Deoxyribonucleic Acid Yield Research Articles

A comparison of the efficiency of five different commercial DNA extraction kits for extraction of DNA from faecal samples

Differences in the composition of the gut microbiota have been associated with a range of diseases using culture-independent methods. Reliable extraction of nucleic acid is a key step in identifying the composition of the faecal microbiota. Five widely used commercial deoxyribonucleic acid (DNA) extraction kits (QIAsymphony® Virus/Bacteria Midi Kit (kit QS), ZR Fecal DNA MiniPrep™ (kit Z), QIAamp® DNA Stool Mini Kit (kit QA), Ultraclean® Fecal DNA Isolation Kit (kit U) and PowerSoil® DNA Isolation Kit (kit P)) were evaluated, using human faecal samples. Yield, purity and integrity of total genomic DNA were compared spectrophotometrically and using gel electrophoresis. Three bacteria, commonly found in human faeces were quantified using real time polymerase chain reaction (qPCR) and total bacterial diversity was studied using denaturing gradient gel electrophoresis (DGGE) as well as terminal restriction fragment length polymorphism (T-RFLP). The measurements of DNA yield and purity exhibited variations between the five kits tested in this study. Automated kit QS exhibited the best quality and highest quantity of DNA. All kits were shown to be reproducible with CV values≤0.46 for DNA extraction. qPCR results showed that all kits were uniformly efficient for extracting DNA from the selected target bacteria. DGGE and T-RFLP produced the highest diversity scores for DNA extracted using kit Z (H′=2.30 and 1.27) and kit QS (H′=2.16 and 0.94), which also extracted the highest DNA yields compared to the other kits assessed.

Influence of various environmental conditions on DNA isolation from dental pulp for sex determination using polymerase chain reaction

Objective: To determine the reliablility of sex determination by polymerase chain reaction Materials and Methods: Extracted teeth were subjected to the following treatment: Varying temperature (100, 200, 300, and 400°C), immersing in sea water (20-36 days), and burying at 30-cm depth. After subjecting to specific environmental stress condition, DNA (Deoxy Ribonucleic acid) was extracted from the tooth pulp. Primers Y-chromosome specifi c alphoid centromeric repeat sequences DYZ3, X-chromosome specifi c alphoid centromeric repeat sequence DXZ1 were used for sex determination by PCR. Results: Out of 15 samples subjected to various environmental conditions, DNA was isolated in 13 samples and sex determination was carried out. There was no DNA yield when teeth were subjected to high temperatures (300, 400°C). The DNA analysis after the respective PCR showed accurate sex determination of the 13 samples. Conclusion: our study showed that teeth are reliable source for sex determination even when subjected to different environmental conditions except during extreme high temperatures. Hence, sex determination by PCR analysis is the most reliable method in markedly decayed or preadolescent bodies.

Genomic Deoxyribonucleic Acid Extraction from Post Mortem Fetal Tissue

A major challenge in post-mortem (PM) genetic testing in fetuses is the difficulty in obtaining high quality deoxyribonucleic acid (DNA) due to maceration. We examined the yield and quality of DNA from various PM fetal tissues.Methods: Fresh tissue samples from the muscle, kidney, liver, heart and placenta from 14 consecutive fetuses were collected at the time of autopsy and stored at -20 °C. We extracted DNA using Promega Wizard Genomic DNA Purification Kit and assessed the DNA yield and quality using the Thermo Scientific Nano-drop and by agarose gel electrophoresis. Polymerase chain reaction (PCR) for genomic testing using 'in house' control primers was performed (Ethics approval: 10/H0713/26).Results: The median (inter quartile range) gestation of the fetuses was 22 (17, 31) weeks and the PM interval was 5.5 (3.7, 7.7) days. Muscle, kidney and placenta gave the highest average yields for non-degraded genomic DNA. Liver DNA had lowest quality;this was due to tissue degradation PM interval or gestational age did correlate with DNA yield or quality. Figure [DNA yield from various fetal tissues] Figure [DNA quality (L=liver, K=kidney, H=Heart, M=muscle)] Conclusion: High quality genomic DNA can be extracted from fetal kidney, muscle and placenta at autopsy, despite advanced maceration.

