Effect Of DNA Extraction Methods Research Articles

Environmental DNA extraction method from water for a high and consistent DNA yield

AbstractEnvironmental DNA (eDNA) measurements have been widely applied in the biomonitoring of organisms. However, different DNA extraction methods may cause changes in yield and consistency, resulting in an inaccurate estimation of eDNA, especially when quantitative measurements are performed. This study focused on evaluating the effects of DNA extraction methods on the magnitude and consistency of fish DNA yield from water samples. Samples were collected periodically over a year from river and lake water systems, and reference eDNA was spiked into them. The spiked samples were extracted using three methods: using lysis buffer for blood and cells (AL method) with the DNeasy Blood and Tissue Kit (Qiagen), using lysis buffer for tissue (ATL method), and the microfluidic‐channel method (BC method). DNA yields were compared by determining the copy number using qPCR. The ATL method showed better stability and a higher yield than the AL method. In addition, despite being comparatively simple, the BC method performed extractions stably and at relatively high yields. Based on the long‐term evaluation of eDNA measurements using various methods, we showed that differences in DNA extraction methods may alter DNA yield and stability.

Effects of DNA extraction methods on the real time PCR quantification of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari in chicken feces and ceca contents

Polymerase chain reaction (PCR) method was coupled with a DNA extraction to enumerate Campylobacter spp. from poultry gastrointestinal tract samples. Three experiments were conducted that included: 1) Development of a DNA standard curve related to bacterial DNA primers; 2) Design of a cell/genomic DNA extraction protocol to isolate Campylobacter spp. DNA from complex samples such as poultry feces; and 3) Comparison of PCR quantification to standard plate count methodology. The standard curve using primers for Campylobacter spp. was created for DNA extracted from environmental isolates with a linear range (R2 > 0.95) and with a high specificity for C. coli and C. jejuni recovered from poultry, swine and laboratory isolates. A 2-step extraction process of bacterial DNA from poultry feces was developed in which the cells were first concentrated using a gradient-centrifugation step followed by comparison of 4 DNA extraction methods. Two commercial DNA extraction methods (Zymo Research Quick DNA, and Invitrogen magnetic separation), a traditional phenol-chloroform DNA extraction method using proteinase K to inactivate DNAses, and an in-house isolation method for DNA extraction based on chaotropic salts were used. The middle gradient layer recovered 89% to 98% of the bacteria cells from the sample, with recovery dependent upon the Campylobacter genus. The 4 DNA extractions methods recovered 112 to 302 ug/nL of DNA. Finally, the qPCR and standard plate methods were highly correlated for enumerating Campylobacter spp. in the 2.0 to 8.0-log CFU range. Analyses of the results from this study demonstrate that the combination of the standard curve for Campylobacter spp. DNA primers, the gradient cell concentration method and DNA extraction techniques with qPCR can be used to enumerate Campylobacter spp. from poultry samples with findings similar those of traditional plate count methodology.

Influence of DNA extraction kits on freshwater fungal DNA metabarcoding.

BackgroundEnvironmental DNA (eDNA) metabarcoding is a common technique for efficient biodiversity monitoring, especially of microbes. Recently, the usefulness of aquatic eDNA in monitoring the diversity of both terrestrial and aquatic fungi has been suggested. In eDNA studies, different experimental factors, such as DNA extraction kits or methods, can affect the subsequent analyses and the results of DNA metabarcoding. However, few methodological studies have been carried out on eDNA of fungi, and little is known about how experimental procedures can affect the results of biodiversity analysis. In this study, we focused on the effect of DNA extraction method on fungal DNA metabarcoding using freshwater samples obtained from rivers and lakes.MethodsDNA was extracted from freshwater samples using the DNeasy PowerSoil kit, which is mainly used to extractmicrobial DNA from soil, and the DNeasy Blood & Tissue kit, which is commonly used for eDNA studies on animals. We then compared PCR inhibition and fungal DNA metabarcoding results; i.e., operational taxonomic unit (OTU) number and composition of the extracted samples.ResultsNo PCR inhibition was detected in any of the samples, and no significant differences in the number of OTUs and OTU compositions were detected between the samples processed using different kits. These results indicate that both DNA extraction kits may provide similar diversity results for the river and lake samples evaluated in this study. Therefore, it may be possible to evaluate the diversity of fungi using a unified experimental method, even with samples obtained for diversity studies on other taxa such as those of animals.

