Yield Of Extracted DNA Research Articles

Abstract 4446: Assessment of tissue composition with digital pathology in colorectal cancer

Abstract Background: The tumor microenvironment is a key feature to understand cancer biology and may be used clinically. Quantification of tissue composition is usually based either on visual pathological review (VPR) or deconvolution of whole genome molecular data. Although the former is a direct measurement it has modest reproducibility while the latter is an indirect measurement of unclear accuracy, expensive and not always available. Here we test digital pathology coupled with machine learning as a new tool to assess tissue composition. Methods: As part of the Stratification in COloRecTal cancer (S:CORT) programme, a set of over 500 colorectal cancer (CRC) archival paraffin blocks from resections and biopsies were sequentially sectioned for Hematoxylin and Eosin staining (H&E), RNA extraction, a second H&E and DNA extraction. RNA expression microarrays, targeted DNA sequencing and DNA methylation arrays were applied. Tissue composition from the H&Es was obtained by VPR of expert pathologists and by a deep neural net (DNN) algorithm after supervised training on >1,500 tissue areas from S:CORT, TCGA, TEM and CORGI CRC cohorts. Tumor purity estimates were obtained from RNA and methylation arrays. Results: DNN estimates including area and cell counts were obtained for tumor, desmoplastic stroma, inflamed stroma, mucin/hypocellular stroma, muscle, necrosis and white space. An average of 6.8x105 total cells (range: 1.2x104-2.8x106) and 1.2x105 (range: 7.2x104-1.8x106) were classified for resections and biopsies respectively. Analyses performed twice on the same H&Es obtained matching results (r=1.0). Comparison of the paired first and second H&E showed very high correlations (r~0.9) and total cell counts correlated with DNA and RNA extraction yields (r~0.6). Tumor purity estimates by VPR mildly correlated with DNN (r~0.5) but they were underestimated and very variable. As a result, copy number adjusted by VPR purity tended to be overestimated compared to adjustment with DNN estimates. The improved performance of DNN is reflected in an accurate capture of non-linear association between area and cell counts in invasive cancer. In contrast, tumor purity estimates derived from RNA or DNA methylation arrays showed better correlations compared with DNN (r~0.6) but both overestimated purity in cases with low cell counts by up to a three-fold difference. Conclusions: Tissue composition analysis with DNN allows analytical robustness, automatization and standardization and provides very high reproducibility at single cell resolution. DNN-based estimation of tumor purity is more accurate than VPR or extrapolation from molecular data derived from genome-wide omic platforms which tend to under and overestimate tumor purity respectively. DNN could be used to better plan and asses downstream molecular analyses and to investigate tissue-based metrics as potential clinical biomarkers in clinical trials. Citation Format: Enric Domingo, Aikaterini Chatzipli, Susan Richman, Andrew Blake, Claire Hardy, Celina Whalley, Keara Redmon, Ian Tomlinson, Philip Dunne, Steven Walker, Andrew Beggs, Ultan McDermott, Graeme I. Murray, Leslie M. Samuel, Matthew Seymour, Philip Quirke, Tim Maughan, Viktor H. Koelzer. Assessment of tissue composition with digital pathology in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4446.

How to Process Sputum Samples and Extract Bacterial DNA for Microbiota Analysis.

Different steps and conditions for DNA extraction for microbiota analysis in sputum have been reported in the literature. We aimed at testing both dithiothreitol (DTT) and enzymatic treatments of sputum samples and identifying the most suitable DNA extraction technique for the microbiota analysis of sputum. Sputum treatments with and without DTT were compared in terms of their median levels and the coefficient of variation between replicates of both DNA extraction yield and real-time PCR for the 16S rRNA gene. Treatments with and without lysozyme and lysostaphin were compared in terms of their median levels of real-time PCR for S. aureus. Two enzyme-based and three beads-based techniques for DNA extraction were compared in terms of their DNA extraction yield, real-time PCR for the 16S rRNA gene and microbiota analysis. DTT treatment decreased the coefficient of variation between replicates of both DNA extraction yield and real-time PCR. Lysostaphin (either 0.18 or 0.36 mg/mL) and lysozyme treatments increased S. aureus detection. One enzyme-based kit offered the highest DNA yield and 16S rRNA gene real-time PCR with no significant differences in terms of alpha-diversity indexes. A condition using both DTT and lysostaphin/lysozyme treatments along with an enzymatic kit seems to be preferred for the microbiota analysis of sputum samples.

