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Sample records for dna extraction step

  1. An improved protocol and a new grinding device for extraction of genomic DNA from microorganisms by a two-step extraction procedure.

    PubMed

    Zhang, S S; Chen, D; Lu, Q

    2012-01-01

    Current protocols to extract genomic DNA from microorganisms are still laborious, tedious and costly, especially for the species with thick cell walls. In order to improve the effectiveness of extracting DNA from microbial samples, a novel protocol, defined as two-step extraction method, along with an improved tissue-grinding device, was developed. The protocol included two steps, disruption of microbial cells or spores by grinding the sample together with silica sand in a new device and extraction of DNA with an effective buffer containing cell lysis chemicals. The device was prepared by using a commercial electric mini-grinder, adapted with a grinding stone, and a sample cup processed by lathing from a polytetrafluoroethylene rod. We tested the method with vegetative cells of four microbial species and two microbial spores that have thick cell walls and are therefore hard to process; these included Escherichia coli JM109, Bacillus subtilis WB600, Sacchromyces cerevisiae INVSc1, Trichoderma viride AS3.3711, and the spores of S. cerevisiae and T. viride, respectively, representing Gram-positive bacteria, Gram-negative bacteria, yeast, filamentous fungi. We found that this new method and device extracted usable quantities of genomic DNA from the samples. The DNA fragments that were extracted exceeded 23 kb. The target sequences up to about 5 kb were successfully and exclusively amplified by PCR using extracted DNA as the template. In addition, the DNA extraction was finalized within 1.5 h. Thus, we conclude that this two-step extraction method is an effective and improved protocol for extraction of genomic DNA from microbial samples. PMID:22653603

  2. DNA Extraction: Organic and Solid-Phase.

    PubMed

    Altayari, Wafa

    2016-01-01

    DNA extraction remains a critical step in DNA profiling of biological material recovered from scenes of crime. In the forensic community several methods have gained popularity, including Chelex(®), organic extraction, and solid-phase extraction. While some laboratories streamlined their processes and only use one method we have retained several methods and continue to use these for different sample types. In this chapter we present three methods that have been used for several years in our laboratory. PMID:27259731

  3. Extraction of Chromosomal DNA from Schizosaccharomyces pombe.

    PubMed

    Murray, Johanne M; Watson, Adam T; Carr, Antony M

    2016-01-01

    Extraction of DNA from Schizosaccharomyces pombe cells is required for various uses, including templating polymerase chain reactions (PCRs), Southern blotting, library construction, and high-throughput sequencing. To purify high-quality DNA, the cell wall is removed by digestion with Zymolyase or Lyticase and the resulting spheroplasts lysed using sodium dodecyl sulfate (SDS). Cell debris, SDS, and SDS-protein complexes are subsequently precipitated by the addition of potassium acetate and removed by centrifugation. Finally, DNA is precipitated using isopropanol. At this stage, purity is usually sufficient for PCR. However, for more sensitive procedures, such as restriction enzyme digestion, additional purification steps, including proteinase K digestion and phenol-chloroform extraction, are recommended. All of these steps are described in detail here. PMID:27140918

  4. DNA INTERSTRAND CROSSLINK REPAIR IN MAMMALIAN CELLS: STEP BY STEP

    PubMed Central

    Muniandy, Parameswary; Liu, Jia; Majumdar, Alokes; Liu, Su-ting; Seidman, Michael M.

    2009-01-01

    Interstrand DNA crosslinks (ICLs) are formed by natural products of metabolism and by chemotherapeutic reagents. Work in E. coli identified a two cycle repair scheme involving incisions on one strand on either side of the ICL (unhooking) producing a gapped intermediate with the incised oligonucleotide attached to the intact strand. The gap is filled by recombinational repair or lesion bypass synthesis. The remaining monoadduct is then removed by Nucleotide Excision Repair (NER). Despite considerable effort, our understanding of each step in mammalian cells is still quite limited. In part this reflects the variety of crosslinking compounds, each with distinct structural features, used by different investigators. Also, multiple repair pathways are involved, variably operative during the cell cycle. G1 phase repair requires functions from NER, although the mechanism of recognition has not been determined. Repair can be initiated by encounters with the transcriptional apparatus, or a replication fork. In the case of the latter, the reconstruction of a replication fork, stalled or broken by collision with an ICL, adds to the complexity of the repair process. The enzymology of unhooking, the identity of the lesion bypass polymerases required to fill the first repair gap, and the functions involved in the second repair cycle are all subjects of active inquiry. Here we will review current understanding of each step in ICL repair in mammalian cells. PMID:20039786

  5. Automated DNA extraction from pollen in honey.

    PubMed

    Guertler, Patrick; Eicheldinger, Adelina; Muschler, Paul; Goerlich, Ottmar; Busch, Ulrich

    2014-04-15

    In recent years, honey has become subject of DNA analysis due to potential risks evoked by microorganisms, allergens or genetically modified organisms. However, so far, only a few DNA extraction procedures are available, mostly time-consuming and laborious. Therefore, we developed an automated DNA extraction method from pollen in honey based on a CTAB buffer-based DNA extraction using the Maxwell 16 instrument and the Maxwell 16 FFS Nucleic Acid Extraction System, Custom-Kit. We altered several components and extraction parameters and compared the optimised method with a manual CTAB buffer-based DNA isolation method. The automated DNA extraction was faster and resulted in higher DNA yield and sufficient DNA purity. Real-time PCR results obtained after automated DNA extraction are comparable to results after manual DNA extraction. No PCR inhibition was observed. The applicability of this method was further successfully confirmed by analysis of different routine honey samples. PMID:24295710

  6. DNA Extraction Techniques for Use in Education

    ERIC Educational Resources Information Center

    Hearn, R. P.; Arblaster, K. E.

    2010-01-01

    DNA extraction provides a hands-on introduction to DNA and enables students to gain real life experience and practical knowledge of DNA. Students gain a sense of ownership and are more enthusiastic when they use their own DNA. A cost effective, simple protocol for DNA extraction and visualization was devised. Buccal mucosal epithelia provide a…

  7. A two-step electrodialysis method for DNA purification from polluted metallic environmental samples.

    PubMed

    Rodríguez-Mejía, José Luis; Martínez-Anaya, Claudia; Folch-Mallol, Jorge Luis; Dantán-González, Edgar

    2008-08-01

    Extracting DNA from samples of polluted environments using standard methods often results in low yields of poor-quality material unsuited to subsequent manipulation and analysis by molecular biological techniques. Here, we report a novel two-step electrodialysis-based method for the extraction of DNA from environmental samples. This technique permits the rapid and efficient isolation of high-quality DNA based on its acidic nature, and without the requirement for phenol-chloroform-isoamyl alcohol cleanup and ethanol precipitation steps. Subsequent PCR, endonuclease restriction, and cloning reactions were successfully performed utilizing DNA obtained by electrodialysis, whereas some or all of these techniques failed using DNA extracted with two alternative methods. We also show that his technique is applicable to purify DNA from a range of polluted and nonpolluted samples. PMID:18601228

  8. Optimisation of DNA extraction from the crustacean Daphnia

    PubMed Central

    Athanasio, Camila Gonçalves; Chipman, James K.; Viant, Mark R.

    2016-01-01

    Daphnia are key model organisms for mechanistic studies of phenotypic plasticity, adaptation and microevolution, which have led to an increasing demand for genomics resources. A key step in any genomics analysis, such as high-throughput sequencing, is the availability of sufficient and high quality DNA. Although commercial kits exist to extract genomic DNA from several species, preparation of high quality DNA from Daphnia spp. and other chitinous species can be challenging. Here, we optimise methods for tissue homogenisation, DNA extraction and quantification customised for different downstream analyses (e.g., LC-MS/MS, Hiseq, mate pair sequencing or Nanopore). We demonstrate that if Daphnia magna are homogenised as whole animals (including the carapace), absorbance-based DNA quantification methods significantly over-estimate the amount of DNA, resulting in using insufficient starting material for experiments, such as preparation of sequencing libraries. This is attributed to the high refractive index of chitin in Daphnia’s carapace at 260 nm. Therefore, unless the carapace is removed by overnight proteinase digestion, the extracted DNA should be quantified with fluorescence-based methods. However, overnight proteinase digestion will result in partial fragmentation of DNA therefore the prepared DNA is not suitable for downstream methods that require high molecular weight DNA, such as PacBio, mate pair sequencing and Nanopore. In conclusion, we found that the MasterPure DNA purification kit, coupled with grinding of frozen tissue, is the best method for extraction of high molecular weight DNA as long as the extracted DNA is quantified with fluorescence-based methods. This method generated high yield and high molecular weight DNA (3.10 ± 0.63 ng/µg dry mass, fragments >60 kb), free of organic contaminants (phenol, chloroform) and is suitable for large number of downstream analyses. PMID:27190714

  9. Optimisation of DNA extraction from the crustacean Daphnia.

    PubMed

    Athanasio, Camila Gonçalves; Chipman, James K; Viant, Mark R; Mirbahai, Leda

    2016-01-01

    Daphnia are key model organisms for mechanistic studies of phenotypic plasticity, adaptation and microevolution, which have led to an increasing demand for genomics resources. A key step in any genomics analysis, such as high-throughput sequencing, is the availability of sufficient and high quality DNA. Although commercial kits exist to extract genomic DNA from several species, preparation of high quality DNA from Daphnia spp. and other chitinous species can be challenging. Here, we optimise methods for tissue homogenisation, DNA extraction and quantification customised for different downstream analyses (e.g., LC-MS/MS, Hiseq, mate pair sequencing or Nanopore). We demonstrate that if Daphnia magna are homogenised as whole animals (including the carapace), absorbance-based DNA quantification methods significantly over-estimate the amount of DNA, resulting in using insufficient starting material for experiments, such as preparation of sequencing libraries. This is attributed to the high refractive index of chitin in Daphnia's carapace at 260 nm. Therefore, unless the carapace is removed by overnight proteinase digestion, the extracted DNA should be quantified with fluorescence-based methods. However, overnight proteinase digestion will result in partial fragmentation of DNA therefore the prepared DNA is not suitable for downstream methods that require high molecular weight DNA, such as PacBio, mate pair sequencing and Nanopore. In conclusion, we found that the MasterPure DNA purification kit, coupled with grinding of frozen tissue, is the best method for extraction of high molecular weight DNA as long as the extracted DNA is quantified with fluorescence-based methods. This method generated high yield and high molecular weight DNA (3.10 ± 0.63 ng/µg dry mass, fragments >60 kb), free of organic contaminants (phenol, chloroform) and is suitable for large number of downstream analyses. PMID:27190714

  10. A Modified SDS-Based DNA Extraction Method for High Quality Environmental DNA from Seafloor Environments

    PubMed Central

    Natarajan, Vengadesh Perumal; Zhang, Xinxu; Morono, Yuki; Inagaki, Fumio; Wang, Fengping

    2016-01-01

    Recovering high quality genomic DNA from environmental samples is a crucial primary step to understand the genetic, metabolic, and evolutionary characteristics of microbial communities through molecular ecological approaches. However, it is often challenging because of the difficulty of effective cell lysis without fragmenting the genomic DNA. This work aims to improve the previous SDS-based DNA extraction methods for high-biomass seafloor samples, such as pelagic sediments and metal sulfide chimney, to obtain high quality and high molecular weight of the genomic DNA applicable for the subsequent molecular ecological analyses. In this regard, we standardized a modified SDS-based DNA extraction method (M-SDS), and its performance was then compared to those extracted by a recently developed hot-alkaline DNA extraction method (HA) and a commercial DNA extraction kit. Consequently, the M-SDS method resulted in higher DNA yield and cell lysis efficiency, lower DNA shearing, and higher diversity scores than other two methods, providing a comprehensive DNA assemblage of the microbial community on the seafloor depositional environment. PMID:27446026

  11. A Modified SDS-Based DNA Extraction Method for High Quality Environmental DNA from Seafloor Environments.

    PubMed

    Natarajan, Vengadesh Perumal; Zhang, Xinxu; Morono, Yuki; Inagaki, Fumio; Wang, Fengping

    2016-01-01

    Recovering high quality genomic DNA from environmental samples is a crucial primary step to understand the genetic, metabolic, and evolutionary characteristics of microbial communities through molecular ecological approaches. However, it is often challenging because of the difficulty of effective cell lysis without fragmenting the genomic DNA. This work aims to improve the previous SDS-based DNA extraction methods for high-biomass seafloor samples, such as pelagic sediments and metal sulfide chimney, to obtain high quality and high molecular weight of the genomic DNA applicable for the subsequent molecular ecological analyses. In this regard, we standardized a modified SDS-based DNA extraction method (M-SDS), and its performance was then compared to those extracted by a recently developed hot-alkaline DNA extraction method (HA) and a commercial DNA extraction kit. Consequently, the M-SDS method resulted in higher DNA yield and cell lysis efficiency, lower DNA shearing, and higher diversity scores than other two methods, providing a comprehensive DNA assemblage of the microbial community on the seafloor depositional environment. PMID:27446026

  12. A single protocol for extraction of gDNA from bacteria and yeast.

    PubMed

    Vingataramin, Laurie; Frost, Eric H

    2015-03-01

    Guanidine thiocyanate breakage of microorganisms has been the standard initial step in genomic DNA (gDNA) extraction of microbial DNA for two decades, despite the requirement for pretreatments to extract DNA from microorganisms other than Gram-negative bacteria. We report a quick and low-cost gDNA extraction protocol called EtNa that is efficient for bacteria and yeast over a broad range of concentrations. EtNa is based on a hot alkaline ethanol lysis. The solution can be immediately centrifuged to yield a crude gDNA extract suitable for PCR, or it can be directly applied to a silica column for purification. PMID:25757544

  13. Microcoding: the second step in DNA barcoding

    PubMed Central

    Summerbell, R.C; Lévesque, C.A; Seifert, K.A; Bovers, M; Fell, J.W; Diaz, M.R; Boekhout, T; de Hoog, G.S; Stalpers, J; Crous, P.W

    2005-01-01

    After the process of DNA barcoding has become well advanced in a group of organisms, as it has in the economically important fungi, the question then arises as to whether shorter and literally more barcode-like DNA segments should be utilized to facilitate rapid identification and, where applicable, detection. Through appropriate software analysis of typical full-length barcodes (generally over 500 base pairs long), uniquely distinctive oligonucleotide ‘microcodes’ of less than 25 bp can be found that allow rapid identification of circa 100–200 species on various array-like platforms. Microarrays can in principle fulfill the function of microcode-based species identification but, because of their high cost and low level of reusability, they tend to be less cost-effective. Two alternative platforms in current use in fungal identification are reusable nylon-based macroarrays and the Luminex system of specific, colour-coded DNA detection beads analysed by means of a flow cytometer. When the most efficient means of rapid barcode-based species identification is sought, a choice can be made either for one of these methodologies or for basic high-throughput sequencing, depending on the strategic outlook of the investigator and on current costs. Arrays and functionally similar platforms may have a particular advantage when a biologically complex material such as soil or a human respiratory secretion sample is analysed to give a census of relevant species present. PMID:16214747

  14. DNA extraction techniques for use in education.

    PubMed

    Hearn, R P; Arblaster, K E

    2010-05-01

    DNA extraction provides a hands-on introduction to DNA and enables students to gain real life experience and practical knowledge of DNA. Students gain a sense of ownership and are more enthusiastic when they use their own DNA. A cost effective, simple protocol for DNA extraction and visualization was devised. Buccal mucosal epithelia provide a readily available source of cells for DNA extraction and can be harvested in a painless, noninvasive manner. Seven criteria were established to evaluate the protocol: Safety, DNA yield, DNA quality/stability, cost, user friendliness, reliability, and time. To identify the optimum conditions for each stage of the protocol (cell harvest, lysis, purification, and precipitation), each was investigated separately, and an adaptation of the fast-boiling protocol was used for the remaining stages. A validation study was undertaken with the optimized protocol to assess its performance when conducted by a group of students in a classroom setting. The optimum protocol used an isotonic Lucozade Hydro Active Fitness Water (HAFW) mouthwash. Lysis was achieved using a TE (10 mM Tris-HCl, 1 mM EDTA, pH 8) + 1% Sodium Dodecyl Sulphate (SDS) buffer. Protein was then digested using Proteinase K (Qiagen Inc., UK) at 56°C for 10 min. The DNA was then precipitated with sodium chloride and absolute ethanol. This protocol achieved an increase in DNA yield using readily available equipment and reagents at a lower per capita cost and is simple to use. PMID:21567818

  15. Leaf tissue sampling and DNA extraction protocols.

    PubMed

    Semagn, Kassa

    2014-01-01

    Taxonomists must be familiar with a number of issues in collecting and transporting samples using freezing methods (liquid nitrogen and dry ice), desiccants (silica gel and blotter paper), and preservatives (CTAB, ethanol, and isopropanol), with each method having its own merits and limitations. For most molecular studies, a reasonably good quality and quantity of DNA is required, which can only be obtained using standard DNA extraction protocols. There are many DNA extraction protocols that vary from simple and quick ones that yield low-quality DNA but good enough for routine analyses to the laborious and time-consuming standard methods that usually produce high quality and quantities of DNA. The protocol to be chosen will depend on the quality and quantity of DNA needed, the nature of samples, and the presence of natural substances that may interfere with the extraction and subsequent analysis. The protocol described in this chapter has been tested for extracting DNA from eight species and provided very good quality and quantity of DNA for different applications, including those genotyping methods that use restriction enzymes. PMID:24415469

  16. The First Attested Extraction of Ancient DNA in Legumes (Fabaceae).

    PubMed

    Mikić, Aleksandar M

    2015-01-01

    Ancient DNA (aDNA) is any DNA extracted from ancient specimens, important for diverse evolutionary researches. The major obstacles in aDNA studies are mutations, contamination and fragmentation. Its studies may be crucial for crop history if integrated with human aDNA research and historical linguistics, both general and relating to agriculture. Legumes (Fabaceae) are one of the richest end economically most important plant families, not only from Neolithic onwards, since they were used as food by Neanderthals and Paleolithic modern man. The idea of extracting and analyzing legume aDNA was considered beneficial for both basic science and applied research, with an emphasis on genetic resources and plant breeding. The first reported successful and attested extraction of the legume aDNA was done from the sample of charred seeds of pea (Pisum sativum) and bitter vetch (Vicia ervilia) from Hissar, southeast Serbia, dated to 1,350-1,000 Before Christ. A modified version of cetyltrimethylammonium bromide (CTAB) method and the commercial kit for DNA extraction QIAGEN DNAesy yielded several ng μl(-1) of aDNA of both species and, after the whole genome amplification and with a fragment of nuclear ribosomal DNA gene 26S rDNA, resulted in the detection of the aDNA among the PCR products. A comparative analysis of four informative chloroplast DNA regions (trnSG, trnK, matK, and rbcL) among the modern wild and cultivated pea taxa demonstrated not only that the extracted aDNA was genuine, on the basis of mutation rate, but also that the ancient Hissar pea was most likely an early domesticated crop, related to the modern wild pea of a neighboring region. It is anticipated that this premier extraction of legume aDNA may provide taxonomists with the answers to diverse questions, such as leaf development in legumes, as well as with novel data on the single steps in domesticating legume crops worldwide. PMID:26635833

  17. The First Attested Extraction of Ancient DNA in Legumes (Fabaceae)

    PubMed Central

    Mikić, Aleksandar M.

    2015-01-01

    Ancient DNA (aDNA) is any DNA extracted from ancient specimens, important for diverse evolutionary researches. The major obstacles in aDNA studies are mutations, contamination and fragmentation. Its studies may be crucial for crop history if integrated with human aDNA research and historical linguistics, both general and relating to agriculture. Legumes (Fabaceae) are one of the richest end economically most important plant families, not only from Neolithic onwards, since they were used as food by Neanderthals and Paleolithic modern man. The idea of extracting and analyzing legume aDNA was considered beneficial for both basic science and applied research, with an emphasis on genetic resources and plant breeding. The first reported successful and attested extraction of the legume aDNA was done from the sample of charred seeds of pea (Pisum sativum) and bitter vetch (Vicia ervilia) from Hissar, southeast Serbia, dated to 1,350–1,000 Before Christ. A modified version of cetyltrimethylammonium bromide (CTAB) method and the commercial kit for DNA extraction QIAGEN DNAesy yielded several ng μl-1 of aDNA of both species and, after the whole genome amplification and with a fragment of nuclear ribosomal DNA gene 26S rDNA, resulted in the detection of the aDNA among the PCR products. A comparative analysis of four informative chloroplast DNA regions (trnSG, trnK, matK, and rbcL) among the modern wild and cultivated pea taxa demonstrated not only that the extracted aDNA was genuine, on the basis of mutation rate, but also that the ancient Hissar pea was most likely an early domesticated crop, related to the modern wild pea of a neighboring region. It is anticipated that this premier extraction of legume aDNA may provide taxonomists with the answers to diverse questions, such as leaf development in legumes, as well as with novel data on the single steps in domesticating legume crops worldwide. PMID:26635833

  18. Comparison of commercial DNA extraction kits for isolation and purification of bacterial and eukaryotic DNA from PAH-contaminated soils.

    PubMed

    Mahmoudi, Nagissa; Slater, Greg F; Fulthorpe, Roberta R

    2011-08-01

    Molecular characterization of the microbial populations of soils and sediments contaminated with polycyclic aromatic hydrocarbons (PAHs) is often a first step in assessing intrinsic biodegradation potential. However, soils are problematic for molecular analysis owing to the presence of organic matter, such as humic acids. Furthermore, the presence of contaminants, such as PAHs, can cause further challenges to DNA extraction, quantification, and amplification. The goal of our study was to compare the effectiveness of four commercial soil DNA extraction kits (UltraClean Soil DNA Isolation kit, PowerSoil DNA Isolation kit, PowerMax Soil DNA Isolation kit, and FastDNA SPIN kit) to extract pure, high-quality bacterial and eukaryotic DNA from PAH-contaminated soils. Six different contaminated soils were used to determine if there were any biases among the kits due to soil properties or level of contamination. Extracted DNA was used as a template for bacterial 16S rDNA and eukaryotic 18S rDNA amplifications, and PCR products were subsequently analyzed using denaturing gel gradient electrophoresis (DGGE). We found that the FastDNA SPIN kit provided significantly higher DNA yields for all soils; however, it also resulted in the highest levels of humic acid contamination. Soil texture and organic carbon content of the soil did not affect the DNA yield of any kit. Moreover, a liquid-liquid extraction of the DNA extracts found no residual PAHs, indicating that all kits were effective at removing contaminants in the extraction process. Although the PowerSoil DNA Isolation kit gave relatively low DNA yields, it provided the highest quality DNA based on successful amplification of both bacterial and eukaryotic DNA for all six soils. DGGE fingerprints among the kits were dramatically different for both bacterial and eukaryotic DNA. The PowerSoil DNA Isolation kit revealed multiple bands for each soil and provided the most consistent DGGE profiles among replicates for both

  19. Microscope Titration and Extraction of DNA from Liver.

    ERIC Educational Resources Information Center

    Mayo, Lois T.; And Others

    1993-01-01

    Describes a simple and inexpensive, one-period activity to extract DNA to make the study of DNA less abstract. A microscope titration is used to determine when cells are ready for DNA extraction. (PR)

  20. Plant and metagenomic DNA extraction of mucilaginous seeds.

    PubMed

    Ramos, Simone N M; Salazar, Marcela M; Pereira, Gonçalo A G; Efraim, Priscilla

    2014-01-01

    The pulp surrounding the seeds of some fruits is rich in mucilage, carbohydrates, etc. Some seeds are rich in proteins and polyphenols. Fruit seeds, like cacao (Theobroma cacao) and cupuassu (Theobroma grandiflorum), are subjected to fermentation to develop flavor. During fermentation, ethanol is produced [2-6]. All of these compounds are considered as interfering substances that hinder the DNA extraction [4-8]. Protocols commonly used in the DNA extraction in samples of plant origin were used, but without success. Thus, a protocol for DNA samples under different conditions that can be used for similar samples was developed and applied with success. The protocol initially described for RNA samples by Zeng et al. [9] and with changes proposed by Provost et al. [5] was adapted for extracting DNA samples from those described. However, several modifications have been proposed:•Samples were initially washed with petroleum ether for fat phase removal.•RNAse was added to the extraction buffer, while spermidin was removed.•Additional steps of extraction with 5 M NaCl, saturated NaCl and CTAB (10%) were included and precipitation was carried out with isopropanol, followed by washing with ethanol. PMID:26150956

  1. Extracting DNA from submerged pine wood.

    PubMed

    Reynolds, M Megan; Williams, Claire G

    2004-10-01

    A DNA extraction protocol for submerged pine logs was developed with the following properties: (i) high molecular weight DNA, (ii) PCR amplification of chloroplast and nuclear sequences, and (iii) high sequence homology to voucher pine specimens. The DNA extraction protocol was modified from a cetyltrimehtylammonium bromide (CTAB) protocol by adding stringent electrophoretic purification, proteinase K, RNAse, polyvinyl pyrrolidone (PVP), and Gene Releaser. Chloroplast rbcL (ribulose-1,5-bisphosphate carboxylase) could be amplified. Nuclear ribosomal sequences had >95% homology to Pinus taeda and Pinus palustris. Microsatellite polymorphism for PtTX2082 matched 2 of 14 known P. taeda alleles. Our results show DNA analysis for submerged conifer wood is feasible. PMID:15499414

  2. Comparison of four commercial DNA extraction kits for the recovery of Bacillus spp. spore DNA from spiked powder samples.

    PubMed

    Mölsä, Markos; Kalin-Mänttäri, Laura; Tonteri, Elina; Hemmilä, Heidi; Nikkari, Simo

    2016-09-01

    Bacillus spp. include human pathogens such as Bacillus anthracis, the causative agent of anthrax and a biothreat agent. Bacillus spp. form spores that are physically highly resistant and may remain active over sample handling. We tested four commercial DNA extraction kits (QIAamp DNA Mini Kit, RTP Pathogen Kit, ZR Fungal/Bacterial DNA MiniPrep, and genesig Easy DNA/RNA Extraction kit) for sample inactivation and DNA recovery from two powders (icing sugar and potato flour) spiked with Bacillus thuringiensis spores. The DNA was analysed using a B. thuringiensis-specific real-time PCR assay. The detection limit was 3×10(1)CFU of spiked B. thuringiensis spores with the QIAamp DNA Mini, RTP Pathogen, and genesig Easy DNA/RNA Extraction kits, and 3×10(3)CFU with the ZR Fungal/Bacterial DNA MiniPrep kit. The results showed that manual extraction kits are effective and safe for fast and easy DNA extraction from powder samples even in field conditions. Adding a DNA filtration step to the extraction protocol ensures the removal of Bacillus spp. spores from DNA samples without affecting sensitivity. PMID:27435532

  3. Extraction of DNA from Forensic Biological Samples for Genotyping.

    PubMed

    Stray, J E; Liu, J Y; Brevnov, M G; Shewale, J G

    2010-07-01

    Biological forensic samples constitute evidence with probative organic matter. Evidence believed to contain DNA is typically processed for extraction and purification of its nucleic acid content. Forensic DNA samples are composed of two things, a tissue and the substrate it resides on. Compositionally, a sample may contain almost anything and for each, the type, integrity, and content of both tissue and substrate will vary, as will the contaminant levels. This fact makes the success of extraction one of the most unpredictable steps in genotypic analysis. The development of robust genotyping systems and analysis platforms for short tandem repeat (STR) and mitochondrial DNA sequencing and the acceptance of results generated by these methods in the court system, resulted in a high demand for DNA testing. The increasing variety of sample submissions created a need to isolate DNA from forensic samples that may be compromised or contain low levels of biological material. In the past decade, several robust chemistries and isolation methods have been developed to safely and reliably recover DNA from a wide array of sample types in high yield and free of PCR inhibitors. In addition, high-throughput automated workflows have been developed to meet the demand for processing increasing numbers of samples. This review summarizes a number of the most widely adopted methods and the best practices for DNA isolation from forensic biological samples, including manual, semiautomated, and fully automated platforms. PMID:26242594

  4. Step-wise supercritical extraction of carbonaceous residua

    DOEpatents

    Warzinski, Robert P.

