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.

Comparison of DNA Extraction and Purification Methods from Different Soils for Metagenomic Sequencing

2014 ◽  
Vol 955-959 ◽  
pp. 306-309 ◽  
Author(s):  
Lin Hui Wu ◽  
Jian Li Liu ◽  
Jing Zeng ◽  
Ji Zhao
Keyword(s):  
Nucleic Acids ◽  
Dna Extraction ◽  
Dna Isolation ◽  
Metagenomic Sequencing ◽  
Soil Dna ◽  
Dna Yield ◽  
Extraction And Purification ◽  
Purification Methods ◽  
The Impact ◽  
Different Soils

There is an increased interest in the extraction of nucleic acids from various environmental samples, since only a minority of naturally occurring microbes can be cultured using standard techniques. Nucleic acids extraction and purification from soils are extremely challenging due to the low biomass, high organic contents and high variability of soil types. This has been regarded as one of the major difficulties that hamper the development of soil microbial ecology study. No commercial nucleic acids kits currently available are capable of preparing the DNAs without modifications. The cost can be very high for DNA extraction from extreme environmental soil samples, such as soils that have extreme high or low pHs. In this work, we developed and optimized soil DNA extraction and purification methods on different soils and compared the impact of three different DNA extraction protocols on DNA yield and purity. For the three different types of soil we used, direct extraction obtained the highest DNA recover rate, but required more cleanup steps. MoBio PowerSoil® DNA Isolation Kit yields less but do not require as many downstream cleaning steps. Both of the two methods obtained a more abundant microbial community than Meta-G-NomeTMDNA Isolation Kit.

An efficient protocol for the isolation of fungal DNA for strains obtained from oil contaminated fields

2012 ◽  
Vol 2 (4) ◽  
pp. 162-165
Author(s):  
Bhavya Ravi ◽  
Madhulika Rai ◽  
Sandhya Mehrotra ◽  
Rajesh Mehrotra
Keyword(s):  
Contaminated Soils ◽  
Petroleum Hydrocarbons ◽  
Dna Isolation ◽  
Cost Effective ◽  
Industrial Applications ◽  
Glass Beads ◽  
Natural Ecosystem ◽  
Dna Yield ◽  
Genomic Study ◽  
Fungal Dna

A natural ecosystem contaminated with petroleum hydrocarbons is likely to favor the growth of taxonomically diverse microbes having the ability to degrade these organic compounds. They can be exploited for purposes like bioremediation of oil contaminated soils and to obtain enzymes like lipases having important industrial applications. In this paper, a novel “IBG” (Improved ‘Bust and Grab’) protocol has been reported for the isolation of fungal DNA from strains collected from oil contaminated fields. Conventional methods for DNA isolation from fungi require the use of enzymes, liquid nitrogen, glass beads etc. The method reported here circumvents the use of enzymes or glass beads and is cost effective and can be used while handling large number of samples. The DNA yield obtained by the IBG protocol is significant and of good quality. The good quality DNA isolated by IBG protocol can be used for the quick and cost effective isolation of fungal genomic DNA facilitating the genomic study of microbes obtained from oil contaminated fields.

scholarly journals To beat or not to beat a tick: Comparison of DNA extraction methods from ticks (Ixodes scapularis)

2015 ◽  
Author(s):  
Alyssa D Ammazzalorso ◽  
Christine P Zolnik ◽  
Thomas J Daniels ◽  
Sergios-Orestis Kolokotronis
Keyword(s):  
Dna Extraction ◽  
Disease Transmission ◽  
Dna Isolation ◽  
Ixodes Scapularis ◽  
Genetic Material ◽  
The United States ◽  
Qualitative Assessment ◽  
Single Individual ◽  
Cross Sectional ◽  
Dna Yield