Assessing effects of different processing procedures on the yield of Treponema pallidum DNA from blood

In this work, we employed real-time PCR analysis targeting tp0574 to investigate the effects of different processing procedures on the yield of T. pallidum DNA from blood to improve assay sensitivity. The T. pallidum DNA yields following red blood cell lysis pretreatment were 40.4 times greater from whole blood and 32.4 times greater from residual hematocytes than yields without pretreatment. For the simulated whole-blood experiments, the T. pallidum DNA yields from the lower layer were 2.8, 4.6, 7.3, 12.6, 15.24, 16.7, 65.1 and 73.1 times those from the upper layer following centrifugation at 500×, 1000×, 2000×, 4000×, 5000×, 7000×, 10,000× and 20,000 × g, respectively. However, the T. pallidum DNA yields from blood clots were only 1.0% at different centrifugal forces. The experiment with infected rabbit blood showed results similar to those mentioned above. In addition, sample processing time (within 48 h) and storage temperature (4 °C and 25 °C) did not affect T. pallidum DNA extraction efficiency. The T. pallidum DNA yield can be significantly improved by red blood cell lysis pretreatment and appropriate centrifugation. Furthermore, the T. pallidum DNA extraction yield is greater from whole blood or residual hematocytes from anti-coagulated blood than from plasma, serum or blood clots.

GDNA extraction yield and methylation status of blood samples are affected by long-term storage conditions.

Epigenetics is believed to provide great chances for a better understanding of the development and treatment of many diseases where the analysis of genomic DNA has so far failed to provide conclusive answers. Methylcytosine is a frequently used quantitative marker of epigenetic studies. Since immediate analysis of sampled material is in most cases not possible, storage time and conditions are critical aspects regarding the quality of genomic DNA and reliability of analysis. Blood is frequently used for such analyses. We, therefore, collected blood samples of ten volunteers and stored them under various conditions for ten months: -70°C, -20°C, 2–8°C and room temperature. An additional aliquot was frozen at -70°C and thawed once a week at room temperature. We then compared the DNA extraction yields and methylation status in relation to storage time and conditions. We found significantly lower DNA extraction yields (up to -97.45%; p ≤ 0.001) as well as significantly higher methylation levels after ten months of storage (up to +42.0%; p ≤ 0.001). These results suggest that storage time has an important influence on DNA analyses of blood samples for all storage conditions. This might be due to differences in stability of methylated and non-methylated DNA. Our study indicates that storage conditions and time may be a critical factor for epigenetic methylation studies and require rigorous validation. For reliable analyses we, therefore, recommend to perform epigenetic analysis directly after sample collection.

Assessment of alternatives to environmental toxic formalin for DNA conservation in biological specimens.

One essential step of museum and clinical specimen preservation is immersion in a fixative fluid to prevent degradation. Formalin is the most largely used fixative, but its benefit is balanced with its toxic and carcinogenic status. Moreover, because formalin-fixation impairs nucleic acids recovery and quality, current museum wet collections and formalin-fixed, paraffin-embedded clinical samples do not represent optimal tanks of molecular information. Our study has been developed to compare formalin to two alternative fixatives (RCL2® and ethanol) in a context of molecular exploitation. Based on a unique protocol, we created mammalian fixed collections, simulated the impact of time on preservation using an artificial ageing treatment and followed the evolution of specimens' DNA quality. DNA extraction yield, purity, visual integrity and qualitative and quantitative ability to amplify the Cox1 gene were assessed. Our results show that both RCL2 and ethanol exhibit better performances than formalin. They do not impair DNA extraction yield, and more importantly, DNA alteration is delayed over the preservation step. The use of RCL2 or ethanol as fixative in biological collections may insure a better exploitation of the genetic resources they propose.

