
Final Reaction ◽  
Staphylococcal Enterotoxins ◽  
Final Reaction Volume

This study describes a rapid procedure for the isolation of genomic DNA from Staphylococcus aureus that yielded a good amount of high quality DNA for the amplification of staphylococcal enterotoxins genes (A, B, C, D, and E) and the TSST-1 gene as well as enzymatic restriction (HaeIII) from environmental isolates. With this method, it was possible to detect these genes in a sample containing as little as 10(5) cells with positive PCR reactions obtained from approximately 10pg of DNA in a final reaction volume of 25µl.

scholarly journals Genetic diversity among forty coffee varieties assessed by RAPD markers associated with restriction digestion

2005 ◽  
Vol 48 (4) ◽  
pp. 511-521 ◽  
Author(s):  
Leandro Eugênio Cardamoni Diniz ◽  
Claudete de Fátima Ruas ◽  
Valdemar de Paula Carvalho ◽  
Fabrício Medeiros Torres ◽  
Eduardo Augusto Ruas ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Genetic Variation ◽  
Genetic Variability ◽  
Clustering Analysis ◽  
Genomic Dna ◽  
Rapd Markers ◽  
Random Amplified Polymorphic Dna ◽  
Pcr Amplification ◽  
Restriction Digestion ◽  
Agronomic Features

The genetic variability of 40 accessions of_C. arabica was evaluated using a combination of the random amplified polymorphic DNA (RAPD) technique and restriction digestion of genomic DNA. The genetic variability and the relatedness among all accessions were initially evaluated using 195 RAPD primers which revealed a very low level of genetic variation. To improve the efficiency in the detection of polymorphism, the genomic DNA of all accessions were submitted to digestion with restriction endonucleases prior to PCR amplification. A total of 24 primers combined with restriction digestion of DNA rendered 318 bands, of which 266 (83.65%) were polymorphic. The associations among genotypes were estimated using UPGMA-clustering analysis. The accessions were properly clustered according to pedigree and agronomic features. The ability to distinguish among coffee accessions was greater for RAPD plus restriction digestion than for RAPD alone, providing evidences that the combination of the techniques was very efficient for the estimation of genetic relationship among_C. arabica genotypes.

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.

Optimization of DNA isolation and PCR parameters for RAPD analysis of Gonyostomum semen (Raphidophyceae)

2012 ◽  
Vol 18 (1) ◽  
pp. 40-45
Author(s):  
Elena Servienė ◽  
Irena Kemežienė ◽  
Jūratė Kasperovičienė ◽  
Brigita Čapukoitienė ◽  
Vida Rančelienė ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Genetic Variability ◽  
Genomic Dna ◽  
Dna Isolation ◽  
Rapd Analysis ◽  
Dna Purification ◽  
High Quality ◽  
Purification Method ◽  
Rapd Pcr ◽  
Gonyostomum Semen

Abstract Servienė E., Kemežienė I., Kasperovičienė J., čapukoitienė B., Rančelienė V., Koreivienė J., 2012: Optimization of DNA isolation and PCR parameters for RAPD analysis of Gonyostomum semen (Raphidophyceae) [DNR izoliavimas ir PGR parametrų optimizavimas Gonyostomum semen (Raphidophyceae) dumblių RAPD analizei]. - Bot. Lith., 18(1): 40-45. The genomic DNA purification method for Gonyostomum semen algae was optimized by applying different DNA purification techniques and rational modifications. This method allowed to obtain high quality DNA preparations suitable for the phylogenetic analysis and genetic variability investigation of algae. DNA isolated by this method yielded strong and reliable amplification products showing their applicability for RAPD-PCR using random decamer primers. In the present study, the RAPD protocol was optimized for the evaluation of Gonyostomum biodiversity.

Study on the Isolation of High-Quality DNA and RNA from Aloe

2012 ◽  
Vol 455-456 ◽  
pp. 449-454
Author(s):  
Yong Si Zhang ◽  
Jun Li ◽  
Xiao Hong Liu
Keyword(s):  
Secondary Metabolites ◽  
Genomic Dna ◽  
Herbal Drug ◽  
High Quality ◽  
Dna And Rna ◽  
Improved Methods

. Aloe, an important folk herbal drug, includes abundant polysaccharides and secondary metabolites which bringing about the difficulty of isolating high-quality DNA or RNA. In this paper, one and two improved methods were used to isolate the genomic DNA and RNA from the leaf of Aloe, respectively. The obtained samples presented good quality and integrality, and thus, they could be further used for many downstream molecular experiments. This reported protocols on extraction of DNA and RNA offered a valuable reference for other related studies.

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