Background. Blacklegged ticks (Ixodes scapularis) are important disease vectors in the United States, known to transmit a variety of pathogens to humans, including bacteria, protozoa, and viruses. Their importance as a disease vector necessitates reliable and comparable methods for extracting microbial DNA from ticks. Furthermore, to explore the population genetics or genomics of this tick, appropriate DNA extraction techniques are needed for both the vector and its microbes. Although a few studies have investigated different methods of DNA isolation from ticks, they are limited in the number and types of DNA extraction and lack species-specific quantification of DNA yield. Methods. Here we determined the most efficient and consistent method of DNA extraction from two different developmental stages of I. scapularis – nymph and adult - that are the most important for disease transmission. We used various methods of physical disruption of the hard, chitinous exoskeleton, as well as commercial and non-commercial DNA isolation kits. To gauge the effectiveness of these methods we quantified the DNA yield and confirmed the DNA quality via PCR of both tick and microbial genetic material. Results. DNA extraction using the Thermo GeneJET Genomic DNA Purification kit resulted in the highest DNA yields and the strongest, most consistent PCR amplification. We also found that physical disruption of the tick exoskeleton was most effective using cross-sectional cutting compared to any type of bead-beating matrices used. Storing ticks at -80°C resulted in considerably higher DNA yields than those from ticks stored in ethanol. Discussion. We contrasted a variety of readily available methods of DNA extraction from single individual blacklegged ticks and presented the results through a quantitative and qualitative assessment.

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 To beat or not to beat a tick: Comparison of DNA extraction methods from ticks (Ixodes scapularis)

2015 ◽  
Author(s):  
Alyssa D Ammazzalorso ◽  
Christine P Zolnik ◽  
Thomas J Daniels ◽  
Sergios-Orestis Kolokotronis
Keyword(s):  
Dna Extraction ◽  
Disease Transmission ◽  
Dna Isolation ◽  
Ixodes Scapularis ◽  
Genetic Material ◽  
The United States ◽  
Qualitative Assessment ◽  
Single Individual ◽  
Cross Sectional ◽  
Dna Yield

Background. Blacklegged ticks (Ixodes scapularis) are important disease vectors in the United States, known to transmit a variety of pathogens to humans, including bacteria, protozoa, and viruses. Their importance as a disease vector necessitates reliable and comparable methods for extracting microbial DNA from ticks. Furthermore, to explore the population genetics or genomics of this tick, appropriate DNA extraction techniques are needed for both the vector and its microbes. Although a few studies have investigated different methods of DNA isolation from ticks, they are limited in the number and types of DNA extraction and lack species-specific quantification of DNA yield. Methods. Here we determined the most efficient and consistent method of DNA extraction from two different developmental stages of I. scapularis – nymph and adult - that are the most important for disease transmission. We used various methods of physical disruption of the hard, chitinous exoskeleton, as well as commercial and non-commercial DNA isolation kits. To gauge the effectiveness of these methods we quantified the DNA yield and confirmed the DNA quality via PCR of both tick and microbial genetic material. Results. DNA extraction using the Thermo GeneJET Genomic DNA Purification kit resulted in the highest DNA yields and the strongest, most consistent PCR amplification. We also found that physical disruption of the tick exoskeleton was most effective using cross-sectional cutting compared to any type of bead-beating matrices used. Storing ticks at -80°C resulted in considerably higher DNA yields than those from ticks stored in ethanol. Discussion. We contrasted a variety of readily available methods of DNA extraction from single individual blacklegged ticks and presented the results through a quantitative and qualitative assessment.

scholarly journals A Rapid and Inexpensive Method of DNA Extraction from Palmyra Palm (Borassus flabellifer)

2019 ◽  
pp. 1-5
Author(s):  
Soni Kumari ◽  
Ruby Rani ◽  
Jitesh Kumar ◽  
Ravi Ranjan Kumar ◽  
Tushar Ranjan
Keyword(s):  
Dna Extraction ◽  
Dna Isolation ◽  
Pcr Amplification ◽  
Tween 20 ◽  
Inexpensive Method ◽  
Rapid Procedure ◽  
Dna Yield ◽  
Lysis Buffer ◽  
Yield And Purity ◽  
Different Levels