    1987-01-01

    A method of fractionating a mixture containing high boiling carbonaceous material and normally solid mineral matter includes processing with a plurality of different supercritical solvents. The mixture is treated with a first solvent of high critical temperature and solvent capacity to extract a large fraction as solute. The solute is released as liquid from solvent and successively treated with other supercritical solvents of different critical values to extract fractions of differing properties. Fractionation can be supplemented by solute reflux over a temperature gradient, pressure let down in steps and extractions at varying temperature and pressure values.

  5. Rapid alkaline extraction method for the isolation of plasmid DNA

    SciTech Connect

    Birnboim, H.C.

    1983-01-01

    Plasmids are double-stranded circular DNA molecules that have the property of self-replication, independent of chromosomal DNA. Although the presence of a plasmid in a bacterial cell may be detected genetically as a change in phenotype, often it is necessary to isolate plasmid DNA for molecular studies, such as size determination, restriction enzyme mapping, and nucleotide sequencing, or for the construction of new hybrid plasmids. The degree of purification required will depend upon the intended use. Less purified plasmid DNA is often satisfactory for recombinant DNA studies, and a large number of shorter and simpler methods have been developed. This chapter describes one such method that uses an alkaline extraction step. It is rapid enough to be used as a screening method, permitting 50-100 or more samples to be extracted in a few hours. The DNA is sufficiently pure to be digestible by restriction enzymes, an important advantage for screening. A preparative version that allows isolation of larger quantities of more highly purified material is also described.

  6. Bioaerosol DNA Extraction Technique from Air Filters Collected from Marine and Freshwater Locations

    NASA Astrophysics Data System (ADS)

    Beckwith, M.; Crandall, S. G.; Barnes, A.; Paytan, A.

    2015-12-01

    Bioaerosols are composed of microorganisms suspended in air. Among these organisms include bacteria, fungi, virus, and protists. Microbes introduced into the atmosphere can drift, primarily by wind, into natural environments different from their point of origin. Although bioaerosols can impact atmospheric dynamics as well as the ecology and biogeochemistry of terrestrial systems, very little is known about the composition of bioaerosols collected from marine and freshwater environments. The first step to determine composition of airborne microbes is to successfully extract environmental DNA from air filters. We asked 1) can DNA be extracted from quartz (SiO2) air filters? and 2) how can we optimize the DNA yield for downstream metagenomic sequencing? Aerosol filters were collected and archived on a weekly basis from aquatic sites (USA, Bermuda, Israel) over the course of 10 years. We successfully extracted DNA from a subsample of ~ 20 filters. We modified a DNA extraction protocol (Qiagen) by adding a beadbeating step to mechanically shear cell walls in order to optimize our DNA product. We quantified our DNA yield using a spectrophotometer (Nanodrop 1000). Results indicate that DNA can indeed be extracted from quartz filters. The additional beadbeating step helped increase our yield - up to twice as much DNA product was obtained compared to when this step was omitted. Moreover, bioaerosol DNA content does vary across time. For instance, the DNA extracted from filters from Lake Tahoe, USA collected near the end of June decreased from 9.9 ng/μL in 2007 to 3.8 ng/μL in 2008. Further next-generation sequencing analysis of our extracted DNA will be performed to determine the composition of these microbes. We will also model the meteorological and chemical factors that are good predictors for microbial composition for our samples over time and space.

  7. [Research advances on DNA extraction methods from peripheral blood mononuclear cells].

    PubMed

    Wang, Xiao-Ying; Yu, Chen-Xi

    2014-10-01

    DNA extraction is a basic technology of molecular biology. The purity and the integrality of DNA structure are necessary for different experiments of gene engineering. As commonly used materials in the clinical detection, the fast, efficient isolation and extraction of genomic DNA from peripheral blood mononuclear cells is very important for the inspection and analysis of clinical blood. At present, there are many methods for extracting DNA, such as phenol-chloroform method, salting out method, centrifugal adsorption column chromatography method (artificial methods), magnetic beads (semi-automatic method) and DNA extraction kit. In this article, a brief review of the principle for existing DNA blood extraction method, the specific steps and the assessment of the specific methods briefly are summarized. PMID:25338615

  8. Improvement of extraction efficiency from a slow extraction synchrotron by applying the constant spiral step condition

    NASA Astrophysics Data System (ADS)

    Ebina, Futaro; Umezawa, Masumi; Hiramoto, Kazuo

    2012-09-01

    An operating condition for high extraction efficiency of a synchrotron with a slow extraction method is presented. With this condition, the separation between a particle injected into the electrostatic septum and a particle circulating in the synchrotron (the spiral step) becomes independent of the momentum of the particle (the constant spiral step condition). In a synchrotron with the Hardt condition, the spiral step varies according to the momentum of the extracted particle. So the extraction efficiency of the circulating beam with large momentum deviation becomes low because of the beam loss at the entrance of the electrostatic septum (ES). In a synchrotron with the constant spiral step condition, the beam loss at the entrance of the ES decreases. Therefore, the extraction efficiency of the synchrotron with the constant spiral step condition can become higher than that with the Hardt condition despite the loss of particles with large momentum deviation in the middle of the ES. Analogous to the Hardt condition, setting the horizontal chromaticity of the synchrotron to an appropriate value provides the constant spiral step condition. Particle tracking simulation results indicate that the extraction efficiency of the synchrotron with the constant spiral step condition is slightly higher than that with the Hardt condition. Adequate extraction efficiency is achieved with the horizontal chromaticity set between the Hardt condition and the constant spiral step condition. The magnetic strength of the sextupole magnets in the synchrotron should be set high enough to get horizontal chromaticity of the ring at any value in this range. Furthermore, it is desirable to design a synchrotron so that the constant spiral step condition becomes close to the Hardt condition.

  9. Methods for microbial DNA extraction from soil for PCR amplification.

    PubMed

    Yeates, C; Gillings, M R; Davison, A D; Altavilla, N; Veal, D A

    1998-05-14

    Amplification of DNA from soil is often inhibited by co-purified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types (1). DNA is also suitable for PCR amplification using various DNA targets. DNA was extracted from 100g of soil using direct lysis with glass beads and SDS followed by potassium acetate precipitation, polyethylene glycol precipitation, phenol extraction and isopropanol precipitation. This method was compared to other DNA extraction methods with regard to DNA purity and size. PMID:12734590

  10. COMPARISON OF DNA EXTRACTION METHODS ON DAIRY CONSTRUCTED WETLAND WASTEWATER

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Direct DNA extraction from environmental samples is a useful and culture-independent method for the examination of microbial diversity. To date, there is little information on the effectiveness of commercial DNA extraction kits on wastewater. We compared two commercial DNA extraction kits for amount...

  11. Extraction of DNA from Human Skeletal Material.

    PubMed

    Pajnič, Irena Zupanič

    2016-01-01

    In recent years the recovery and analysis of DNA from skeletal remains has been applied to several contexts ranging from disaster victim identification to the identification of the victims of conflict. Here are described procedures for processing the bone and tooth samples including mechanical and chemical cleaning, cutting and powdering in the presence of liquid nitrogen, complete demineralization of bone and tooth powder, DNA extraction, DNA purification using magnetic beads, and the precautions and strategies implemented to avoid and detect contamination. It has proven highly successful in the analysis of bones and teeth from Second World War victims' skeletal remains that have been excavated from mass graves in Slovenia and is also suitable for genetic identification of relatively fresh human remains. PMID:27259733

  12. An improved protocol for DNA extraction from alkaline soil and sediment samples for constructing metagenomic libraries.

    PubMed

    Verma, Digvijay; Satyanarayana, T

    2011-09-01

    An improved single-step protocol has been developed for extracting pure community humic substance-free DNA from alkaline soils and sediments. The method is based on direct cell lysis in the presence of powdered activated charcoal and polyvinylpolypyrrolidone followed by precipitation with polyethyleneglycol and isopropanol. The strategy allows simultaneous isolation and purification of DNA while minimizing the loss of DNA with respect to other available protocols for metagenomic DNA extraction. Moreover, the purity levels are significant, which are difficult to attain with any of the methods reported in the literature for DNA extraction from soils. The DNA thus extracted was free from humic substances and, therefore, could be processed for restriction digestion, PCR amplification as well as for the construction of metagenomic libraries. PMID:21519906

  13. Arduino-based automation of a DNA extraction system.

    PubMed

    Kim, Kyung-Won; Lee, Mi-So; Ryu, Mun-Ho; Kim, Jong-Won

    2015-01-01

    There have been many studies to detect infectious diseases with the molecular genetic method. This study presents an automation process for a DNA extraction system based on microfluidics and magnetic bead, which is part of a portable molecular genetic test system. This DNA extraction system consists of a cartridge with chambers, syringes, four linear stepper actuators, and a rotary stepper actuator. The actuators provide a sequence of steps in the DNA extraction process, such as transporting, mixing, and washing for the gene specimen, magnetic bead, and reagent solutions. The proposed automation system consists of a PC-based host application and an Arduino-based controller. The host application compiles a G code sequence file and interfaces with the controller to execute the compiled sequence. The controller executes stepper motor axis motion, time delay, and input-output manipulation. It drives the stepper motor with an open library, which provides a smooth linear acceleration profile. The controller also provides a homing sequence to establish the motor's reference position, and hard limit checking to prevent any over-travelling. The proposed system was implemented and its functionality was investigated, especially regarding positioning accuracy and velocity profile. PMID:26409535

  14. A rapid and efficient assay for extracting DNA from fungi

    USGS Publications Warehouse

    Griffin, Dale W.; Kellogg, C.A.; Peak, K.K.; Shinn, E.A.

    2002-01-01

    Aims: A method for the rapid extraction of fungal DNA from small quantities of tissue in a batch-processing format was investigated. Methods and Results: Tissue (< 3.0 mg) was scraped from freshly-grown fungal isolates. The tissue was suspended in buffer AP1 and subjected to seven rounds of freeze/thaw using a crushed dry ice/ethanol bath and a boiling water bath. After a 30 min boiling step, the tissue was quickly ground against the wall of the microfuge tube using a sterile pipette tip. The Qiagen DNeasy Plant Tissue Kit protocol was then used to purify the DNA for PCR/ sequencing applications. Conclusions: The method allowed batch DNA extraction from multiple fungal isolates using a simple yet rapid and reliable assay. Significance and Impact of the Study: Use of this assay will allow researchers to obtain DNA from fungi quickly for use in molecular assays that previously required specialized instrumentation, was time-consuming or was not conducive to batch processing.

  15. Comparison of DNA Extraction Methods from Small Samples of Newborn Screening Cards Suitable for Retrospective Perinatal Viral Research

    PubMed Central

    McMichael, Gai L.; Highet, Amanda R.; Gibson, Catherine S.; Goldwater, Paul N.; O'Callaghan, Michael E.; Alvino, Emily R.; MacLennan, Alastair H.

    2011-01-01

    Reliable detection of viral DNA in stored newborn screening cards (NSC) would give important insight into possible silent infection during pregnancy and around birth. We sought a DNA extraction method with sufficient sensitivity to detect low copy numbers of viral DNA from small punch samples of NSC. Blank NSC were spotted with seronegative EDTA-blood and seropositive EBV EDTA-blood. DNA was extracted with commercial and noncommercial DNA extraction methods and quantified on a spectrofluorometer using a PicoGreen dsDNA quantification kit. Serial dilutions of purified viral DNA controls determined the sensitivity of the amplification protocol, and seropositive EBV EDTA-blood amplified by nested PCR (nPCR) validated the DNA extraction methods. There were considerable differences between the commercial and noncommercial DNA extraction methods (P=0.014; P=0.016). Commercial kits compared favorably, but the QIamp DNA micro kit with an added forensic filter step was marginally more sensitive. The mean DNA yield from this method was 3 ng/μl. The limit of detection was 10 viral genome copies in a 50-μl reaction. EBV nPCR detection in neat and 1:10 diluted DNA extracts could be replicated reliably. We conclude that the QIamp Micro DNA extraction method with the added forensic spin-filter step was suitable for retrospective DNA viral assays from NSC. PMID:21455476

  16. High Efficiency DNA Extraction by Graphite Oxide/Cellulose/Magnetite Composites Under Na+ Free System

    NASA Astrophysics Data System (ADS)

    Akceoglu, Garbis Atam; Li, Oi Lun; Saito, Nagahiro

    2016-04-01

    DNA extraction is the key step at various research areas like biotechnology, diagnostic development, paternity determination, and forensic science . Solid support extraction is the most common method for DNA purification. In this method, Na+ ions have often been applied as binding buffers in order to obtain high extraction efficiency and high quality of DNA; however, the presence of Na+ ions might be interfering with the downstream DNA applications. In this study, we proposed graphite oxide (GO)/magnetite composite/cellulose as an innovative material for Na+-free DNA extraction. The total wt.% of GO was fixed at 4.15% in the GO/cellulose/magnetite composite . The concentration of magnetite within the composites were controlled at 0-3.98 wt.%. The extraction yield of DNA increased with increasing weight percentage of magnetite. The highest yield was achieved at 3.98 wt.% magnetite, where the extraction efficiency was reported to be 338.5 ng/µl. The absorbance ratios between 260 nm and 280 nm (A260/A280) of the DNA elution volume was demonstrated as 1.81, indicating the extracted DNA consisted of high purity. The mechanism of adsorption of DNA was provided by (1) π-π interaction between the aromatic ring in GO and nucleobases of DNA molecule, and (2) surface charge interaction between the positive charge magnetite and anions such as phosphates within the DNA molecules. The results proved that the GO/cellulose/magnetite composite provides a Na+-free method for selective DNA extraction with high extraction efficiency of pure DNA.

  17. A simple, reliable, and fast protocol for thraustochytrid DNA extraction.

    PubMed

    Mo, C; Rinkevich, B

    2001-03-01

    DNA extraction of thraustochytrids, common marine unicellular organisms, is usually accomplished by either the cetyltrimethylammonium bromide (CTAB) or proteinase K protocols. A novel lysis buffer protocol for thraustochytrid total DNA extraction is described. The average isolated total DNA is 20 to 40 kb, and DNA samples are suitable for a variety of uses including 18S-ribosomal DNA polymerase chain reaction, restriction enzyme digestions, and amplified fragment length polymorphism analyses. The new protocol is also faster than the other protocols. PMID:14961371

  18. Purification of total DNA extracted from activated sludge.

    PubMed

    Shan, Guobin; Jin, Wenbiao; Lam, Edward K H; Xing, Xinhui

    2008-01-01

    Purification of the total DNA extracted from activated sludge samples was studied. The effects of extraction buffers and lysis treatments (lysozyme, sodium dodecyl sulfate (SDS), sonication, mechanical mill and thermal shock) on yield and purity of the total DNA extracted from activated sludge were investigated. It was found that SDS and mechanical mill were the most effective ways for cell lysis, and both gave the highest DNA yields, while by SDS and thermal shock, the purest DNA extract could be obtained. The combination of SDS with other lysis treatment, such as sonication and thermal shock, could apparently increase the DNA yields but also result in severe shearing. For the purification of the crude DNA extract, polyvinyl polypyrrolidone was used for the removal of humic contaminants. Cetyltrimethyl ammonium bromide, potassium acetate and phenol/chloroform were used to remove proteins and polysaccharides from crude DNA. Crude DNA was further purified by isopropanol precipitation. Thus, a suitable protocol was proposed for DNA extraction, yielding about 49.9 mg (total DNA)/g volatile suspended solids, and the DNA extracts were successfully used in PCR amplifications for 16S rDNA and 16S rDNA V3 region. The PCR products of 16S rDNA V3 region allowed the DGGE analysis (denatured gradient gel electrophoresis) to be possible. PMID:18572527

  19. Genomic DNA extraction from medicinal plants available in Malaysia using a TriOmic(TM) improved extraction kit.

    PubMed

    Mohd-Hairul, A R; Sade, A B; Yiap, B C; Raha, A R

    2011-01-01

    DNA extraction was carried out on 32 medicinal plant samples available in Malaysia using the TriOmic(TM) extraction kit. Amounts of 0.1 g flowers or young leaves were ground with liquid nitrogen, lysed at 65°C in RY1(plus) buffer and followed by RNAse treatment. Then, RY2 buffer was added to the samples and mixed completely by vortexing before removal of cell debris by centrifugation. Supernatants were transferred to fresh microcentrifuge tubes and 0.1 volume RY3 buffer was added to each of the transferred supernatant. The mixtures were applied to spin columns followed by a centrifugation step to remove buffers and other residues. Washing step was carried out twice by applying 70% ethanol to the spin columns. Genomic DNA of the samples was recovered by applying 50 μL TE buffer to the membrane of each spin column, followed by a centrifugation step at room temperature. A modification of the TriOmic(TM) extraction procedure was carried out by adding chloroform:isoamyl alcohol (24:1) steps in the extraction procedure. The genomic DNA extracted from most of the 32 samples showed an increase of total yield when chloroform:isoamyl alcohol (24:1) steps were applied in the TriOmicTM extraction procedure. This preliminary study is very important for molecular studies of medicinal plants available in Malaysia since the DNA extraction can be completed in a shorter period of time (within 1 h) compared to manual extraction, which entails applying phenol, chloroform and ethanol precipitation, and requires 1-2 days to complete. PMID:22095601

  20. Thermodynamics of the DNA Damage Repair Steps of Human 8-Oxoguanine DNA Glycosylase

    PubMed Central

    Kuznetsov, Nikita A.; Kuznetsova, Alexandra A.; Vorobjev, Yuri N.; Krasnoperov, Lev N.; Fedorova, Olga S.

    2014-01-01

    Human 8-oxoguanine DNA glycosylase (hOGG1) is a key enzyme responsible for initiating the base excision repair of 7,8-dihydro-8-oxoguanosine (oxoG). In this study a thermodynamic analysis of the interaction of hOGG1 with specific and non-specific DNA-substrates is performed based on stopped-flow kinetic data. The standard Gibbs energies, enthalpies and entropies of specific stages of the repair process were determined via kinetic measurements over a temperature range using the van’t Hoff approach. The three steps which are accompanied with changes in the DNA conformations were detected via 2-aminopurine fluorescence in the process of binding and recognition of damaged oxoG base by hOGG1. The thermodynamic analysis has demonstrated that the initial step of the DNA substrates binding is mainly governed by energy due to favorable interactions in the process of formation of the recognition contacts, which results in negative enthalpy change, as well as due to partial desolvation of the surface between the DNA and enzyme, which results in positive entropy change. Discrimination of non-specific G base versus specific oxoG base is occurring in the second step of the oxoG-substrate binding. This step requires energy consumption which is compensated by the positive entropy contribution. The third binding step is the final adjustment of the enzyme/substrate complex to achieve the catalytically competent state which is characterized by large endothermicity compensated by a significant increase of entropy originated from the dehydration of the DNA grooves. PMID:24911585

  1. Viral DNA Packaging: One Step at a Time

    NASA Astrophysics Data System (ADS)

    Bustamante, Carlos; Moffitt, Jeffrey R.

    During its life-cycle the bacteriophage φ29 actively packages its dsDNA genome into a proteinacious capsid, compressing its genome to near crystalline densities against large electrostatic, elastic, and entropic forces. This remarkable process is accomplished by a nano-scale, molecular DNA pump - a complex assembly of three protein and nucleic acid rings which utilizes the free energy released in ATP hydrolysis to perform the mechanical work necessary to overcome these large energetic barriers. We have developed a single molecule optical tweezers assay which has allowed us to probe the detailed mechanism of this packaging motor. By following the rate of packaging of a single bacteriophage as the capsid is filled with genome and as a function of optically applied load, we find that the compression of the genome results in the build-up of an internal force, on the order of ˜ 55 pN, due to the compressed genome. The ability to work against such large forces makes the packaging motor one of the strongest known molecular motors. By titrating the concentration of ATP, ADP, and inorganic phosphate at different opposing load, we are able to determine features of the mechanochemistry of this motor - the coupling between the mechanical and chemical cycles. We find that force is generated not upon binding of ATP, but rather upon release of hydrolysis products. Finally, by improving the resolution of the optical tweezers assay, we are able to observe the discrete increments of DNA encapsidated each cycle of the packaging motor. We find that DNA is packaged in 10-bp increments preceded by the binding of multiple ATPs. The application of large external forces slows the packaging rate of the motor, revealing that the 10-bp steps are actually composed of four 2.5-bp steps which occur in rapid succession. These data show that the individual subunits of the pentameric ring-ATPase at the core of the packaging motor are highly coordinated, with the binding of ATP and the

  2. Food Fish Identification from DNA Extraction through Sequence Analysis

    ERIC Educational Resources Information Center

    Hallen-Adams, Heather E.

    2015-01-01

    This experiment exposed 3rd and 4th y undergraduates and graduate students taking a course in advanced food analysis to DNA extraction, polymerase chain reaction (PCR), and DNA sequence analysis. Students provided their own fish sample, purchased from local grocery stores, and the class as a whole extracted DNA, which was then subjected to PCR,…

  3. One-step enzyme extraction and immobilization for biocatalysis applications.

    PubMed

    Cassimjee, Karim Engelmark; Kourist, Robert; Lindberg, Diana; Wittrup Larsen, Marianne; Thanh, Nguyen Hong; Widersten, Mikael; Bornscheuer, Uwe T; Berglund, Per

    2011-04-01

    An extraction/immobilization method for HIs(6) -tagged enzymes for use in synthesis applications is presented. By modifying silica oxide beads to be able to accommodate metal ions, the enzyme was tethered to the beads after adsorption of Co(II). The beads were successfully used for direct extraction of C. antarctica lipase B (CalB) from a periplasmic preparation with a minimum of 58% activity yield, creating a quick one-step extraction-immobilization protocol. This method, named HisSi Immobilization, was evaluated with five different enzymes [Candida antarctica lipase B (CalB), Bacillus subtilis lipase A (BslA), Bacillus subtilis esterase (BS2), Pseudomonas fluorescence esterase (PFE), and Solanum tuberosum epoxide hydrolase 1 (StEH1)]. Immobilized CalB was effectively employed in organic solvent (cyclohexane and acetonitrile) in a transacylation reaction and in aqueous buffer for ester hydrolysis. For the remaining enzymes some activity in organic solvent could be shown, whereas the non-immobilized enzymes were found inactive. The protocol presented in this work provides a facile immobilization method by utilization of the common His(6) -tag, offering specific and defined means of binding a protein in a specific location, which is applicable for a wide range of enzymes. PMID:21381205

  4. Direct DNA extraction method of an obligate parasitic fungus from infected plant tissue.

    PubMed

    Liu, L; Wang, C L; Peng, W Y; Yang, J; Lan, M Q; Zhang, B; Li, J B; Zhu, Y Y; Li, C Y

    2015-01-01

    Powdery mildew and rust fungi are obligate parasites that cannot live without host organisms. They are difficult to culture in synthetic medium in the laboratory. Genomic DNA extraction is one of the basic molecular techniques used to study the genetic structure of populations. In this study, 2 different DNA extraction methods, Chelex-100 and cetyltrimethylammonium bromide (CTAB), were used to extract DNA from euonymus powdery mildew and Puccinia striiformis f. sp Tritici. Polymerase chain reaction was carried out with a race-specific-marker rDNA-internal transcribed spacer sequence. Both DNA extraction methods were compared and analyzed. The results showed that both Chelex-100 and CTAB were effective for extracting genomic DNA from infected plant tissue. However, less DNA was required for the Chelex-100 method than for the CTAB method, and the Chelex-100 method involved fewer steps, was simpler and safer, and did not require organic solvents compared to the CTAB method. DNA quality was evaluated by polymerase chain reaction, and the results showed that genomic DNA extracted using the Chelex-100 method was better than that using CTAB method, and was sufficient for studying the genetic structure of population. PMID:26782502

  5. A fully automatable enzymatic method for DNA extraction from plant tissues

    PubMed Central

    Manen, Jean-François; Sinitsyna, Olga; Aeschbach, Lorène; Markov, Alexander V; Sinitsyn, Arkady

    2005-01-01

    Background DNA extraction from plant tissues, unlike DNA isolation from mammalian tissues, remains difficult due to the presence of a rigid cell wall around the plant cells. Currently used methods inevitably require a laborious mechanical grinding step, necessary to disrupt the cell wall for the release of DNA. Results Using a cocktail of different carbohydrases, a method was developed that enables a complete digestion of the plant cell walls and subsequent DNA release. Optimized conditions for the digestion reaction minimize DNA shearing and digestion, and maximize DNA release from the plant cell. The method gave good results in 125 of the 156 tested species. Conclusion In combination with conventional DNA isolation techniques, the new enzymatic method allows to obtain high-yield, high-molecular weight DNA, which can be used for many applications, including genome characterization by AFLP, RAPD and SSR. Automation of the protocol (from leaf disks to DNA) is possible with existing workstations. PMID:16269076

  6. An alternative protocol for DNA extraction from formalin fixed and paraffin wax embedded tissue

    PubMed Central

    Coura, R; Prolla, J C; Meurer, L; Ashton-Prolla, P

    2005-01-01

    Background: DNA extraction from paraffin wax embedded tissue requires special protocols, and most described methods report an amplification success rate of 60–80%. Aims: To propose a simple and inexpensive protocol consisting of xylene/ethanol dewaxing, followed by a kit based extraction. Method: Xylene/ethanol dewaxing was followed by a long rehydration step and a kit based DNA extraction step. Results: This method produced a 100% amplification success rate for fragments of 121 to 227 bp for tamponated formalin fixed paraffin wax embedded tissue. Conclusion: This cost effective and non-laborious protocol can successfully extract DNA from tamponated formalin fixed paraffin wax embedded tissue and should facilitate the molecular analysis of a large number of archival specimens in retrospective studies. PMID:16049299

  7. Extraction of high-quality genomic DNA from latex-containing plants.

    PubMed

    Michiels, An; Van den Ende, Wim; Tucker, Mark; Van Riet, Liesbet; Van Laere, André

    2003-04-01

    The isolation of intact, high-molecular-mass genomic DNA is essential for many molecular biology applications including long PCR, endonuclease restriction digestion, Southern blot analysis, and genomic library construction. Many protocols are available for the extraction of DNA from plant material. However, for latex-containing Asteraceae (Cichorioideae) species, standard protocols and commercially available kits do not produce efficient yields of high-quality amplifiable DNA. A cetyltrimethylammonium bromide protocol has been optimized for isolation of genomic DNA from latex-containing plants. Key steps in the modified protocol are the use of etiolated leaf tissue for extraction and an overnight 25 degrees C isopropanol precipitation step. The purified DNA has excellent spectral qualities, is efficiently digested by restriction endonucleases, and is suitable for long-fragment PCR amplification. PMID:12672415

  8. DNA walks one step at a time in electrophoresis

    NASA Astrophysics Data System (ADS)

    Guan, Juan; Wang, Bo; Granick, Steve

    2011-03-01

    Testing the classical view that in DNA gel electrophoresis, long polymer chains navigate through their gel environment via reptation, we reach a different conclusion: this driven motion proceeds by stick-slip. Our single-molecule experiments visualize fluorescent-labeled lambda-DNA, whose intramolecular conformations are resolved with 30 ms resolution using home-written software. Combining hundreds to thousands of trajectories under amplitudes of electric field ranging from zero to large, we quantify the full statistical distribution of motion with unprecedented statistics. Pauses are seen between steps of driven motion, probably reflecting that the chain is trapped inside the gel matrix. The pausing time is exponentially distributed and decreases with increasing electric field strength, suggesting that the jerky behavior is an activated process, facilitated by electric field. We propose a stretch-assisted mechanism: that the energy barrier to move through the gel environment is first overcome by a leading segment, the ensuing intramolecular stress from stretching causing lagging segments to recoil and follow along.

