Total DNA Yield Research Articles

Simple and Reliable DNA Extraction Method for the Dark Pigmented Fungus, Cercospora sojina

This study used a modified cetyltrimethylammonium bromide (CTAB) method to efficiently extract DNA from the plant pathogenic fungus Cercospora sojina. Total DNA yield obtained by this method was approximately 1 mg/g of mycelia (fresh weight), and the mean ratio of A260/A280 and A260/A230 were 2.04 and 2.1, respectively. The results of random amplified polymorphic DNA (RAPD) analysis, digestion with restriction enzymes, and Southern hybridization indicated that polysaccharides were effectively removed by this method, and the resulting DNA was sufficient for use in subsequent molecular analysis.

Clinical trial participant characteristics and saliva and DNA metrics

BackgroundClinical trial and epidemiological studies need high quality biospecimens from a representative sample of participants to investigate genetic influences on treatment response and disease. Obtaining blood biospecimens presents logistical and financial challenges. As a result, saliva biospecimen collection is becoming more frequent because of the ease of collection and lower cost. This article describes an assessment of saliva biospecimen samples collected through the mail, trial participant demographic and behavioral characteristics, and their association with saliva and DNA quantity and quality.MethodsSaliva biospecimens were collected using the Oragene® DNA Self-Collection Kits from participants in a National Cancer Institute funded smoking cessation trial. Saliva biospecimens from 565 individuals were visually inspected for clarity prior to and after DNA extraction. DNA samples were then quantified by UV absorbance, PicoGreen®, and qPCR. Genotyping was performed on 11 SNPs using TaqMan® SNP assays and two VNTR assays. Univariate, correlation, and analysis of variance analyses were conducted to observe the relationship between saliva sample and participant characteristics.ResultsThe biospecimen kit return rate was 58.5% among those invited to participate (n = 967) and 47.1% among all possible COMPASS participants (n = 1202). Significant gender differences were observed with males providing larger saliva volume (4.7 vs. 4.5 ml, p = 0.019), samples that were more likely to be judged as cloudy (39.5% vs. 24.9%, p < 0.001), and samples with greater DNA yield as measured by UV (190.0 vs. 138.5, p = 0.002), but reduced % human DNA content (73.2 vs. 77.6 p = 0.005) than females. Other participant characteristics (age, self-identified ethnicity, baseline cigarettes per day) were associated with saliva clarity. Saliva volume and saliva and DNA clarity were positively correlated with total DNA yield by all three quantification measurements (all r > 0.21, P < 0.001), but negatively correlated with % human DNA content (saliva volume r = -0.148 and all P < 0.010). Genotyping completion rate was not influenced by saliva or DNA clarity.ConclusionFindings from this study show that demographic and behavioral characteristics of smoking cessation trial participants have significant associations with saliva and DNA metrics, but not with the performance of TaqMan® SNP or VNTR genotyping assays.Trial registrationCOMPASS; registered as NCT00301145 at clinicaltrials.gov.

Determinants of DNA yield and purity collected with buccal cell samples

Buccal cells are an important source of DNA in epidemiological studies, but little is known about factors that influence amount and purity of DNA. We assessed these factors in a self-administered buccal cell collection procedure, obtained with three cotton swabs. In 2,451 patients DNA yield and in 1,033 patients DNA purity was assessed. Total DNA yield ranged from 0.08 to 1078.0 μg (median 54.3 μg; mean 82.2 μg ± SD 92.6). The median UV 260:280 ratio, was 1.95. Samples from men yielded significantly more DNA (median 58.7 μg) than those from women (median 44.2 μg). Diuretic drug users had significantly lower purity (median 1.92) compared to other antihypertensive drug users (1.95). One technician obtained significantly lower DNA yields. Older age was associated with lower DNA purity. In conclusion, DNA yield from buccal swabs was higher in men and DNA purity was associated with age and the use of diuretics.

Participation among breast cancer patients in a prospective cancer registry and quality of specimens collected for a tissue repository: the Expanded Breast Cancer Registry and Tissue Repository (EBCR) experience.