Evaluation of three <em>Brettanomyces</em> qPCR commercial kits: results from an interlaboratory study

<p style="text-align: justify;"><em>Brettanomyces bruxellensis</em> is well adapted to high ethanol concentrations and low pH which allows it to develop in difficult environments, such as wine. <em>B. bruxellensis</em> is mainly found in red wine and is regarded as a spoilage yeast due to its production of ethylphenols and other compounds responsible for organoleptic defects. The detection and quantification of this yeast is essential to preventing wine spoilage. Several specific detection and quantification kits based on real time quantitative PCR are commercially available. Although these kits are frequently used by private enological and research laboratories, no scientific report on the reliability and performance of these kits, including inter-laboratory and inter-assay comparisons have been published. The aim of this work was to compare available kits to quantify <em>B</em>. <em>bruxellensis</em> in red wine to classical method (plate counting on selective medium) in an interlaboratory study. Three different commercial kits were tested on three different wines from Bordeaux, Côtes du Rhône, and Burgundy inoculated with <em>B</em>. <em>bruxellensis </em>at four different concentrations. Five naturally contaminated wines from different French wine regions were also tested. Our results suggest that all the kits tested probably over or underestimate the quantity of <em>B</em>. <em>bruxellensis</em> in red wine and, under specific conditions, give false positives. Quantification may be very heterogeneous depending on the wine, laboratory, or population level. Underestimations or false negative results may have serious consequences for winemakers. Overestimation may be partly due to the quantification of dead cells qPCR.</p><p style="text-align: justify;">This study highlights that quantification of<em> B</em>. <em>bruxellensis</em> in red wine using commercial kits requires a high level of expertise in molecular biology. We recommend that all users use a microbiological internal control to validate DNA extraction yield.</p>

RAPID AND EFFICIENT METHOD FOR ENVIRONMENTAL DNA EXTRACTION AND PURIFICATION FROM SOIL

Large proportion of microbial population in the world is unculturable. Extraction of total DNA from soil is usually a crucial step considering to the difficulties of study the uncultivable microorganisms. Humic acid is considered as the main inhibitory agent in the environmental DNA studies. Here, we introduced a rapid and efficient method for DNA extraction and purification from soil. Yield of DNA extraction by the presented method was 130 ng/µl. Three conventional methods of DNA extraction including liquid nitrogen incursion, bead beating and sonication were performed as control methods. Yield of DNA extraction by these methods were 110, 90 and 50 ng/µl, respectively. A rapid and efficient one step DNA purification method was introduced instead of hazardous conventional phenol-chloroform methods. Humic acid removal percentage by the introduced method was 95.8 % that is comparable with 97 % gained by the conventional gel extraction method and yield of DNA after purification was 84 % and 73 %, respectively. This study could be useful in molecular ecology and metagenomics study as a fast and reliable method.

Blood DNA Yield but Not Integrity or Methylation Is Impacted After Long-Term Storage.

Collection of human whole blood for genomic DNA extraction is part of numerous clinical studies. Since DNA extraction cannot always be performed at the time of sample collection, whole blood samples may be stored for years before being processed. The use of appropriate storage conditions is then critical to obtain DNA in sufficient quantity and of adequate quality in order to obtain reliable results from the subsequent molecular biological analyses. In this study, EDTA whole blood samples were collected from 8 healthy volunteers, and different durations (up to 1 year) and temperatures (room temperature, 4°C, -20°C, and -80°C) of storage were compared. The effect of the addition of a DNA preservative agent was also assessed before and after storage. DNA concentrations measured by UV spectrophotometry and spectrofluorometry were used to calculate DNA extraction yields and double-strand DNA ratios. DNA integrity was controlled by agarose gel electrophoresis and long-range polymerase chain reaction. The impact of storage conditions on DNA methylation was also evaluated. Results showed that certain storage conditions have a significant impact on the DNA extraction yield but little or no effect on DNA integrity and methylation. Storage of EDTA blood at -80°C guarantees high-quality DNA with a good yield. Higher DNA extraction yields were obtained with the addition of a DNA preservative agent before thawing EDTA blood stored at -20°C or -80°C. Long-term storage at room temperature in the presence of a DNA preservative agent also appeared to be a reliable procedure.

