Nucleic Acid Yield Research Articles

Validation of Internal Controls for Extraction and Amplification of Nucleic Acids from Enteric Viruses in Water Samples

Inhibitors that reduce viral nucleic acid extraction efficiency and interfere with cDNA synthesis and/or polymerase activity affect the molecular detection of viruses in aquatic environments. To overcome these significant problems, we developed a methodology for assessing nucleic acid yields and DNA amplification efficiencies for environmental water samples. This involved adding particles of adenovirus type 5 and murine norovirus and newly developed primer-sharing controls, which are amplified with the same primer pairs and result in the same amplicon sizes as the targets, to these samples. We found that nucleic acid loss during the extraction process, rather than reverse transcription-PCR (RT-PCR) inhibition, more significantly attributed to underestimation of the presence of viral genomes in the environmental water samples tested in this study. Our success rate for satisfactorily amplifying viral RNAs and DNAs by RT-PCR was higher than that for obtaining adequate nucleic acid preparations. We found that inhibitory properties were greatest when we used larger sample volumes. A magnetic silica bead-based RNA extraction method effectively removed inhibitors that interfere with viral nucleic acid extraction and RT-PCR. To our knowledge, this is the first study to assess the inhibitory properties of environmental water samples by using both control virus particles and primer-sharing controls.

Comparison of methods in the recovery of nucleic acids from archival formalin-fixed paraffin-embedded autopsy tissues

Archival formalin-fixed paraffin-embedded (FFPE) human tissue collections are typically in poor states of storage across the developing world. With advances in biomolecular techniques, these extraordinary and virtually untapped resources have become an essential part of retrospective epidemiological studies. To successfully use such tissues in genomic studies, scientists require high nucleic acid yields and purity. In spite of the increasing number of FFPE tissue kits available, few studies have analyzed their applicability in recovering high-quality nucleic acids from archived human autopsy samples. Here we provide a study involving 10 major extraction methods used to isolate total nucleic acid from FFPE tissues ranging in age from 3 to 13 years. Although all 10 methods recovered quantifiable amounts of DNA, only 6 recovered quantifiable RNA, varying considerably and generally yielding lower DNA concentrations. Overall, we show quantitatively that TrimGen’s WaxFree method and our in-house phenol–chloroform extraction method recovered the highest yields of amplifiable DNA, with considerable polymerase chain reaction (PCR) inhibition, whereas Ambion’s RecoverAll method recovered the most amplifiable RNA.

Extracting nucleic acids from activated sludge which reflect community population diversity

Critical to most studies in molecular microbial ecology is the application of DNA/RNA extraction methods which can reveal the true level of population biodiversity present in samples from the community under investigation. Activated sludge communities have been studied extensively using molecular methods, but rarely have the nucleic acid isolation methods applied been assessed for their ability to achieve this. This study compares eight published RNA and DNA extraction protocols and one commercially available DNA isolation kit for their capacity to provide high quality nucleic acids that reflect the community composition. Each method was assessed on the basis of nucleic acid yield, purity and integrity, and the ability to provide PCR amplifiable RNA and DNA from known marker populations that varied in their resistance to nucleic acid extraction. Only three consistently provided DNA from each of the marker populations known to be present in the samples from fluorescence in situ hybridisation analysis. The failure of the other methods emphasises the need to validate all DNA/RNA extraction protocols. It is recommended that several validated extraction methods be used and the extracts pooled to further minimise any risk of bias.

Improving molecular detection of Candida DNA in whole blood: comparison of seven fungal DNA extraction protocols using real-time PCR

The limitations of classical diagnostic methods for invasive Candida infections have led to the development of molecular techniques such as real-time PCR to improve diagnosis. However, the detection of low titres of Candida DNA in blood from patients with candidaemia requires the use of extraction methods that efficiently lyse yeast cells and recover small amounts of DNA suitable for amplification. In this study, a Candida-specific real-time PCR assay was used to detect Candida albicans DNA in inoculated whole blood specimens extracted using seven different extraction protocols. The yield and quality of total nucleic acids were estimated using UV absorbance, and specific recovery of C. albicans genomic DNA was estimated quantitatively in comparison with a reference (Qiagen kit/lyticase) method currently in use in our laboratory. The extraction protocols were also compared with respect to sensitivity, cost and time required for completion. The TaqMan PCR assay used to amplify the DNA extracts achieved high levels of specificity, sensitivity and reproducibility. Of the seven extraction protocols evaluated, only the MasterPure yeast DNA extraction reagent kit gave significantly higher total nucleic acid yields than the reference method, although nucleic acid purity was highest using either the reference or YeaStar genomic DNA kit methods. More importantly, the YeaStar method enabled C. albicans DNA to be detected with highest sensitivity over the entire range of copy numbers evaluated, and appears to be an optimal method for extracting Candida DNA from whole blood.