Abstract Abstract #3104 BACKGROUND: We describe herein our experience with the EBCR, an IRB approved protocol, which prospectively collects clinically annotated tissues from a cohort of New Mexican breast cancer patients. This resource provides high quality specimens and detailed epidemiologic/clinical data for correlative studies. Participants understand that the resource will be used for future undetermined research; thus they altruistically participate in hopes of helping others. All specimens are processed immediately after collection, increasing their availability for molecular- and immunological assays. Follow-up clinical data are documented with associated specimens allowing for inter- and intra-individual comparison.&amp;#x2028; MATERIALS AND METHODS: Newly diagnosed breast cancer patients are approached for participation at our Breast Clinic. Eligiblity criteria are: non-metastatic breast cancer, within 12 months of definitive surgery, age &amp;gt; 18 years. After obtaining consent, a breast cancer risk assessment is administered and buccal cells are collected. Blood for serum, plasma and DNA is collected at enrollment then 4-6 months for year 1 and annually thereafter. Tumor and normal breast tissue are collected at time of surgery, with a fine needle aspiration (FNA) performed and the cells stored in RNAlater. Quarterly, EBCR staff update each participant's clinical history.&amp;#x2028; RESULTS: Since opening in February 2006, 224 patients have been enrolled, representing 86% participation among eligible patients approached. The average age is 52.7 years; 77% reside in the Albuquerque metro area. Self-reported racial/ethnic distribution among participants are: 51% non-Hispanic white, 35% Hispanic, 5% Native American, 3% African-American and 5% other. The distribution for those who declined participation (n=34) was: 35% non-Hispanic white, 38% Hispanic, 12% Native American, 9% African-American and 6% Asian. Typically, patients declining participation were those who appeared to be overwhelmed with their diagnosis or with treatment plans discussed at the same visit.&amp;#x2028; We have collected blood and buccal cells from 88% and 98% of participants at enrollment, respectively. Serum and plasma was extracted from blood and frozen within an hour of draw. On average, the total DNA yield from blood and mouthwash was 138.7 ug/ml and 92.8 ug/ml, respectively. Starting at arterial clamp time, FNA's of breast tissue were collected within 5 minutes, and fresh breast tissue was flash frozen within 1 hour to decrease degradation of key metabolites and RNA.&amp;#x2028; CONCLUSION: Participation among breast cancer patients in this prospective breast specific registry and tissue repository is robust, particularly among racial/ethnic minorities. From our experience, participation rates can be improved if the protocol is presented after patients had time to process their diagnosis and are not distracted by recently presented information. Specimens collected in the manner described provide premium specimens to conduct innovative and responsible research. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3104.

Comparison between DNA obtained from buccal cells of the upper and lower gutter area

This study compared quantitatively and qualitatively the DNA extracted from buccal cells collected from the upper or lower gutter areas. Buccal cells were collected from the upper (n=15) and lower gutter (n=15) region from 15 volunteers using a special cytobrush (Gentra), totaling 2 collections from each individual. DNA was extracted from the samples according to the manufacturer's instructions. The DNA obtained was qualitatively and quantitatively evaluated by 2 calibrated blind examiners using spectrophotometry and analysis of DNA bands (0.8% agarose gel electrophoresis). Data was statistically analyzed by one-way ANOVA (alpha=0.05). Means and standard derivation (SD) for total DNA yield from the upper and lower gutter area were 12.2 microg (12.0) and 9.4 microg (8.5), respectively (p=0.821). There was higher (p<0.05) DNA purity for the upper gutter (1.79; 0.05) when compared to lower gutter area (1.66; 0.10). Regarding to the DNA quality, no differences were observed between the 2 location sites, but all samples showed similar degree of degradation. In conclusion, it would be recommendable that buccal cells for DNA extraction be collected from the upper gutter area in the attempt to increase DNA purity.