An Improved Quantitative Real-Time PCR Assay for the Enumeration of Heterosigma akashiwo (Raphidophyceae) Cysts Using a DNA Debris Removal Method and a Cyst-Based Standard Curve.

The identification and quantification of Heterosigma akashiwo cysts in sediments by light microscopy can be difficult due to the small size and morphology of the cysts, which are often indistinguishable from those of other types of algae. Quantitative real-time PCR (qPCR) based assays represent a potentially efficient method for quantifying the abundance of H. akashiwo cysts, although standard curves must be based on cyst DNA rather than on vegetative cell DNA due to differences in gene copy number and DNA extraction yield between these two cell types. Furthermore, qPCR on sediment samples can be complicated by the presence of extracellular DNA debris. To solve these problems, we constructed a cyst-based standard curve and developed a simple method for removing DNA debris from sediment samples. This cyst-based standard curve was compared with a standard curve based on vegetative cells, as vegetative cells may have twice the gene copy number of cysts. To remove DNA debris from the sediment, we developed a simple method involving dilution with distilled water and heating at 75°C. A total of 18 sediment samples were used to evaluate this method. Cyst abundance determined using the qPCR assay without DNA debris removal yielded results up to 51-fold greater than with direct counting. By contrast, a highly significant correlation was observed between cyst abundance determined by direct counting and the qPCR assay in conjunction with DNA debris removal (r2 = 0.72, slope = 1.07, p < 0.001). Therefore, this improved qPCR method should be a powerful tool for the accurate quantification of H. akashiwo cysts in sediment samples.

A grave in my garden. Genetic identification of Spanish civil war victims buried in two mass graves in Espinosa de los Monteros (Burgos, Spain)

We report the genetic study of 13 victims of the Spanish Civil war buried in two mass graves in Espinosa de los Monteros (Burgos, Spain). The grave with the highest number of individuals (grave 1; N=9) was found in the garden of a private house of the village. The other grave (grave 2) was only 200m away and the remains of four women were unexpectedly found. Skeletal remains, mainly teeth, were better preserved in graves 1 than 2. Mean DNA extraction yield was 0.17ng/μl. In order to carry out the genetic identification of the remains, buccal swabs from 11 family relatives were available. The choice of genetic markers analyzed relied on the different levels of biological kinship to be elucidated. For 75% of the post-mortem samples, ≥12 autosomal STR profiles were obtained. Y chromosome STRs, as well as mitochondrial DNA control region, were also analyzed in order to study paternal and maternal lineages, respectively. Besides, X chromosome STRs were typed in one case to verify a paternal grandmother–granddaughter relationship. We successfully identified 9 of 13 individuals buried in the two mass graves of Espinosa de los Monteros, 6 in grave 1 and 3 in grave 2. The search of further relatives’ samples would be of interest to perform more comparative analyses, and eventually do not leave any of the victims without a name.

Adsorption and desorption of DNA tuned by hydroxyl groups in graphite oxides-based solid extraction material

The extraction of DNA is the most crucial method used in molecular biology. Up to date silica matrices has been widely applied as solid support for selective DNA adsorption and extraction. However, since adsorption force of SiOH functional groups is much greater than that of desorption force, the DNA extraction efficiency of silica surfaces is limited. In order to increase the DNA extraction yield, a new surface with different functional groups which possess of greater desorption property is required. In this study, we proposed cellulose/graphite oxide (GO) composite as an alternative material for DNA adsorption and extraction. GO/Cellulose composite provides the major adsorption and desorption of DNA by COH, which belongs to alkyl or phenol type of OH functional group. Compared to SiOH, COH is less polarized and reactive, therefore the composite might provide a higher desorption of DNA during the elution process. The GO/cellulose composite were prepared in spherical structure by mixing urea, cellulose, NaOH, Graphite oxide and water. The concentration of GO within the composites were controlled to be 0–4.15wt.%. The extraction yield of DNA increased with increasing weight percentage of GO. The highest yield was achieved at 4.15wt.% GO, where the extraction efficiency was reported as 660.4ng/μl when applying 2M GuHCl as the binding buffer. The absorbance ratios between 260nm and 280nm (A260/A280) of the DNA elution was demonstrated as 1.86, indicating the extracted DNA consisted of high purity. The results proved that GO/cellulose composite provides a simple method for selective DNA extraction with high extraction efficiency of pure DNA.