Effects of DNA preservation solution and DNA extraction methods on microbial community profiling of soil

Microbial community profiling using high-throughput sequencing relies in part on the preservation of the DNA and the effectiveness of the DNA extraction method. This study aimed at understanding to what extent these parameters affect the profiling. We obtained samples treated with and without a preservation solution. Also, we compared DNA extraction kits from Qiagen and Zymo-Research. The types of samples were defined strains, both as single species and mixtures, as well as undefined indigenous microbial communities from soil. We show that the use of a preservation solution resulted in substantial changes in the 16S rRNA gene profiles either due to an overrepresentation of Gram-positive bacteria or to an underrepresentation of Gram-negative bacteria. In addition, 16S rRNA gene profiles were substantially different depending on the type of kit that was used for extraction. The kit from Zymo extracted DNA from different types of bacteria in roughly equal amounts. In contrast, the kit from Qiagen preferentially extracted DNA from Gram-negative bacteria while DNA from Gram-positive bacteria was extracted less effectively. These differences in kit performance strongly influenced the interpretation of our microbial ecology studies.

Improving the yield of environmental DNA from filtered aquatic samples

Analysis of environmental DNA (eDNA) through non-invasive sampling continues to gain popularity in the surveillance of organisms. Methodological improvement to ensure maximum DNA recovery from the samples would benefit future studies. We investigated the effects of DNA extraction methods and filter preservation methods on eDNA yield by analyzing field-collected water samples for eDNA of the Eastern Hellbender (Cryptobranchus a. alleganiensis). We tested whether the use of bead beating during DNA extraction, which likely facilitates removal of cells trapped on filters, would increase eDNA yield. We also examined whether preservation of filters in ethanol or storage at − 20 °C before extraction would yield more eDNA. Bead beating and preservation at − 20 °C significantly increased the estimated amount of eDNA.

Effect of DNA extraction methods on the detection of porcine ingredients in halal cosmetics using real-time PCR

In recent years, halal cosmetics have attracted considerable attention worldwide. We developed a real-time PCR assay based on the mitochondrial gene ndh5 for rapid detection of porcine ingredients in halal cosmetic products. We also compared several DNA extraction methods for the most efficient approach in different types of cosmetics. Porcine template DNA was spiked into three types of cosmetics (liquid-type and powder-type mask packs, and cream) and extracted with five commercial DNA extraction kits and the CTAB method. The extraction efficiency of each method was evaluated by determining the detection limits of real-time PCR assay. The lowest detection limit of real-time PCR for each cosmetic product was as follows: 2.28 × 100 copies for liquid-type mask pack when the Power Prep™ DNA extraction kit and TIANamp Genomic DNA kit were used, 2.28 × 101 copies for powder-type mask pack when QIAamp DNA stool mini kit and the Power Prep™ DNA extraction kit were used, and 2.28 × 100 copies for cream when the Power Prep™ DNA extraction kit was used. The pig-specific real-time PCR assay facilitated the detection of trace amounts of the template DNA in cosmetics, and an appropriate DNA extraction method was used depending on the type of cosmetics.