Aims: The study aims to highlight the simple optimisation, inexpensive and rapid procedure for DNA isolation from tough leaves (Palmyra palm) without compromising the yield and purity of DNA. Study Design: Leaf of palmyra palm (Borassus flabellifer) was used to conduct the experiment followed by laboratory analysis, DNA extraction and PCR amplification. Results and Discussion: The results showed that different buffers examined for the extraction of DNA provided significantly different levels of yield and purity. DNA isolated by lysis buffers C showed satisfactory amplifications in PCR. The fingerprint we obtained by using the DNA extracted by these buffers provided higher resolution than those using buffers. Conclusion: This study suggests that grinding of Palmyra palm leaves with sterile sand or cover slips and inclusion of SDS, Tween 20, and NaCl (1.4 M) in the lysis buffer without the costly use of liquid nitrogen, PVP and β mercaptoethanol, provides a DNA yield of sufficient purity, suitable for PCR amplification and subsequent use.

Extraction of DNA from a Wide Range of Objects by Treatment with Ammonium Salts

2020 ◽  
Vol 36 (6) ◽  
pp. 98-106
Author(s):  
E.I. Levitin ◽  
B.V. Sviridov ◽  
O.V. Piksasova ◽  
T.E. Shustikova
Keyword(s):  
Dna Extraction ◽  
Dna Isolation ◽  
Tissue Samples ◽  
New Approach ◽  
Cell Wall Disruption ◽  
Wide Range ◽  
Low Salt ◽  
Mammalian Blood ◽  
Plasmid Extraction ◽  
Two Hybrid

Currently, simple, rapid, and efficient techniques for DNA isolation from a wide range of organisms are in demand in biotechnology and bioinformatics. A key (and often limiting) step is the cell wall disruption and subsequent DNA extraction from the disintegrated cells. We have developed a new approach to DNA isolation from organisms with robust cell walls. The protocol includes the following steps: treatment of cells or tissue samples with ammonium acetate followed by cell lysis in low-salt buffer with the addition of SDS. Further DNA extraction is carried out according to standard methods. This approach is efficient for high-molecular native DNA isolation from bacteria, ascomycetes, yeast, and mammalian blood; it is also useful for express analysis of environmental microbial isolates and for plasmid extraction for two-hybrid library screening. express method for DNA isolation; ammonium salt treatment (в русских ключевых такой порядок), osmotic breakage of cells This study was financially supported by the NRC "Kurchatov Institute"-GOSNIIGENETIKA Kurchatov Genomic Center.

scholarly journals Amplicon-sequencing of raw milk microbiota: impact of DNA extraction and library-PCR

2021 ◽  
Author(s):  
Annemarie Siebert ◽  
Katharina Hofmann ◽  
Lena Staib ◽  
Etienne V. Doll ◽  
Siegfried Scherer ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Dna Extraction ◽  
Raw Milk ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Microbiome Analysis ◽  
Eukaryotic Dna ◽  
Selective Lysis ◽  
The Impact

Abstract The highly complex raw milk matrix challenges the sample preparation for amplicon-sequencing due to low bacterial counts and high amounts of eukaryotic DNA originating from the cow. In this study, we optimized the extraction of bacterial DNA from raw milk for microbiome analysis and evaluated the impact of cycle numbers in the library-PCR. The selective lysis of eukaryotic cells by proteinase K and digestion of released DNA before bacterial lysis resulted in a high reduction of mostly eukaryotic DNA and increased the proportion of bacterial DNA. Comparative microbiome analysis showed that a combined enzymatic and mechanical lysis procedure using the DNeasy® PowerFood® Microbial Kit with a modified protocol was best suitable to achieve high DNA quantities after library-PCR and broad coverage of detected bacterial biodiversity. Increasing cycle numbers during library-PCR systematically altered results for species and beta-diversity with a tendency to overrepresentation or underrepresentation of particular taxa. To limit PCR bias, high cycle numbers should thus be avoided. An optimized DNA extraction yielding sufficient bacterial DNA and enabling higher PCR efficiency is fundamental for successful library preparation. We suggest that a protocol using ethylenediaminetetraacetic acid (EDTA) to resolve casein micelles, selective lysis of somatic cells, extraction of bacterial DNA with a combination of mechanical and enzymatic lysis, and restriction of PCR cycles for analysis of raw milk microbiomes is optimal even for samples with low bacterial numbers. Key points • Sample preparation for high-throughput 16S rRNA gene sequencing of raw milk microbiota. • Reduction of eukaryotic DNA by enzymatic digestion. • Shift of detected microbiome caused by high cycle numbers in library-PCR.