  9. Comparative analysis of protocols for DNA extraction from soybean caterpillars.

    PubMed

    Palma, J; Valmorbida, I; da Costa, I F D; Guedes, J V C

    2016-01-01

    Genomic DNA extraction is crucial for molecular research, including diagnostic and genome characterization of different organisms. The aim of this study was to comparatively analyze protocols of DNA extraction based on cell lysis by sarcosyl, cetyltrimethylammonium bromide, and sodium dodecyl sulfate, and to determine the most efficient method applicable to soybean caterpillars. DNA was extracted from specimens of Chrysodeixis includens and Spodoptera eridania using the aforementioned three methods. DNA quantification was performed using spectrophotometry and high molecular weight DNA ladders. The purity of the extracted DNA was determined by calculating the A260/A280 ratio. Cost and time for each DNA extraction method were estimated and analyzed statistically. The amount of DNA extracted by these three methods was sufficient for PCR amplification. The sarcosyl method yielded DNA of higher purity, because it generated a clearer pellet without viscosity, and yielded high quality amplification products of the COI gene I. The sarcosyl method showed lower cost per extraction and did not differ from the other methods with respect to preparation times. Cell lysis by sarcosyl represents the best method for DNA extraction in terms of yield, quality, and cost effectiveness. PMID:27173218

  10. Rapid extraction of DNA from archival clinical specimens: our experiences.

    PubMed

    Poljak, M; Seme, K; Gale, N

    2000-01-01

    The analysis of DNA extracted from archival clinical specimens using polymerase chain reaction represents the basis of a variety of research and diagnostic protocols in medicine. However, the selection of optimal DNA extraction method is critical if such an analysis is to be successful. Recently, we have evaluated a number of rapid DNA extraction protocols in order to find the most suitable method for routine processing of the most common archival materials in pathological and cytological laboratories: paraffin-embedded tissues and Papanicolaou- or Giemsa-stained smears. Our results demonstrate that rapid DNA extraction methods have comparable DNA extraction efficiencies with standard DNA isolation protocols on archival clinical specimens with the exception of Giemsa-stained smears. PMID:10653137

  11. Microbial food safety: Potential of DNA extraction methods for use in diagnostic metagenomics.

    PubMed

    Josefsen, Mathilde H; Andersen, Sandra C; Christensen, Julia; Hoorfar, Jeffrey

    2015-07-01

    The efficiency of ten widely applied DNA extraction protocols was evaluated for suitability for diagnostic metagenomics. The protocols were selected based on a thorough literature study. Chicken fecal samples inoculated with about 1×10(3) and 1×10(6) CFU/g Campylobacter jejuni were used as a model. The evaluation was performed based on total DNA yield measured by fluorometry, and quality and quantity of C. jejuni DNA measured by real-time PCR. There was up to a 25-fold variance between the lowest (NucliSens miniMAG, BIOMÉRIEUX) and highest (PowerLyzer PowerSoil DNA Isolation Kit, MO BIO Laboratories) yielding protocols. The PowerLyzer PowerSoil DNA Isolation Kit performed significantly better than all other protocols tested. Selected protocols were modified, i.e., extended heating and homogenization, resulting in increased yields of total DNA. For QIAamp Fast DNA Stool Mini Kit (Qiagen) a 7-fold increase in total DNA was observed following the protocol for human DNA analysis and including a 5 min heating step at 70°C. For the PowerLyzer PowerSoil and the PowerFecal DNA Isolation Kit (MO BIO Laboratories) the total DNA fold increase was 1.6 to 1.8 when including an extra 10 min of bead-vortexing. There was no correlation between the yield of total DNA and the amount of PCR-amplifiable DNA from C. jejuni. The protocols resulting in the highest yield of total DNA did not show correspondingly increased levels of C. jejuni DNA as determined by PCR. In conclusion, substantial variation in the efficiency of the protocols to extract DNA was observed. The highest DNA yield was obtained with the PowerLyzer PowerSoil DNA Isolation Kit, whereas the FastDNA SPIN Kit for Feces (MP Biomedicals) resulted in the highest amount of PCR-amplifiable C. jejuni DNA. PMID:25937085

  12. A comparison of DNA extraction methods using Petunia hybrida tissues.

    PubMed

    Tamari, Farshad; Hinkley, Craig S; Ramprashad, Naderia

    2013-09-01

    Extraction of DNA from plant tissue is often problematic, as many plants contain high levels of secondary metabolites that can interfere with downstream applications, such as the PCR. Removal of these secondary metabolites usually requires further purification of the DNA using organic solvents or other toxic substances. In this study, we have compared two methods of DNA purification: the cetyltrimethylammonium bromide (CTAB) method that uses the ionic detergent hexadecyltrimethylammonium bromide and chloroform-isoamyl alcohol and the Edwards method that uses the anionic detergent SDS and isopropyl alcohol. Our results show that the Edwards method works better than the CTAB method for extracting DNA from tissues of Petunia hybrida. For six of the eight tissues, the Edwards method yielded more DNA than the CTAB method. In four of the tissues, this difference was statistically significant, and the Edwards method yielded 27-80% more DNA than the CTAB method. Among the different tissues tested, we found that buds, 4 days before anthesis, had the highest DNA concentrations and that buds and reproductive tissue, in general, yielded higher DNA concentrations than other tissues. In addition, DNA extracted using the Edwards method was more consistently PCR-amplified than that of CTAB-extracted DNA. Based on these results, we recommend using the Edwards method to extract DNA from plant tissues and to use buds and reproductive structures for highest DNA yields. PMID:23997658

  13. A DNA extraction protocol for improved DNA yield from individual mosquitoes.

    PubMed

    Nieman, Catelyn C; Yamasaki, Youki; Collier, Travis C; Lee, Yoosook

    2015-01-01

    Typical DNA extraction protocols from commercially available kits provide an adequate amount of DNA from a single individual mosquito sufficient for PCR-based assays. However, next-generation sequencing applications and high-throughput SNP genotyping assays exposed the limitation of DNA quantity one usually gets from a single individual mosquito. Whole genome amplification could alleviate the issue but it also creates bias in genome representation. While trying to find alternative DNA extraction protocols for improved DNA yield, we found that a combination of the tissue lysis protocol from Life Technologies and the DNA extraction protocol from Qiagen yielded a higher DNA amount than the protocol using the Qiagen or Life Technologies kit only. We have not rigorously tested all the possible combinations of extraction protocols; we also only tested this on mosquito samples. Therefore, our finding should be noted as a suggestion for improving people's own DNA extraction protocols and not as an advertisement of a commercially available product. PMID:26937269

  14. One-stop Genomic DNA Extraction by Salicylic Acid Coated Magnetic Nanoparticles

    PubMed Central

    Zhou, Zhongwu; Kadam, Ulhas; Irudayaraj, Joseph

    2014-01-01

    Salicylic acid coated magnetic nanoparticles were prepared via a modified, one-step synthesis and used for a one-stop extraction of genomic DNA from mammalian cells. The synthesized magnetic particles were used for magnetic separation of cells from the media by non-specific binding of the particles, as well as extraction of genomic DNA from the lysate. The quantity and quality were confirmed by agarose gel electrophoresis and polymerase chain reaction. The entire process of extraction and isolation can be completed within 30 min. Compared to traditional methods based on centrifugation and filtration, the established method is fast, simple, reliable, and environmentally-friendly. PMID:23911528

  15. Theoretical investigation on the performance of DNA electrophoresis under programmed step electric field strength: Two-step condition.

    PubMed

    Ni, Yi; Liu, Chenchen; Chen, Qinmiao; Zhu, Xifang; Dou, Xiaoming

    2015-10-01

    Programmed step electric field strength is a simple-to-use technique that has already been reported to be effective to enhance the efficiency or speed of DNA electrophoresis. However, a global understanding and the details of this technique are still vague. In this paper, we investigated the influence of programmed step electric field strength by theoretical calculation and concentrated on a basic format named as two-step electric field strength. Both subtypes of two-step electric field strength conditions were considered. The important parameters, such as peak spacing, peak width, resolution, and migration time, were calculated in theory to understand the performance of DNA electrophoresis under programmed step electric field strength. The influence of two-step electric field strength on DNA electrophoresis was clearly revealed on a diagram of resolution versus migration time. Both resolution and speed of DNA electrophoresis under two-step electric field strength conditions are simply expressed by the shape of curves in the diagram. The possible shapes of curve were explored by calculation and shown in this paper. The subtype II of two-step electric field strength brings drastic variation on the resolution. Its limitations of enhancement and deterioration of resolution were predicted in theory. PMID:26289302

  16. Extraction of high molecular weight DNA from microbial mats.

    PubMed

    Bey, Benjamin S; Fichot, Erin B; Norman, R Sean

    2011-01-01

    Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes. The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 μg of HMW DNA (35-50 kb) per gram of mat sample, with an A(260/280) ratio of 1.6. Furthermore, amplification of 16S rRNA genes suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging. PMID:21775955

  17. [DNA extraction methods of compost for molecular ecology analysis].

    PubMed

    Yang, Zhao-Hui; Xiao, Yong; Zeng, Guang-Ming; Liu, Yun-Guo; Deng, Jiu-Hua

    2006-08-01

    Molecular ecology provides new techniques for studying compost microbes, and the DNA extraction is the basis of molecular techniques. Because of the contamination of humic acids, it turns to be more difficult for compost microbial DNA extraction. Three different approaches, named as lysozyme lysis, ultrasonic lysis and proteinase K lysis with CTAB, were used to extract the total DNA from compost. The detection performed on a nucleic acids and protein analyzer showed that all the three approaches produced high DNA yields. The agarose gel electrophoresis showed that the DNA fragments extracted from compost had a length of about 23 kb. A eubacterial 16S rRNA gene targeted primer pair (27F and 1 495R) was used for PCR amplification, and all the samples got almost the full length 16S rDNA sequence (about 1.5 kb). After digested by restriction endonucleases (Hae Ill and Alu I), the restriction map showed relatively identical microbial diversity in the DNA, which was extracted by the three different approaches. All the compost microbial DNA extracted by the three different approaches could be used for molecular ecological study, and researchers should choose the right approach for extracting microbial DNA from compost based on the facts. PMID:17111621

  18. A simple Chelex protocol for DNA extraction from Anopheles spp.

    PubMed

    Musapa, Mulenga; Kumwenda, Taida; Mkulama, Mtawa; Chishimba, Sandra; Norris, Douglas E; Thuma, Philip E; Mharakurwa, Sungano

    2013-01-01

    Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens. We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km(2) vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 μl of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 μl was retained while the other 10 μl was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol(9,10). The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction(9). Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality, a PCR for identification of Anopheles gambiae sibling species(10) and a nested PCR for typing of Plasmodium falciparum infection

  19. Microwave-accelerated method for ultra-rapid extraction of Neisseria gonorrhoeae DNA for downstream detection.

    PubMed

    Melendez, Johan H; Santaus, Tonya M; Brinsley, Gregory; Kiang, Daniel; Mali, Buddha; Hardick, Justin; Gaydos, Charlotte A; Geddes, Chris D

    2016-10-01

    Nucleic acid-based detection of gonorrhea infections typically require a two-step process involving isolation of the nucleic acid, followed by detection of the genomic target often involving polymerase chain reaction (PCR)-based approaches. In an effort to improve on current detection approaches, we have developed a unique two-step microwave-accelerated approach for rapid extraction and detection of Neisseria gonorrhoeae (gonorrhea, GC) DNA. Our approach is based on the use of highly focused microwave radiation to rapidly lyse bacterial cells, release, and subsequently fragment microbial DNA. The DNA target is then detected by a process known as microwave-accelerated metal-enhanced fluorescence (MAMEF), an ultra-sensitive direct DNA detection analytical technique. In the current study, we show that highly focused microwaves at 2.45 GHz, using 12.3-mm gold film equilateral triangles, are able to rapidly lyse both bacteria cells and fragment DNA in a time- and microwave power-dependent manner. Detection of the extracted DNA can be performed by MAMEF, without the need for DNA amplification, in less than 10 min total time or by other PCR-based approaches. Collectively, the use of a microwave-accelerated method for the release and detection of DNA represents a significant step forward toward the development of a point-of-care (POC) platform for detection of gonorrhea infections. PMID:27325503

  20. Preparation of DNA-containing extract for PCR amplification

    DOEpatents

    Dunbar, John M.; Kuske, Cheryl R.

    2006-07-11

    Environmental samples typically include impurities that interfere with PCR amplification and DNA quantitation. Samples of soil, river water, and aerosol were taken from the environment and added to an aqueous buffer (with or without detergent). Cells from the sample are lysed, releasing their DNA into the buffer. After removing insoluble cell components, the remaining soluble DNA-containing extract is treated with N-phenacylthiazolium bromide, which causes rapid precipitation of impurities. Centrifugation provides a supernatant that can be used or diluted for PCR amplification of DNA, or further purified. The method may provide a DNA-containing extract sufficiently pure for PCR amplification within 5–10 minutes.

  1. Comparison of microvolume DNA quantification methods for use with volume-sensitive environmental DNA extracts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Accurate DNA concentration estimates from environmental samples using minimal sample volumes are essential for most downstream applications. To compare the efficacy of microvolume quantification methods, DNA was extracted from soil, compost, and pure culture samples, and quantified using two absorba...

  2. Extraction of high molecular weight DNA from microbial mats.

    PubMed

    Bey, Benjamin S; Fichot, Erin B; Dayama, Gargi; Decho, Alan W; Norman, R Sean

    2010-09-01

    Due to the presence of inhibitors such as extracellular polymeric substances (EPSs) and salts, most microbial mat studies have relied on harsh methods of direct DNA extraction that result in DNA fragments too small for large-insert vector cloning. High molecular weight (HMW) DNA is crucial in functional metagenomic studies, because large fragments present greater access to genes of interest. Here we report improved methodologies for extracting HMW DNA from EPS-rich hypersaline microbial mats. The protocol uses a combination of microbial cell separation with mechanical and chemical methods for DNA extraction and purification followed by precipitation with polyethylene glycol (PEG). The protocol yields >2 µg HMW DNA (>48 kb) per gram of mat sample, with A260:280 ratios >1.7. In addition, 16S rRNA gene analysis using denaturing gradient gel electrophoresis and pyrosequencing showed that this protocol extracts representative DNA from microbial mat communities and results in higher overall calculated diversity indices compared with three other standard methods of DNA extraction. Our results show the importance of validating the DNA extraction methods used in metagenomic studies to ensure optimal recovery of microbial richness. PMID:20854264

  3. DNA, RNA, and Protein Extraction: The Past and The Present

    PubMed Central

    Tan, Siun Chee; Yiap, Beow Chin

    2009-01-01

    Extraction of DNA, RNA, and protein is the basic method used in molecular biology. These biomolecules can be isolated from any biological material for subsequent downstream processes, analytical, or preparative purposes. In the past, the process of extraction and purification of nucleic acids used to be complicated, time-consuming, labor-intensive, and limited in terms of overall throughput. Currently, there are many specialized methods that can be used to extract pure biomolecules, such as solution-based and column-based protocols. Manual method has certainly come a long way over time with various commercial offerings which included complete kits containing most of the components needed to isolate nucleic acid, but most of them require repeated centrifugation steps, followed by removal of supernatants depending on the type of specimen and additional mechanical treatment. Automated systems designed for medium-to-large laboratories have grown in demand over recent years. It is an alternative to labor-intensive manual methods. The technology should allow a high throughput of samples; the yield, purity, reproducibility, and scalability of the biomolecules as well as the speed, accuracy, and reliability of the assay should be maximal, while minimizing the risk of cross-contamination. PMID:20011662

  4. One-step cloning and chromosomal integration of DNA.

    PubMed

    St-Pierre, François; Cui, Lun; Priest, David G; Endy, Drew; Dodd, Ian B; Shearwin, Keith E

    2013-09-20

    We describe "clonetegration", a method for integrating DNA into prokaryotic chromosomes that approaches the simplicity of cloning DNA within extrachromosomal vectors. Compared to existing techniques, clonetegration drastically decreases the time and effort needed for integration of single or multiple DNA fragments. Additionally, clonetegration facilitates cloning and expression of genetic elements that are impossible to propagate within typical multicopy plasmids. PMID:24050148

  5. Stepped electrophoresis for movement and concentration of DNA

    DOEpatents

    Miles, Robin R.; Wang, Amy Wei-Yun; Mariella, Jr., Raymond P.

    2005-03-15

    A fluidic channel patterned with a series of thin-film electrodes makes it possible to move and concentrate DNA in a fluid passing through the fluidic channel. The DNA has an inherent negative charge and by applying a voltage between adjacent electrodes the DNA is caused to move. By using a series of electrodes, when one electrode voltage or charge is made negative with respect to adjacent electrodes, the DNA is repelled away from this electrode and attached to a positive charged electrode of the series. By sequentially making the next electrode of the series negative, the DNA can be moved to and concentrated over the remaining positive electrodes.

  6. Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR.

    PubMed

    Fredricks, David N; Smith, Caitlin; Meier, Amalia

    2005-10-01

    The detection of fungal pathogens in clinical samples by PCR requires the use of extraction methods that efficiently lyse fungal cells and recover DNA suitable for amplification. We used quantitative PCR assays to measure the recovery of DNA from two important fungal pathogens subjected to six DNA extraction methods. Aspergillus fumigatus conidia or Candida albicans yeast cells were added to bronchoalveolar lavage fluid and subjected to DNA extraction in order to assess the recovery of DNA from a defined number of fungal propagules. In order to simulate hyphal growth in tissue, Aspergillus fumigatus conidia were allowed to form mycelia in tissue culture media and then harvested for DNA extraction. Differences among the DNA yields from the six extraction methods were highly significant (P<0.0001) in each of the three experimental systems. An extraction method based on enzymatic lysis of fungal cell walls (yeast cell lysis plus the use of GNOME kits) produced high levels of fungal DNA with Candida albicans but low levels of fungal DNA with Aspergillus fumigatus conidia or hyphae. Extraction methods employing mechanical agitation with beads produced the highest yields with Aspergillus hyphae. The Master Pure yeast method produced high levels of DNA from C. albicans but only moderate yields from A. fumigatus. A reagent from one extraction method was contaminated with fungal DNA, including DNA from Aspergillus and Candida species. In conclusion, the six extraction methods produce markedly differing yields of fungal DNA and thus can significantly affect the results of fungal PCR assays. No single extraction method was optimal for all organisms. PMID:16207973

  7. Genomic DNA extraction from cells by electroporation on an integrated microfluidic platform

    PubMed Central

    Geng, Tao; Bao, Ning; Sriranganathanw, Nammalwar; Li, Liwu; Lu, Chang

    2012-01-01

    The vast majority of genetic analysis of cells involves chemical lysis for release of DNA molecules. However, chemical reagents required in the lysis interfere with downstream molecular biology and often require removal after the step. Electrical lysis based on irreversible electroporation is a promising technique to prepare samples for genetic analysis due to its purely physical nature, fast speed, and simple operation. However, there has been no experimental confirmation on whether electrical lysis extracts genomic DNA from cells in a reproducible and efficient fashion in comparison to chemical lysis, especially for eukaryotic cells that have most of DNA enclosed in the nucleus. In this work, we construct an integrated microfluidic chip that physically traps a low number of cells, lyses the cells using electrical pulses rapidly, then purifies and concentrates genomic DNA. We demonstrate that electrical lysis offers high efficiency for DNA extraction from both eukaryotic cells (up to ~36% for Chinese hamster ovary cells) and bacterial cells (up to ~45% for Salmonella typhimurium) that is comparable to the widely-used chemical lysis. The DNA extraction efficiency has dependence on both electric parameters and relative amount of beads used for DNA adsorption. We envision that electroporation-based DNA extraction will find use in ultrasensitive assays that benefit from minimal dilution and simple procedure. PMID:23061629

  8. The influence of substrate on DNA transfer and extraction efficiency.

    PubMed

    Verdon, Timothy J; Mitchell, R John; van Oorschot, Roland A H

    2013-01-01

    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

  9. Hot-Alkaline DNA Extraction Method for Deep-Subseafloor Archaeal Communities

    PubMed Central

    Terada, Takeshi; Hoshino, Tatsuhiko; Inagaki, Fumio

    2014-01-01

    A prerequisite for DNA-based microbial community analysis is even and effective cell disruption for DNA extraction. With a commonly used DNA extraction kit, roughly two-thirds of subseafloor sediment microbial cells remain intact on average (i.e., the cells are not disrupted), indicating that microbial community analyses may be biased at the DNA extraction step, prior to subsequent molecular analyses. To address this issue, we standardized a new DNA extraction method using alkaline treatment and heating. Upon treatment with 1 M NaOH at 98°C for 20 min, over 98% of microbial cells in subseafloor sediment samples collected at different depths were disrupted. However, DNA integrity tests showed that such strong alkaline and heat treatment also cleaved DNA molecules into short fragments that could not be amplified by PCR. Subsequently, we optimized the alkaline and temperature conditions to minimize DNA fragmentation and retain high cell disruption efficiency. The best conditions produced a cell disruption rate of 50 to 80% in subseafloor sediment samples from various depths and retained sufficient DNA integrity for amplification of the complete 16S rRNA gene (i.e., ∼1,500 bp). The optimized method also yielded higher DNA concentrations in all samples tested compared with extractions using a conventional kit-based approach. Comparative molecular analysis using real-time PCR and pyrosequencing of bacterial and archaeal 16S rRNA genes showed that the new method produced an increase in archaeal DNA and its diversity, suggesting that it provides better analytical coverage of subseafloor microbial communities than conventional methods. PMID:24441163

  10. Extracting DNA of nematodes communities from Argentine Pampas agricultural soils.

    PubMed

    Mondino, Eduardo A; Covacevich, Fernanda; Studdert, Guillermo A; Pimentel, João P; Berbara, Ricardo L L

    2015-01-01

    We examined four strategies (Tris/EDTA, sodium dodecyl sulfate, Chelex 100 resin and cetyltrimethylammonium bromide -CTAB-) for extracting nucleic acid (DNA) from communities of nematodes. Nematodes were isolated from an agricultural area under different management of long-term crop rotation experiment from Argentina during three seasons. After DNA extraction, Polymerase Chain Reaction-amplifications were performed and considered as indicators of successful DNA extraction. The CTAB combined with proteinase K and phenol-chloroform-isoamyl alcohol was the unique successful method because positive amplifications were obtained by using both eukaryotic and nematode specific primers. This work could contribute to biodiversity studies of nematodes on agroecosystems. PMID:26131632

  11. Optimization of DNA Extraction from Deep-sea Basalt

    NASA Astrophysics Data System (ADS)

    Wang, H.; Edwards, K. J.

    2007-12-01

    Studies on the microorganisms that inhabit deep-sea basalt can provide information on this dark ecosystem, which will contribution to our understanding of mass transformation and energy flow in the deep ocean. However, molecular methods for use with metal- and clay-rich rock materials such as basalt have not been suitably developed at present, yet are critically required in order to be able to fully evaluate the basalt biotope. For example, inefficient DNA extraction might lead to loss of information about important components of this community, and misinterpretation about the total community diversity and function. In order to investigate the effects of sample pretreated method, particle size, different DNA extraction methods and cell density on extracted DNA yields, two basalt samples were collected from the East Pacific Rise 9° N during research cruise AT11- 20 in Nov 2004. Basalt samples were crushed to different particle size, washed with ddH2O and 100% ethanol respectively, and autoclaved. Marinobacter aquaeolei cultures with different cell densities were inoculated into differently treated basalt samples. Pure culture and basalt samples without inoculation were used as positive and negative control to evaluate the extracting efficiency. FastDNA spin for soil kit, GeneClean for ancient DNA kit and UltraCleanTM soil DNA Kit are used for DNA extraction. Results showed that DNA yields increased with culture density. FastDNA spin for soil kit gave the highest DNA yields, which is almost 10 times more than that of UltraCleanTM soil DNA Kit. Ethanol washing and ddH2O washing did not make big difference to DNA yields. Mineral composition and surface areas might also affect DNA yields.

  12. Extraction of DNA from plant and fungus tissues in situ

    PubMed Central

    2012-01-01

    Background When samples are collected in the field and transported to the lab, degradation of the nucleic acids contained in the samples is frequently observed. Immediate extraction and precipitation of the nucleic acids reduces degradation to a minimum, thus preserving accurate sequence information. An extraction method to obtain high quality DNA in field studies is described. Findings DNA extracted immediately after sampling was compared to DNA extracted after allowing the sampled tissues to air dry at 21°C for 48 or 72 hours. While DNA extracted from fresh tissues exhibited little degradation, DNA extracted from all tissues exposed to 21°C air for 48 or 72 hours exhibited varying degrees of degradation. Yield was higher for extractions from fresh tissues in most cases. Four microcentrifuges were compared for DNA yield: one standard electric laboratory microcentrifuge (max rcf = 16,000×g), two battery-operated microcentrifuges (max rcf = 5,000 and 3,000 ×g), and one manually-operated microcentrifuge (max rcf = 120×g). Yields for all centrifuges were similar. DNA extracted under simulated field conditions was similar in yield and quality to DNA extracted in the laboratory using the same equipment. Conclusions This CTAB (cetyltrimethylammonium bromide) DNA extraction method employs battery-operated and manually-operated equipment to isolate high quality DNA in the field. The method was tested on plant and fungus tissues, and may be adapted for other types of organisms. The method produced high quality DNA in laboratory tests and under simulated field conditions. The field extraction method should prove useful for working in remote sites, where ice, dry ice, and liquid nitrogen are unavailable; where degradation is likely to occur due to the long distances between the sample site and the laboratory; and in instances where other DNA preservation and transportation methods have been unsuccessful. It may be possible to adapt this method for genomic

  13. PCR amplification of crude microbial DNA extracted from soil.

    PubMed

    Yeates, C; Gillings, M R; Davison, A D; Altavilla, N; Veal, D A

    1997-10-01

    A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types. DNA was extracted from 100 g of soil using direct lysis with glass beads and sodium dodecyl sulphate (SDS) followed by polyethylene glycol precipitation, potassium acetate precipitation, phenol extraction and isopropanol precipitation. The crude extract could be used in PCR directed at high-copy number (bacterial small subunit rRNA) and single-copy (fungal beta-tubulin) genes. PMID:9351282

  14. A novel method of genomic DNA extraction for Cactaceae1

    PubMed Central

    Fehlberg, Shannon D.; Allen, Jessica M.; Church, Kathleen

    2013-01-01

    • Premise of the study: Genetic studies of Cactaceae can at times be impeded by difficult sampling logistics and/or high mucilage content in tissues. Simplifying sampling and DNA isolation through the use of cactus spines has not previously been investigated. • Methods and Results: Several protocols for extracting DNA from spines were tested and modified to maximize yield, amplification, and sequencing. Sampling of and extraction from spines resulted in a simplified protocol overall and complete avoidance of mucilage as compared to typical tissue extractions. Sequences from one nuclear and three plastid regions were obtained across eight genera and 20 species of cacti using DNA extracted from spines. • Conclusions: Genomic DNA useful for amplification and sequencing can be obtained from cactus spines. The protocols described here are valuable for any cactus species, but are particularly useful for investigators interested in sampling living collections, extensive field sampling, and/or conservation genetic studies. PMID:25202521

  15. Rapid extraction of genomic DNA from saliva for HLA typing on microarray based on magnetic nanobeads

    NASA Astrophysics Data System (ADS)

    Xie, Xin; Zhang, Xu; Yu, Bingbin; Gao, Huafang; Zhang, Huan; Fei, Weiyang

    2004-09-01

    A series of simplified protocols are developed for extracting genomic DNA from saliva by using the magnetic nanobeads as absorbents. In these protocols, both the enrichment of the target cells and the adsorption of DNA can be achieved simultaneously by our functionally modified magnetic beads in one step, and the DNA-nanobeads complex can be used as PCR templates. HLA typing based on an oligonucleotide array was conducted by hybridization with the PCR products. The result shows that the protocols are robust and sensitive.