Evaluation of pre-analytical factors affecting plasma DNA analysis

Pre-analytical factors can significantly affect circulating cell-free DNA (cfDNA) analysis. However, there are few robust methods to rapidly assess sample quality and the impact of pre-analytical processing. To address this gap and to evaluate effects of DNA extraction methods and blood collection tubes on cfDNA yield and fragment size, we developed a multiplexed droplet digital PCR (ddPCR) assay with 5 short and 4 long amplicons targeting single copy genomic loci. Using this assay, we compared 7 cfDNA extraction kits and found cfDNA yield and fragment size vary significantly. We also compared 3 blood collection protocols using plasma samples from 23 healthy volunteers (EDTA tubes processed within 1 hour and Cell-free DNA Blood Collection Tubes processed within 24 and 72 hours) and found no significant differences in cfDNA yield, fragment size and background noise between these protocols. In 219 clinical samples, cfDNA fragments were shorter in plasma samples processed immediately after venipuncture compared to archived samples, suggesting contribution of background DNA by lysed peripheral blood cells. In summary, we have described a multiplexed ddPCR assay to assess quality of cfDNA samples prior to downstream molecular analyses and we have evaluated potential sources of pre-analytical variation in cfDNA studies.

Effect of endogenous reference genes on digital PCR assessment of genetically engineered canola events

Droplet digital PCR (ddPCR) has been used for absolute quantification of genetically engineered (GE) events. Absolute quantification of GE events by duplex ddPCR requires the use of appropriate primers and probes for target and reference gene sequences in order to accurately determine the amount of GE materials. Single copy reference genes are generally preferred for absolute quantification of GE events by ddPCR. Study has not been conducted on a comparison of reference genes for absolute quantification of GE canola events by ddPCR. The suitability of four endogenous reference sequences (HMG-I/Y, FatA(A), CruA and Ccf) for absolute quantification of GE canola events by ddPCR was investigated. The effect of DNA extraction methods and DNA quality on the assessment of reference gene copy numbers was also investigated. ddPCR results were affected by the use of single vs. two copy reference genes. The single copy, FatA(A), reference gene was found to be stable and suitable for absolute quantification of GE canola events by ddPCR. For the copy numbers measured, the HMG-I/Y reference gene was less consistent than FatA(A) reference gene. The expected ddPCR values were underestimated when CruA and Ccf (two copy endogenous Cruciferin sequences) were used because of high number of copies. It is important to make an adjustment if two copy reference genes are used for ddPCR in order to obtain accurate results. On the other hand, real-time quantitative PCR results were not affected by the use of single vs. two copy reference genes.

The Effect of DNA Extraction Methods on Observed Microbial Communities from Fibrous and Liquid Rumen Fractions of Dairy Cows.

DNA based methods have been widely used to study the complexity of the rumen microbiota, and it is well known that the method of DNA extraction is a critical step in enabling accurate assessment of this complexity. Rumen fluid (RF) and fibrous content (FC) fractions differ substantially in terms of their physical nature and associated microorganisms. The aim of this study was therefore to assess the effect of four DNA extraction methods (RBB, PBB, FDSS, PQIAmini) differing in cell lysis and/or DNA recovery methods on the observed microbial diversity in RF and FC fractions using samples from four rumen cannulated dairy cows fed 100% grass silage (GS100), 67% GS and 33% maize silage (GS67MS33), 33% GS and 67% MS (GS33MS67), or 100% MS (MS100). An ANOVA statistical test was applied on DNA quality and yield measurements, and it was found that the DNA yield was significantly affected by extraction method (p < 0.001) and fraction (p < 0.001). The 260/280 ratio was not affected by extraction (p = 0.08) but was affected by fraction (p = 0.03). On the other hand, the 260/230 ratio was affected by extraction method (p < 0.001) but not affected by fraction (p = 0.8). However, all four extraction procedures yielded DNA suitable for further analysis of bacterial, archaeal and anaerobic fungal communities using quantitative PCR and pyrosequencing of relevant taxonomic markers. Redundancy analysis (RDA) of bacterial 16S rRNA gene sequence data at the family level showed that there was a significant effect of rumen fraction (p = 0.012), and that PBB (p = 0.012) and FDSS (p = 0.024) also significantly contributed to explaining the observed variation in bacterial community composition. Whilst the DNA extraction method affected the apparent bacterial community composition, no single extraction method could be concluded to be ineffective. No obvious effect of DNA extraction method on the anaerobic fungi or archaea was observed, although fraction effects were evident for both. In summary, the comprehensive assessment of observed communities of bacteria, archaea and anaerobic fungi described here provides insight into a rational basis for selecting an optimal methodology to obtain a representative picture of the rumen microbiota.