High resolution melting analysis of DNA microsatellites in olive pastes and virgin olive oils obtained by talc addition

Talc (hydrated magnesium silicate) is a physical coadjuvant that can be employed in the production of extra virgin olive oil to increase yield. The adsorbent properties of talc could hamper DNA recovery, leading to false negatives in DNA analysis. The aim of this work was to verify the effect of talc addition on olive oil DNA by targeting four selected microsatellites. Olive processing trials were carried out at two different levels of talc (1 and 2%) and without talc (control). DNA extraction yield and purity level were ascertained, and High Resolution Melting (HRM) analysis of microsatellites was subsequently applied to the extracted DNA. Neither the DNA extraction yield nor the A260/230 and A260/280 ratios showed significant differences between control and talc‐treated samples. Higher values of A260/230 and lower yields were observed in olive oils than in olive pastes. The DNA microsatellites analyzed showed identical HRM profiles in all the samples, excluding any effect of talc and confirming the genetic homogeneity of the olive lot processed (cv. Coratina).Practical applications: High added‐value mono‐cultivar extra virgin olive oils are niche products very appreciated by consumers. DNA analysis is able to reveal mixing with cultivars different from those declared in the label. The use of talc in olive oil industries has progressively increased to improve yield. The results obtained prove that the DNA‐based methods set up until now to check the authenticity of extra virgin olive oil can be effectively carried out also in oils produced by using talc. In addition, the results are among the first applications of HRM to the food sector and evidence the feasibility of this technique along the olive oil chain for checking both olive pastes and oils.Talc (hydrated magnesium silicate) is a physical coadjuvant that can be employed in the production of extra virgin olive oil to increase yield. The adsorbent properties of talc could hamper DNA recovery, leading to false negatives in DNA analysis. By means of High Resolution Melting (HRM) analysis, DNA samples from talc‐treated olive pastes and oils were compared with untreated control based on the shape of temperature‐shifted melting curves. For the same microsatellite, the different samples analyzed showed almost identical HRM profiles. This result excluded any effect of talc addition during olive processing in the analysis of DNA microsatellites of olive oil.

Yeast DNA recovery during the secondary fermentation step of Lombardy sparkling wines produced by Champenoise method

The possibility of recovering amplifiable DNA from sparkling wine produced by Champenoise method was investigated. Indeed, the sensory refining of this product occurs through the yeast autolysis with the release of cellular components, including nucleic acids. An efficient and reliable method to purify DNA is provided coupling two purification treatments: organic solvents and magnetic beads that have never been applied before on wine. The yield of DNA extraction ranged between 10 and 60 %. Starting from 150 mL spiked samples, up to 0.1–0.6 pg of yeast DNA was detected, potentially corresponding to about 4–24 cells of a diploid strain of S. cerevisiae. After development of new primers, the performance of the protocol was validated in real-time PCR by targeting the ITS region of Saccharomyces cerevisiae, one of the main species involved in the secondary fermentation. The monitoring of sparkling wines during the ageing on lees was carried out in four wineries in Franciacorta and Oltrepo Pavese areas up to 2 years from the tirage to wine samples ready for the market as well. Results show that DNA extraction and amplification are properly obtained all along the times, for the species identification; however, after 16 months of ageing, a pre-amplification step needed to be introduced to the procedure for finding a specific signal. Because of the strong DNA degradation, the typing of the inoculated strain or the grape cultivar was not achieved in commercialized wine samples.

