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.

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.

scholarly journals Profiling soil microbial communities with next-generation sequencing: the influence of DNA kit selection and technician technical expertise

2017 ◽  
Author(s):  
Taha Soliman ◽  
Sung-Yin Yang ◽  
Tomoko Yamazaki ◽  
Holger Jenke-Kodama
Keyword(s):  
Next Generation Sequencing ◽  
Microbial Communities ◽  
Dna Extraction ◽  
Dna Isolation ◽  
Soil Microbial Communities ◽  
Next Generation ◽  
Microbial Composition ◽  
Okinawa Island ◽  
The Impact ◽  
Generation Sequencing

Structure and diversity of microbial communities are an important research topic in biology, since microbes play essential roles in the ecology of various environments. Different DNA isolation protocols can lead to data bias and can affect results of next-generation sequencing. To evaluate the impact of protocols for DNA isolation from soil samples and also the influence of individual handling of samples, we compared results obtained by two researchers (R and T) using two different DNA extraction kits: (1) MO BIO PowerSoil® DNA Isolation kit (MO_R and MO_T) and (2) NucleoSpin® Soil kit (MN_R and MN_T). Samples were collected from six different sites on Okinawa Island, Japan. For all sites, differences in the results of microbial composition analyses (bacteria, archaea, fungi, and other eukaryotes), obtained by the two researchers using the two kits, were analyzed. For both researchers, the MN kit gave significantly higher yields of genomic DNA at all sites compared to the MO kit (ANOVA; P <0.006). In addition, operational taxonomic units for some phyla and classes were missed in some cases: Micrarchaea were detected only in the MN_T and MO_R analyses; the bacterial phylum Armatimonadetes was detected only in MO_R and MO_T; and WIM5 of the phylum Amoebozoa of eukaryotes was found only in the MO_T analysis. Our results suggest the possibility of handling bias; therefore, it is crucial that replicated DNA extraction be performed by at least two technicians for thorough microbial analyses and to obtain accurate estimates of microbial diversity.

scholarly journals Profiling soil microbial communities with next-generation sequencing: the influence of DNA kit selection and technician technical expertise

2017 ◽  
Author(s):  
Taha Soliman ◽  
Sung-Yin Yang ◽  
Tomoko Yamazaki ◽  
Holger Jenke-Kodama
Keyword(s):  
Next Generation Sequencing ◽  
Microbial Communities ◽  
Dna Extraction ◽  
Dna Isolation ◽  
Soil Microbial Communities ◽  
Next Generation ◽  
Microbial Composition ◽  
Okinawa Island ◽  
The Impact ◽  
Generation Sequencing

Structure and diversity of microbial communities are an important research topic in biology, since microbes play essential roles in the ecology of various environments. Different DNA isolation protocols can lead to data bias and can affect results of next-generation sequencing. To evaluate the impact of protocols for DNA isolation from soil samples and also the influence of individual handling of samples, we compared results obtained by two researchers (R and T) using two different DNA extraction kits: (1) MO BIO PowerSoil® DNA Isolation kit (MO_R and MO_T) and (2) NucleoSpin® Soil kit (MN_R and MN_T). Samples were collected from six different sites on Okinawa Island, Japan. For all sites, differences in the results of microbial composition analyses (bacteria, archaea, fungi, and other eukaryotes), obtained by the two researchers using the two kits, were analyzed. For both researchers, the MN kit gave significantly higher yields of genomic DNA at all sites compared to the MO kit (ANOVA; P <0.006). In addition, operational taxonomic units for some phyla and classes were missed in some cases: Micrarchaea were detected only in the MN_T and MO_R analyses; the bacterial phylum Armatimonadetes was detected only in MO_R and MO_T; and WIM5 of the phylum Amoebozoa of eukaryotes was found only in the MO_T analysis. Our results suggest the possibility of handling bias; therefore, it is crucial that replicated DNA extraction be performed by at least two technicians for thorough microbial analyses and to obtain accurate estimates of microbial diversity.

A comparison of DNA extraction and purification methods to detect Escherichia coli O157:H7 in cattle manure

2003 ◽  
Vol 54 (2) ◽  
pp. 165-175 ◽  
Author(s):  
Teegan Trochimchuk ◽  
John Fotheringham ◽  
Edward Topp ◽  
Heidi Schraft ◽  
Kam Tin Leung
Keyword(s):  
Escherichia Coli ◽  
Dna Extraction ◽  
Cattle Manure ◽  
Escherichia Coli O157 ◽  
Extraction And Purification ◽  
Purification Methods

scholarly journals Effects of DNA Extraction and Purification Methods on Real-Time Quantitative PCR Analysis of Roundup Ready Soybean

2009 ◽  
Vol 92 (4) ◽  
pp. 1136-1144 ◽  
Author(s):  
Tigst Demeke ◽  
Indira Ratnayaka ◽  
Anh Phan
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Quantitative Pcr ◽  
Real Time Pcr ◽  
Roundup Ready ◽  
Dna Recovery ◽  
Real Time Quantitative Pcr ◽  
Extraction And Purification ◽  
Purification Methods ◽  
Accuracy And Repeatability