Use of UV methods for measurement of protein and nucleic acid concentrations.

Use of UV methods for measurement of protein and nucleic acid concentrations.

Further analysis of nucleic acids in purified scrapie prion preparations by improved return refocusing gel electrophoresis.

Although increasingly unlikely, the possibility of a scrapie-specific nucleic acid carried by infectious prion particles is still unresolved. Return refocusing gel electrophoresis was developed to detect homogeneous and heterogeneous nucleic acids extracted from highly purified scrapie prion preparations. This method was improved with respect to the size range from 13 to 1100 nucleotides (nt) over which analyses could be performed. The yield of nucleic acid, particularly of small DNA oligonucleotides and polyadenylated RNA, was determined after deproteinization and two-phase extraction. Despite extensive nuclease digestions some small polynucleotides remained. Although a scrapie-specific nucleic acid cannot be excluded, the results further define the possible characteristics of a hypothetical molecule. If homogeneous in size, such a molecule would be less than 80 nt in length at a particle-to-infectivity ratio near unity, if heterogeneous, scrapie-specific nucleic acids would have to include molecules smaller than 240 nt.

Isolation and Analysis of Genomic DNA from Single Seeds

Our objective was to adapt a plant DNA minipreparation procedure for the rapid isolation of total nucleic acid from single legume seeds. Single‐seed extractions from cultivars of soybean (Glycine max L. Merr.) and frenchbean (Phaseolus vulgaris L.) were characterized for total nucleic acid yield, DNA content, and restriction fragment length polymorphisms (RFLPs). This procedure can employed in the analysis of a large number of individual seeds by genomic blotting and molecular hybridization. while probing seed lots with the same complementary DNA (cDNA) fragment, we found that ostensibly pure soybean seed lines contained significant levels of identical RFLPs while frenchbean seed lines from plants with dramatic morphological differences had no detectable RFLPs for the same sequence. When isolated genomic DNAs were used to detect restriction fragment length polymorphisms (RFLPs), it was possible to make limited distinctions among cultivars of both soybean and frenchbean seed lots. This technique met the criteria of simplicity, rapidity, and low cost, and yielded DNA fragments of sufficient purity and length for RFLP analysis.

An improved version of the diethyl pyrocarbonate method for extracting ribosomal nucleic acids

An improved version of the diethyl pyrocarbonate method is described which ensures high nucleic acid yields without preferential loss of cytoplasmic heavy ribosomal RNA.

Fractionation of nucleic acids isolated by the phenol- and DEP-methods from leaf tissues at different stages of development

The phenol method and a new method based on the use of diethylpyrocarbonate (DEP) as a nuclease inhibitor were compared with respect to the yield of total nucleic acid extractable from barley and tobacco plants in different stages of development. The percentage distribution of different nucleic acid species in the extracts prepared by the two methods was also studied by sucrose density gradient centrifugation and MAK column chromatography. The nucleic acid yield in the extracts from first leaves of 11-day-old barley seedlings was higher with the DEP method than with the phenol method. With barley the same percentage distribution of the different nucleic acid species was obtained with both methods. By contrast, with tobacco (except for very young tissues) a preferential loss of hrRNA was observed when using the DEP method. The ageing of barley leaf tissues was characterized by a virtual increase in DNA apparently due to a preferential breakdown of rRNA. This pattern, characteristic of ageing tissues, was more pronounced with attached leaves than with detached, “artificially aged” tissues.

Further Observations on the Increased Yield of Nucleic Acid from Irradiated Yeast

DURING the course of investigations intended to extend previously reported observations1 of increased yields of nucleic-acid-like substances from yeast irradiated with full ultra-violet radiation under conditions resulting in prolonged injury to the yeast cells, certain irregularities in the yields were noted. It now appears that these may be attributed to lack of quantitative yields in the final step of the chemical procedure wherein nucleic acid is precipitated.

Increased Yield of Nucleic Acid–like Substances from Irradiated Yeast

EVIDENCE that yeast irradiated with full ultraviolet light produces nucleic acid–like proliferation–promoting factors1 led us to compare the yield of nucleic acid from irradiated and non–irradiated yeast in the following experiments.