Abstract 304: Feasibility study of next-generation sequencing on residual formalin-fixed paraffin-embedded tissues

Abstract Next Generation Sequencing (NGS) technologies are being used for detection of somatic mutations in tumors and studies of germline variation. However, most NGS studies used DNA isolated either from whole blood or fresh frozen tissue specimens. Meanwhile, the tissue specimens available from most National Cancer Institute (NCI) funded cohorts and the Surveillance, Epidemiology and End Results (SEER) registries (http://seer.cancer.gov/biospecimen/) are primarily formalin fixed paraffin embedded (FFPE). There are limited data, on a small number of FFPE tissue samples, which suggest NGS is feasible. Much less is known about the feasibility of these technologies for large scale studies or using older FFPE specimens (e.g. 5-30 years old). The main objective of this project was to conduct a pilot study to determine whether the DNA obtained from archival FFPE tissue is of sufficient quality and quantity to conduct NGS. Sixty high-grade serous ovarian adenocarcinomas from FFPE tissues which were between 7 and 31 years old were obtained from three SEER registries. DNA was extracted, quantified, quality assessed, and subjected to whole exome sequencing. DNA extraction (yields and quality) and whole exome sequencing (depths of coverage and exome coverage obtained) results from this study will be presented. Ultimately, data derived from this analysis could serve as the basis for determining the utility of archival FFPE biospecimens for characterization and discovery projects utilizing NGS technologies instead of relying on frozen biospecimens. Citation Format: Danielle Mercatante Carrick, Sean Altekruse, Corrine Camalier, Wendy Cozen, Brenda Hernandez, Charles Lynch, Paul McGregor, Michele G. Mehaffey, Lisa McShane, JoyAnn Phillips Rohan, Mickey Williams, Elizabeth M. Gillanders, Leah E. Mechanic, Sheri Schully. Feasibility study of next-generation sequencing on residual formalin-fixed paraffin-embedded tissues. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 304. doi:10.1158/1538-7445.AM2014-304

Rapid extraction and preservation of genomic DNA from human samples

Simple and rapid extraction of human genomic DNA remains a bottleneck for genome analysis and disease diagnosis. Current methods using microfilters require cumbersome, multiple handling steps in part because salt conditions must be controlled for attraction and elution of DNA in porous silica. We report a novel extraction method of human genomic DNA from buccal swab and saliva samples. DNA is attracted onto a gold-coated microchip by an electric field and capillary action while the captured DNA is eluted by thermal heating at 70 °C. A prototype device was designed to handle four microchips, and a compatible protocol was developed. The extracted DNA using microchips was characterized by qPCR for different sample volumes, using different lengths of PCR amplicon, and nuclear and mitochondrial genes. In comparison with a commercial kit, an equivalent yield of DNA extraction was achieved with fewer steps. Room-temperature preservation for 1 month was demonstrated for captured DNA, facilitating straightforward collection, delivery, and handling of genomic DNA in an environment-friendly protocol.

Poplar stems show opposite epigenetic patterns during winter dormancy and vegetative growth

In this study the identification of epigenetic marks of closed and open chromatin states has been performed by immunofluorescence in vibratome-thick sections from poplar stems, followed by quantification of the signal in a representative population of individual nuclei on a confocal microscope. Unlike other methods often used for detection of global DNA methylation levels, the procedure hereby proposed can be applied to a wide range of specimens regardless of the purity and yield of genomic DNA extraction and in a tissue-dependent manner. Using antibodies to 5-methylcytidine and acetylated lysine 8 of histone H4, as reliable indicators of transcription incompatible and compatible chromatin states, respectively, the influence of epigenetic regulation on differential gene expression during the growth and arrest periods in xylem and phloem tissues was assessed. The fluorescence signal of 5-methylcytosine was significantly higher in winter as compared with summer. Conversely, the fluorescence signal for acetylated Lys 8 of histone H4 was significantly higher in summer than in winter in both tissues examined. Collectively, these results put forward an epigenetic control of winter dormancy in poplar stems.