Long-term impact of acid resin waste deposits on soil quality of forest areas II. Biological indicators

In this study, we evaluated the effects of two acid resin deposits on the soil microbiota of forest areas by means of biomass, microbial activity-related estimations and simple biological ratios. The determinations carried out included: total DNA yield, basal respiration, intracellular enzyme activities (dehydrogenase and catalase) and extracellular enzyme activities involved in the cycles of C (β-glucosidase and chitinase), N (protease) and P (acid-phosphatase). The calculated ratios were: total DNA/total N; basal respiration/total DNA; dehydrogenase/total DNA and catalase/total DNA. Total DNA yield was used to estimate soil microbial biomass. Results showed that microbial biomass and activity were severely inhibited in the deposits, whilst resin effects on contaminated zones were variable and site-dependant. Correlation analysis showed no clear effect of contaminants on biomass and activities outside the deposits, but a strong interdependence with natural organic matter related parameters such as total N. In contrast, by using simple ratios we could detect more stressful conditions in terms of organic matter turnover and basal metabolism in contaminated areas compared to their uncontaminated counterparts. These results stress that developed ecosystems such as forests can buffer the effects of pollutants and preserve high functionality via natural attenuation mechanisms, but also that acid resins can be toxic to biological targets negatively affecting soil dynamics. Acid resin deposits can therefore act as contaminant sources adversely altering soil processes and reducing the environmental quality of affected areas despite the solid nature of these wastes.

Evaluation of the use of freezer mill to improve DNA retrieval from dried cotton swabs

This investigation evaluated the use of a freezer mill to improve retrieval of DNA from dried cotton swabs (using 100 μl saliva) compared to uncrushed swabs and whole saliva by measuring DNA yield and profile average peak height. Three treatments were tested; short, medium (as for a bone sample) and extended. The samples subjected to the freezer mill had the powder that remained on the freezer mill components collected (using a water and agitation method). All freezer mill samples returned a lower average DNA yield than either uncrushed swabs or whole saliva. The powder from the crushed swabs comprised an average of 35% of the total DNA yield, whereas the powder from the components comprised 65%. Allele drop-out was observed in samples exposed to extended treatments. Both short and medium treatments provided significantly higher peak heights than uncrushed cotton swabs with equal quantities of DNA ( P < 0.05). Using a freezer mill on dried cotton swabs does not increase the DNA yield. This investigation suggests collecting powder from the freezer mill components will increase DNA yield, especially from trace samples.

Comparason of extraction methods for PCR detection of Burkholderia cepacia complex (BCC) from cystic fibrosis patients

AbstractDirect detection of Burkholderia cepacia complex (BCC) and its genomovars from sputum by molecular tests emerges as a method for rapid identification. In this study, four DNA extraction methods were evaluated for the identification for BCC from sputum of CF patients. Sputa from 28 CF patients were aliquoted and spiked with BCC reference strain. Boiling, phenol-chloroform, CTAB methods and a commercial spin column kit was used for DNA extraction. Total DNA yields were determined by spectrophotometry and single-round recA PCR was used for detection of BCC. No significant difference was observed in DNA yields from different extraction methods. Lower limit of detection for recA PCR was determined as 106 cfu/ml. Amplification was observed in 7/16 (43.7%) of sputa for boiling, 8/16 (50%) of sputa for CTAB and 13/16 (81.2%) of sputa for phenol-chloroform method and spin column kit in the assay sensitivity range determined in the study. Phenol-chloroform and commercial spin column kit were found to be better suited for DNA purification from sputum of CF patients for BCC identification. Diagnostic impact of single-round recA PCR directly from sputum was limited to chronically-infected patients.

Isolation of Microarray-Grade Total RNA, MicroRNA, and DNA from a Single PAXgene Blood RNA Tube

We have developed a procedure for isolation of microRNA and genomic DNA in addition to total RNA from whole blood stabilized in PAXgene Blood RNA tubes. The procedure is based on automatic extraction on a BioRobot MDx and includes isolation of DNA from a fraction of the stabilized blood and recovery of small RNA species that are otherwise lost. The procedure presented here is suitable for large-scale experiments and is amenable to further automation. Procured total RNA and DNA was tested using Affymetrix Expression and single-nucleotide polymorphism GeneChips, respectively, and isolated microRNA was tested using spotted locked nucleic acid-based microarrays. We conclude that the yield and quality of total RNA, microRNA, and DNA from a single PAXgene blood RNA tube is sufficient for downstream microarray analysis.