  16. Mycobacterial DNA extraction for whole-genome sequencing from early positive liquid (MGIT) cultures.

    PubMed

    Votintseva, Antonina A; Pankhurst, Louise J; Anson, Luke W; Morgan, Marcus R; Gascoyne-Binzi, Deborah; Walker, Timothy M; Quan, T Phuong; Wyllie, David H; Del Ojo Elias, Carlos; Wilcox, Mark; Walker, A Sarah; Peto, Tim E A; Crook, Derrick W

    2015-04-01

    We developed a low-cost and reliable method of DNA extraction from as little as 1 ml of early positive mycobacterial growth indicator tube (MGIT) cultures that is suitable for whole-genome sequencing to identify mycobacterial species and predict antibiotic resistance in clinical samples. The DNA extraction method is based on ethanol precipitation supplemented by pretreatment steps with a MolYsis kit or saline wash for the removal of human DNA and a final DNA cleanup step with solid-phase reversible immobilization beads. The protocol yielded ≥0.2 ng/μl of DNA for 90% (MolYsis kit) and 83% (saline wash) of positive MGIT cultures. A total of 144 (94%) of the 154 samples sequenced on the MiSeq platform (Illumina) achieved the target of 1 million reads, with <5% of reads derived from human or nasopharyngeal flora for 88% and 91% of samples, respectively. A total of 59 (98%) of 60 samples that were identified by the national mycobacterial reference laboratory (NMRL) as Mycobacterium tuberculosis were successfully mapped to the H37Rv reference, with >90% coverage achieved. The DNA extraction protocol, therefore, will facilitate fast and accurate identification of mycobacterial species and resistance using a range of bioinformatics tools. PMID:25631807

  17. EXTRACTION AND PURIFICATION OF MICROBIAL DNA FROM SEDIMENTS

    EPA Science Inventory

    A new method for the isolation of intracellular and extracellular DNA from a range of sediment types has been developed. The method is based upon the direct lysis of cells in the sediment, extraction of released DNA from the sediments and its subsequent purification by CsC1-EtBr ...

  18. Extraction of DNA from the plant Kalanchoë daigremontiana.

    PubMed

    Garcês, Helena; Sinha, Neelima

    2009-10-01

    This protocol describes how to isolate genomic DNA from leaves and stems of Kalanchoë daigremontiana. The procedure can be applied to adult leaves, but it is best to use younger leaves because they have fewer secondary metabolites and polysaccharides, which can interfere with the DNA extraction. The resulting DNA can be used for polymerase chain reactions (PCRs), Southern blots, or other applications. PMID:20147049

  19. A simple and efficient method for PCR amplifiable DNA extraction from ancient bones

    PubMed Central

    Kalmár, Tibor; Bachrati, Csanád Z.; Marcsik, Antónia; Raskó, István

    2000-01-01

    A simple and effective modified ethanol precipitation-based protocol is described for the preparation of DNA from ancient human bones. This method is fast and requires neither hazardous chemicals nor special devices. After the powdering and incubating of the bone samples Dextran Blue was added as a carrier for removing the PCR inhibitors with selective ethanol precipitation. This method could eliminate the time-consuming separate decalcification step, dialysis, application of centrifugation-driven microconcentrators and the second consecutive PCR amplification. The efficiency of this procedure was demonstrated on ten 500–1200-year-old human bones from four different Hungarian burial sites. A mitochondrial specific primer pair was used to obtain sequence information from the purified ancient DNA. The PCR amplification, after our DNA extraction protocol, was successful from each of the 10 bone samples investigated. The results demonstrate that extraction of DNA from ancient bone samples with this new approach increases the success rate of PCR amplification. PMID:10871390

  20. Translocation step size and mechanism of the RecBC DNA helicase.

    PubMed

    Bianco, P R; Kowalczykowski, S C

    2000-05-18

    DNA helicases are ubiquitous enzymes that unwind double-stranded DNA. They are a diverse group of proteins that move in a linear fashion along a one-dimensional polymer lattice--DNA--by using a mechanism that couples nucleoside triphosphate hydrolysis to both translocation and double-stranded DNA unwinding to produce separate strands of DNA. The RecBC enzyme is a processive DNA helicase that functions in homologous recombination in Escherichia coli; it unwinds up to 6,250 base pairs per binding event and hydrolyses slightly more than one ATP molecule per base pair unwound. Here we show, by using a series of gapped oligonucleotide substrates, that this enzyme translocates along only one strand of duplex DNA in the 3'-->5' direction. The translocating enzyme will traverse, or 'step' across, single-stranded DNA gaps in defined steps that are 23 (+/-2) nucleotides in length. This step is much larger than the amount of double-stranded DNA that can be unwound using the free energy derived from hydrolysis of one molecule of ATP, implying that translocation and DNA unwinding are separate events. We propose that the RecBC enzyme both translocates and unwinds by a quantized, two-step, inchworm-like mechanism that may have parallels for translocation by other linear motor proteins. PMID:10830968

  1. Impedimetric DNA detection--steps forward to sensorial application.

    PubMed

    Riedel, Marc; Kartchemnik, Julia; Schöning, Michael J; Lisdat, Fred

    2014-08-01

    This study describes a label-free impedimetric sensor based on short ssDNA recognition elements for the detection of hybridization events. We concentrate on the elucidation of the influence of target length and recognition sequence position on the sensorial performance. The impedimetric measurements are performed in the presence of the redox system ferri-/ferrocyanide and show an increase in charge transfer resistance upon hybridization of ssDNA to the sensor surface. Investigations on the impedimetric signal stability demonstrate a clear influence of the buffers used during the sensor preparation and the choice of the passivating mercaptoalcanol compound. A stable sensor system has been developed, enabling a reproducible detection of 25mer target DNA in the low nanomolar range. After hybridization, a sensor regeneration can be reached with deionized water by adjustment of effective convection conditions, ensuring a sensor reusability. By investigations of longer targets with overhangs exposed to the solution, we can demonstrate applicability of the impedimetric detection for longer ssDNA. However, a decreasing charge transfer resistance change (ΔR(ct)) is found by extending the overhang. As a strategy to increase the impedance change for longer target strands, the position of the recognition sequence can be designed in a way that a small overhang is exposed to the electrode surface. This is found to result in an increase in the relative R(ct) change. These results suggest that DNA and consequently negative charge near the electrode possess a larger impact on the impedimetric signal than DNA further away. PMID:24999077

  2. Evaluation of DNA extraction methods for freshwater eukaryotic microalgae.

    PubMed

    Eland, Lucy E; Davenport, Russell; Mota, Cesar R

    2012-10-15

    The use of molecular methods to investigate microalgal communities of natural and engineered freshwater resources is in its infancy, with the majority of previous studies carried out by microscopy. Inefficient or differential DNA extraction of microalgal community members can lead to bias in downstream community analysis. Three commercially available DNA extraction kits have been tested on a range of pure culture freshwater algal species with diverse cell walls and mixed algal cultures taken from eutrophic waste stabilization ponds (WSP). DNA yield and quality were evaluated, along with DNA suitability for amplification of 18S rRNA gene fragments by polymerase chain reaction (PCR). QiagenDNeasy(®) Blood and Tissue kit (QBT), was found to give the highest DNA yields and quality. Denaturant Gradient Gel Electrophoresis (DGGE) was used to assess the diversity of communities from which DNA was extracted. No significant differences were found among kits when assessing diversity. QBT is recommended for use with WSP samples, a conclusion confirmed by further testing on communities from two tropical WSP systems. The fixation of microalgal samples with ethanol prior to DNA extraction was found to reduce yields as well as diversity and is not recommended. PMID:22853974

  3. High-throughput Method of One-Step DNA Isolation for PCR Diagnostics of Mycobacterium tuberculosis.

    PubMed

    Kapustin, D V; Prostyakova, A I; Alexeev, Ya I; Varlamov, D A; Zubov, V P; Zavriev, S K

    2014-04-01

    The efficiency of one-step and multi-step protocols of DNA isolation from lysed sputum samples containing the Mycobacterium tuberculosis complex has been compared. DNA was isolated using spin-cartridges containing a special silica-based sorbent modified with fluoroplast and polyaniline, or using an automated isolation system. One-step isolation using the obtained sorbent has been shown to ensure a significantly lower DNA loss and higher sensitivity in the PCR detection of Mycobacterium tuberculosis as compared to a system based on sorption and desorption of nucleic acids during the isolation. PMID:25093111

  4. High-throughput DNA extraction of forensic adhesive tapes.

    PubMed

    Forsberg, Christina; Jansson, Linda; Ansell, Ricky; Hedman, Johannes

    2016-09-01

    Tape-lifting has since its introduction in the early 2000's become a well-established sampling method in forensic DNA analysis. Sampling is quick and straightforward while the following DNA extraction is more challenging due to the "stickiness", rigidity and size of the tape. We have developed, validated and implemented a simple and efficient direct lysis DNA extraction protocol for adhesive tapes that requires limited manual labour. The method uses Chelex beads and is applied with SceneSafe FAST tape. This direct lysis protocol provided higher mean DNA yields than PrepFiler Express BTA on Automate Express, although the differences were not significant when using clothes worn in a controlled fashion as reference material (p=0.13 and p=0.34 for T-shirts and button-down shirts, respectively). Through in-house validation we show that the method is fit-for-purpose for application in casework, as it provides high DNA yields and amplifiability, as well as good reproducibility and DNA extract stability. After implementation in casework, the proportion of extracts with DNA concentrations above 0.01ng/μL increased from 71% to 76%. Apart from providing higher DNA yields compared with the previous method, the introduction of the developed direct lysis protocol also reduced the amount of manual labour by half and doubled the potential throughput for tapes at the laboratory. Generally, simplified manual protocols can serve as a cost-effective alternative to sophisticated automation solutions when the aim is to enable high-throughput DNA extraction of complex crime scene samples. PMID:27448236

  5. Patterning of a nanoporous membrane for multi-sample DNA extraction

    NASA Astrophysics Data System (ADS)

    Kim, Jungkyu; Voelkerding, Karl V.; Gale, Bruce K.

    2006-01-01

    A novel method for patterning a nanoporous aluminum oxide membrane was developed using SU-8 to allow for extraction of DNA from multiple samples simultaneously. To facilitate the patterning process, the spin curve for SU-8 2015 on the membrane was determined. The correlation profile of the SU-8 spin curve was similar in form to curves of SU-8 spun on silicon wafers, but with a uniform decrease in thickness across the curve. Characterization of the patterned SU-8 regarding step height and surface roughness was determined using pin-based surface profilometry. The patterned aluminum oxide membrane displayed acceptable flatness and precise pattern replication. Thickness uniformity was also observed to be approximately ±2.8% for a 29.3 µm thick pattern. SEM imaging was used to examine the exposed membrane surface for any visible changes or damage to the membrane caused by the patterning process, with none observed. DNA extraction was performed on the patterned membrane using multiple wells to show the ability of collecting multiple DNA samples simultaneously. DNA, marked with a fluorescent dye, was collected on the membrane and successfully observed using a fluorescence microscope. The techniques developed in this work have application to lab-on-a-chip type systems that include DNA extraction steps.

  6. Microfluidic enzymatic DNA extraction on a hybrid polyester-toner-PMMA device.

    PubMed

    Thompson, Brandon L; Birch, Christopher; Li, Jingyi; DuVall, Jacquelyn A; Le Roux, Delphine; Nelson, Daniel A; Tsuei, An-Chi; Mills, Daniel L; Krauss, Shannon T; Root, Brian E; Landers, James P

    2016-08-01

    To date, the forensic community regards solid phase extraction (SPE) as the most effective methodology for the purification of DNA for use in short tandem repeat (STR) polymerase chain reaction (PCR) amplification. While a dominant methodology, SPE protocols generally necessitate the use of PCR inhibitors (guanidine, IPA) and, in addition, can demand timescales of up to 30 min due to the necessary load, wash and elution steps. The recent discovery and characterization of the EA1 protease has allowed the user to enzymatically extract (not purify) DNA, dramatically simplifying the task of producing a PCR-ready template. Despite this, this procedure has yet to make a significant impact on microfluidic technologies. Here, we describe a microfluidic device that implements the EA1 enzyme for DNA extraction by incorporating it into a hybrid microdevice comprising laminated polyester (Pe) and PMMA layers. The PMMA layer provides a macro-to-micro interface for introducing the biological sample into the microfluidic architecture, whilst also possessing the necessary dimensions to function as the swab acceptor. Pre-loaded reagents are then introduced to the swab chamber centrifugally, initiating DNA extraction at 75 °C. The extraction of DNA occurs in timescales of less than 3 min and any external hardware associated with the transportation of reagents by pneumatic pumping is eliminated. Finally, multiplexing is demonstrated with a circular device containing eight separate chambers for the simultaneous processing of eight buccal swab samples. The studies here provide DNA concentrations up to 10 ng μL(-1) with a 100% success rate in less than 3 minutes. The STR profiles generated using these extracted samples demonstrate that the DNA is of PCR forensic-quality and adequate for human identification. PMID:27250903

  7. Evaluation of DNA extraction methods for Bacillus anthracis spores spiked to food and feed matrices at biosafety level 3 conditions.

    PubMed

    Wielinga, Peter R; de Heer, Lianne; de Groot, Astrid; Hamidjaja, Raditijo A; Bruggeman, Geert; Jordan, Kieran; van Rotterdam, Bart J

    2011-11-01

    The DNA extraction efficiency from milk, whey, soy, corn gluten meal, wheat powders and heat-treated corn grain that were spiked with Bacillus anthracis and Bacillus thuringiensis spores was determined. Two steps were critical: lysis of the spores and binding of the free DNA to the DNA binding magnetic beads in the presence of the interfering powders. For the guanidine-thiocyanate based Nuclisens lysis buffer from Biomerieux we found that between 15 and 30% of the spores survived the lysis step. As most lysis buffers in DNA/RNA extraction kits are guanidine based it is likely that other lysis buffers will show a similar partial lysis of the Bacillus spores. Our results show that soybean flour and wheat flour inhibited the DNA extraction process strongest, leading to unreliable DNA extractions when using too much of the matrix. For corn gluten meal, heat-treated corn grain and milk powders, DNA extraction efficiencies in the presence of 100mg and 10mg of powder resulted in 70%-95% reduced DNA recoveries. The inhibition was, however, reliable and intermediate compared to the inhibition by soy and wheat. Whey powder had the lowest inhibitory effect on DNA-extraction efficiency and recoveries of 70-100% could be reached when using 10mg of powder. The results show that reducing the amount of matrix leads to better DNA-extraction efficiencies, particularly for strongly inhibiting powders such as soy and wheat. Based on these results, a standard protocol to directly isolate DNA from micro-organisms present in complex matrixes such as food and feed powders was designed. PMID:21864928

  8. Comparison of five DNA extraction methods for molecular analysis of Jerusalem artichoke (Helianthus tuberosus).

    PubMed

    Mornkham, T; Wangsomnuk, P P; Wangsomnuk, P; Jogloy, S; Pattanothai, A; Fu, Y B

    2012-01-01

    DNA extraction is an essential step for molecular analysis of an organism, but it is difficult to acquire a sufficient amount of pure DNA from plant tissue with high levels of phenolic compounds, carbohydrates, proteins, and secondary metabolites. Jerusalem artichoke (Helianthus tuberosus) has high levels of such substances. We compared five commonly used methods of extracting genomic DNA in tests made with leaves and seed of four Jerusalem artichoke genotypes: 1) modified method of Tai and Tanksley, 2) method of Doyle and Doyle, 3) method of Porebski, 4) modified method of Štorchová, and 5) Plant DNA Kit of Omega Bio-tek. The quality and quantity of extracted DNAs were assessed by photometric assay, electrophoresis on 1% agarose gel and a PCR-based technique. The modified method of Tai and Tanksley was found to be superior for both young leaves and seed. The quality of the extracted DNA was confirmed by sequence-related amplified polymorphism. This information will be useful for molecular analyses of Jerusalem artichoke and other related Helianthus species. PMID:22535392

  9. Statistical assessment of DNA extraction reagent lot variability in real-time quantitative PCR

    USGS Publications Warehouse

    Bushon, R.N.; Kephart, C.M.; Koltun, G.F.; Francy, D.S.; Schaefer, F. W., III; Lindquist, H.D. Alan

    2010-01-01

    Aims: The aim of this study was to evaluate the variability in lots of a DNA extraction kit using real-time PCR assays for Bacillus anthracis, Francisella tularensis and Vibrio cholerae. Methods and Results: Replicate aliquots of three bacteria were processed in duplicate with three different lots of a commercial DNA extraction kit. This experiment was repeated in triplicate. Results showed that cycle threshold values were statistically different among the different lots. Conclusions: Differences in DNA extraction reagent lots were found to be a significant source of variability for qPCR results. Steps should be taken to ensure the quality and consistency of reagents. Minimally, we propose that standard curves should be constructed for each new lot of extraction reagents, so that lot-to-lot variation is accounted for in data interpretation. Significance and Impact of the Study: This study highlights the importance of evaluating variability in DNA extraction procedures, especially when different reagent lots are used. Consideration of this variability in data interpretation should be an integral part of studies investigating environmental samples with unknown concentrations of organisms. ?? 2010 The Society for Applied Microbiology.

  10. Single-step DNA immobilization on antifouling self-assembled monolayers covalently bound to silicon (111).

    PubMed

    Böcking, Till; Kilian, Kristopher A; Gaus, Katharina; Gooding, J Justin

    2006-04-11

    Hydrosilylation of alkenes with epoxide-terminated tri(ethylene oxide) moieties on Si-H surfaces yields homogeneous monolayers for the efficient coupling of biomolecules. The wetting properties of the epoxide-functionalized surface allow for the spotting of solutions of biomolecules, making the surface amenable to microarraying. Immobilization of thiolated DNA was achieved in a single step to fabricate biorecognition interfaces showing the hybridization of complementary DNA at low concentrations and negligible binding of noncomplementary DNA. PMID:16584219

  11. Microfluidic DNA extraction using a patterned aluminum oxide membrane

    NASA Astrophysics Data System (ADS)

    Kim, Jungkyu; Gale, Bruce K.

    2006-01-01

    A DNA extraction system was designed and fabricated using an AOM (aluminum oxide membrane) with 200 nm pores and PDMS microfluidic channels. The membrane was patterned using soft lithography techniques and SU-8 photolithography on the membrane. After making the pattern with SU-8, the AOM was observed using an SEM (scanning electro microscope) to verify the AOM structure was not damaged. From the SEM images, the AOM structure was not different after modification with SU-8. To complete the system, a PDMS mold for the microfluidic channels was made by soft lithography. Using the SU-8 mold, PDMS microchannels were cast using PDMS with a low polymer to curing agent ratio to provide adhesion between the patterned membrane and microfluidic channel. Then, the patterned membrane was sandwiched between PDMS microfluidic channels in a parallel format. The completed system was tested with 10ug of Lambda DNA mixed with the fluorescent dye SYBR Green I. Following DNA extraction, the surface of each well was examined with fluorescence microscopy while embedded in the microfluidic system. Extracted and immobilized DNA on the AOM was observed in almost every separation well. This microsystem, referred to as a membrane-on-a-chip, has potential applications in high-throughput DNA extraction and analysis, with the possibility of being integrated into polymer-based microfluidic systems.

  12. A simple method for extracting DNA from old skeletal material.

    PubMed

    Cattaneo, C; Smillie, D M; Gelsthorpe, K; Piccinini, A; Gelsthorpe, A R; Sokol, R J

    1995-07-28

    Extraction of DNA from old skeletal material is of great importance in the identification of human remains, but is particularly difficult because the methods currently employed, especially those using phenol/chloroform, are not always satisfactory. A simple technique based on the removal of non-nucleic acid material by salting out (precipitation) with saturated sodium acetate is described; the presence of DNA in the extract being confirmed by amplification of selected sequences of the HLA-DRB1 gene using the polymerase chain reaction (PCR). The method was applied to fresh bone (five femoral heads and six vertebral bodies) and to bone from two forensic cases, 3 and 9 months post-mortem, respectively. Parallel extractions using the phenol/chloroform technique were performed on all samples in order to compare the efficiency of the two methods. Using sodium acetate precipitation, amplifiable DNA was consistently extracted from fresh bone, as well as from the two forensic cases. With the phenol/chloroform method, amplification was successful in only 7 out of 11 instances with the fresh bone samples and failed in both forensic cases. The studies also showed that an effective way of removing PCR inhibitors is to subject the extract to agarose gel electrophoresis, isolate the high molecular weight area and re-extract the DNA from the gel by boiling. It was concluded that the sodium acetate method is a valid alternative to established techniques for extracting DNA from bone and that it offers the advantages of being simple, quick, inexpensive and avoids using hazardous reagents. PMID:7557753

  13. DNA purification using dynamic solid-phase extraction on a rotationally-driven polyethylene-terephthalate microdevice.

    PubMed

    Jackson, K R; Borba, J C; Meija, M; Mills, D L; Haverstick, D M; Olson, K E; Aranda, R; Garner, G T; Carrilho, E; Landers, J P

    2016-09-21

    We report the development of a disposable polyester toner centrifugal device for semi-automated, dynamic solid phase DNA extraction (dSPE) from whole blood samples. The integration of a novel adhesive and hydrophobic valving with a simple and low cost microfabrication method allowed for sequential addition of reagents without the need for external equipment for fluid flow control. The spin-dSPE method yielded an average extraction efficiency of ∼45% from 0.6 μL of whole blood. The device performed single sample extractions or accommodate up to four samples for simultaneous DNA extraction, with PCR-readiness DNA confirmed by effective amplification of a β-globin gene. The purity of the DNA was challenged by a multiplex amplification with 16 targeted amplification sites. Successful multiplexed amplification could routinely be obtained using the purified DNA collected post an on-chip extraction, with the results comparable to those obtained with commercial DNA extraction methods. This proof-of-principle work represents a significant step towards a fully-automated low cost DNA extraction device. PMID:27590539

  14. Inter-laboratory evaluation of the ISO standard 11063 "Soil quality - Method to directly extract DNA from soil samples".

    PubMed

    Petric, I; Philippot, L; Abbate, C; Bispo, A; Chesnot, T; Hallin, S; Laval, K; Lebeau, T; Lemanceau, P; Leyval, C; Lindström, K; Pandard, P; Romero, E; Sarr, A; Schloter, M; Simonet, P; Smalla, K; Wilke, B-M; Martin-Laurent, F

    2011-03-01

    Extracting DNA directly from micro-organisms living in soil is a crucial step for the molecular analysis of soil microbial communities. However, the use of a plethora of different soil DNA extraction protocols, each with its own bias, makes accurate data comparison difficult. To overcome this problem, a method for soil DNA extraction was proposed to the International Organization for Standardization (ISO) in 2006. This method was evaluated by 13 independent European laboratories actively participating in national and international ring tests. The reproducibility of the standardized method for molecular analyses was evaluated by comparing the amount of DNA extracted, as well as the abundance and genetic structure of the total bacterial community in the DNA extracted from 12 different soils by the 13 laboratories. High quality DNA was successfully extracted from all 12 soils, despite different physical and chemical characteristics and a range of origins from arable soils, through forests to industrial sites. Quantification of the 16S rRNA gene abundances by real time PCR and analysis of the total bacterial community structure by automated ribosomal intergenic spacer analysis (A-RISA) showed acceptable to good levels of reproducibility. Based on the results of both ring-tests, the method was unanimously approved by the ISO as an international standard method and the normative protocol will now be disseminated within the scientific community. Standardization of a soil DNA extraction method will improve data comparison, facilitating our understanding of soil microbial diversity and soil quality monitoring. PMID:21256879

  15. Application of the NucliSENS easyMAG system for nucleic acid extraction: optimization of DNA extraction for molecular diagnosis of parasitic and fungal diseases.

    PubMed

    Jeddi, Fakhri; Piarroux, Renaud; Mary, Charles

    2013-01-01

    During the last 20 years, molecular biology techniques have propelled the diagnosis of parasitic diseases into a new era, as regards assay speed, sensitivity, and parasite characterization. However, DNA extraction remains a critical step and should be adapted for diagnostic and epidemiological studies. The aim of this report was to document the constraints associated with DNA extraction for the diagnosis of parasitic diseases and illustrate the adaptation of an automated extraction system, NucliSENS easyMAG, to these constraints, with a critical analysis of system performance. Proteinase K digestion of samples is unnecessary with the exception of solid tissue preparation. Mechanically grinding samples prior to cell lysis enhances the DNA extraction rate of fungal cells. The effect of host-derived nucleic acids on the extraction efficiency of parasite DNA varies with sample host cell density. The optimal cell number for precise parasite quantification ranges from 10 to 100,000 cells. Using the NucliSENS easyMAG technique, the co-extraction of inhibitors is reduced, with an exception for whole blood, which requires supplementary extraction steps to eliminate inhibitors. PMID:24331004

  16. Toenails as an alternative source material for the extraction of DNA from decomposed human remains.

    PubMed

    Schlenker, Andrew; Grimble, Katelyn; Azim, Arani; Owen, Rebecca; Hartman, Dadna

    2016-01-01

    The DNA identification of decomposed human remains for coronial investigations at the Victorian Institute of Forensic Medicine routinely requires the retrieval and processing of a bone sample obtained from the deceased. Bone is a difficult sample type to work with as it requires surgical removal from the deceased, refrigerated storage, and additional processing steps prior to DNA analysis in comparison to other samples types such as buccal swabs or blood stains. In an attempt to overcome the issues posed by bone, a DNA extraction method utilising toenails as an alternate source material was optimised and trialled. Two DNA extraction methods were optimised for digestion of toenail material, with the method utilising the QIAGEN DNA Investigator Kit selected for a casework trial. Single source DNA profiles, matching those of the conventional samples taken, were obtained for toenail samples collected from 28 of 30 coronial cases available for this study. Of these, 26 toenail samples produced full profiles. Although the overall DNA profile quality from the toenails was less than that of the conventional sample, the profiles from toenails met the reporting requirements for identification. Based on the results obtained, the Victorian Institute of Forensic Medicine will be implementing toenails as the primary sample type for collection from decomposed remains when blood is not a suitable sample type. PMID:26610200

  17. Cell lysis and DNA extraction in microfabricated devices

    NASA Astrophysics Data System (ADS)

    Prinz, Christelle; Tegenfeldt, Jonas; Austin, Robert

    2002-03-01

    We are developing a microfabricated device to lyse single cells and extract the DNA. The chip consists of two parts: a diffuse mixer combined with a dielectrophoretic trap. We are working with E. coli which have been made osmoticaly unstable before loading into the chip. The cells are lysed by osmotic shock in the mixer. The lysate is then passed to the dielectrophoretic trap. Attempts to separate the genomic DNA from the lysate fragments by selectively trapping the DNA using dielectrophoresis have been made. We have encountered cell sticking problems and are investingating surface modifications using Polyethylene glycol to solve this problem.