Effect of DNA Extraction Methods on the Apparent Structure of Yak Rumen Microbial Communities as Revealed by 16S rDNA Sequencing

To more efficiently identify the microbial community of the yak rumen, the standardization of DNA extraction is key to ensure fidelity while studying environmental microbial communities. In this study, we systematically compared the efficiency of several extraction methods based on DNA yield, purity, and 16S rDNA sequencing to determine the optimal DNA extraction methods whose DNA products reflect complete bacterial communities. The results indicate that method 6 (hexadecyltrimethylammomium bromide-lysozyme-physical lysis by bead beating) is recommended for the DNA isolation of the rumen microbial community due to its high yield, operational taxonomic unit, bacterial diversity, and excellent cell-breaking capability. The results also indicate that the bead-beating step is necessary to effectively break down the cell walls of all of the microbes, especially Gram-positive bacteria. Another aim of this study was to preliminarily analyze the bacterial community via 16S rDNA sequencing. The microbial community spanned approximately 21 phyla, 35 classes, 75 families, and 112 genera. A comparative analysis showed some variations in the microbial community between yaks and cattle that may be attributed to diet and environmental differences. Interestingly, numerous uncultured or unclassified bacteria were found in yak rumen, suggesting that further research is required to determine the specific functional and ecological roles of these bacteria in yak rumen. In summary, the investigation of the optimal DNA extraction methods and the preliminary evaluation of the bacterial community composition of yak rumen support further identification of the specificity of the rumen microbial community in yak and the discovery of distinct gene resources.

Influence of DNA Extraction Method, 16S rRNA Targeted Hypervariable Regions, and Sample Origin on Microbial Diversity Detected by 454 Pyrosequencing in Marine Chemosynthetic Ecosystems

Next-generation sequencing (NGS) opens up exciting possibilities for improving our knowledge of environmental microbial diversity, allowing rapid and cost-effective identification of both cultivated and uncultivated microorganisms. However, library preparation, sequencing, and analysis of the results can provide inaccurate representations of the studied community compositions. Therefore, all these steps need to be taken into account carefully. Here we evaluated the effects of DNA extraction methods, targeted 16S rRNA hypervariable regions, and sample origins on the diverse microbes detected by 454 pyrosequencing in marine cold seep and hydrothermal vent sediments. To assign the reads with enough taxonomic precision, we built a database with about 2,500 sequences from Archaea and Bacteria from deep-sea marine sediments, affiliated according to reference publications in the field. Thanks to statistical and diversity analyses as well as inference of operational taxonomic unit (OTU) networks, we show that (i) while DNA extraction methods do not seem to affect the results for some samples, they can lead to dramatic changes for others; and (ii) the choice of amplification and sequencing primers also considerably affects the microbial community detected in the samples. Thereby, very different proportions of pyrosequencing reads were obtained for some microbial lineages, such as the archaeal ANME-1, ANME-2c, and MBG-D and deltaproteobacterial subgroups. This work clearly indicates that the results from sequencing-based analyses, such as pyrosequencing, should be interpreted very carefully. Therefore, the combination of NGS with complementary approaches, such as fluorescence in situ hybridization (FISH)/catalyzed reporter deposition (CARD)-FISH or quantitative PCR (Q-PCR), would be desirable to gain a more comprehensive picture of environmental microbial communities.