A simple and cost-effective protocol for DNA isolation from buccal epithelial cells

Buccal cells provide a convenient source of DNA for epidemiological studies. The goal of this study was to develop a convenient method to obtain buccal cells from mouthwash samples to be used as a source of DNA, and to evaluate the stability of the DNA in mouthwash solution over time. The procedures used in the method described in this paper avoid the use of any organic solvents. This is achieved by salting out the cellular proteins by dehydration and precipitation with a saturated ammonium acetate solution. The protocol described here is fast, simple to perform, sensitive, economical and several samples can be processed at the same time. The analyses provide consistent evidence that DNA extracted by this methodology is sufficient for several PCR amplifications. The total DNA yield ranged from 5 to 93 microg (median 15 microg, mean 20.71 microg). DNA can be extracted and PCR amplified after storage of mouthwash solution at room temperature for periods of up to 30 days.

Improvement in fetal DNA extraction from maternal plasma. Evaluation of the NucliSens Magnetic Extraction system and the QIAamp DSP Virus Kit in comparison with the QIAamp DNA Blood Mini Kit

Prenatal diagnostic assays have been developed using free fetal DNA circulating in the maternal blood of pregnant women. Efficient DNA extraction is crucial for a robust analysis. To improve fetal DNA yield, we tested two manual extraction methods--the NucliSens Magnetic Extraction (NMAG) system and the QIAamp DSP Virus Kit (QDSP)--against our current standard method, the widely used QIAamp DNA Blood Mini Kit (QDNA). The fetal DNA yield of the two extraction systems was evaluated using the RHD exon 7 as target in DNA extracts of 75 plasma samples from pregnant RhD-negative women, known to have given birth to RhD-positive infanto. The total DNA yield was evaluated in 23 samples, targeting GAPDH. The fetal DNA yield was improved by a mean factor of 1.7 using the NMAG system, and improved by a mean factor of 1.5 using the QDSP. The total DNA yield was improved by a mean factor of 2.3 using the NMAG system, and by a mean factor of 1.3 using the QDSP. Both extraction systems tested were superior to our standard with regard to DNA yield. This improvement may have a great impact on the success of genotyping in early pregnancy.

ISOLATION OF LISTERIA MONOCYTOGENES DNA FROM MEAT PRODUCTS FOR QUANTITATIVE DETECTION BY REAL‐TIME PCR

ABSTRACT We report a study of two different procedures for extraction of Listeria monocytogenes genomic DNA from cooked pork ham products, to be coupled to a specific real‐time (RTi‐) PCR assay, an open formula protocol based on the Chelex‐100 resin and the Wizard DNA kit. The reliability of each DNA extraction was evaluated by performing three independent experiments using eight replicates. The quality and quantity of the L. monocytogenes DNA were tested using both a fluorescence‐based DNA measuring protocol and a specific quantitative RTi‐PCR assay. The total DNA yield was similar for both extraction protocols. However, the Wizard‐based protocol produced a better quality of L. monocytogenes DNA, as it enhanced the RTi‐PCR results (approximately one order of magnitude greater than the Chelex‐based protocol). Thus, the protocol based on the Wizard DNA kit was more suitable for L. monocytogenes DNA extraction from meat samples when sensitive and accurate results are needed.

Quality and Quantity of Saliva DNA Obtained from the Self-administrated Oragene Method—A Pilot Study on the Cohort of Swedish Men

Self-collection of saliva has the potential to provide molecular epidemiologic studies with DNA in a user-friendly way. We evaluated the new Oragene saliva collection method and requested saliva samples by mail from 611 men (ages 53-87 years). We obtained a response rate of, on average, 80% [varying from 89% (ages 67-71 years) to 71% (ages 77-87 years)]. DNA was extracted from 90 randomly selected samples, and its usefulness was evaluated with respect to quality, quantity, and whole-genome amplification (WGA). Visual inspection of DNA on agarose gels showed high molecular weight DNA (>23 kb) and no degradation. Total DNA yield measured with PicoGreen ranged from 1.2 to 169.7 mug, with a mean of 40.3 mug (SD, 36.5 mug) and a median of 29.4 mug. Human DNA yield was estimated by real-time PCR of the human prothrombin gene to account for 68% (SD, 20%) of total DNA. We did WGA on 81 saliva DNA samples by using the GenomiPhi DNA kit and genotyped both saliva DNA and WGA DNA for 10 single-nucleotide polymorphisms randomly selected from the human genome. Overall genotyping success rate was 96% for saliva DNA and 95% for WGA DNA; 79% of saliva DNA samples and 79% of WGA DNA samples were successfully genotyped for all 10 single-nucleotide polymorphisms. For the 10 specific assays, the success rates ranged between 88% and 100%. Almost complete genotypic concordance (99.7%) was observed between saliva DNA and WGA DNA. In conclusion, Oragene saliva DNA in this study collected from men is of high quality and can be used as an alternative to blood DNA in molecular epidemiologic studies.