  18. Development of a fast DNA extraction method for sea food and marine species identification.

    PubMed

    Tagliavia, Marcello; Nicosia, Aldo; Salamone, Monica; Biondo, Girolama; Bennici, Carmelo Daniele; Mazzola, Salvatore; Cuttitta, Angela

    2016-07-15

    The authentication of food components is one of the key issues in food safety. Similarly taxonomy, population and conservation genetics as well as food web structure analysis, also rely on genetic analyses including the DNA barcoding technology. In this scenario we developed a fast DNA extraction method without any purification step from fresh and processed seafood, suitable for any PCR analysis. The protocol allows the fast DNA amplification from any sample, including fresh, stored and processed seafood and from any waste of industrial fish processing, independently of the sample storage method. Therefore, this procedure is particularly suitable for the fast processing of samples and to carry out investigations for the authentication of seafood by means of DNA analysis. PMID:26948627

  19. Method of preparing an equimolar DNA mixture for one-step DNA assembly of over 50 fragments.

    PubMed

    Tsuge, Kenji; Sato, Yukari; Kobayashi, Yuka; Gondo, Maiko; Hasebe, Masako; Togashi, Takashi; Tomita, Masaru; Itaya, Mitsuhiro

    2015-01-01

    In the era of synthetic biology, techniques for rapidly constructing a designer long DNA from short DNA fragments are desired. To realize this, we attempted to establish a method for one-step DNA assembly of unprecedentedly large numbers of fragments. The basic technology is the Ordered Gene Assembly in Bacillus subtilis (OGAB) method, which uses the plasmid transformation system of B. subtilis. Since this method doesn't require circular ligation products but needs tandem repeat ligation products, the degree of deviation in the molar concentration of the material DNAs is the only determinant that affects the efficiency of DNA assembly. The strict standardization of the size of plasmids that clone the DNA block and the measurement of the block in the state of intact plasmid improve the reliability of this step, with the coefficient of variation of the molar concentrations becoming 7%. By coupling this method with the OGAB method, one-step assembly of more than 50 DNA fragments becomes feasible. PMID:25990947

  20. Method of preparing an equimolar DNA mixture for one-step DNA assembly of over 50 fragments

    PubMed Central

    Tsuge, Kenji; Sato, Yukari; Kobayashi, Yuka; Gondo, Maiko; Hasebe, Masako; Togashi, Takashi; Tomita, Masaru; Itaya, Mitsuhiro

    2015-01-01

    In the era of synthetic biology, techniques for rapidly constructing a designer long DNA from short DNA fragments are desired. To realize this, we attempted to establish a method for one-step DNA assembly of unprecedentedly large numbers of fragments. The basic technology is the Ordered Gene Assembly in Bacillus subtilis (OGAB) method, which uses the plasmid transformation system of B. subtilis. Since this method doesn’t require circular ligation products but needs tandem repeat ligation products, the degree of deviation in the molar concentration of the material DNAs is the only determinant that affects the efficiency of DNA assembly. The strict standardization of the size of plasmids that clone the DNA block and the measurement of the block in the state of intact plasmid improve the reliability of this step, with the coefficient of variation of the molar concentrations becoming 7%. By coupling this method with the OGAB method, one-step assembly of more than 50 DNA fragments becomes feasible. PMID:25990947

  1. A method suitable for DNA extraction from humus-rich soil.

    PubMed

    Miao, Tianjin; Gao, Song; Jiang, Shengwei; Kan, Guoshi; Liu, Pengju; Wu, Xianming; An, Yingfeng; Yao, Shuo

    2014-11-01

    A rapid and convenient method for extracting DNA from soil is presented. Soil DNA is extracted by direct cell lysis in the presence of EDTA, SDS, phenol, chloroform and isoamyl alcohol (3-methyl-1-butanol) followed by precipitation with 2-propanol. The extracted DNA is purified by modified DNA purification kit and DNA gel extraction kit. With this method, DNA extracted from humus-rich dark brown forest soil was free from humic substances and, therefore, could be used for efficient PCR amplification and restriction digestion. In contrast, DNA sample extracted with the traditional CTAB-based method had lower yield and purity, and no DNA could be extracted from the same soil sample with a commonly-used commercial soil DNA isolation kit. In addition, this method is time-saving and convenient, providing an efficient choice especially for DNA extraction from humus-rich soils. PMID:24980851

  2. Impact of the DNA extraction method on 2-LTR DNA circle recovery from HIV-1 infected cells

    PubMed Central

    Badralmaa, Yunden; Natarajan, Ven

    2013-01-01

    Detection of episomal 2-LTR DNA circles is used as a marker for the ongoing virus replication in patients infected with HIV-1, and efficient extraction of episomal DNA is critical for accurate estimation of the 2-LTR circles. The impact of different methods of DNA extraction on the recovery of 2-LTR circles was compared using mitochondrial DNA extracted as an internal control. The bacterial plasmid DNA isolation method extracted less than 10% of cellular DNA, 40% of mitochondrial DNA and 12-20 % of the input 2-LTR DNA. The total DNA isolation method recovered about 70% of mitochondrial DNA and 45% of the input 2-LTR DNA. The total nucleic acid isolation method recovered 90% of mitochondrial DNA and 60% of the input 2-LTR DNA. Similar results were obtained when the DNA was extracted from HIV-1 infected cells. Plasmid DNA isolation could not distinguish between 12 and 25 copies of 2-LTR DNA per million cells, whereas the total nucleic acid isolation showed a consistent and statistically significant difference between 12 and 25 copies. In conclusion, the total nucleic acid isolation method is more efficient than the plasmid DNA isolation method in recovering mitochondrial DNA and 2-LTR DNA circles from HIV-1 infected cells. PMID:23773807

  3. A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types

    PubMed Central

    Lever, Mark A.; Torti, Andrea; Eickenbusch, Philip; Michaud, Alexander B.; Šantl-Temkiv, Tina; Jørgensen, Bo Barker

    2015-01-01

    A method for the extraction of nucleic acids from a wide range of environmental samples was developed. This method consists of several modules, which can be individually modified to maximize yields in extractions of DNA and RNA or separations of DNA pools. Modules were designed based on elaborate tests, in which permutations of all nucleic acid extraction steps were compared. The final modular protocol is suitable for extractions from igneous rock, air, water, and sediments. Sediments range from high-biomass, organic rich coastal samples to samples from the most oligotrophic region of the world's oceans and the deepest borehole ever studied by scientific ocean drilling. Extraction yields of DNA and RNA are higher than with widely used commercial kits, indicating an advantage to optimizing extraction procedures to match specific sample characteristics. The ability to separate soluble extracellular DNA pools without cell lysis from intracellular and particle-complexed DNA pools may enable new insights into the cycling and preservation of DNA in environmental samples in the future. A general protocol is outlined, along with recommendations for optimizing this general protocol for specific sample types and research goals. PMID:26042110

  4. A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types.

    PubMed

    Lever, Mark A; Torti, Andrea; Eickenbusch, Philip; Michaud, Alexander B; Šantl-Temkiv, Tina; Jørgensen, Bo Barker

    2015-01-01

    A method for the extraction of nucleic acids from a wide range of environmental samples was developed. This method consists of several modules, which can be individually modified to maximize yields in extractions of DNA and RNA or separations of DNA pools. Modules were designed based on elaborate tests, in which permutations of all nucleic acid extraction steps were compared. The final modular protocol is suitable for extractions from igneous rock, air, water, and sediments. Sediments range from high-biomass, organic rich coastal samples to samples from the most oligotrophic region of the world's oceans and the deepest borehole ever studied by scientific ocean drilling. Extraction yields of DNA and RNA are higher than with widely used commercial kits, indicating an advantage to optimizing extraction procedures to match specific sample characteristics. The ability to separate soluble extracellular DNA pools without cell lysis from intracellular and particle-complexed DNA pools may enable new insights into the cycling and preservation of DNA in environmental samples in the future. A general protocol is outlined, along with recommendations for optimizing this general protocol for specific sample types and research goals. PMID:26042110

  5. Filtration Isolation of Nucleic Acids: A Simple and Rapid DNA Extraction Method.

    PubMed

    McFall, Sally M; Neto, Mário F; Reed, Jennifer L; Wagner, Robin L

    2016-01-01

    FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 µl whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets. PMID:27583575

  6. Direct Extraction and Amplification of DNA from Soil.

    ERIC Educational Resources Information Center

    Trevors, Jack T.; Leung, K.

    1998-01-01

    Presents an exercise that describes the direct extraction and purification of DNA from a small soil sample. Also discusses the subsequent amplification of a 343-bp Tn7 transposate A gene fragment (tnsA) from a strain of Pseudomonas aureofaciens 3732RNL11. Contains 21 references. (DDR)

  7. Extraction and Elongation of Genomic DNA from a Single Cell

    NASA Astrophysics Data System (ADS)

    Prinz, Christelle; Tegenfeldt, Jonas; Austin, Robert

    2001-03-01

    We are developing ways to use microfabricated electrode arrays to extract genomic DNA from single E. coli cells and then move the genomic material into an dielectrophoretic trap for clean-up and fractionation. We will present some preliminary data and discuss some of basic polymer physics that impact on these experiments.

  8. Quantitative cell lysis of indigenous microorganisms and rapid extraction of microbial DNA from sediment.

    PubMed Central

    Moré, M I; Herrick, J B; Silva, M C; Ghiorse, W C; Madsen, E L

    1994-01-01

    This study reports improvements in two of the key steps, lysis of indigenous cells and DNA purification, required for achieving a rapid nonselective protocol for extracting nucleic acids directly from sodium dodecyl sulfate (SDS)-treated sediment rich in organic matter. Incorporation of bead-mill homogenization into the DNA extraction procedure doubled the densitometrically determined DNA yield (11.8 micrograms of DNA.g [dry weight] of sediment-1) relative to incorporation of three cycles of freezing and thawing (5.2 micrograms of DNA.g [dry weight] of sediment-1). The improved DNA extraction efficiency was attributed to increased cell lysis, measured by viable counts of sediment microorganisms which showed that 2 and 8%, respectively, survived the bead-mill homogenization and freeze-thaw procedures. Corresponding measurements of suspensions of viable Bacillus endospores demonstrated that 2 and 94% of the initial number survived. Conventional, laser scanning epifluorescence phase-contrast, and differential interference-contrast microscopy revealed that small coccoid bacterial cells (1.2 to 0.3 micron long) were left intact after combined SDS and bead-mill homogenization of sediment samples. Estimates of the residual fraction of the fluorescently stained cell numbers indicated that 6% (2.2 x 10(8) cells.g [dry weight] of sediment-1) of the original population (3.8 x 10(9) cells.g [dry weight] of sediment-1) remained after treatment with SDS and bead-mill homogenization. Thus, lysis of total cells was less efficient than that of cells which could be cultured. The extracted DNA was used to successfully amplify nahR, the regulatory gene for naphthalene catabolism in Pseudomonas putida G7, by PCR.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8017936

  9. Numerical extraction of Cole-Cole impedance parameters from step response

    NASA Astrophysics Data System (ADS)

    Freeborn, Todd J.; Maundy, Brent; Elwakil, Ahmed

    In this paper we propose a numerical method to extract the four parameters (Ro, R∞, C, and α) that characterize a single-dispersion Cole-Cole impedance model from its step response, based on modelling the step response using fractional calculus, without requiring direct measurement of the real and imaginary impedance parts. MATLAB simulations using Cole-Cole impedances of fruit tissues with α<1, PSPICE simulations and experimental results using a Cole-Cole model with α=1 are given to verify the method. Extracted impedance parameters show less than 0.1% relative error in simulation and less than 10% error in experimental results for the extracted impedance parameters.

  10. Biochemical reconstitution of abasic DNA lesion replication in Xenopus extracts

    PubMed Central

    Liao, Shuren; Matsumoto, Yoshihiro; Yan, Hong

    2007-01-01

    Cellular DNA is under constant attack from numerous exogenous and endogenous agents. The resulting DNA lesions, if not repaired timely, could stall DNA replication, leading to genome instability. To better understand the mechanism of DNA lesion replication at the biochemical level, we have attempted to reconstitute this process in Xenopus egg extracts, the only eukaryotic in vitro system that relies solely on cellular proteins for DNA replication. By using a plasmid DNA that carries a site-specific apurinic/apyrimidinic (AP) lesion as template, we have found that DNA replication is stalled one nucleotide before the lesion. The stalling is temporary and the lesion is eventually replicated by both an error-prone mechanism and an error-free mechanism. This is the first biochemical system that recapitulates efficiently and faithfully all major aspects of DNA lesion replication. It has provided the first direct evidence for the existence of an error-free lesion replication mechanism and also demonstrated that the error-prone mechanism is a major contributor to lesion replication. PMID:17702761

  11. Inaccurate DNA Synthesis in Cell Extracts of Yeast Producing Active Human DNA Polymerase Iota

    PubMed Central

    Makarova, Alena V.; Grabow, Corinn; Gening, Leonid V.; Tarantul, Vyacheslav Z.; Tahirov, Tahir H.; Bessho, Tadayoshi; Pavlov, Youri I.

    2011-01-01

    Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn2+ ions, can bypass some DNA lesions and misincorporates “G” opposite template “T” more frequently than incorporates the correct “A.” We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of “G” versus “A” method of Gening, abbreviated as “misGvA”). We provide unambiguous proof of the “misGvA” approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The “misGvA” activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts. PMID:21304950

  12. Development of microfluidic modules for DNA purification via phenol extraction and analyte concentration using transverse electrokinetics

    NASA Astrophysics Data System (ADS)

    Morales, Mercedes C.

    bounding walls. The miniaturization of DNA phenol extraction and the novel electrokinetic trapping techniques presented in this research are the initial steps towards an efficient DNA sample preparation chip which could be integrated with post-extraction DNA manipulations for genomic analysis modules such as capillary electrophoretic separations.

  13. Ultrasensitive DNA detection based on two-step quantitative amplification on magnetic nanoparticles.

    PubMed

    Jin, Mingliang; Liu, Xia; van den Berg, Albert; Zhou, Guofu; Shui, Lingling

    2016-08-19

    Sensitive detection of a specific deoxyribo nucleic acid (DNA) sequence is important for biomedical applications. In this report, a two-step amplification strategy is developed based on magnetic nanoparticles (MNPs) to achieve ultrasensitive DNA fluorescence detection. The first level amplification is obtained from multiple binding sites on MNPs to achieve thousands of probe DNA molecules on one nanoparticle surface. The second level amplification is gained by enzymatic reaction to achieve fluorescence signal enhancement. MNPs functionalized by probe DNA (DNAp) are bound to target DNA (t-DNA) molecules with a ratio of 1:1 on a substrate with capture DNA (DNAc). After the MNPs with DNAp are released from the substrate, alkaline phosphatase (AP) is labelled to MNPs via hybridization reaction between DNAp on MNPs and detection DNAs (DNAd) with AP. The AP on MNPs catalyses non-fluorescent 4-methylumbelliferyl phosphate (4-MUP) to fluorescent 4-methylumbelliferone (4-MU) with high intensity. Finally, fluorescence intensity of the 4-MU is detected by a conventional fluorescence spectrophotometer. With this two-step amplification strategy, the limit of detection (LOD) of 2.8 × 10(-18) mol l(-1) for t-DNA has been achieved. PMID:27378514

  14. Ultrasensitive DNA detection based on two-step quantitative amplification on magnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Jin, Mingliang; Liu, Xia; van den Berg, Albert; Zhou, Guofu; Shui, Lingling

    2016-08-01

    Sensitive detection of a specific deoxyribo nucleic acid (DNA) sequence is important for biomedical applications. In this report, a two-step amplification strategy is developed based on magnetic nanoparticles (MNPs) to achieve ultrasensitive DNA fluorescence detection. The first level amplification is obtained from multiple binding sites on MNPs to achieve thousands of probe DNA molecules on one nanoparticle surface. The second level amplification is gained by enzymatic reaction to achieve fluorescence signal enhancement. MNPs functionalized by probe DNA (DNAp) are bound to target DNA (t-DNA) molecules with a ratio of 1:1 on a substrate with capture DNA (DNAc). After the MNPs with DNAp are released from the substrate, alkaline phosphatase (AP) is labelled to MNPs via hybridization reaction between DNAp on MNPs and detection DNAs (DNAd) with AP. The AP on MNPs catalyses non-fluorescent 4-methylumbelliferyl phosphate (4-MUP) to fluorescent 4-methylumbelliferone (4-MU) with high intensity. Finally, fluorescence intensity of the 4-MU is detected by a conventional fluorescence spectrophotometer. With this two-step amplification strategy, the limit of detection (LOD) of 2.8 × 10‑18 mol l‑1 for t-DNA has been achieved.

  15. Stacking interactions in RNA and DNA: Roll-slide energy hyperspace for ten unique dinucleotide steps.

    PubMed

    Mukherjee, Sanchita; Kailasam, Senthilkumar; Bansal, Manju; Bhattacharyya, Dhananjay

    2015-03-01

    Understanding dinucleotide sequence directed structures of nuleic acids and their variability from experimental observation remained ineffective due to unavailability of statistically meaningful data. We have attempted to understand this from energy scan along twist, roll, and slide degrees of freedom which are mostly dependent on dinucleotide sequence using ab initio density functional theory. We have carried out stacking energy analysis in these dinucleotide parameter phase space for all ten unique dinucleotide steps in DNA and RNA using DFT-D by ωB97X-D/6-31G(2d,2p), which appears to satisfactorily explain conformational preferences for AU/AU step in our recent study. We show that values of roll, slide, and twist of most of the dinucleotide sequences in crystal structures fall in the low energy region. The minimum energy regions with large twist values are associated with the roll and slide values of B-DNA, whereas, smaller twist values correspond to higher stability to RNA and A-DNA like conformations. Incorporation of solvent effect by CPCM method could explain the preference shown by some sequences to occur in B-DNA or A-DNA conformations. Conformational preference of BII sub-state in B-DNA is preferentially displayed mainly by pyrimidine-purine steps and partly by purine-purine steps. The purine-pyrimidine steps show largest effect of 5-methyl group of thymine in stacking energy and the introduction of solvent reduces this effect significantly. These predicted structures and variabilities can explain the effect of sequence on DNA and RNA functionality. PMID:25257334

  16. Simulated radioactive decontamination of biological samples using a portable DNA extraction instrument for rapid DNA profiling.

    PubMed

    Frégeau, Chantal J; Dalpé, Claude

    2016-02-01

    A portable DNA extraction instrument was evaluated for its ability to decontaminate blood and saliva samples deposited on different surfaces (metal, plastic and glass) contaminated with stable isotopes of cobalt (Co), cesium (Cs), and strontium (Sr) as equivalents to their radiogenic (60)Co, (137)Cs, and (90)Sr isotopes, respectively, that could be released during a nuclear weapon accident or a radiological dispersal device (RDD) detonation. Despite the very high contamination levels tested in this study, successful removal of greater than 99.996% of the Co, Cs, Sr contaminants was achieved based on inductively coupled plasma-mass spectrometry (ICP-MS) and neutron activation analyses carried out on all liquids (including DNA eluates) and solid waste produced during automated DNA extraction. The remaining amounts of Co, Cs and Sr in the DNA eluates, when converted to dose rates (corresponding to (60)Co, (137)Cs and (90)Sr), were determined to be below the recommended dose limits for the general public in most of the scenarios tested. The presence of Co, Cs and Sr contaminants in the cell lysates had no adverse impact on the binding of DNA onto the magnetic DNA IQ™ beads. DNA yields were similar to uncontaminated controls. The remaining Co, Cs and Sr in the DNA eluates did not interfere with real-time PCR DNA quantification. In addition, the quality of the AmpFlSTR(®) Identifiler(®) profiles derived in 26min using an accelerated protocol was very good and comparable to controls. This study emphasizes the use of an accelerated process involving a portable DNA extraction instrument to significantly reduce radioactive dose rates to allow contaminated samples to be processed safely in a forensic mobile laboratory to expedite the identification of individuals potentially involved in the dispersal of nuclear or other radioactive materials. PMID:26773226

  17. The fate of the chemical warfare agent during DNA extraction.

    PubMed

    Wilkinson, Della A; Hulst, Albert G; de Reuver, Leo P J; van Krimpen, Simon H; van Baar, Ben M L

    2007-11-01

    Forensic laboratories do not have the infrastructure to process or store contaminated DNA samples that have been recovered from a crime scene contaminated with chemical or biological warfare agents. Previous research has shown that DNA profiles can be recovered from blood exposed to several chemical warfare agents after the agent has been removed. The fate of four toxic agents, sulfur mustard, sodium 2-fluoroacetate, sarin, and diazinon, in a lysis buffer used in Promega DNA IQ extraction protocol was studied to determine if extraction would render the samples safe. Two independent analytical methods were used per agent, selected from GC-MS, 1H NMR, 19F NMR, (31)P NMR, or LC-ES MS. The methods were validated before use. Determinations were carried out in a semi-quantitative way, by direct comparison to standards. Agent levels in the elution buffer were found to be below the detectable limits for mustard, sarin, sodium 2-fluoroacetate or low (<0.02 mg/mL) for diazinon. Therefore, once extracted these DNA samples could be safely processed in a forensic laboratory. PMID:18093062

  18. One-step large-scale deposition of salt-free DNA origami nanostructures

    PubMed Central

    Linko, Veikko; Shen, Boxuan; Tapio, Kosti; Toppari, J. Jussi; Kostiainen, Mauri A.; Tuukkanen, Sampo

    2015-01-01

    DNA origami nanostructures have tremendous potential to serve as versatile platforms in self-assembly -based nanofabrication and in highly parallel nanoscale patterning. However, uniform deposition and reliable anchoring of DNA nanostructures often requires specific conditions, such as pre-treatment of the chosen substrate or a fine-tuned salt concentration for the deposition buffer. In addition, currently available deposition techniques are suitable merely for small scales. In this article, we exploit a spray-coating technique in order to resolve the aforementioned issues in the deposition of different 2D and 3D DNA origami nanostructures. We show that purified DNA origamis can be controllably deposited on silicon and glass substrates by the proposed method. The results are verified using either atomic force microscopy or fluorescence microscopy depending on the shape of the DNA origami. DNA origamis are successfully deposited onto untreated substrates with surface coverage of about 4 objects/mm2. Further, the DNA nanostructures maintain their shape even if the salt residues are removed from the DNA origami fabrication buffer after the folding procedure. We believe that the presented one-step spray-coating method will find use in various fields of material sciences, especially in the development of DNA biochips and in the fabrication of metamaterials and plasmonic devices through DNA metallisation. PMID:26492833

  19. One-step large-scale deposition of salt-free DNA origami nanostructures

    NASA Astrophysics Data System (ADS)

    Linko, Veikko; Shen, Boxuan; Tapio, Kosti; Toppari, J. Jussi; Kostiainen, Mauri A.; Tuukkanen, Sampo

    2015-10-01

    DNA origami nanostructures have tremendous potential to serve as versatile platforms in self-assembly -based nanofabrication and in highly parallel nanoscale patterning. However, uniform deposition and reliable anchoring of DNA nanostructures often requires specific conditions, such as pre-treatment of the chosen substrate or a fine-tuned salt concentration for the deposition buffer. In addition, currently available deposition techniques are suitable merely for small scales. In this article, we exploit a spray-coating technique in order to resolve the aforementioned issues in the deposition of different 2D and 3D DNA origami nanostructures. We show that purified DNA origamis can be controllably deposited on silicon and glass substrates by the proposed method. The results are verified using either atomic force microscopy or fluorescence microscopy depending on the shape of the DNA origami. DNA origamis are successfully deposited onto untreated substrates with surface coverage of about 4 objects/mm2. Further, the DNA nanostructures maintain their shape even if the salt residues are removed from the DNA origami fabrication buffer after the folding procedure. We believe that the presented one-step spray-coating method will find use in various fields of material sciences, especially in the development of DNA biochips and in the fabrication of metamaterials and plasmonic devices through DNA metallisation.

  20. One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX

    PubMed Central

    Nitsche, Andreas; Kurth, Andreas; Dunkhorst, Anna; Pänke, Oliver; Sielaff, Hendrik; Junge, Wolfgang; Muth, Doreen; Scheller, Frieder; Stöcklein, Walter; Dahmen, Claudia; Pauli, Georg; Kage, Andreas

    2007-01-01

    Background As a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures. Results The selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner. Conclusion The MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method. PMID:17697378

  1. Data on single-step purification method for dye-labeled DNA sequencing.

    PubMed

    Fujikura, Kohei

    2016-06-01

    Dye-labelled DNA sequencing is one of the most common and robust technique required for molecular biology since 1977 (Sanger, 1977) [1]. I have recently provided the single-step purification method for dye-labeled sequencing products, which is based on the removal of the washing step in EDTA/ethanol precipitation (Fujikura, 2015) [2]. Here I assess and report the accumulated data of the modified method on the larger scale in practice. PMID:27077088

  2. Study of microtip-based extraction and purification of DNA from human samples for portable devices

    NASA Astrophysics Data System (ADS)

    Fotouhi, Gareth

    recovery of DNA to 45% efficiency. Furthermore, the 225°C-cured PEI-coated microtips recover more DNA than gold-coated microtips when the surface is washed. Heat-cured (225°C) PEI-coated microtips are used for the recovery of human genomic DNA from whole blood. A washing protocol is developed to remove inhibiting particles bound to the PEI-coated microtip surface after DNA extraction. From 1.25 muL of whole blood, an average of 1.83 ng of human genomic DNA is captured, purified, and released using a 225°C-cured PEI-coated microtip in less than 30 minutes. The extracted DNA is profiled by short tandem repeat analysis (STR). For forensic and medical applications, genomic DNA is extracted from dried samples using heat-cured PEI-coated microtips that are integrated into an automated device. DNA extraction from dried samples is critical for forensics. The use of dried samples in the medical field is increasing because dried samples are convenient for storage, biosafety, and contamination. The main challenge is the time required to properly extract DNA in a purified form. Typically, a 1 hour incubation period is required to complete this process. Overnight incubation is sometimes necessary. To address this challenge, a pre-extraction washing step is investigated to remove inhibiting particles from dried blood spots (DBS) before DNA is released from dried form into solution for microtip extraction. The developed protocol is expanded to extract DNA from a variety of dried samples including nasal swabs, buccal swabs, and other forensic samples. In comparison to a commercial kit, the microtip-based extraction reduced the processing time from 1.5 hours to 30 minutes or less with an equivalent concentration of extracted DNA from dried blood spots. The developed assay will benefit genetic studies on newborn screening, forensic investigation, and POC diagnostics.

  3. DNA extraction from protozoan oocysts/cysts in feces for diagnostic PCR.

    PubMed

    Hawash, Yousry

    2014-06-01

    PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. QIAamp® DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer's protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit's protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume (50-100 µl) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, ≈ 2 oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis. PMID:25031466

  4. The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy

    PubMed Central

    Kim, Jiho; Doose, Sören; Neuweiler, Hannes; Sauer, Markus

    2006-01-01

    Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes. We then studied the kinetics of complex formation between MR121 and dG residues site-specifically incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin folding we investigated complex formation in ssDNA carrying one or two complementary base pairs (dC–dG pairs) that could hybridize to form a short stem. Our data show that incorporation of a single dC–dG pair leads to non-exponential decays for opening and closing kinetics and reduces rate constants by one to two orders of magnitude. We found positive activation enthalpies independent of the number of dC–dG pairs. These results imply that the rate limiting step of DNA hairpin folding is not determined by loop dynamics, or by mismatches in the stem, but rather by interactions between stem and loop nucleotides. PMID:16687657

  5. A hybrid DNA extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples.

    PubMed

    Rothrock, Michael J; Hiett, Kelli L; Gamble, John; Caudill, Andrew C; Cicconi-Hogan, Kellie M; Caporaso, J Gregory

    2014-01-01

    The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the "gold standard" enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples. PMID:25548939

  6. A simplified genomic DNA extraction protocol for pre-germination genotyping in rice.

    PubMed

    Duan, Y B; Zhao, F L; Chen, H D; Li, H; Ni, D H; Wei, P C; Sheng, W; Teng, J T; Zhang, A M; Xue, J P

    2015-01-01

    Genotyping is a critical step for molecular marker-assisted selection in rice. Rice genomic DNA samples for genotyping are typically isolated from living tissues such as seedlings. This requires the germination of all candidate seeds and extraction of DNA from the seedlings. Currently, an ideal individual is selected from a very large number of plants, which is time- and labor-consuming, requiring several transplantations of materials and sampling processes. In this study, we developed a simplified genomic DNA extraction protocol in rice by using amylase to treat half-seeds. The yields of genomic DNA from a half-seed of Indica and Japonica rice were greater than 203.8 ± 32.5 and 143.2 ± 25.5 ng, respectively, and the 260/280 nm absorbance ratio was 1.75-2.10. The DNA was confirmed to be sufficient for polymerase chain reaction amplification and can be used in a marker-assisted selection program. PMID:26125841

  7. Detection of Streptococcus mutans Genomic DNA in Human DNA Samples Extracted from Saliva and Blood

    PubMed Central

    Vieira, Alexandre R.; Deeley, Kathleen B.; Callahan, Nicholas F.; Noel, Jacqueline B.; Anjomshoaa, Ida; Carricato, Wendy M.; Schulhof, Louise P.; DeSensi, Rebecca S.; Gandhi, Pooja; Resick, Judith M.; Brandon, Carla A.; Rozhon, Christopher; Patir, Asli; Yildirim, Mine; Poletta, Fernando A.; Mereb, Juan C.; Letra, Ariadne; Menezes, Renato; Wendell, Steven; Lopez-Camelo, Jorge S.; Castilla, Eduardo E.; Orioli, Iêda M.; Seymen, Figen; Weyant, Robert J.; Crout, Richard; McNeil, Daniel W.; Modesto, Adriana; Marazita, Mary L.