Giardia intestinalis: DNA extraction approaches to improve PCR results

Difficulty in disrupting cysts of Giardia intestinalis, a cosmopolitan protozoan parasite, decreases the yield of DNA extracted and reduces the effectiveness of the polymerase chain reaction (PCR). To improve the detection of the Giardia Glutamate Dehydrogenase (gdh) gene, we re-evaluated the effects of deoxyribonucleic acid (DNA) extraction methods. Purified and concentrated cysts from 33 fecal samples were disrupted using conventional methods, and DNA extraction was conducted using two protocols: the QIAamp Stool Mini Kit and phenol/chloroform/isoamyl alcohol (PCI). PCR amplification was successful for 12 extracted DNA samples (36%) using PCI following a glass bead and freeze/thaw pretreatment and for all 33 samples (100%) using the QIAamp Stool Mini Kit following the aforementioned pretreatment. Consequently, the pretreatment of cysts with glass beads and freeze/thaw cycles followed by extraction of DNA with the QIAamp Stool Mini kit was the more effective protocol.

DNA charge transport over 34 nm

Molecular wires show promise in nanoscale electronics, but the synthesis of uniform, long conductive molecules is a significant challenge. Deoxyribonucleic acid (DNA) of precise length, by contrast, is synthesized easily, but its conductivity over the distances required for nanoscale devices has not been explored. Here we demonstrate DNA charge transport (CT) over 34 nm in 100-mer monolayers on gold. Multiplexed gold electrodes modified with 100-mer DNA yield sizable electrochemical signals from a distal, covalent Nile Blue redox probe. Significant signal attenuation upon incorporation of a single base-pair mismatch demonstrates that CT is DNA-mediated. Efficient cleavage of these 100-mers by a restriction enzyme indicates that the DNA adopts a native conformation accessible to protein binding. Similar electron-transfer rates measured through 100-mer and 17-mer monolayers are consistent with rate-limiting electron tunnelling through the saturated carbon linker. This DNA-mediated CT distance of 34 nm surpasses that of most reports of molecular wires.

Use of quantitative polymerase chain reaction analysis to compare quantity and stability of isolated murine DNA

Deoxyribonucleic acid (DNA) can be extracted from different tissue sources for genotyping of mice. Methods of collecting tissue vary with respect to their perceived invasiveness, and in some cases, tissue is collected multiple times in order to verify a genotype. The authors' goal was to refine and optimize tissue collection methods with quantitative measures for subsequent genotyping. To do this, the authors used real-time quantitative polymerase chain reaction (qPCR) analysis to quantify DNA extracted from fecal pellets, hair, buccal swab samples, ear punch samples and tail biopsy samples and then compared the quantities of DNA obtained by using either previously published protocols or commercial kits to extract DNA. They found that 2-mm tail biopsy samples yielded significantly more DNA than did the other sources and that commercial extraction kits generally yielded more DNA than did published protocols. The authors also assessed the stability of DNA extracted from the various tissue sources by repeating the qPCR analysis after the samples had been frozen and stored for 44 months. Although the quantities of DNA in the stored samples had decreased, all the samples could still be used for PCR-based genotyping. The authors' work supports the collection of a single minimal biopsy sample of 2 mm of tail or ear tissue, above other sources, for highest yield of DNA for genotyping. With appropriate storage, DNA remains usable for PCR-based genotyping for years without a need for repeated animal sampling.

Maximizing deoxyribonucleic acid yield from dried blood spots.

One source of deoxyribonucleic acid (DNA) for genetic studies is the utilization of dried blood spots stored on paper cards (Guthrie cards) collected shortly after birth. These cards represent an important source of material for epidemiologic and population-based genetic studies. Extraction of DNA from these cards can lead to variable amounts of recovered DNA. We report here results of our efforts to maximize yield from this valuable, but nonrenewable, resource. Commercial methods of DNA extraction from blood cards were used, and protocol modifications were introduced that enhanced DNA yield. Use of a commercial solvent prior to DNA extraction steps gave greater yields than extraction without the solvent. Modification of the elution step by use of prewarmed extraction buffer and a soaking step at an elevated temperature increased yield by 6- to 10-fold. The modified DNA extraction method yielded as much as 660 ng of DNA from a single 5-mm-diameter punch of a blood spot card. The DNA performed well in downstream, polymerase chain reaction-based applications.