Optimizing buccal cell DNA yields in mothers and infants for human leukocyte antigen genotyping.

Buccal cells provide a convenient source of DNA for epidemiologic studies. Mouthwash rinses yield a higher quality and quantity of DNA than cytobrushes but are not practical for collection from infants. Although cytobrushes yield sufficient DNA for most genotyping analyses, human leukocyte antigen (HLA) analysis can require 1,000-fold more DNA. In Iowa City, Iowa, in 2002, the authors tested two cytobrush collection methods to optimize total DNA yield and purity for HLA genotyping in mothers and infants: 1) brushing the left and right inner cheeks (standard method) and 2) brushing the upper and lower "gutters", that is, the space between the gums and the inner lips/cheeks along the front and sides of the mouth (test method). Storage and mailing experiments were performed to define conditions for optimizing DNA yield and purity. Mothers' gutter samples yielded significantly higher total amounts of DNA (mean yield = 15.0 micro g/two brushes) than cheek samples (mean yield = 7.6 micro g/two brushes) (paired t test: p < 0.001), while DNA yields from cheek and gutter collections from infants were equivalent. Cytobrushes stored and/or mailed in paper envelopes yielded significantly more and higher-purity DNA than brushes in plastic bags or tubes. Cytobrush sampling of the mouth's gutter areas can enhance DNA yield in mothers but not in young infants. DNA yields can be further optimized by controlling mailing and storage conditions.

Influence of Growth in a Food Medium on the Detection of Escherichia coli O157:H7 by Polymerase Chain Reaction

The effects of storage time and growth in broth culture and in a food medium on the efficiency of Escherichia coli O157: H7 DNA extraction and on the sensitivity of polymerase chain reaction (PCR) detection of E. coli O157:H7 were investigated. Detection limits were evaluated with dilution series PCR targeting the slt-II gene. The relationship between cell density and DNA yield was generally log-linear for pure cultures of E. coli O157:H7. When the bacteria were suspended in skim milk at a density of 106 CFU/ml, held at 4°C, and sampled at 24-h intervals, cell density, total DNA yield, and PCR detection limits remained stable throughout the 96-h storage period. However, when E. coli O157:H7 was grown in skim milk to a final cell density of 106 CFU/ml, PCR amplification efficiency was drastically reduced, although overall DNA yields from these samples were consistent with those for the samples in which E. coli O157:H7 growth was static over 96 h of storage at 4°C. This result is most likely due to poor DNA purity, which was consistently observed when DNA was extracted from food matrices in which the pathogen was grown rather than stored. The results of this investigation underscore the likelihood that multiple components may drastically affect DNA extraction and PCR amplification efficiency in the detection of pathogens in the food matrix. It is clear that before nucleic acid amplification technologies are widely applied to food systems, it would be prudent to test their efficacy in multiple food matrices and under conditions in which the bacterial population is both static and actively growing.

Comparison of seven DNA extraction and amplification protocols in historical herbarium specimens of juncaceae

Seven DNA extraction protocols were used to obtain DNA from herbarium specimens ofJuncus andLuzula (Juncaceae) of various ages. DNA of historical samples is difficult to extract, and the extracts are seldom of good quality. The quality of DNA obtained was estimated by using a spectrophotometer to measure the A260/280 absorbance ratio. The total DNA yield was measured by a fluorometer. The results indicate the success of using both mixer mill grinding and a DNeasy Plant Kit. Another extraction protocol (grinding with mortar and pestle, using liquid nitrogen) yielded DNA from many samples. Modified CTAB extraction, with a lengthy precipitation, usually provided good amounts of DNA. Other protocols did not give satisfactory results.