    2011-01-01

    Caries is a multifactorial disease, and studies aiming to unravel the factors modulating its etiology must consider all known predisposing factors. One major factor is bacterial colonization, and Streptococcus mutans is the main microorganism associated with the initiation of the disease. In our studies, we have access to DNA samples extracted from human saliva and blood. In this report, we tested a real-time PCR assay developed to detect copies of genomic DNA from Streptococcus mutans in 1,424 DNA samples from humans. Our results suggest that we can determine the presence of genomic DNA copies of Streptococcus mutans in both DNA samples from caries-free and caries-affected individuals. However, we were not able to detect the presence of genomic DNA copies of Streptococcus mutans in any DNA samples extracted from peripheral blood, which suggests the assay may not be sensitive enough for this goal. Values of the threshold cycle of the real-time PCR reaction correlate with higher levels of caries experience in children, but this correlation could not be detected for adults. PMID:21731912

  8. Direct extraction of genomic DNA from maize with aqueous ionic liquid buffer systems for applications in genetically modified organisms analysis.

    PubMed

    Gonzalez García, Eric; Ressmann, Anna K; Gaertner, Peter; Zirbs, Ronald; Mach, Robert L; Krska, Rudolf; Bica, Katharina; Brunner, Kurt

    2014-12-01

    To date, the extraction of genomic DNA is considered a bottleneck in the process of genetically modified organisms (GMOs) detection. Conventional DNA isolation methods are associated with long extraction times and multiple pipetting and centrifugation steps, which makes the entire procedure not only tedious and complicated but also prone to sample cross-contamination. In recent times, ionic liquids have emerged as innovative solvents for biomass processing, due to their outstanding properties for dissolution of biomass and biopolymers. In this study, a novel, easily applicable, and time-efficient method for the direct extraction of genomic DNA from biomass based on aqueous-ionic liquid solutions was developed. The straightforward protocol relies on extraction of maize in a 10 % solution of ionic liquids in aqueous phosphate buffer for 5 min at room temperature, followed by a denaturation step at 95 °C for 10 min and a simple filtration to remove residual biopolymers. A set of 22 ionic liquids was tested in a buffer system and 1-ethyl-3-methylimidazolium dimethylphosphate, as well as the environmentally benign choline formate, were identified as ideal candidates. With this strategy, the quality of the genomic DNA extracted was significantly improved and the extraction protocol was notably simplified compared with a well-established method. PMID:25381609

  9. Magnetic controlling of migration of DNA and proteins using one-step modified gold nanoparticles.

    PubMed

    Xu, Lu; Feng, Lei; Dong, Shuli; Hao, Jingcheng

    2015-06-01

    A protocol was developed for preparing magnetic gold nanoparticles via one-step modification with a paramagnetic cationic surfactant. These magnetic gold nanoparticles can bind to and manipulate a low strength magnetic field-based delivery of DNA and proteins powerfully and non-invasively. PMID:25847127

  10. Optimization of DNA Extractions from Iron-rich Microbial Mats

    NASA Astrophysics Data System (ADS)

    Fullerton, H.; Hilton, T. S.; Moyer, C. L.

    2013-12-01

    Iron is the fourth most abundant element in the Earth's crust and is potentially one of the most abundant energy sources on the earth as an electron donor for chemolithoautotrophicgrowth coupled to Fe(II) oxidation. Many microbes have adapted to this energy source. One such bacterial class are the Zetaproteobacteria, which dominate Iron-rich microbial mats at Loihi seamount. Although cell counts are very high (up to 5.3x108 cells/ml), efficient DNA yields are low in comparison. In this study we compared extraction efficiency across different methods and with the addition of various buffers. Regardless of protocol (i.e., kit), the addition of sodium citrate drastically increased the DNA yield. The addition of sodium citrate did not alter community structure as determined by T-RFLP and qPCR. Citrate is a well-known ferric iron chelator and will bind ferrous as well. The chelated iron is then unable to participate in the Fenton reaction and this stops the generation of hydroxyl radicals which in turn can react and degrade the extracted DNA. We have utilized this relationship to allow us to obtain nearly an order of magnitude more microbial community DNA per sample, which should also have implications when processing low biomass samples, e.g., from the deep subsurface.

  11. Assessing the Utility of Soil DNA Extraction Kits for Increasing DNA Yields and Eliminating PCR Inhibitors from Buried Skeletal Remains.

    PubMed

    Hebda, Lisa M; Foran, David R

    2015-09-01

    DNA identification of human remains is often necessary when decedents are skeletonized; however, poor DNA recovery and polymerase chain reaction (PCR) inhibition are frequently encountered, a situation exacerbated by burial. In this research, the utility of integrating soil DNA isolation kits into buried skeletal DNA analysis was evaluated and compared to a standard human DNA extraction kit and organic extraction. The soil kits successfully extracted skeletal DNA at quantities similar to standard methods, although the two kits tested, which differ mechanistically, were not equivalent. Further, the PCR inhibitors calcium and humic acid were effectively removed using the soil kits, whereas collagen was less so. Finally, concordant control region sequences were obtained from human skeletal remains using all four methods. Based on these comparisons, soil DNA isolation kits, which quickened the extraction process, proved to be a viable extraction technique for skeletal remains that resulted in positive identification of a decedent. PMID:26258388

  12. Evaluation of methods to improve the extraction and recovery of DNA from cotton swabs for forensic analysis.

    PubMed

    Adamowicz, Michael S; Stasulli, Dominique M; Sobestanovich, Emily M; Bille, Todd W

    2014-01-01

    Samples for forensic DNA analysis are often collected from a wide variety of objects using cotton or nylon tipped swabs. Testing has shown that significant quantities of DNA are retained on the swab, however, and subsequently lost. When processing evidentiary samples, the recovery of the maximum amount of available DNA is critical, potentially dictating whether a usable profile can be derived from a piece of evidence or not. The QIAamp DNA Investigator extraction kit was used with its recommended protocol for swabs (one hour incubation at 56°C) as a baseline. Results indicate that over 50% of the recoverable DNA may be retained on the cotton swab tip, or otherwise lost, for both blood and buccal cell samples when using this protocol. The protocol's incubation time and temperature were altered, as was incubating while shaking or stationary to test for increases in recovery efficiency. An additional step was then tested that included periodic re-suspension of the swab tip in the extraction buffer during incubation. Aliquots of liquid blood or a buccal cell suspension were deposited and dried on cotton swabs and compared with swab-less controls. The concentration of DNA in each extract was quantified and STR analysis was performed to assess the quality of the extracted DNA. Stationary incubations and those performed at 65°C did not result in significant gains in DNA yield. Samples incubated for 24 hours yielded less DNA. Increased yields were observed with three and 18 hour incubation periods. Increases in DNA yields were also observed using a swab re-suspension method for both cell types. The swab re-suspension method yielded an average two-fold increase in recovered DNA yield with buccal cells and an average three-fold increase with blood cells. These findings demonstrate that more of the DNA collected on swabs can be recovered with specific protocol alterations. PMID:25549111

  13. Evaluation of Methods to Improve the Extraction and Recovery of DNA from Cotton Swabs for Forensic Analysis

    PubMed Central

    Adamowicz, Michael S.; Stasulli, Dominique M.; Sobestanovich, Emily M.; Bille, Todd W.

    2014-01-01

    Samples for forensic DNA analysis are often collected from a wide variety of objects using cotton or nylon tipped swabs. Testing has shown that significant quantities of DNA are retained on the swab, however, and subsequently lost. When processing evidentiary samples, the recovery of the maximum amount of available DNA is critical, potentially dictating whether a usable profile can be derived from a piece of evidence or not. The QIAamp DNA Investigator extraction kit was used with its recommended protocol for swabs (one hour incubation at 56°C) as a baseline. Results indicate that over 50% of the recoverable DNA may be retained on the cotton swab tip, or otherwise lost, for both blood and buccal cell samples when using this protocol. The protocol’s incubation time and temperature were altered, as was incubating while shaking or stationary to test for increases in recovery efficiency. An additional step was then tested that included periodic re-suspension of the swab tip in the extraction buffer during incubation. Aliquots of liquid blood or a buccal cell suspension were deposited and dried on cotton swabs and compared with swab-less controls. The concentration of DNA in each extract was quantified and STR analysis was performed to assess the quality of the extracted DNA. Stationary incubations and those performed at 65°C did not result in significant gains in DNA yield. Samples incubated for 24 hours yielded less DNA. Increased yields were observed with three and 18 hour incubation periods. Increases in DNA yields were also observed using a swab re-suspension method for both cell types. The swab re-suspension method yielded an average two-fold increase in recovered DNA yield with buccal cells and an average three-fold increase with blood cells. These findings demonstrate that more of the DNA collected on swabs can be recovered with specific protocol alterations. PMID:25549111

  14. The Mechanism of the Translocation Step in DNA Replication by DNA Polymerase I: A Computer Simulation Analysis

    SciTech Connect

    Golosov, Andrei A.; Warren, Joshua J.; Beese, Lorena S.; Karplus, Martin

    2010-11-03

    High-fidelity DNA polymerases copy DNA rapidly and accurately by adding correct deoxynucleotide triphosphates to a growing primer strand of DNA. Following nucleotide incorporation, a series of conformational changes translocate the DNA substrate by one base pair step, readying the polymerase for the next round of incorporation. Molecular dynamics simulations indicate that the translocation consists globally of a polymerase fingers-opening transition, followed by the DNA displacement and the insertion of the template base into the preinsertion site. They also show that the pyrophosphate release facilitates the opening transition and that the universally conserved Y714 plays a key role in coupling polymerase opening to DNA translocation. The transition involves several metastable intermediates in one of which the O helix is bent in the vicinity of G711. Completion of the translocation appears to require a gating motion of the O1 helix, perhaps facilitated by the presence of G715. These roles are consistent with the high level of conservation of Y714 and the two glycine residues at these positions. It is likely that a corresponding mechanism is applicable to other polymerases.

  15. Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase

    PubMed Central

    Morin, José A.; Cao, Francisco J.; Lázaro, José M.; Arias-Gonzalez, J. Ricardo; Valpuesta, José M.; Carrascosa, José L.; Salas, Margarita; Ibarra, Borja

    2015-01-01

    During DNA replication replicative polymerases move in discrete mechanical steps along the DNA template. To address how the chemical cycle is coupled to mechanical motion of the enzyme, here we use optical tweezers to study the translocation mechanism of individual bacteriophage Phi29 DNA polymerases during processive DNA replication. We determine the main kinetic parameters of the nucleotide incorporation cycle and their dependence on external load and nucleotide (dNTP) concentration. The data is inconsistent with power stroke models for translocation, instead supports a loose-coupling mechanism between chemical catalysis and mechanical translocation during DNA replication. According to this mechanism the DNA polymerase works by alternating between a dNTP/PPi-free state, which diffuses thermally between pre- and post-translocated states, and a dNTP/PPi-bound state where dNTP binding stabilizes the post-translocated state. We show how this thermal ratchet mechanism is used by the polymerase to generate work against large opposing loads (∼50 pN). PMID:25800740

  16. Improved rapid and efficient method for Staphylococcus aureus DNA extraction from milk for identification of mastitis pathogens.

    PubMed

    Unno, Hirotaka; Inada, Mika; Nakamura, Akiyoshi; Hashimoto, Michie; Ito, Keiko; Hashimoto, Koji; Nikaido, Masaru; Hayashi, Tomohito; Hata, Eiji; Katsuda, Ken; Kiku, Yoshio; Tagawa, Yuichi; Kawai, Kazuhiro

    2015-08-01

    A rapid and efficient DNA extraction method was developed for detecting mastitis pathogens in milk. The first critical step involved cell wall disruption by bead-beating, as physical disruption using beads was more effective for DNA extraction from Gram-positive bacteria, such as Staphylococcus aureus, than enzymatic disruption using proteinase K. The second critical step involves the use of acetic acid and ammonium sulfate in the purification process, as these reagents effectively and efficiently remove the lipids and proteins in milk. Using these methods, DNA suitable for loop-mediated isothermal amplification was obtained within 30 min. Also, the rapid and sensitive detection of S. aureus in milk was possible at levels as low as 200 cfu/ml. PMID:25843742

  17. Comparison of six methods of extracting Mycobacterium tuberculosis DNA from processed sputum for testing by quantitative real-time PCR.

    PubMed

    Aldous, Wade K; Pounder, June I; Cloud, Joann L; Woods, Gail L

    2005-05-01

    Six methods of extracting Mycobacterium tuberculosis DNA from sputum for testing by quantitative PCR were compared: Tris-EDTA (TE) buffer, PrepMan Ultra, 2% sodium dodecyl sulfate (SDS)-10% Triton X with and without sonication, Infectio Diagnostics, Inc. (IDI) lysing tubes, and QIAGEN QIAamp DNA mini kit; all included a 15-min boiling step. Pooled digested and decontaminated sputum was spiked with M. tuberculosis ATCC 27294. Each extraction method was repeated eight times. Quantitative PCR was performed on the Smart Cycler and Rotor-Gene 3000 using primers targeting an 83-bp fragment of IS6110. An minor grove binding Eclipse probe with a fluorescent label was used for detection. An internal control was included to detect amplification inhibition. The limit of detection of M. tuberculosis DNA was 0.5 fg with both instruments. Calculated DNA concentrations (picograms) extracted using IDI, PrepMan, QIAGEN, and TE were 42.8, 30.4, 28.2, and 7.4, respectively, when run on the Smart Cycler, and 51.7, 20.1, 14.9, and 8.6, respectively, when run on Rotor-Gene. All extractions using SDS/Triton X with or without sonication were inhibited. Of the extraction methods evaluated, IDI lysis tubes provided the greatest yield of mycobacterial DNA, and the procedure can be completed in less than 1 h versus 2.5-3 h for the QIAGEN extraction. PMID:15872286

  18. Procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder

    SciTech Connect

    Marko, M.A.; Chipperfield, R.; Birnboim, H.C.

    1982-01-01

    A preparative procedure for obtaining highly purified plasmid DNA from bacterial cells is described. The method is adapted from our earlier procedure, which gave partially purified plasmid in a form suitable for rapid screening of a large number of samples. In the present method, all detectable RNA, chromosomal DNA, and protein are removed without the use of enzymes, phenol extraction, dialysis, or equilibrium centrifugation. Binding of plasmid DNA to glass powder in the presence of 6 M sodium perchlorate is used for the final purification step.

  19. Forensic DNA-typing of dog hair: DNA-extraction and PCR amplification.

    PubMed

    Pfeiffer, I; Völkel, I; Täubert, H; Brenig, B

    2004-05-10

    The forensic application of DNA-typing for the identification of dog hair provides objective evidence in the characterisation of traces found at crime scenes. During the past few years forensic dog identity testing has been improved considerably using multiplex PCR systems. However, DNA-typing from samples of one up to 10 dog hairs is often problematic in forensic science. A single dog hair contains very small quantities of DNA or the hair sample consists of hairs with roots of bad quality or even of broken hairshafts without roots. Here we describe an experimental study about dog hairs by means of a Ca(2+) improved DNA-extraction method, quantification and amplification. PMID:15062955

  20. The RSC chromatin remodelling ATPase translocates DNA with high force and small step size

    PubMed Central

    Sirinakis, George; Clapier, Cedric R; Gao, Ying; Viswanathan, Ramya; Cairns, Bradley R; Zhang, Yongli

    2011-01-01

    ATP-dependent chromatin remodelling complexes use the energy of ATP hydrolysis to reposition and reconfigure nucleosomes. Despite their diverse functions, all remodellers share highly conserved ATPase domains, many shown to translocate DNA. Understanding remodelling requires biophysical knowledge of the DNA translocation process: how the ATPase moves DNA and generates force, and how translocation and force generation are coupled on nucleosomes. Here, we characterize the real-time activity of a minimal RSC translocase ‘motor' on bare DNA, using high-resolution optical tweezers and a ‘tethered' translocase system. We observe on dsDNA a processivity of ∼35 bp, a speed of ∼25 bp/s, and a step size of 2.0 (±0.4, s.e.m.) bp. Surprisingly, the motor is capable of moving against high force, up to 30 pN, making it one of the most force-resistant motors known. We also provide evidence for DNA ‘buckling' at initiation. These observations reveal the ATPase as a powerful DNA translocating motor capable of disrupting DNA–histone interactions by mechanical force. PMID:21552204

  1. Two-step mechanism involving active-site conformational changes regulates human telomerase DNA binding.

    PubMed

    Tomlinson, Christopher G; Moye, Aaron L; Holien, Jessica K; Parker, Michael W; Cohen, Scott B; Bryan, Tracy M

    2015-01-15

    The ribonucleoprotein enzyme telomerase maintains telomeres and is essential for cellular immortality in most cancers. Insight into the telomerase mechanism can be gained from syndromes such as dyskeratosis congenita, in which mutation of telomerase components manifests in telomere dysfunction. We carried out detailed kinetic and thermodynamic analyses of wild-type telomerase and two disease-associated mutations in the reverse transcriptase domain. Differences in dissociation rates between primers with different 3' ends were independent of DNA affinities, revealing that initial binding of telomerase to telomeric DNA occurs through a previously undescribed two-step mechanism involving enzyme conformational changes. Both mutations affected DNA binding, but through different mechanisms: P704S specifically affected protein conformational changes during DNA binding, whereas R865H showed defects in binding to the 3' region of the DNA. To gain further insight at the structural level, we generated the first homology model of the human telomerase reverse transcriptase domain; the positions of P704S and R865H corroborate their observed mechanistic defects, providing validation for the structural model. Our data reveal the importance of protein interactions with the 3' end of telomeric DNA and the role of protein conformational change in telomerase DNA binding, and highlight naturally occurring disease mutations as a rich source of mechanistic insight. PMID:25365545

  2. Nonspecific DNA Binding and Coordination of the First Two Steps of Base Excision Repair

    PubMed Central

    Baldwin, Michael R.; O'Brien, Patrick J.

    2010-01-01

    The base excision repair (BER) pathway repairs a wide variety of damaged nucleobases in DNA. This pathway is initiated by a DNA repair glycosylase, which locates the site of damage and catalyzes the excision of the damaged nucleobase. The resulting abasic site is further processed by apurinic/apyrimidinic site endonuclease 1 (APE1) to create a single strand nick with the 3'-hydroxyl that serves as a primer for DNA repair synthesis. Since an abasic site is highly mutagenic it is critical that the steps of the BER pathway be coordinated. Most human glycosylases bind tightly to their abasic product. APE1 displaces the bound glycosylase, thereby stimulating multiple turnover base excision. It has been proposed that direct protein-protein interactions are involved in the stimulation by APE1, but no common interaction motifs have been identified among the glycosylases that are stimulated by APE1. We characterized the APE1 stimulation of alkyladenine DNA glycosylase (AAG) using a variety of symmetric and asymmetric lesion-containing oligonucleotides. Efficient stimulation on a wide variety of substrates favors a model whereby both AAG and APE1 can simultaneously bind to DNA, but may not interact directly. Rather, nonspecific DNA binding by both AAG and APE1 enables APE1 to replace AAG at the abasic site. AAG is not displaced into solution, but remains bound to an adjacent undamaged site. We propose that nonspecific DNA binding interactions allow transient exposure of the abasic site so that it can be captured by APE1. PMID:20701268

  3. An alternate method for extracting DNA from environmentally challenged teeth for improved DNA analysis.

    PubMed

    Hughes-Stamm, Sheree; Warnke, Frauke; van Daal, Angela

    2016-01-01

    A grinding-free method to extract DNA from teeth via a direct minimal-invasive retrograde approach to the pulp cavity and dentine was compared to a standard grinding/pulverisation method. This alternate method uses endodontic dental files to access the root canals and pulp cavity for tissue and dentine harvest via the apical end of the roots and avoids mechanical damage to the crown and root morphology. In contrast, other methods require pulverisation of the whole root or tooth, transection or destruction of the occlusal surface to gain access to the DNA in the root canals and pulp chamber. This study compared two methods for preparing dentine powder from the roots of environmentally challenged teeth for forensic DNA analysis. We found that although the filing method was more laborious, and produced less dentine powder, the amount of amplifiable DNA per milligram of powder was substantially higher with the filing method compared to grinding the entire root. In addition, the number of short tandem repeat (STR) alleles detected and the peak height ratios of the STR profiles were notably higher. Although several other methods of extracting DNA-rich tissue from the pulp chamber of teeth have previously been reported, the method presented in this study is minimally invasive, thereby allowing the preservation of tooth and crown morphology. PMID:26832373

  4. A two-step supercritical fluid extraction of polycyclic aromatic hydrocarbons from roadside soil samples.

    PubMed

    Lojková, Lea; Sedláková, Jitka; Kubán, Vlastimil

    2005-11-01

    A two-step procedure for the supercritical fluid extraction (SFE) of polycyclic aromatic hydrocarbons from soil samples was developed. The procedure consists of a static supercritical fluid treatment in a closed extraction cell at a high temperature (T=250 or 340degreesC for 20 min) and an SFE with a solvent trapping. During the static phase, the sample is exposed to a supercritical organic solvent (methanol, toluene, dichloromethane, ACN, acetone, and hexane). The solvent penetrates particles of the matrix to substitute strongly bonded molecules and dissolves the analytes in the supercritical phase. At ambient temperature, supercritical fluids became liquid and lost their solvation abilities. Most of the analytes condense on the surface of the particles or on the extraction cell walls without forming strong bonds or penetrating deep into the matrix. Thus, the pretreatment liberates the analytes and they behave similar to those in freshly spiked samples. The common SFE with toluene-modified CO2 as an extraction fluid follows the static phase. With the use of the most suitable extraction phases (toluene, ACN), the extraction efficiency of the combined procedure is much higher (approximately100%). The results of the combined procedure are compared to the SFE procedure of the same untreated sample (difference less than 5%) and to the Soxhlet extraction. The extracts were analyzed using a GC with the flame ionization detection. PMID:16318201

  5. Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction.

    PubMed

    Patel, Palak G; Selvarajah, Shamini; Boursalie, Suzanne; How, Nathan E; Ejdelman, Joshua; Guerard, Karl-Philippe; Bartlett, John M; Lapointe, Jacques; Park, Paul C; Okello, John B A; Berman, David M

    2016-01-01

    Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation. PMID:27583817

  6. Optimization of a one-step heat-inducible in vivo mini DNA vector production system.

    PubMed

    Nafissi, Nafiseh; Sum, Chi Hong; Wettig, Shawn; Slavcev, Roderick A

    2014-01-01

    While safer than their viral counterparts, conventional circular covalently closed (CCC) plasmid DNA vectors offer a limited safety profile. They often result in the transfer of unwanted prokaryotic sequences, antibiotic resistance genes, and bacterial origins of replication that may lead to unwanted immunostimulatory responses. Furthermore, such vectors may impart the potential for chromosomal integration, thus potentiating oncogenesis. Linear covalently closed (LCC), bacterial sequence free DNA vectors have shown promising clinical improvements in vitro and in vivo. However, the generation of such minivectors has been limited by in vitro enzymatic reactions hindering their downstream application in clinical trials. We previously characterized an in vivo temperature-inducible expression system, governed by the phage λ pL promoter and regulated by the thermolabile λ CI[Ts]857 repressor to produce recombinant protelomerase enzymes in E. coli. In this expression system, induction of recombinant protelomerase was achieved by increasing culture temperature above the 37°C threshold temperature. Overexpression of protelomerase led to enzymatic reactions, acting on genetically engineered multi-target sites called "Super Sequences" that serve to convert conventional CCC plasmid DNA into LCC DNA minivectors. Temperature up-shift, however, can result in intracellular stress responses and may alter plasmid replication rates; both of which may be detrimental to LCC minivector production. We sought to optimize our one-step in vivo DNA minivector production system under various induction schedules in combination with genetic modifications influencing plasmid replication, processing rates, and cellular heat stress responses. We assessed different culture growth techniques, growth media compositions, heat induction scheduling and temperature, induction duration, post-induction temperature, and E. coli genetic background to improve the productivity and scalability of our system

  7. Comparison of Boiling and Robotics Automation Method in DNA Extraction for Metagenomic Sequencing of Human Oral Microbes.

    PubMed

    Yamagishi, Junya; Sato, Yukuto; Shinozaki, Natsuko; Ye, Bin; Tsuboi, Akito; Nagasaki, Masao; Yamashita, Riu

    2016-01-01

    The rapid improvement of next-generation sequencing performance now enables us to analyze huge sample sets with more than ten thousand specimens. However, DNA extraction can still be a limiting step in such metagenomic approaches. In this study, we analyzed human oral microbes to compare the performance of three DNA extraction methods: PowerSoil (a method widely used in this field), QIAsymphony (a robotics method), and a simple boiling method. Dental plaque was initially collected from three volunteers in the pilot study and then expanded to 12 volunteers in the follow-up study. Bacterial flora was estimated by sequencing the V4 region of 16S rRNA following species-level profiling. Our results indicate that the efficiency of PowerSoil and QIAsymphony was comparable to the boiling method. Therefore, the boiling method may be a promising alternative because of its simplicity, cost effectiveness, and short handling time. Moreover, this method was reliable for estimating bacterial species and could be used in the future to examine the correlation between oral flora and health status. Despite this, differences in the efficiency of DNA extraction for various bacterial species were observed among the three methods. Based on these findings, there is no "gold standard" for DNA extraction. In future, we suggest that the DNA extraction method should be selected on a case-by-case basis considering the aims and specimens of the study. PMID:27104353

  8. Comparison of Boiling and Robotics Automation Method in DNA Extraction for Metagenomic Sequencing of Human Oral Microbes

    PubMed Central

    Shinozaki, Natsuko; Ye, Bin; Tsuboi, Akito; Nagasaki, Masao; Yamashita, Riu

    2016-01-01

    The rapid improvement of next-generation sequencing performance now enables us to analyze huge sample sets with more than ten thousand specimens. However, DNA extraction can still be a limiting step in such metagenomic approaches. In this study, we analyzed human oral microbes to compare the performance of three DNA extraction methods: PowerSoil (a method widely used in this field), QIAsymphony (a robotics method), and a simple boiling method. Dental plaque was initially collected from three volunteers in the pilot study and then expanded to 12 volunteers in the follow-up study. Bacterial flora was estimated by sequencing the V4 region of 16S rRNA following species-level profiling. Our results indicate that the efficiency of PowerSoil and QIAsymphony was comparable to the boiling method. Therefore, the boiling method may be a promising alternative because of its simplicity, cost effectiveness, and short handling time. Moreover, this method was reliable for estimating bacterial species and could be used in the future to examine the correlation between oral flora and health status. Despite this, differences in the efficiency of DNA extraction for various bacterial species were observed among the three methods. Based on these findings, there is no “gold standard” for DNA extraction. In future, we suggest that the DNA extraction method should be selected on a case-by-case basis considering the aims and specimens of the study. PMID:27104353

  9. 2-step purification of the Ku DNA repair protein expressed in Escherichia coli

    PubMed Central

    2007-01-01

    The Ku protein is involved in DNA double-strand break repair by non-homologous end-joining (NHEJ), which is crucial to the maintenance of genomic integrity in mammals. To study the role of Ku in NHEJ we developed a bicistronic E. coli expression system for the Ku70 and Ku80 subunits. Association of the Ku70 and Ku80 subunits buries a substantial amount of surface area (~9000Å2 [1]), which suggests that herterodimerization may be important for protein stability. N-terminally his6-tagged Ku80 was soluble in the presence, but not in the absence, of bicistronically expressed untagged Ku70. In a 2-step purification, metal chelating affinity chromatography was followed by step-gradient elution from heparin-agarose. Co-purification of equimolar amounts of his6-tagged Ku80 and untagged Ku70 was observed, which indicated heterodimerization. Recombinant Ku bound dsDNA, activated the catalytic subunit of the DNA-dependent kinase (DNA-PKcs) and functioned in NHEJ reactions in vitro. Our results demonstrate that while the heterodimeric interface of Ku is extensive it is nonetheless possible to produce biologically active Ku protein in E. coli. PMID:17110127

  10. [Study of DNA extraction methods for testing for genetically modified organisms in soyproducts].