Comparing Three DNA Extraction Procedures for Cost, Efficiency, and DNA Yield

An efficient deoxyribonucleic acid (DNA) extraction procedure that yields large quantities of DNA would provide adequate DNA for a large number of different analytical procedures. This study was conducted to compare three DNA extraction procedures for cost, time efficiency, and DNA content while extracting DNA from Kentucky bluegrass (Poa pratensis L.). Three students at the Univ. of Illinois with varying levels of DNA extraction experience conducted DNA extractions using Plant DNeasy™ Mini Kits, Plant DNAzol® Reagent, and a PEX/CTAB buffer. Costs varied significantly with cost (US$) per DNA sample of $3.04 for the DNeasy™ method, $0.99 for the DNAzol® method, and $0.39 for the PEX/CTAB extraction. The DNAzol® method was the fastest; although extracting 2.8 ng less DNA than the DNeasy™ method, it did not require the use of hazardous organic solvents, and random amplified polymorphic DNA (RAPD) markers were satisfactory for DNA fingerprinting of Kentucky bluegrass cultivars. The PEX/CTAB method, which did not include a tissue homogenization step, did not have reproducible banding patterns due to miniscule and inconsistent quantities of DNA extracted, or possibly due to inadequate purification. The investigator with the least DNA extraction experience was the slowest, while extracting 75% more DNA. All three methods are easily adapted to laboratories having personnel with different levels of experience. The DNAzol® Reagent method should save time and money, with reproducible results when many individual plant samples need to be identified. Chemical names used: potassium ethyl xanthogenate (PEX); cetyltrimethyl ammonium bromide (CTAB)

Genotypic analysis of flow-sorted and microdissected head and neck squamous lesions by whole-genome amplification.

To investigate the utility of primer extension preamplification (PEP) in the genetic analysis of head and neck squamous tumorigenesis, microsatellite analysis was performed on matched deoxyribonucleic acid (DNA) samples extracted from 32 flow-sorted and microdissected specimens before and after PEP. Eighteen fresh and nine archival specimens were taken from invasive carcinomas, and five specimens were obtained from microdissected archival premalignant squamous epithelial lesions. Identical microsatellite patterns were observed in 276 (87%) of the 319 paired PEP and non-PEP genotypes with sufficient DNA. Overall, 13 (4%) of the PEP and 28 (8.8%) of the non-PEP fresh tissue samples failed specific microsatellite amplification. All 14 PEP-archival specimens were successfully amplified. Sorted cells showed a higher incidence (42.8%) of loss of heterozygosity (LOH) in both PEP and non-PEP samples compared with their unsorted counterparts. The results of this study indicate that (a) PEP is a simple and reliable technique for enhancing the DNA yield from small specimens; (b) flow sorting, in certain cases, improves the interpretation of genetic results; and (c) PEP may be used to compensate for PCR failure of unamplified DNA specimens in these lesions.

Hypoxia-induced brain angiogenesis in the adult rat.

1. Prolonged hypoxia increases the brain vascularity. Here we studied the protein and deoxyribonucleic acid (DNA) content of isolated cerebral microvessels in hypoxic and control rats. 2. Adult male Wistar rats that were subjected to hypobaric hypoxia at 50.5 kPa (0.5 atm) for periods of 1, 2, or 3 weeks and normoxic littermate controls were used. Cerebral microvessels were harvested by bulk isolation from the pooled cortical mantles of three to six rats in each group. The isolated microvessels were assayed for their protein and DNA content. 3. Microvessel protein yield increased from 0.31 to 0.45 mg of microvessel protein (g wet wt)-1 of cerebral cortex after 1 week of hypoxia, but did not increase further during up to 3 weeks of hypoxia. In contrast, microvessel DNA yield did not increase during the first week of hypoxia, but increased significantly after 2 weeks of hypoxia and continued to increase up to 56 micrograms of microvessel DNA (g cerebral cortex)-1 at 3 weeks of hypoxia (normoxic mean was 32 micrograms DNA g-1). 4. The cell size index (mg protein:mg DNA) of isolated cerebral microvessels increased after 1 week of hypoxia, suggesting microvascular hypertrophy, but returned to control by the second week of hypoxia and decreased to below control levels by the third week of hypoxia, suggesting microvascular hyperplasia. These results indicate that the increased vascularity of the brain in hypobaric hypoxia progresses from an early phase of microvascular hypertrophy to later microvascular hyperplasia.