Effect of DNA Extraction Methods and Sampling Techniques on the Apparent Structure of Cow and Sheep Rumen Microbial Communities

Molecular microbial ecology techniques are widely used to study the composition of the rumen microbiota and to increase understanding of the roles they play. Therefore, sampling and DNA extraction methods that result in adequate yields of microbial DNA that also accurately represents the microbial community are crucial. Fifteen different methods were used to extract DNA from cow and sheep rumen samples. The DNA yield and quality, and its suitability for downstream PCR amplifications varied considerably, depending on the DNA extraction method used. DNA extracts from nine extraction methods that passed these first quality criteria were evaluated further by quantitative PCR enumeration of microbial marker loci. Absolute microbial numbers, determined on the same rumen samples, differed by more than 100-fold, depending on the DNA extraction method used. The apparent compositions of the archaeal, bacterial, ciliate protozoal, and fungal communities in identical rumen samples were assessed using 454 Titanium pyrosequencing. Significant differences in microbial community composition were observed between extraction methods, for example in the relative abundances of members of the phyla Bacteroidetes and Firmicutes. Microbial communities in parallel samples collected from cows by oral stomach-tubing or through a rumen fistula, and in liquid and solid rumen digesta fractions, were compared using one of the DNA extraction methods. Community representations were generally similar, regardless of the rumen sampling technique used, but significant differences in the abundances of some microbial taxa such as the Clostridiales and the Methanobrevibacter ruminantium clade were observed. The apparent microbial community composition differed between rumen sample fractions, and Prevotellaceae were most abundant in the liquid fraction. DNA extraction methods that involved phenol-chloroform extraction and mechanical lysis steps tended to be more comparable. However, comparison of data from studies in which different sampling techniques, different rumen sample fractions or different DNA extraction methods were used should be avoided.

Effect of DNA Extraction Method on the Apparent Microbial Diversity of Soil

Four extraction methods, including a novel one, were compared for their efficiencies in producing DNA from three contrasting agricultural soils. Molecular analyses (PCR-denaturing gradient gel electrophoresis [DGGE] and clone libraries) focusing on different microbial groups were used as assessment criteria. Per soil, the DNA yields differed between extraction methods. Clear effects of method on apparent richness and community structure were found. Actinobacterial diversity based on soil DNA produced by two divergent methods revealed that a hitherto-undescribed group was obtained by the novel method.

Toward Metrological Traceability for DNA Fragment Ratios in GM Quantification. 1. Effect of DNA Extraction Methods on the Quantitative Determination of Bt176 Corn by Real-Time PCR

An international CCQM-P60 pilot study involving eight national metrological institutes was organized to investigate if the quantification of genetically modified (GM) corn powder by real-time PCR was affected by the DNA extraction method applied. Four commonly used extraction methods were compared for the extraction of DNA from a GM Bt176 corn powder. The CTAB-based method yielded the highest DNA template quantity and quality. A difference in the 260 nm/230 nm absorbance ratio was observed among the different extraction methods. Real-time amplification of sequences specific for endogenous genes zein and hmg as well as transgenic sequences within the cryIA(b) gene and a fragment covering the junction between the transformed DNA and the plant genome were used to determine the GM percentage. The detection of the transgenic gene was affected by the quantity and quality of template used for the PCR reaction. The Bt176 percentages measured on diluted or purified templates were statistically different depending on the extraction method applied.

The effect of extraction methods. The kind of organ samples and the examination delay on the DNA yields and typing.

This study investigated the effect of DNA extraction methods, examination delay and the kind of organs samples to the DNA yields and typing. Thirty autopsy cases with postmortem period less than 12 hours were used as the sample resources. The DNA was successfully extracted from cerebral cortex, liver, spleen, lymph nodes, kidney, psoas muscle and prostate gland by Bar and Kirby methods. The spectrophotometric measurement showed that the spleen, lymph nodes, kidney and liver provided more DNA rather than the other organs. The agarose gel electrophoresis showed that the majority of the samples had High Molecular Weight-DNA (HMW-DNA) with variable degree of degradation. All of these DNA were successfully typed on the D1S80 locus using the PCR according to Kasai method. The spleen samples were collected from the same cases and stored at -20 degrees C for 1 to 6 weeks before the DNA extraction was performed. The analysis of the DNA extracted from these samples showed that the DNA yields and typing did not change significantly among the samples with examination delay up to 6 weeks after the sample collection. The comparison between the Bar and Kirby methods showed that the Kirby method resulted in more DNA yields with the same purity of DNA, but less HMW-DNA compared with Bar method.