    PubMed

    Moriuchi, Rie; Monma, Kimio; Kamata, Kunihiro; Ibe, Akihiro

    2008-01-01

    In order to evaluate three different methods for DNA extraction (CTAB, DNeasy Plant Mini Kit and Wizard DNA Clean-up Resin system), the yields of DNA extracted from soyproducts and the copy numbers of lectin genes amplified by quantitative PCR were compared. Fermented foods, such as miso and nattou, gave poor yields of DNA and low copy numbers with any method. However atsu-age and kinugoshi-tofu gave high-quality results with all methods. Kinako gave a high yield of DNA, but poor amplification. Boiled soybeans and soymilk showed in poor amplification. It is important to choose the appropriate DNA extraction method for each product. PMID:19029786

  11. Two-step voltage dual electromembrane extraction: A new approach to simultaneous extraction of acidic and basic drugs.

    PubMed

    Asadi, Sakine; Nojavan, Saeed

    2016-06-01

    In the present work, acidic and basic drugs were simultaneously extracted by a novel method of high efficiency herein referred to as two-step voltage dual electromembrane extraction (TSV-DEME). Optimizing effective parameters such as composition of organic liquid membrane, pH values of donor and acceptor solutions, voltage and duration of each step, the method had its figures of merit investigated in pure water, human plasma, wastewater, and breast milk samples. Simultaneous extraction of acidic and basic drugs was done by applying potentials of 150 V and 400 V for 6 min and 19 min as the first and second steps, respectively. The model compounds were extracted from 4 mL of sample solution (pH = 6) into 20 μL of each acceptor solution (32 mM NaOH for acidic drugs and 32 mM HCL for basic drugs). 1-Octanol was immobilized within the pores of a porous hollow fiber of polypropylene, as the supported liquid membrane (SLM) for acidic drugs, and 2-ethyle hexanol, as the SLM for basic drugs. The proposed TSV-DEME technique provided good linearity with the resulting correlation coefficients ranging from 0.993 to 0.998 over a concentration range of 1-1000 ng mL(-1). The limit of detections of the drugs were found to range within 0.3-1.5 ng mL(-1), while the corresponding repeatability ranged from 7.7 to 15.5% (n = 4). The proposed method was further compared to simple dual electromembrane extraction (DEME), indicating significantly higher recoveries for TSV-DEME procedure (38.1-68%), as compared to those of simple DEME procedure (17.7-46%). Finally, the optimized TSV-DEME was applied to extract and quantify model compounds in breast milk, wastewater, and plasma samples. PMID:27155299

  12. Optimization of isopropanol and ammonium sulfate precipitation steps in the purification of plasmid DNA.

    PubMed

    Freitas, S S; Santos, J A L; Prazeres, D M F

    2006-01-01

    Large-scale processes used to manufacture grams of plasmid DNA (pDNA) should be cGMP compliant, economically feasible, and environmentally friendly. Alcohol and salt precipitation techniques are frequently used in plasmid DNA (pDNA) downstream processing, as concentration and prepurification steps, respectively. This work describes a study of a standard 2-propanol (IsopOH; 0.7 v/v) and ammonium sulfate (AS; 2.5 M) precipitation. When inserted in a full process, this tandem precipitation scheme represents a high economic and environmental impact due to the large amounts of the two precipitant agents and their environmental relevance. Thus, major goals of the study were the minimization of precipitants and the selection of the best operating conditions for high pDNA recovery and purity. The pDNA concentration in the starting Escherichia coli alkaline lysate strongly affected the efficiency of IsopOH precipitation as a concentration step. The results showed that although an IsopOH concentration of at least 0.6 (v/v) was required to maximize recovery when using lysates with less than 80 microg pDNA/mL, concentrations as low as 0.4 v/v could be used with more concentrated lysates (170 microg pDNA/mL). Following resuspension of pDNA pellets generated by 0.6 v/v IsopOH, precipitation at 4 degrees C with 2.4 M AS consistently resulted in recoveries higher than 80% and in removal of more than 90% of the impurities (essentially RNA). An experimental design further indicated that AS concentrations could be reduced down to 2.0 M, resulting in an acceptable purity (21-23%) without compromising recovery (84-86%). Plasmid recovery and purity after the sequential IsopOH/AS precipitation could be further improved by increasing the concentration factor (CF) upon IsopOH precipitation from 2 up to 25. Under these conditions, IsopOH and AS concentrations of 0.60 v/v and 1.6 M resulted in high recovery (approximately 100%) and purity (32%). In conclusion, it is possible to reduce

  13. Facile and rapid DNA extraction and purification from food matrices using IFAST (immiscible filtration assisted by surface tension).

    PubMed

    Strotman, Lindsay N; Lin, Guangyun; Berry, Scott M; Johnson, Eric A; Beebe, David J

    2012-09-01

    Extraction and purification of DNA is a prerequisite to detection and analytical techniques. While DNA sample preparation methods have improved over the last few decades, current methods are still time consuming and labor intensive. Here we demonstrate a technology termed IFAST (Immiscible Filtration Assisted by Surface Tension), that relies on immiscible phase filtration to reduce the time and effort required to purify DNA. IFAST replaces the multiple wash and centrifugation steps required by traditional DNA sample preparation methods with a single step. To operate, DNA from lysed cells is bound to paramagnetic particles (PMPs) and drawn through an immiscible fluid phase barrier (i.e. oil) by an external handheld magnet. Purified DNA is then eluted from the PMPs. Here, detection of Clostridium botulinum type A (BoNT/A) in food matrices (milk, orange juice), a bioterrorism concern, was used as a model system to establish IFAST's utility in detection assays. Data validated that the DNA purified by IFAST was functional as a qPCR template to amplify the bont/A gene. The sensitivity limit of IFAST was comparable to the commercially available Invitrogen ChargeSwitch® method. Notably, pathogen detection via IFAST required only 8.5 μL of sample and was accomplished in five-fold less time. The simplicity, rapidity and portability of IFAST offer significant advantages when compared to existing DNA sample preparation methods. PMID:22814365

  14. Improved Methods of Carnivore Faecal Sample Preservation, DNA Extraction and Quantification for Accurate Genotyping of Wild Tigers

    PubMed Central

    Harika, Katakam; Mahla, Ranjeet Singh; Shivaji, Sisinthy

    2012-01-01

    Background Non-invasively collected samples allow a variety of genetic studies on endangered and elusive species. However due to low amplification success and high genotyping error rates fewer samples can be identified up to the individual level. Number of PCRs needed to obtain reliable genotypes also noticeably increase. Methods We developed a quantitative PCR assay to measure and grade amplifiable nuclear DNA in feline faecal extracts. We determined DNA degradation in experimentally aged faecal samples and tested a suite of pre-PCR protocols to considerably improve DNA retrieval. Results Average DNA concentrations of Grade I, II and III extracts were 982pg/µl, 9.5pg/µl and 0.4pg/µl respectively. Nearly 10% of extracts had no amplifiable DNA. Microsatellite PCR success and allelic dropout rates were 92% and 1.5% in Grade I, 79% and 5% in Grade II, and 54% and 16% in Grade III respectively. Our results on experimentally aged faecal samples showed that ageing has a significant effect on quantity and quality of amplifiable DNA (p<0.001). Maximum DNA degradation occurs within 3 days of exposure to direct sunlight. DNA concentrations of Day 1 samples stored by ethanol and silica methods for a month varied significantly from fresh Day 1 extracts (p<0.1 and p<0.001). This difference was not significant when samples were preserved by two-step method (p>0.05). DNA concentrations of fresh tiger and leopard faecal extracts without addition of carrier RNA were 816.5pg/µl (±115.5) and 690.1pg/µl (±207.1), while concentrations with addition of carrier RNA were 49414.5pg/µl (±9370.6) and 20982.7pg/µl (±6835.8) respectively. Conclusions Our results indicate that carnivore faecal samples should be collected as freshly as possible, are better preserved by two-step method and should be extracted with addition of carrier RNA. We recommend quantification of template DNA as this facilitates several downstream protocols. PMID:23071624

  15. Comparative study of methods for extraction and purification of environmental DNA from soil and sludge samples.

    PubMed

    Roh, Changhyun; Villatte, Francois; Kim, Byung-Gee; Schmid, Rolf D

    2006-08-01

    An important prerequisite for successful construction of a metagenome library is an efficient procedure for extracting DNA from environmental samples. We compared three indirect and four direct extraction methods, including a commercial kit, in terms of DNA yield, purity, and time requirement. A special focus was on methods that are appropriate for the extraction of environmental DNA (eDNA) from very limited sample sizes (0.1 g) to enable a highly parallel approach. Direct extraction procedures yielded on average 100-fold higher DNA amounts than indirect ones. A drawback of direct extraction was the small fragment size of approx 12 kb. The quality of the extracted DNA was evaluated by the ability of different restriction enzymes to digest the eDNA. Only the commercial kit and a direct extraction method using freeze-thaw cell lysis in combination with an in-gel patch electrophoresis with hydroxyapatite to remove humic acid substances yielded DNA, which was completely digested by all restriction enzymes. Moreover, only DNA extracted by these two procedures could be used as template for the amplification of fragments of several 16S rDNA, 18S rDNA groups under standard polymerase chain reaction conditions. PMID:16943632

  16. Comparisons of direct extraction methods of microbial DNA from different paddy soils.

    PubMed

    Islam, Md Rashedul; Sultana, Tahera; Melvin Joe, M; Cho, Jang-Cheon; Sa, Tongmin

    2012-07-01

    Molecular analyses for the study of soil microbial communities often depend on the direct extraction of DNA from soils. The present work compares the effectiveness of three different methods of extracting microbial DNA from seven different paddy soils. Comparison among different DNA extraction methods against different paddy soil samples revealed a marked variation in DNA yields from 3.18-20.17 μg DNA/g of dry soil. However, irrespective of the soil samples and extraction methods the DNA fragment size was >10 kb. Among the methods evaluated, method-C (chemical-enzymatic-mechanical) had better cell lysis efficiency and DNA yield. After purification of crude DNA by Purification Kit, A260/A230 and A260/A280 ratios of the DNA obtained by method-C reached up to 2.27 and 1.89, respectively, sustaining the efficacy of this technique in removing humic acid, protein and other contaminants. Results of the comprehensive evaluation of DNA extraction methods suggest that method-C is superior to other two methods (chemical-enzymatic and chemical-mechanical), and was the best choice for extraction of total DNA from soil samples. Since soil type and microbial community characteristics influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods according to experimental goals. PMID:23961194

  17. DNA Extraction by Isotachophoresis in a Microfluidic Channel

    SciTech Connect

    Stephenson, S J

    2011-08-10

    Biological assays have many applications. For example, forensics personnel and medical professionals use these tests to diagnose diseases and track their progression or identify pathogens and the host response to them. One limitation of these tests, however, is that most of them target only one piece of the sample - such as bacterial DNA - and other components (e.g. host genomic DNA) get in the way, even though they may be useful for different tests. To address this problem, it would be useful to extract several different substances from a complex biological sample - such as blood - in an inexpensive and efficient manner. This summer, I worked with Maxim Shusteff at Lawrence Livermore National Lab on the Rapid Automated Sample Prep project. The goal of the project is to solve the aforementioned problem by creating a system that uses a series of different extraction methods to extract cells, bacteria, and DNA from a complex biological sample. Biological assays can then be run on purified output samples. In this device, an operator could input a complex sample such as blood or saliva, and would receive separate outputs of cells, bacteria, viruses, and DNA. I had the opportunity to work this summer with isotachophoresis (ITP), a technique that can be used to extract nucleic acids from a sample. This technique is intended to be the last stage of the purification device. Isotachophoresis separates particles based on different electrophoretic mobilities. This technique is convenient for out application because free solution DNA mobility is approximately equal for DNA longer than 300 base pairs in length. The sample of interest - in our case DNA - is fed into the chip with streams of leading electrolyte (LE) and trailing electrolyte (TE). When an electric field is applied, the species migrate based on their electrophoretic mobilities. Because the ions in the leading electrolyte have a high electrophoretic mobility, they race ahead of the slower sample and trailing

  18. A simple and cost-effective method of DNA extraction from small formalin-fixed paraffin-embedded tissue for molecular oncologic testing

    PubMed Central

    2014-01-01

    Background Extraction of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue is a critical step in molecular oncologic testing. As molecular oncology testing becomes more important for prognostic and therapeutic decision making and tissue specimens become smaller due to earlier detection of suspicious lesions and the use of fine needle aspiration methods for tissue collection, it becomes more challenging for the typical molecular pathology laboratory to obtain reliable test results. We developed a DNA extraction method to obtain sufficient quantity and high quality genomic DNA from limited FFPE tissue for molecular oncology testing using a combination of H&E stained slides, a matrix capture method and the Qiagen DNA column. Methods Three DNA extraction methods were compared: our standard procedure of manually scraping tissue from unstained slides followed by DNA extraction using the QIAamp FFPE column (Qiagen, Valencia, CA), a glue capture method (Pinpoint Solution, Zymo Research Corp, Inc) on H&E stained slides followed by DNA extraction using either the QIAamp column or the column included with the Pinpoint kit (Zymo Research). The DNA extraction protocol was optimized. Statistical analysis was performed using the paired two-sample student’s t-test. Results The combination of the matrix capture method with the QIAamp column gave an equivalent amount of DNA as our standard extraction method using the unstained slides and a 4.6-fold higher DNA yield than using the Zymo column included in the Pinpoint Slide Solution kit. Several molecular tests were performed and DNA purified using the new method gave the same results as for the previous methods. Conclusions Using H&E stained slides allows visual confirmation of tumor cells during microdissection. The Pinpoint solution made removal of specific tissue from the slides easier and reduced the risk of contamination and tissue loss. This DNA extraction method is simple, cost-effective, and blends with our current

  19. Comparison of different protocols for the extraction of microbial DNA from reef corals

    PubMed Central

    Santos, H.F.; Carmo, F.L.; Leite, D.C.A.; Jesus, H.E.; Maalouf, P. De Carvalho; Almeida, C.; Soriano, A.U.; Altomari, D.; Suhett, L.; Vólaro, V.; Valoni, E.; Francisco, M.; Vieira, J.; Rocha, R.; Sardinha, B.L.; Mendes, L.B.; João, R.R.; Lacava, B.; Jesus, R.F.; Sebastian, G.V.; Pessoa, A.; van Elsas, J.D.; Rezende, R.P.; Pires, D.O.; Duarte, G.; Castro, C.B.; Rosado, A.S.; Peixoto, R.S.

    2012-01-01

    This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals. PMID:24031859

  20. Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction.

    PubMed Central

    Jackson, D P; Lewis, F A; Taylor, G R; Boylston, A W; Quirke, P

    1990-01-01

    Several DNA extraction techniques were quantitatively and qualitatively compared using both fresh and paraffin wax embedded tissue and their suitability investigated for providing DNA and RNA for the polymerase chain reaction (PCR). A one hour incubation with proteinase K was the most efficient DNA extraction procedure for fresh tissue. For paraffin wax embedded tissue a five day incubation with proteinase K was required to produce good yields of DNA. Incubation with sodium dodecyl sulphate produced very poor yields, while boiling produced 20% as much DNA as long enzyme digestion. DNA extracted by these methods was suitable for the PCR amplification of a single copy gene. Proteinase K digestion also produced considerable amounts of RNA which has previously been shown to be suitable for PCR analysis. A delay before fixation had no effect on the amount of DNA obtained while fixation in Carnoy's reagent results in a much better preservation of DNA than formalin fixation, allowing greater yields to be extracted. Images PMID:1696290

  1. Fast protocol for extraction of DNA from Prosopis spp leaves (plant adapted to arid environment) without liquid nitrogen.

    PubMed

    Michel-López, C Y; González-Mendoza, D; Grimaldo-Juarez, O

    2013-01-01

    The extraction of high-quality genomic DNA from Prosopis spp for polymerase chain reaction (PCR) amplification is complicated, owing to the presence of a high percentage of secondary metabolites that bind to or co-precipitate with nucleic acids. In the present study, we report a modified sodium dodecyl sulfate/phenol protocol that eliminates the use of liquid nitrogen in the maceration process, β-mercaptoethanol in the buffer extraction, and the ethanol precipitation step. The A₂₆₀/A₂₈₀ absorbance ratios of the isolated DNA were approximately 2.0 to 1.9, suggesting that the DNA fraction was pure and can be used for further PCR analysis. The DNA isolated by this protocol is of sufficient quality for molecular applications; this technique could be applied to other organisms that have similar substances that hinder DNA extraction. Finally, this proposal represents an alternative fast, cheap, and effective method for the isolation of genomic DNA from fresh leaves of Prosopis spp, even in low-technology laboratories. PMID:24089098

  2. Specific requirement for ATP at an early step of in vitro transcription of human mitochondrial DNA

    SciTech Connect

    Narasimhan, N.; Attardi, G.

    1987-06-01

    The ATP concentrations allowing transcription of heavy- and light-strand of human mtDNA in a HeLa cell mitochrondrial lysate were found to cover a broad range, with a maximum around 2.5 mM, and with reproducible differences in the ATP response curves for the two transcription events. Direct measurements showed that nonspecific ATP degradation during the assay did not account for the high ATP requirement. 5'-Adenylyl imidodiphosphate (p(NH)ppA), an ATP analog with a nonhydrolyzable ..beta..-..gamma.. bond, was unable to substitute for ATP in supporting mtDNA transcription but greatly stimulated this transcription in the presence of a low concentration of exogenous APT, measured with (/sup 32/P)-labeled nucleotides. Evidence was obtained indicating that p(NH)ppA did not support an early event in mtDNA transcription (formation of preinitiation complex or initiation), whereas this analog could substitute effectively for ATP in the subsequent elongation steps. These results pointed to a specific requirement for ATP at an early step of the transcription process.

  3. PcrA helicase dismantles RecA filaments by reeling in DNA in uniform steps

    PubMed Central

    Park, Jeehae; Myong, Sua; Niedziela-Majka, Anita; Yu, Jin; Lohman, Timothy M.; Ha, Taekjip

    2010-01-01

    Translocation of helicase-like proteins on nucleic acids underlies key cellular functions. However, it is still unclear how translocation can drive removal of DNA bound proteins, and basic properties like the elementary step size remain controversial. Using single molecule fluorescence analysis on a prototypical superfamily 1 helicase, Bacillus stearothermophilus PcrA, we discovered that PcrA preferentially translocates on the DNA lagging strand instead of unwinding the template duplex. PcrA anchors itself to the template duplex using the 2B subdomain and reels in the lagging strand, extruding a single stranded loop. Static disorder limited previous ensemble studies of PcrA stepping mechanism. Here, highly repetitive looping revealed that PcrA translocates in uniform steps of 1 nt. This reeling-in activity requires the open conformation of PcrA and can rapidly dismantle a preformed RecA filament even at low PcrA concentrations suggesting a mode of action for eliminating potentially deleterious recombination intermediates. PMID:20723756

  4. PcrA helicase dismantles RecA filaments by reeling in DNA in uniform steps.

    PubMed

    Park, Jeehae; Myong, Sua; Niedziela-Majka, Anita; Lee, Kyung Suk; Yu, Jin; Lohman, Timothy M; Ha, Taekjip

    2010-08-20

    Translocation of helicase-like proteins on nucleic acids underlies key cellular functions. However, it is still unclear how translocation can drive removal of DNA-bound proteins, and basic properties like the elementary step size remain controversial. Using single-molecule fluorescence analysis on a prototypical superfamily 1 helicase, Bacillus stearothermophilus PcrA, we discovered that PcrA preferentially translocates on the DNA lagging strand instead of unwinding the template duplex. PcrA anchors itself to the template duplex using the 2B subdomain and reels in the lagging strand, extruding a single-stranded loop. Static disorder limited previous ensemble studies of a PcrA stepping mechanism. Here, highly repetitive looping revealed that PcrA translocates in uniform steps of 1 nt. This reeling-in activity requires the open conformation of PcrA and can rapidly dismantle a preformed RecA filament even at low PcrA concentrations, suggesting a mode of action for eliminating potentially deleterious recombination intermediates. PMID:20723756

  5. Evaluation of DNA extraction methods for the analysis of microbial community in biological activated carbon.

    PubMed

    Zheng, Lu; Gao, Naiyun; Deng, Yang

    2012-01-01

    It is difficult to isolate DNA from biological activated carbon (BAC) samples used in water treatment plants, owing to the scarcity of microorganisms in BAC samples. The aim of this study was to identify DNA extraction methods suitable for a long-term, comprehensive ecological analysis of BAC microbial communities. To identify a procedure that can produce high molecular weight DNA, maximizes detectable diversity and is relatively free from contaminants, the microwave extraction method, the cetyltrimethylammonium bromide (CTAB) extraction method, a commercial DNA extraction kit, and the ultrasonic extraction method were used for the extraction of DNA from BAC samples. Spectrophotometry, agarose gel electrophoresis and polymerase chain reaction (PCR)-restriction fragment length polymorphisms (RFLP) analysis were conducted to compare the yield and quality of DNA obtained using these methods. The results showed that the CTAB method produce the highest yield and genetic diversity of DNA from BAC samples, but DNA purity was slightly less than that obtained with the DNA extraction-kit method. This study provides a theoretical basis for establishing and selecting DNA extraction methods for BAC samples. PMID:22629615

  6. Effective removal of co-purified inhibitors from extracted DNA samples using synchronous coefficient of drag alteration (SCODA) technology.

    PubMed

    Schmedes, Sarah; Marshall, Pamela; King, Jonathan L; Budowle, Bruce

    2013-07-01

    Various types of biological samples present challenges for extraction of DNA suitable for subsequent molecular analyses. Commonly used extraction methods, such as silica membrane columns and phenol-chloroform, while highly successful may still fail to provide a sufficiently pure DNA extract with some samples. Synchronous coefficient of drag alteration (SCODA), implemented in Boreal Genomics' Aurora Nucleic Acid Extraction System (Boreal Genomics, Vancouver, BC), is a new technology that offers the potential to remove inhibitors effectively while simultaneously concentrating DNA. In this initial study, SCODA was tested for its ability to remove various concentrations of forensically and medically relevant polymerase chain reaction (PCR) inhibitors naturally found in tissue, hair, blood, plant, and soil samples. SCODA was used to purify and concentrate DNA from intentionally contaminated DNA samples containing known concentrations of hematin, humic acid, melanin, and tannic acid. The internal positive control (IPC) provided in the Quantifiler™ Human DNA Quantification Kit (Life Technologies, Foster City, CA) and short tandem repeat (STR) profiling (AmpFℓSTR® Identifiler® Plus PCR Amplification Kit; Life Technologies, Foster City, CA) were used to measure inhibition effects and hence purification. SCODA methodology yielded overall higher efficiency of purification of highly contaminated samples compared with the QIAquick® PCR Purification Kit (Qiagen, Valencia, CA). SCODA-purified DNA yielded no cycle shift of the IPC for each sample and yielded greater allele percentage recovery and relative fluorescence unit values compared with the QIAquick® purification method. The Aurora provided an automated, minimal-step approach to successfully remove inhibitors and concentrate DNA from challenged samples. PMID:23254459

  7. Tenrec phylogeny and the noninvasive extraction of nuclear DNA.

    PubMed

    Asher, Robert J; Hofreiter, Michael

    2006-04-01

    Due in part to scarcity of material, no published study has yet cladistically addressed the systematics of living and fossil Tenrecidae (Mammalia, Afrotheria). Using a noninvasive technique for sampling nuclear DNA from museum specimens, we investigate the evolution of the Tenrecidae and assess the extent to which tenrecids fit patterns of relationships proposed for other terrestrial mammals on Madagascar. Application of several tree-reconstruction techniques on sequences of the nuclear growth hormone receptor gene and morphological data for all recognized tenrecid genera supports monophyly of Malagasy tenrecids to the exclusion of the two living African genera. However, both parsimony and Bayesian methods favor a close relationship between fossil African tenrecs and the Malagasy Geogale, supporting the hypothesis of island paraphyly, but not polyphyly. More generally, the noninvasive extraction technique can be applied with minimal risk to rare/unique specimens and, by better utilizing museum collections for genetic work, can greatly mitigate field expenses and disturbance of natural populations. PMID:16522569

  8. A kinetic analysis of strand breaks on large DNA induced by cigarette smoke extract

    NASA Astrophysics Data System (ADS)

    Kurita, Hirofumi; Takata, Tatsuya; Yasuda, Hachiro; Takashima, Kazunori; Mizuno, Akira

    2010-06-01

    We report a kinetic analysis of strand breakages on large DNA molecules induced by cigarette smoke extract (CSE), an extract of soluble cigarette smoke components. Previously, this DNA damage was analyzed by agarose gel electrophoresis, whereas we used fluorescence to kinetically analyze damage to individual DNA molecules. CSE caused a marked change in length of DNA molecules. The rate of CSE-induced double-strand breakage on large random-coiled DNA molecules was determined using a simple theoretical model, allowing the facile estimation of the rate of double-strand breaks on large DNA molecules.