scholarly journals Assessing Quality and Functionality of DNA from Fresh and Archival Dried Blood Spots and Recommendations for Quality Control Guidelines

2007 ◽  
Vol 53 (8) ◽  
pp. 1401-1407 ◽  
Author(s):  
Malin Ida Linnea Sjöholm ◽  
Joakim Dillner ◽  
Joyce Carlson
Keyword(s):  
Dna Extraction ◽  
Multiple Displacement Amplification ◽  
Dried Blood Spots ◽  
Extraction Methods ◽  
Population Based ◽  
Alkaline Lysis ◽  
Screening Programs ◽  
Blood Spots ◽  
Dna Yield ◽  
Chelex 100

Abstract Background: Dried blood spots (DBS) are a convenient and inexpensive method for biobanking. Although many countries have established population-based DBS biobanks from neonatal screening programs, the quality and usefulness of DNA from DBS have not been extensively assessed. Methods: We compared 4 common DNA extraction methods (Qiagen, EZNA, Chelex 100, and alkaline lysis) in a pilot study using fresh DBS with known lymphocyte count. We assessed suitability for multiple displacement amplification (MDA) and subsequent single-nucleotide polymorphism (SNP) analyses. We selected the EZNA method for DNA extraction from archival samples up to 27 years old, stored at room temperature or −20 °C, and SNP analyses were performed after MDA. Results: Extraction using alkaline lysis failed in most tests, and Chelex 100 was unsuccessful in real-time PCR, whereas the EZNA and Qiagen methods were successful by all evaluated quality indices. DNA extraction by EZNA, MDA, and SNP analyses were successful for the archival samples stored at −20 °C. Conclusion: Routine protocols for evaluation of the quality and functional integrity of DNA based on DNA yield, DNA size, and quantification of amplifiable DNA allow use of sufficient template for MDA and successful SNP analyses from both primary DBS extract and MDA product. A single 3-mm disc can yield sufficient DNA for several thousand SNP analyses. DNA from DBS is thus suitable for genetic epidemiology studies.

scholarly journals Molecular diagnosis of Hepatozoon canis in symptomatic dogs in the city of Goiania, Goiás, Brazil

2016 ◽  
Vol 68 (6) ◽  
pp. 1431-1439
Author(s):  
S.C. Duarte ◽  
J.A. Parente ◽  
O.J. Silveira Neto ◽  
V.S. Jayme ◽  
T.S.A. Bastos ◽  
...  
Keyword(s):  
Laboratory Diagnosis ◽  
Extraction Process ◽  
Hepatozoon Canis ◽  
Phylogenetic Study ◽  
Blood Smears ◽  
Two Samples ◽  
Pcr Products ◽  
Hepatozoon Americanum ◽  
The City ◽  
Veterinary Hospital

ABSTRACT More than 300 species have been described in the genus Hepatozoon, occurring in different vertebrates. Among these, only Hepatozoon canis and Hepatozoon americanum are seen in dogs. Different methods may be used for laboratory diagnosis. The most common of these is direct parasitological examination of parasite stages in blood smears. The aim of this investigation was to conduct a phylogenetic study on Hepatozoon isolates from symptomatic dogs in the city of Goiânia, Goiás, Brazil. Blood samples were obtained from 40 symptomatic dogs that had been referred to the Veterinary Hospital of the Federal University of Goiás. Among these, only two samples were positive for Hepatozoon spp. using the direct parasitological method. These samples were then subjected to a DNA extraction process and amplification of a fragment of the 18S rRNA by means of PCR. Subsequently, the PCR products from each sample were purified and sequenced. The sequences obtained were then analyzed using the BLASTn algorithm, which identified both sequences of this study as Hepatozoon canis. By applying the Mega4 software, it was confirmed that these isolates of H. canis from dogs in Goiânia are similar to other reference isolates of the same species from other regions of Brazil and worldwide.

Sign in / Sign up

Export Citation Format

Share Document