Abstract The quality of DNA affects the accuracy and repeatability of quantitative PCR results. Different DNA extraction and purification methods were compared for quantification of Roundup Ready (RR) soybean (event 40-3-2) by real-time PCR. DNA was extracted using cetylmethylammonium bromide (CTAB), DNeasy Plant Mini Kit, and Wizard Magnetic DNA purification system for food. CTAB-extracted DNA was also purified using the Zymo (DNA Clean & Concentrator 25 kit), Qtip 100 (Qiagen Genomic-Tip 100/G), and QIAEX II Gel Extraction Kit. The CTAB extraction method provided the largest amount of DNA, and the Zymo purification kit resulted in the highest percentage of DNA recovery. The Abs260/280 and Abs260/230 ratios were less than the expected values for some of the DNA extraction and purification methods used, indicating the presence of substances that could inhibit PCR reactions. Real-time quantitative PCR results were affected by the DNA extraction and purification methods used. Further purification or dilution of the CTAB DNA was required for successful quantification of RR soybean. Less variability of quantitative PCR results was observed among experiments and replications for DNA extracted and/or purified by CTAB, CTAB+Zymo, CTAB+Qtip 100, and DNeasy methods. Correct and repeatable results for real-time PCR quantification of RR soybean were achieved using CTAB DNA purified with Zymo and Qtip 100 methods.

scholarly journals Profiling soil microbial communities with next-generation sequencing: the influence of DNA kit selection and technician technical expertise

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e4178 ◽  
Author(s):  
Taha Soliman ◽  
Sung-Yin Yang ◽  
Tomoko Yamazaki ◽  
Holger Jenke-Kodama
Keyword(s):  
Next Generation Sequencing ◽  
Microbial Communities ◽  
Dna Extraction ◽  
Dna Isolation ◽  
Soil Microbial Communities ◽  
Next Generation ◽  
Microbial Composition ◽  
Okinawa Island ◽  
The Impact ◽  
Generation Sequencing

Structure and diversity of microbial communities are an important research topic in biology, since microbes play essential roles in the ecology of various environments. Different DNA isolation protocols can lead to data bias and can affect results of next-generation sequencing. To evaluate the impact of protocols for DNA isolation from soil samples and also the influence of individual handling of samples, we compared results obtained by two researchers (R and T) using two different DNA extraction kits: (1) MO BIO PowerSoil®DNA Isolation kit (MO_R and MO_T) and (2) NucleoSpin®Soil kit (MN_R and MN_T). Samples were collected from six different sites on Okinawa Island, Japan. For all sites, differences in the results of microbial composition analyses (bacteria, archaea, fungi, and other eukaryotes), obtained by the two researchers using the two kits, were analyzed. For both researchers, the MN kit gave significantly higher yields of genomic DNA at all sites compared to the MO kit (ANOVA;P < 0.006). In addition, operational taxonomic units for some phyla and classes were missed in some cases: Micrarchaea were detected only in the MN_T and MO_R analyses; the bacterial phylum Armatimonadetes was detected only in MO_R and MO_T; and WIM5 of the phylum Amoebozoa of eukaryotes was found only in the MO_T analysis. Our results suggest the possibility of handling bias; therefore, it is crucial that replicated DNA extraction be performed by at least two technicians for thorough microbial analyses and to obtain accurate estimates of microbial diversity.

scholarly journals Innovative Methods for Soil DNA Purification Tested in Soils with Widely Differing Characteristics

2008 ◽  
Vol 74 (9) ◽  
pp. 2902-2907 ◽  
Author(s):  
Marketa Sagova-Mareckova ◽  
Ladislav Cermak ◽  
Jitka Novotna ◽  
Kamila Plhackova ◽  
Jana Forstova ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Soil Ph ◽  
Vegetation Cover ◽  
Organic Matter Content ◽  
Clay Content ◽  
Matter Content ◽  
Soil Dna ◽  
Innovative Methods ◽  
Soil Dna Extraction ◽  
Extraction And Purification

ABSTRACT Seven methods of soil DNA extraction and purification were tested in a set of 14 soils differing in bedrock, texture, pH, salinity, moisture, organic matter content, and vegetation cover. The methods introduced in this study included pretreatment of soil with CaCO3 or purification of extracted DNA by CaCl2. The performance of innovated methods was compared to that of the commercial kit Mo Bio PowerSoil and the phenol-chloroform-based method of D. N. Miller, J. E. Bryant, E. L. Madsen, and W. C. Ghiorse (Appl. Environ. Microbiol. 65:4715-4724, 1999). This study demonstrated significant differences between the tested methods in terms of DNA yield, PCR performance, and recovered bacterial diversity. The differences in DNA yields were correlated to vegetation cover, soil pH, and clay content. The differences in PCR performances were correlated to vegetation cover and soil pH. The innovative methods improved PCR performance in our set of soils, in particular for forest acidic soils. PCR was successful in 95% of cases by the method using CaCl2 purification and in 93% of cases by the method based on CaCO3 pretreatment, but only in 79% by Mo Bio PowerSoil, for our range of soils. Also, the innovative methods recovered a higher percentage of actinomycete diversity from a subset of three soils. Recommendations include the assessment of soil characteristics prior to selecting the optimal protocol for soil DNA extraction and purification.

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.

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