Extraction, Evaluation, and Amplification of DNA from Decalcified and Undecalcified United States Civil War Bone

Deoxyribonucleic acid (DNA) was extracted from documented skeletal specimens of U.S. Civil War soldiers to determine the need for decalcification prior to extraction. The polymerase chain reaction (PCR) was performed to determine if the calcification state had an effect on the ability to amplify the extracts and to determine how successful amplification would be with these aged specimens. Bone samples were pulverized to a fine powder and divided into two sets. One set of samples was decalcified and the other set left undecalcified. Both sets were extracted using an organic procedure. The results demonstrate that decalcification is not a necessary step in the extraction process and that the yield of DNA is generally two times greater when decalcification is omitted. Furthermore, the calcification state had no effect on the ability to perform the PCR. Although the extracted DNA was very degraded, a 410 base pair (bp) segment of the mitochondrial DNA (mtDNA) control region was amplified. These results suggest that DNA can be extracted and amplified from 125 year old bone without decalcification, which may assist in the identity of modern and historic forensic specimens.

Evaluation of Four Deoxyribonucleic Acid (DNA) Extraction Protocols for DNA Yield and Variation in Restriction Fragment Length Polymorphism (RFLP) Sizes Under Varying Gel Conditions

This study originated from discussions and recommendations of the Technical Working Group on DNA Analysis Methods (TWGDAM). Four bloodstain deoxyribonucleic acid (DNA) extraction protocols and five semen stain DNA extraction protocols were evaluated. Nine laboratories participated in the extraction of DNA from 20 bloodstains and 20 semen stains using each protocol. All blood and semen stains originated from a single donor and were prepared under uniform conditions to permit the direct comparison of DNA yields and restriction fragment lengths. The extracted DNA from approximately 600 bloodstains and 700 semen stains was quantified by yield gel analysis and a slot blot hybridization technique. The extracted DNA was digested and restriction fragment length polymorphism (RFLP) patterns were generated using three single-locus probes. The RFLP sizing data produced from the blood and semen stains were evaluated with respect to (1) DNA extraction method, (2) gel length, (3) agarose type, (4) presence or absence of ethidium bromide in the gel, and (5) fragment sizes obtained from DNA isolated directly from the donor's liquid blood. This study demonstrates conclusively that high-molecular-weight DNA can be isolated using either organic or nonorganic DNA extraction protocols and that the resulting RFLP sizes are highly reproducible regardless of gel length, agarose type, or presence/absence of ethidium bromide.

11] Construction and use of pBR322 plasmids that yield single-stranded DNA for sequencing

This chapter discusses the construction and use of pBR322 plasmids that yield single-stranded deoxyribonucleic acid (DNA) for sequencing. The chapter describes a set of chimeric cloning vectors with regard to the yield of single-stranded DNA produced. The chapter also describes a procedure for the construction of chimeric plasmids in detail. The filamentous male-specific coliphages, Ff (fl, M13, fd), only infect Escherichia coli cells that contain an F pilus and are one of the smallest known single-stranded DNA bacteriophages. DNA replication of the phage involves three principal steps. In the first step, synthesis of the complementary strand converts the single-stranded viral DNA into a double-stranded replicative form (RF). Viral or (+)-strand DNA replication in the second step is initiated by the introduction of a specific nick on the (+) strand of the RF molecule by viral gene II protein. In the third step, the concentration of viral gene V protein, a single-stranded DNA binding protein, reaches a level high enough to coat the newly replicated single-stranded (+) DNA. The yield of single-stranded plasmid DNA varies with the type of helper phage used in the infection. Some of the mutant helper phages have mutations within gene II that alter either the level or activity of gene II protein.

Recombinant DNA studies on stored necropsy brain samples from patients with Huntington's chorea.

An analysis of deoxyribonucleic acid (DNA) in deep frozen brain samples taken from 100 patients with Huntington's chorea after death showed undegraded DNA in 44 cases. Of these, 16 were analysed with G8, a recombinant DNA probe, linked to the Huntington's chorea locus. In all cases unambiguous Southern blots were obtainable. No correlation between the yield of DNA and the principal storage factors was observed. The use of stored brain tissue obtained after death from patients with Huntington's chorea should be considered when analysis of DNA is needed for predictive studies, but DNA should preferably be isolated from the tissue before storage as degradation in these samples can occur.