The influence of substrate on DNA transfer and extraction efficiency

The circumstances surrounding deposition of DNA profiles are increasingly becoming an issue in court proceedings, especially whether or not the deposit was made by primary transfer. In order to improve the currently problematic evaluation of transfer scenarios in court proceedings, we examined the influence a variety of nine substrate types (six varieties of fabric, plywood, tarpaulin, and plastic sheets) has on DNA transfer involving blood. DNA transfer percentages were significantly higher (p=0.03) when the primary substrate was of non-porous material (such as tarpaulin, plastic or, to a lesser degree, wood) and the secondary substrate porous (such as fabrics). These findings on transfer percentages confirm the results of previous studies. Fabric composition was also shown to have a significant (p=0.03) effect on DNA transfer; when experiments were performed with friction from a variety of fabrics to a specific weave of cotton, transfer percentages ranged from 4% (flannelette) to 94% (acetate). The propensity for the same nine substrates to impact upon the efficiency of DNA extraction procedures was also examined. Significant (p=0.03) differences were found among the extraction efficiencies from different materials. When 15μL of blood was deposited on each of the substrates, the lowest quantity of DNA was extracted from plastic (20ng) and the highest quantities extracted from calico and flannelette (650ng). Significant (p<0.05) differences also exist among the DNA extraction yield from different initial blood volumes from all substrates. Also, significantly greater (p<0.05) loss of DNA was seen during concentration of extracts with higher compared to lower initial quantities of DNA. These findings suggest that the efficiency of extraction and concentration impacts upon the final amount of DNA available for analysis and that consideration of these effects should not be ignored. The application of correction factors to adjust for any variation among extraction and concentration efficiencies among substrates is proposed.

An evaluation of commercial DNA extraction kits for the isolation of bacterial spore DNA from soil

To evaluate six commercial DNA extraction kits for their ability to isolate PCR-quality DNA from Bacillus spores in various soil samples. Three soils were inoculated with various amounts of Bacillus cereus spores to simulate an outbreak or intentional release of the threat agent Bacillus anthracis. DNA was isolated from soil samples using six commercial DNA extraction kits. Extraction and purification efficiencies were assessed using a duplex real-time PCR assay that included an internal positive control. The FastDNA(®) SPIN kit for Soil showed the highest DNA extraction yield, while the E.Z.N.A.(®) Soil DNA and PowerSoil(®) DNA Isolation kits showed the highest efficiencies in removing PCR inhibitors from loam soil extracts. The results of this study suggest that commercially available extraction kits can be used to extract PCR-quality DNA from bacterial spores in soil. The selection of an appropriate extraction kit should depend on the characteristics of the soil sample and the intended downstream application. The results of this study aid in the selection of an appropriate DNA extraction kit for a given soil sample. Its application could expedite sample processing for real-time PCR detection of a pathogen in soil.

PCR-DGGE Comparison of Bacterial Community Structure in Fresh and Archived Soils Sampled along a Chihuahuan Desert Elevational Gradient

The polymerase chain reaction coupled with denaturing gradient gel electrophoresis (PCR-DGGE) has been used widely to determine species richness and structure of microbial communities in a variety of environments. Researchers commonly archive soil samples after routine chemical or microbial analyses, and applying PCR-DGGE technology to these historical samples offers evaluation of long-term patterns in bacterial species richness and community structure that was not available with previous technology. However, use of PCR-DGGE to analyze microbial communities of archived soils has been largely unexplored. To evaluate the stability of DGGE patterns in archived soils in comparison with fresh soils, fresh and archived soils from five sites along an elevational gradient in the Chihuahuan Desert were compared using PCR-DGGE of 16S rDNA. DNA from all archived samples was extracted reliably, but DNA in archived soils collected from a closed-canopy oak forest site could not be amplified. DNA extraction yields were lower for most archived soils, but minimal changes in bacterial species richness and structure due to archiving were noted in bacterial community profiles from four sites. Use of archived soils to determine long-term changes in bacterial community structure via PCR-DGGE appears to be a viable option for addressing microbial community dynamics for particular ecosystems or landscapes.

Rapid detection and quantification of Propionibacteriaceae

Background:Propionibacteriaceae (Propioni) are anaerobic bacteria associated with human and animal infections. Present-day methods of diagnosis for Propioni are unsatisfactory due to a lack of sensitivity of culture, time required for...