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 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.

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.

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 Comparison of 12 DNA extraction kits for vertebrate samples

2020 ◽  
pp. 67-77
Author(s):  
I. Martincová ◽  
T. Aghová
Keyword(s):  
Dna Extraction ◽  
Genomic Dna ◽  
Dna Purification ◽  
Research Projects ◽  
High Quality ◽  
Sample Weight ◽  
Crucial Step ◽  
Dna Yield ◽  
Vertebrate Tissue ◽  
Biology Research

Comparison of 12 DNA extraction kits for vertebrate samples Martincová, I. Aghová, T. Abstract Obtaining high quality DNA extractions is a crucial step for molecular biology research projects. At present, numerous protocols are available for vertebrate tissue extractions. In the present study we compared eleven column–based protocols and one HotSHOT protocol using similar conditions (i.e., type of sample, weight of starting material). We evaluated time of extraction, quality and quantity of DNA yield, and price of extraction for a single sample. Based on our analysis, the most successful kits for producing DNA with the highest concentration and purity are the JetQuick® Genomic DNA Purification Kit (Genomed) and the NucleoSpin® Tissue (Macherey–Nagel). Nevertheless, it is highly recommended to test various extraction kits with specific samples to find the optimal kit in all aspects of time, quality and cost for a particular project.

ANALYSIS OF FOUR DIFFERENT METHODS OF DNA EXTRACTION FROM THE TRUE WEEVIL (COLEOPTERA: CURCULIONIDAE)

2018 ◽  
Vol 24 (1) ◽  
Author(s):  
DIVYA SHARMA ◽  
DALIP KUMAR ◽  
RHITOBAN RAY CHOUDHURY
Keyword(s):  
Dna Extraction ◽  
Genomic Dna ◽  
Extraction Method ◽  
Sitophilus Oryzae ◽  
Potassium Acetate ◽  
Single Specimen ◽  
High Quality ◽  
Dna Yield ◽  
Yield And Quality ◽  
Dna Extraction Method

PCR-based markers have been widely used for the analysis of genetic diversity and to avoid ambiguity, molecular characterization is very effective tool for accurate discrimination and identification of a species in insects. Because these studies require analysis of large number of samples, a DNA extraction method that is fast, inexpensive and yields high quality DNA from the preserved samples, needs to be evaluated. A comparative analysis of four methods for DNA extraction from a single specimen of rice weevil, Sitophilus oryzae preserved in 90% alcohol has been communicated. Significantly higher DNA yields were obtained by using SDS-Potassium acetate method followed by CTAB, DNA XPress and Bioline Isolate II genomic DNA kit. Maximum purity (A260/A280- 1.8) was obtained with Bioline Isolate II genomic DNA kit method. The Absorbance ratio was appreciably low with DNA Xpress kit showing the presence of proteins. Bioline Isolate II genomic DNA kit was time efficient and yielded good quality DNA but at a high cost. Based on DNA yield and quality, these evaluations provide a guide for choosing Bioline Isolate II genomic DNA kit method of DNA extraction for rice weevils and optimizing the extraction conditions for rice weevils.

High Microsatellite and SNP Genotyping Success Rates Established in a Large Number of Genomic DNA Samples Extracted From Mouth Swabs and Genotypes

2006 ◽  
Vol 9 (4) ◽  
pp. 501-506 ◽  
Author(s):  
Josine L. Min ◽  
Nico Lakenberg ◽  
Margreet Bakker-Verweij ◽  
Eka Suchiman ◽  
Dorret I. Boomsma ◽  
...  
Keyword(s):  
Success Rate ◽  
Genomic Dna ◽  
Snp Genotyping ◽  
High Yield ◽  
Success Rates ◽  
Nucleotide Polymorphism ◽  
High Quality ◽  
Single Nucleotide ◽  
Dna Yield ◽  
Yield And Quality

AbstractIn this article, we present the genomic DNA yield and the microsatellite and single nucleotide polymorphism (SNP) genotyping success rates of genomic DNA extracted from a large number of mouth swab samples. In total, the median yield and quality was determined in 714 individuals and the success rates in 378,480 genotypings of 915 individuals. The median yield of genomic DNA per mouth swab was 4.1 μg (range 0.1–42.2 μg) and was not reduced when mouth swabs were stored for at least 21 months prior to extraction. A maximum of 20 mouth swabs is collected per participant. Mouth swab samples showed in, respectively, 89% for 390 microsatellites and 99% for 24 SNPs a genotyping success rate higher than 75%. A very low success rate of genotyping (0%–10%) was obtained for 3.2% of the 915 mouth swab samples using microsatellite markers. Only 0.005% of the mouth swab samples showed a geno-typing success rate lower than 75% (range 58%–71%) using SNPs. Our results show that mouth swabs can be easily collected, stored by our conditions for months prior to DNA extraction and result in high yield and high-quality DNA appropriate for genotyping with high success rate including whole genome searches using microsatellites or SNPs.

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.

Comparison of commercial DNA extraction kits for isolation and purification of bacterial and eukaryotic DNA from PAH-contaminated soils

2011 ◽  
Vol 57 (8) ◽  
pp. 623-628 ◽  
Author(s):  
Nagissa Mahmoudi ◽  
Greg F. Slater ◽  
Roberta R. Fulthorpe
Keyword(s):  
Dna Extraction ◽  
Contaminated Soils ◽  
Dna Isolation ◽  
Extraction Process ◽  
Organic Carbon Content ◽  
Soil Dna ◽  
Isolation And Purification ◽  
Dna Yield ◽  
Pcr Products ◽  
Eukaryotic Dna

Molecular characterization of the microbial populations of soils and sediments contaminated with polycyclic aromatic hydrocarbons (PAHs) is often a first step in assessing intrinsic biodegradation potential. However, soils are problematic for molecular analysis owing to the presence of organic matter, such as humic acids. Furthermore, the presence of contaminants, such as PAHs, can cause further challenges to DNA extraction, quantification, and amplification. The goal of our study was to compare the effectiveness of four commercial soil DNA extraction kits (UltraClean Soil DNA Isolation kit, PowerSoil DNA Isolation kit, PowerMax Soil DNA Isolation kit, and FastDNA SPIN kit) to extract pure, high-quality bacterial and eukaryotic DNA from PAH-contaminated soils. Six different contaminated soils were used to determine if there were any biases among the kits due to soil properties or level of contamination. Extracted DNA was used as a template for bacterial 16S rDNA and eukaryotic 18S rDNA amplifications, and PCR products were subsequently analyzed using denaturing gel gradient electrophoresis (DGGE). We found that the FastDNA SPIN kit provided significantly higher DNA yields for all soils; however, it also resulted in the highest levels of humic acid contamination. Soil texture and organic carbon content of the soil did not affect the DNA yield of any kit. Moreover, a liquid–liquid extraction of the DNA extracts found no residual PAHs, indicating that all kits were effective at removing contaminants in the extraction process. Although the PowerSoil DNA Isolation kit gave relatively low DNA yields, it provided the highest quality DNA based on successful amplification of both bacterial and eukaryotic DNA for all six soils. DGGE fingerprints among the kits were dramatically different for both bacterial and eukaryotic DNA. The PowerSoil DNA Isolation kit revealed multiple bands for each soil and provided the most consistent DGGE profiles among replicates for both bacterial and eukaryotic DNA.

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