PCR detection of the human papillomavirus: improved DNA recovery from frozen biopsies.

1. One of the main obstacles to the reliable detection of human papillomavirus in cervical intraepithelial neoplasia is that a minute quantity of infected tissue is often all that is available. 2. In this work, proteinase K-phenol-chloroform-treated sections of frozen cervical biopsies were split in two. Half of the material was precipitated by ethanol in the presence of glycogen, and in the remaining half glycogen was absent. 3. On average a 15-fold increase in total DNA yield was obtained with glycogen. As a result, in 63 cases analysed by PCR for human papillomavirus type 16, we were able to detect nearly 20% more positive samples when glycogen was used. 4. The implications of the improved accuracy of diagnosis are that women requiring a closer follow-up can be identified, and conversely rescreening intervals can be extended for those testing negative for oncogenic human papillomavirus types with more confidence than in the past.

Identification of consumed stone flounder, Kareius bicoloratus (Basilewsky), from the stomach contents of sand shrimp, Crangon affinis (De Haan) using mitochondrial DNA analysis

We have developed a method for the identification of consumed stone flounder, Kareius bicoloratus (Basilewsky) larvae and juveniles from the stomach contents of sand shrimp, Crangon affinis (De Haan). We succeeded in isolating stone flounder DNA from the stomach contents of sand shrimp, using a cell lysis buffer containing 8 M urea. DNA for PCR analysis was obtained from stomach contents even when it was isolated 5 hours after the termination of predation. However, the total DNA yield decreased as the time after predation progressed. Stone flounder was distinguished from other prey in the stomach contents of sand shrimp using PCR with universal primers for fish and restriction analysis of mtDNA. Successful amplification of stone flounder mtDNA was possible up until 4 h after the termination of predation, but no amplification was possible 5 h after the termination of predation. In addition, a part of the D-loop region of stone flounder mtDNA was cloned, sequenced, and used to design species-specific PCR primers that allow amplification for stone flounder. PCR products were not obtained from the total DNA of other possible prey of the sand shrimp: 11 fish species; 2 mysid species; one amphipod species; and 2 polychaete species. In this paper, we discuss the advantages and disadvantages of using the PCR method coupled with restriction analysis, and the species-specific PCR method to identify consumed larval and juvenile fish species in the guts of predators.

DNA extraction conditions fromPorphyra perforata using LiCl

A rapid and economical method of DNA extraction from a red seaweedPorphyra perforata J. Agardh has been developed by the use of lithium chloride. This paper describes the optimization of extraction conditions. Heat treatment of tissues in a solution (0.8 M LiCl, 0.6% Sarkosyl, 10 mM EDTA, 0.2% PVPP, 5% s-mercaptoethanol, pH 9.0) at 55 °C for 10 min extracts DNA that is of sufficient quality to be used as a template for PCR amplification. Total DNA yield was approximately 30 to 50μg g−1 t of partially dried tissue. Total RNA yield was approximately 400μg g−1 of partially dried tissue. Carbohydrate was contained as approximately 40 to 90 mM (expressed as glucose equivalents) from 1 g tissue, and protein contamination calculated as the O.D. 260/280 ratio was in the range of 1.4 to 1.7. The DNA was characterized by high molecular weight larger than 50 kb.

Isolation and analysis of the total genomic DNA from the date palm (P. dactylifera L.) and related species

AbstractThe objective of this study was to adapt a plant DNA preparation procedure for the isolation of biologically active DNA and DNA with a high molecular weight from the date palm and other related palms. Mature leaf tissue extractions of the date palm, Phoenix dactylifera L., the coconut tree, Cocos nucifera, and the Mexican Fan Palm, Washingtonia robusta, were characterized for total genomic DNA yield, purity, integrity, as well as restriction digestion and ligation capabilities. It is demonstrated here that the DNA isolation procedure, modified for use with various palm leaf tissues, met the criteria for simplicity and low costs, and yielded DNA of high molecular weight (∼50 Kbp) and of sufficient purity suitable for molecular studies.