Increase of the yield of DNA from barley nuclei in ethylene glycol by gamma-irradiation.

After gamma-irradiating the nuclei isolated from dry seed-embryo of barley in the presence of varying concentrations of ethylene glycol, macromolecular DNA (deoxyribonucleic acid) was extracted from them. The yields of DNA was determined by the 2 methods. The both method of determination on DNA showed a similar tendency; the higher the exposed dose and the concentration of ethylene glycol, the better the yields.

Possible Relationship Between Yeast Deoxyribonucleases and Deoxyribonucleic Acid Yield

The deoxyribonucleic acid (DNA) yield and deoxyribonuclease (DNase) activity of several yeasts were correlated. Debaryomyces castellii and Debaryomyces franciscae were found to contain active DNases which carry out DNA hydrolysis, whereas the amounts of DNA as determined by extraction with Sarkosyl buffer (pH 7.8) were found to be small. On the other hand, Candida parapsilosis, Saccharomyces carmosousae, and Lodderomyces elongisporus produced no detectable DNases active at pH 7.8, and their DNA yield was correspondingly high. L. elongisporus was found to possess DNase only at pH 4.0.

Pus, deoxyribonucleic acid, and sputum viscosity.

On 100 sputum specimens selected from patients suffering from chronic bronchitis, bronchiectasis, asthma, and cystic fibrosis total deoxyribonucleic acid (DNA) content has been related to macroscopic type, to total dry weight yield, and to the apparent viscosity of the secretion at 1350 s-1: since DNA may be present, either as fibres or within cells, in one-third of the specimens the contribution of each form to the apparent viscosity was assessed. The effect on sputum viscosity of the addition of DNA in vitro has also been studied. Whereas between mucoid, mucopurulent, and purulent macroscopic types a significant difference in total DNA and dry weight yield has been found, viscosity was not significantly correlated with purulence. Similarly, the concentration of either cells or fibres correlated significantly with total DNA but not with viscosity. The in vitro addition of DNA to sputum caused a significant increase in its viscosity, and reasons for the differences between the iv vivo and in vitro effect are discussed. Certain constituents of purulent sputum tend to increase viscosity and others to reduce it, and the influence of these varies in the several diseases studied.

Effect of Gestation and Dry Period on Deoxyribonucleic Acid and Alveolar Characteristics of Bovine Mammary Glands

Udders of 21 lactating and 26 dry cows were analyzed for deoxyribonucleic acid (DNA) concentration and amount. Right and left halves of 21 cows were dried off at different times to give comparisons between dry periods of 15, 45, 60, and 75 days. Udders of five dry cows with dry periods of 8 and 48 days for right and left halves, respectively, were obtained about a week before calving. Histological sections of 26 udders were used to record alveolar density and surface area. The correlation coefiicient between total DNA and milk yield per half udder (r = 0.34) was small but statistically significant (P<.05). Concentration of DNA and alveoli increased in dry udders compared to prepartum and laxtating udders. Involution of udders of nonpregnant or early gestation cows was evidenced by reductions in fresh weight, dry weight, alveolar surface area, and DNA. In prepartum half udders, six weeks’ difference in dry period did not significantly affect dry weight, DNA, or alveolar concentration. Lack of interference with either involution or alveolar development in the udder due to short dry periods indicates that subsequent subnormal lactation may be due to subnormal function rather than structure.

The isolation and composition of the nucleic acids of Aerobacter aerogenes.

SUMMARY: The nucleic acids were isolated from Aerobacter aerogenes by extraction of mechanically disintegrated organisms with 0·14 m-sodium chloride at pH 7·5. Upon subsequent isolation, about 80 % of the deoxyribonucleic acid (DNA) originally contained in the organisms was obtained, but only about 50% of the ribonucleic acid (RNA). The loss of RNA was mainly due to enzymic degradation. By extraction of A. aerogenes with 1 % sodium cholate at 60°, enzymic degradation was virtually eliminated and a good yield of both DNA and RNA obtained. They were relatively undegraded and both showed evidence of the presence of hydrogen bonds. The amounts of the purine and pyrimidine bases in both types of nucleic acid were determined. The DNA and RNA from the normal streptomycin-sensitive strain of A. aerogenes differed significantly in base composition from the corresponding nucleic acids from a strain which had been trained to grow in 5 % (w/v) streptomycin.