  9. DNA Extraction and Amplification from Contemporary Polynesian Bark-Cloth

    PubMed Central

    Moncada, Ximena; Payacán, Claudia; Arriaza, Francisco; Lobos, Sergio; Seelenfreund, Daniela; Seelenfreund, Andrea

    2013-01-01

    Background Paper mulberry has been used for thousands of years in Asia and Oceania for making paper and bark-cloth, respectively. Museums around the world hold valuable collections of Polynesian bark-cloth. Genetic analysis of the plant fibers from which the textiles were made may answer a number of questions of interest related to provenance, authenticity or species used in the manufacture of these textiles. Recovery of nucleic acids from paper mulberry bark-cloth has not been reported before. Methodology We describe a simple method for the extraction of PCR-amplifiable DNA from small samples of contemporary Polynesian bark-cloth (tapa) using two types of nuclear markers. We report the amplification of about 300 bp sequences of the ITS1 region and of a microsatellite marker. Conclusions Sufficient DNA was retrieved from all bark-cloth samples to permit successful PCR amplification. This method shows a means of obtaining useful genetic information from modern bark-cloth samples and opens perspectives for the analyses of small fragments derived from ethnographic materials. PMID:23437166

  10. Extraction of genomic DNA from yeasts for PCR-based applications.

    PubMed

    Lõoke, Marko; Kristjuhan, Kersti; Kristjuhan, Arnold

    2011-05-01

    We have developed a quick and low-cost genomic DNA extraction protocol from yeast cells for PCR-based applications. This method does not require any enzymes, hazardous chemicals, or extreme temperatures, and is especially powerful for simultaneous analysis of a large number of samples. DNA can be efficiently extracted from different yeast species (Kluyveromyces lactis, Hansenula polymorpha, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris, and Saccharomyces cerevisiae). The protocol involves lysis of yeast colonies or cells from liquid culture in a lithium acetate (LiOAc)-SDS solution and subsequent precipitation of DNA with ethanol. Approximately 100 nanograms of total genomic DNA can be extracted from 1 × 10(7) cells. DNA extracted by this method is suitable for a variety of PCR-based applications (including colony PCR, real-time qPCR, and DNA sequencing) for amplification of DNA fragments of ≤ 3500 bp. PMID:21548894

  11. Human beta-globin gene polymorphisms characterized in DNA extracted from ancient bones 12,000 years old.

    PubMed Central

    Béraud-Colomb, E; Roubin, R; Martin, J; Maroc, N; Gardeisen, A; Trabuchet, G; Goosséns, M

    1995-01-01

    Analyzing the nuclear DNA from ancient human bones is an essential step to the understanding of genetic diversity in current populations, provided that such systematic studies are experimentally feasible. This article reports the successful extraction and amplification of nuclear DNA from the beta-globin region from 5 of 10 bone specimens up to 12,000 years old. These have been typed for beta-globin frameworks by sequencing through two variable positions and for a polymorphic (AT) chi (T) gamma microsatellite 500 bp upstream of the beta-globin gene. These specimens of human remains are somewhat older than those analyzed in previous nuclear gene sequencing reports and considerably older than those used to study high-copy-number human mtDNA. These results show that the systematic study of nuclear DNA polymorphisms of ancient populations is feasible. Images Figure 2 Figure 3 PMID:8533755

  12. Human {beta}-globin gene polymorphisms characterized in DNA extracted from ancient bones 12,000 years old

    SciTech Connect

    Beraud-Colomb, E. |; Maroc, N.; Roubin, R.

    1995-12-01

    Analyzing the nuclear DNA from ancient human bones is an essential step to the understanding of genetic diversity in current populations, provided that such systematic studies are experimentally feasible. This article reports the successful extraction and amplification of nuclear DNA from the P-globin region from 5 of 10 bone specimens up to 12,000 years old. These have been typed for P-globin frameworks by sequencing through two variable positions and for a polymorphic (AT){sub x}(T){sub y} microsatellite 500 bp upstream of the P-globin gene. These specimens of human remains are somewhat older than those analyzed in previous nuclear gene sequencing reports and considerably older than those used to study high-copy-number human mtDNA. These results show that the systematic study of nuclear DNA polymorphisms of ancient populations is feasible. 34 refs., 3 figs., 2 tabs.

  13. Innovative Graphite Oxide-Cellulose Based Material Specific for Genomic DNA Extraction

    NASA Astrophysics Data System (ADS)

    Akceoglu, Garbis Atam; Li, Oi Lun; Saito, Nagahiro

    2015-11-01

    Extraction of genomic DNA from various types of samples is often challenging for commercial silica spin column. In this study, we proposed graphite oxide (GO)/cellulose composite as an alternative material for genomic DNA extraction. The purity of DNA and extraction efficiency were compared to that of commercial silica product. In this study, the total weight % of GO was fixed at 4.15% in GO/Cellulose composite. Chewed gum, nail clip, cigarette bud paper, animal tissue and hair sample were used as various genomic DNA sources for extraction experiments. Among all types of samples, the extraction efficiencies were 4 to 12 times higher than that of commercial silica spin column. The absorbance ratio of 260 nm to 280 nm (A260/A280) of all samples ranged between 1.6 and 2.0. The results demonstrated that GO/Cellulose composites might serve as an innovative solid support material for genomic DNA extraction.

  14. Streamlining DNA Barcoding Protocols: Automated DNA Extraction and a New cox1 Primer in Arachnid Systematics

    PubMed Central

    Vidergar, Nina; Toplak, Nataša; Kuntner, Matjaž

    2014-01-01

    Background DNA barcoding is a popular tool in taxonomic and phylogenetic studies, but for most animal lineages protocols for obtaining the barcoding sequences—mitochondrial cytochrome C oxidase subunit I (cox1 AKA CO1)—are not standardized. Our aim was to explore an optimal strategy for arachnids, focusing on the species-richest lineage, spiders by (1) improving an automated DNA extraction protocol, (2) testing the performance of commonly used primer combinations, and (3) developing a new cox1 primer suitable for more efficient alignment and phylogenetic analyses. Methodology We used exemplars of 15 species from all major spider clades, processed a range of spider tissues of varying size and quality, optimized genomic DNA extraction using the MagMAX Express magnetic particle processor—an automated high throughput DNA extraction system—and tested cox1 amplification protocols emphasizing the standard barcoding region using ten routinely employed primer pairs. Results The best results were obtained with the commonly used Folmer primers (LCO1490/HCO2198) that capture the standard barcode region, and with the C1-J-2183/C1-N-2776 primer pair that amplifies its extension. However, C1-J-2183 is designed too close to HCO2198 for well-interpreted, continuous sequence data, and in practice the resulting sequences from the two primer pairs rarely overlap. We therefore designed a new forward primer C1-J-2123 60 base pairs upstream of the C1-J-2183 binding site. The success rate of this new primer (93%) matched that of C1-J-2183. Conclusions The use of C1-J-2123 allows full, indel-free overlap of sequences obtained with the standard Folmer primers and with C1-J-2123 primer pair. Our preliminary tests suggest that in addition to spiders, C1-J-2123 will also perform in other arachnids and several other invertebrates. We provide optimal PCR protocols for these primer sets, and recommend using them for systematic efforts beyond DNA barcoding. PMID:25415202

  15. Comparison of DNA extraction protocols for microbial communities from soil treated with biochar

    PubMed Central

    Leite, D.C.A.; Balieiro, F.C.; Pires, C.A.; Madari, B.E.; Rosado, A.S.; Coutinho, H.L.C.; Peixoto, R.S.

    2014-01-01

    Many studies have evaluated the effects of biochar application on soil structure and plant growth. However, there are very few studies describing the effect of biochar on native soil microbial communities. Microbial analysis of environmental samples requires accurate and reproducible methods for the extraction of DNA from samples. Because of the variety among microbial species and the strong adsorption of the phosphate backbone of the DNA molecule to biochar, extracting and purifying high quality microbial DNA from biochar-amended soil is not a trivial process and can be considerably more difficult than the extraction of DNA from other environmental samples. The aim of this study was to compare the relative efficacies of three commercial DNA extraction kits, the FastDNA® SPIN Kit for Soil (FD kit), the PowerSoil® DNA Isolation Kit (PS kit) and the ZR Soil Microbe DNA Kit Miniprep™ (ZR kit), for extracting microbial genomic DNA from sand treated with different types of biochar. The methods were evaluated by comparing the DNA yields and purity and by analysing the bacterial and fungal community profiles generated by PCR-DGGE. Our results showed that the PCR-DGGE profiles for bacterial and fungal communities were highly affected by the purity and yield of the different DNA extracts. Among the tested kits, the PS kit was the most efficient with respect to the amount and purity of recovered DNA and considering the complexity of the generated DGGE microbial fingerprint from the sand-biochar samples. PMID:24948928

  16. Comparison of DNA extraction protocols for microbial communities from soil treated with biochar.

    PubMed

    Leite, D C A; Balieiro, F C; Pires, C A; Madari, B E; Rosado, A S; Coutinho, H L C; Peixoto, R S

    2014-01-01

    Many studies have evaluated the effects of biochar application on soil structure and plant growth. However, there are very few studies describing the effect of biochar on native soil microbial communities. Microbial analysis of environmental samples requires accurate and reproducible methods for the extraction of DNA from samples. Because of the variety among microbial species and the strong adsorption of the phosphate backbone of the DNA molecule to biochar, extracting and purifying high quality microbial DNA from biochar-amended soil is not a trivial process and can be considerably more difficult than the extraction of DNA from other environmental samples. The aim of this study was to compare the relative efficacies of three commercial DNA extraction kits, the FastDNA® SPIN Kit for Soil (FD kit), the PowerSoil® DNA Isolation Kit (PS kit) and the ZR Soil Microbe DNA Kit Miniprep™ (ZR kit), for extracting microbial genomic DNA from sand treated with different types of biochar. The methods were evaluated by comparing the DNA yields and purity and by analysing the bacterial and fungal community profiles generated by PCR-DGGE. Our results showed that the PCR-DGGE profiles for bacterial and fungal communities were highly affected by the purity and yield of the different DNA extracts. Among the tested kits, the PS kit was the most efficient with respect to the amount and purity of recovered DNA and considering the complexity of the generated DGGE microbial fingerprint from the sand-biochar samples. PMID:24948928

  17. Local surface sampling step estimation for extracting boundaries of planar point clouds

    NASA Astrophysics Data System (ADS)

    Brie, David; Bombardier, Vincent; Baeteman, Grégory; Bennis, Abdelhamid

    2016-09-01

    This paper presents a new approach to estimate the surface sampling step of planar point clouds acquired by Terrestrial Laser Scanner (TLS) which is varying with the distance to the surface and the angular positions. The local surface sampling step is obtained by doing a first order Taylor expansion of planar point coordinates. Then, it is shown how to use it in Delaunay-based boundary point extraction. The resulting approach, which is implemented in the ModiBuilding software, is applied to two facade point clouds of a building. The first is acquired with a single station and the second with two stations. In both cases, the proposed approach performs very accurately and appears to be robust to the variations of the point cloud density.

  18. Carrier molecules and extraction of circulating tumor DNA for next generation sequencing in colorectal cancer.

    PubMed

    Beránek, Martin; Sirák, Igor; Vošmik, Milan; Petera, Jiří; Drastíková, Monika; Palička, Vladimír

    2016-01-01

    The aims of the study were: i) to compare circulating tumor DNA (ctDNA) yields obtained by different manual extraction procedures, ii) to evaluate the addition of various carrier molecules into the plasma to improve ctDNA extraction recovery, and iii) to use next generation sequencing (NGS) technology to analyze KRAS, BRAF, and NRAS somatic mutations in ctDNA from patients with metastatic colorectal cancer. Venous blood was obtained from patients who suffered from metastatic colorectal carcinoma. For plasma ctDNA extraction, the following carriers were tested: carrier RNA, polyadenylic acid, glycogen, linear acrylamide, yeast tRNA, salmon sperm DNA, and herring sperm DNA. Each extract was characterized by quantitative real-time PCR and next generation sequencing. The addition of polyadenylic acid had a significant positive effect on the amount of ctDNA eluted. The sequencing data revealed five cases of ctDNA mutated in KRAS and one patient with a BRAF mutation. An agreement of 86% was found between tumor tissues and ctDNA. Testing somatic mutations in ctDNA seems to be a promising tool to monitor dynamically changing genotypes of tumor cells circulating in the body. The optimized process of ctDNA extraction should help to obtain more reliable sequencing data in patients with metastatic colorectal cancer. PMID:27526306

  19. Water extraction of pyrolysis oil: the first step for the recovery of renewable chemicals.

    PubMed

    Vitasari, Caecilia R; Meindersma, G W; de Haan, André B

    2011-07-01

    The interest in biomass as a source of renewable energy and chemicals has been increasing in keeping up with the transition to a sustainable bio-based economy. An important initial step of chemicals recovery from biomass-derived pyrolysis oil is water extraction where most of polar compounds are isolated in the aqueous phase. This study was done to investigate the effects of stirring rate and water-to-oil ratio on the extraction capability (distribution coefficient and yield), water content, and atomic composition of both aqueous and organic phases. The results show that the stirring rate above 300 rpm has no influence on the equilibrium. Increasing the water-to-oil ratio dilutes the aqueous phase without changing the atomic distribution. Forest residue-derived pyrolysis oil should be extracted at a water-to-oil ratio of 0.65-0.7, whereas pine-derived pyrolysis oil is preferably extracted at the lowest feasible water-to-oil ratio where complete phase separation occurs, which is 0.5 in this study. PMID:21592785

  20. Evaluation of a Modified DNA Extraction Method for Isolation of Cell-Free Fetal DNA from Maternal Serum

    PubMed Central

    Keshavarz, Zeinab; Moezzi, Leili; Ranjbaran, Reza; Aboualizadeh, Farzaneh; Behzad-Behbahani, Abbas; Abdullahi, Masooma; Sharifzadeh, Sedigheh

    2015-01-01

    Background: Discovery of short cell free fetal DNA (cffDNA) fragments in maternal plasma has created major changes in the field of prenatal diagnosis. The use of cffDNA to set up noninvasive prenatal test is limited due to the low concentration of fetal DNA in maternal plasma therefore, employing a high efficiency extraction method leads to more accurate results. The aim of this study was to evaluate the efficiency of Triton/Heat/Phenol (THP) protocol in comparison with the QIAamp DNA Blood mini Kit for cffDNA purification. Methods: In order to evaluate the efficiency of THP protocol, DNA of Rhesus D (RhD) negative pregnant women's plasma was collected, then real-time PCR for RHD exon 7 was performed. The Ct value data of real time PCR obtained by two different methods were compared and after delivery serology test on cord blood was done to validate the real time PCR results. Results: The results indicated significant differences between two extraction methods (p=0.001). The mean±SD of Ct-value using THP protocol was 33.8±1.6 and 36.1±2.47 using QIAamp DNA Blood mini Kit. Conclusion: Our finding demonstrated that THP protocol was more effective than the QIAamp DNA Blood mini Kits for cffDNA extraction and lead to decrease the false negative results. PMID:26140187

  1. A ruthenium dimer complex with a flexible linker slowly threads between DNA bases in two distinct steps

    PubMed Central

    Bahira, Meriem; McCauley, Micah J.; Almaqwashi, Ali A.; Lincoln, Per; Westerlund, Fredrik; Rouzina, Ioulia; Williams, Mark C.

    2015-01-01

    Several multi-component DNA intercalating small molecules have been designed around ruthenium-based intercalating monomers to optimize DNA binding properties for therapeutic use. Here we probe the DNA binding ligand [μ-C4(cpdppz)2(phen)4Ru2]4+, which consists of two Ru(phen)2dppz2+ moieties joined by a flexible linker. To quantify ligand binding, double-stranded DNA is stretched with optical tweezers and exposed to ligand under constant applied force. In contrast to other bis-intercalators, we find that ligand association is described by a two-step process, which consists of fast bimolecular intercalation of the first dppz moiety followed by ∼10-fold slower intercalation of the second dppz moiety. The second step is rate-limited by the requirement for a DNA-ligand conformational change that allows the flexible linker to pass through the DNA duplex. Based on our measured force-dependent binding rates and ligand-induced DNA elongation measurements, we are able to map out the energy landscape and structural dynamics for both ligand binding steps. In addition, we find that at zero force the overall binding process involves fast association (∼10 s), slow dissociation (∼300 s), and very high affinity (Kd ∼10 nM). The methodology developed in this work will be useful for studying the mechanism of DNA binding by other multi-step intercalating ligands and proteins. PMID:26365236

  2. A ruthenium dimer complex with a flexible linker slowly threads between DNA bases in two distinct steps.

    PubMed

    Bahira, Meriem; McCauley, Micah J; Almaqwashi, Ali A; Lincoln, Per; Westerlund, Fredrik; Rouzina, Ioulia; Williams, Mark C

    2015-10-15

    Several multi-component DNA intercalating small molecules have been designed around ruthenium-based intercalating monomers to optimize DNA binding properties for therapeutic use. Here we probe the DNA binding ligand [μ-C4(cpdppz)2(phen)4Ru2](4+), which consists of two Ru(phen)2dppz(2+) moieties joined by a flexible linker. To quantify ligand binding, double-stranded DNA is stretched with optical tweezers and exposed to ligand under constant applied force. In contrast to other bis-intercalators, we find that ligand association is described by a two-step process, which consists of fast bimolecular intercalation of the first dppz moiety followed by ∼10-fold slower intercalation of the second dppz moiety. The second step is rate-limited by the requirement for a DNA-ligand conformational change that allows the flexible linker to pass through the DNA duplex. Based on our measured force-dependent binding rates and ligand-induced DNA elongation measurements, we are able to map out the energy landscape and structural dynamics for both ligand binding steps. In addition, we find that at zero force the overall binding process involves fast association (∼10 s), slow dissociation (∼300 s), and very high affinity (Kd ∼10 nM). The methodology developed in this work will be useful for studying the mechanism of DNA binding by other multi-step intercalating ligands and proteins. PMID:26365236

  3. A high-throughput, high-quality plant genomic DNA extraction protocol.

    PubMed

    Li, H; Li, J; Cong, X H; Duan, Y B; Li, L; Wei, P C; Lu, X Z; Yang, J B

    2013-01-01

    The isolation of high-quality genomic DNA (gDNA) is a crucial technique in plant molecular biology. The quality of gDNA determines the reliability of real-time polymerase chain reaction (PCR) analysis. In this paper, we reported a high-quality gDNA extraction protocol optimized for real-time PCR in a variety of plant species. Performed in a 96-well block, our protocol provides high throughput. Without the need for phenol-chloroform and liquid nitrogen or dry ice, our protocol is safer and more cost-efficient than traditional DNA extraction methods. The method takes 10 mg leaf tissue to yield 5-10 µg high-quality gDNA. Spectral measurement and electrophoresis were used to demonstrate gDNA purity. The extracted DNA was qualified in a restriction enzyme digestion assay and conventional PCR. The real-time PCR amplification was sufficiently sensitive to detect gDNA at very low concentrations (3 pg/µL). The standard curve of gDNA dilutions from our phenol-chloroform-free protocol showed better linearity (R(2) = 0.9967) than the phenol-chloroform protocol (R(2) = 0.9876). The results indicate that the gDNA was of high quality and fit for real-time PCR. This safe, high-throughput plant gDNA extraction protocol could be used to isolate high-quality gDNA for real-time PCR and other downstream molecular applications. PMID:24222228

  4. DNA excision repair in cell extracts from human cell lines exhibiting hypersensitivity to DNA-damaging agents

    SciTech Connect

    Hansson, J.; Keyse, S.M.; Lindahl, T.; Wood, R.D. )

    1991-07-01

    Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurements of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links.

  5. Estimation of the uncertainties of extraction and clean-up steps in pesticide residue analysis of plant commodities.

    PubMed

    Omeroglu, P Yolci; Ambrus, A; Boyacioglu, D

    2013-01-01

    Extraction and clean-up constitute important steps in pesticide residue analysis. For the correct interpretation of analytical results, uncertainties of extraction and clean-up steps should be taken into account when the combined uncertainty of the analytical result is estimated. In the scope of this study, uncertainties of extraction and clean-up steps were investigated by spiking (14)C-labelled chlorpyrifos to analytical portions of tomato, orange, apple, green bean, cucumber, jackfruit, papaya and starfruit. After each step, replicate measurements were carried out with a liquid scintillation counter. Uncertainties in extraction and clean-up steps were estimated separately for every matrix and method combination by using within-laboratory reproducibility standard deviation and were characterised with the CV of recoveries. It was observed that the uncertainty of the ethyl acetate extraction step varied between 0.8% and 5.9%. The relative standard uncertainty of the clean-up step with dispersive SPE used in the method known as QuEChERS was estimated to be around 1.5% for tomato, apple and green beans. The highest variation of 4.8% was observed in cucumber. The uncertainty of the clean-up step with gel permeation chromatography ranged between 5.3% and 13.1%, and it was relatively higher than that obtained with the dispersive SPE method. PMID:23216411

  6. Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells.

    PubMed Central

    Stillman, B W; Gluzman, Y

    1985-01-01

    Soluble extracts prepared from the nucleus and cytoplasm of human 293 cells are capable of efficient replication and supercoiling of added DNA templates that contain the origin of simian virus 40 replication. Extracts prepared from human HeLa cells are less active than similarly prepared extracts from 293 cells for initiation and elongation of nascent DNA strands. DNA synthesis is dependent on addition of purified simian virus 40 tumor (T) antigen, which is isolated by immunoaffinity chromatography of extracts from cells infected with an adenovirus modified to produce large quantities of this protein. In the presence of T antigen and the cytoplasmic extract, replication initiates at the origin and continues bidirectionally. Initiation is completely dependent on functional origin sequences; a plasmid DNA containing an origin mutation known to affect DNA replication in vivo fails to replicate in vitro. Multiple rounds of DNA synthesis occur, as shown by the appearance of heavy-heavy, bromodeoxyuridine-labeled DNA products. The products of this reaction are resolved, but are relaxed, covalently closed DNA circles. Addition of a nuclear extract during DNA synthesis promotes the negative supercoiling of the replicated DNA molecules. Images PMID:3018548

  7. In situ 2D-extraction of DNA wheels by 3D through-solution transport.

    PubMed

    Yonamine, Yusuke; Cervantes-Salguero, Keitel; Nakanishi, Waka; Kawamata, Ibuki; Minami, Kosuke; Komatsu, Hirokazu; Murata, Satoshi; Hill, Jonathan P; Ariga, Katsuhiko

    2015-12-28

    Controlled transfer of DNA nanowheels from a hydrophilic to a hydrophobic surface was achieved by complexation of the nanowheels with a cationic lipid (2C12N(+)). 2D surface-assisted extraction, '2D-extraction', enabled structure-persistent transfer of DNA wheels, which could not be achieved by simple drop-casting. PMID:26583486

  8. DNA extraction protocols from dormant buds of twelve woody plant genera

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Standard plant DNA extraction protocols call for samples of newly expanding leaves and shoots yet analysis is sometimes needed when plants are dormant. We evaluated three DNA extraction protocols using dormant buds from 40 species and four hybrids of 12 genera. Two protocols were from ready-to-use ...

  9. Comparison of DNA extraction methods for sweet corn and processed sweet corns.

    PubMed

    Takabatake, Reona; Noritake, Hiromichi; Noguchi, Akio; Nakamura, Kosuke; Kondo, Kazunari; Akiyama, Hiroshi; Teshima, Reiko; Mano, Junichi; Kitta, Kazumi

    2013-01-01

    DNA was extracted from sweet corn and its processed products using four DNA extraction methods: the CTAB method, the DNeasy Plant Maxi kit, GM Quicker 3, and Genomic-tip 20/G. DNA was successfully extracted from raw sweet corn and baby corn samples using all four methods. Meanwhile, from frozen, canned, and dry pack products, DNA was well extracted using the DNeasy Plant Maxi kit, GM Quicker 3, and Genomic-tip 20/G, but not enough with the CTAB method. The highest yield of DNA was obtained with Genomic-tip 20/G. The degree of degradation of extracted DNA was observed to increase in the order of raw, frozen, canned, dry pack, and baby corn samples. To evaluate the quality of extracted DNA, real-time PCR analyses were conducted using three maize endogenous genes. The DNAs extracted using GM Quicker 3 had high purity, suggesting that GM Quicker 3 would be the most suitable method for DNA extraction from processed sweet corn products. PMID:24025210

  10. A RAPID DNA EXTRACTION METHOD FOR PCR IDENTIFICATION OF FUNGAL INDOOR AIR CONTAMINANTS

    EPA Science Inventory

    Following air sampling, fungal DNA needs to be extracted and purified to a state suitable for laboratory use. Our laboratory has developed a simple method of extraction and purification of fungal DNA appropriate for enzymatic manipulation and polymerase chain reaction (PCR) appli...

  11. EFFECTIVE METHOD TO EXTRACT DNA FROM ENVIRONMENTAL SAMPLES FOR POLYMERASE CHAIN REACTION AMPLIFICATION AND DNA FINGERPRINT ANALYSIS

    EPA Science Inventory

    A rapid direct-extraction method was used to obtain DNA from environmental soil samples. eat, enzymes, and guanidine isothiocyanate were utilized to lyse cells. he DNA was purified by agarose gel electrophoresis, amplified with 16S based primers by use of the polymerase chain rea...

  12. PCR AMPLIFICATION OF WHEAT SEQUENCES FROM DNA EXTRACTED DURING MILLING AND BAKING

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Detection of transgenic events are based upon the identification of novel proteins specific activities, detection of specific DNA sequences. Processing steps have a profound effect upon the proteins and DNA present in the final product. DNA-based analysis has several advantages over protein-based me...

  13. A modified acidic approach for DNA extraction from plant species containing high levels of secondary metabolites.

    PubMed

    Cavallari, M M; Siqueira, M V B M; Val, T M; Pavanelli, J C; Monteiro, M; Grando, C; Pinheiro, J B; Zucchi, M I; Gimenes, M A

    2014-01-01

    Purified genomic DNA can be difficult to obtain from some plant species because of the presence of impurities such as polysaccharides, which are often co-extracted with DNA. In this study, we developed a fast, simple, and low-cost protocol for extracting DNA from plants containing high levels of secondary metabolites. This protocol does not require the use of volatile toxic reagents such as mercaptoethanol, chloroform, or phenol and allows the extraction of high-quality DNA from wild and cultivated tropical species. PMID:25158268

  14. Glycans affect DNA extraction and induce substantial differences in gut metagenomic studies.

    PubMed

    Angelakis, Emmanouil; Bachar, Dipankar; Henrissat, Bernard; Armougom, Fabrice; Audoly, Gilles; Lagier, Jean-Christophe; Robert, Catherine; Raoult, Didier

    2016-01-01

    Exopolysaccharides produced by bacterial species and present in feces are extremely inhibitory to DNA restriction and can cause discrepancies in metagenomic studies. We determined the effects of different DNA extraction methods on the apparent composition of the gut microbiota using Illumina MiSeq deep sequencing technology. DNA was extracted from the stool from an obese female using 10 different methods and the choice of DNA extraction method affected the proportional abundance at the phylum level, species richness (Chao index, 227 to 2,714) and diversity (non parametric Shannon, 1.37 to 4.4). Moreover DNA was extracted from stools obtained from 83 different individuals by the fastest extraction assay and by an extraction assay that degradated exopolysaccharides. The fastest extraction method was able to detect 68% to 100% genera and 42% to 95% species whereas the glycan degradation extraction method was able to detect 56% to 93% genera and 25% to 87% species. To allow a good liberation of DNA from exopolysaccharides commonly presented in stools, we recommend the mechanical lysis of stools plus glycan degradation, used here for the first time. Caution must be taken in the interpretation of current metagenomic studies, as the efficiency of DNA extraction varies widely among stool samples. PMID:27188959

  15. Glycans affect DNA extraction and induce substantial differences in gut metagenomic studies

    PubMed Central

    Angelakis, Emmanouil; Bachar, Dipankar; Henrissat, Bernard; Armougom, Fabrice; Audoly, Gilles; Lagier, Jean-Christophe; Robert, Catherine; Raoult, Didier

    2016-01-01

    Exopolysaccharides produced by bacterial species and present in feces are extremely inhibitory to DNA restriction and can cause discrepancies in metagenomic studies. We determined the effects of different DNA extraction methods on the apparent composition of the gut microbiota using Illumina MiSeq deep sequencing technology. DNA was extracted from the stool from an obese female using 10 different methods and the choice of DNA extraction method affected the proportional abundance at the phylum level, species richness (Chao index, 227 to 2,714) and diversity (non parametric Shannon, 1.37 to 4.4). Moreover DNA was extracted from stools obtained from 83 different individuals by the fastest extraction assay and by an extraction assay that degradated exopolysaccharides. The fastest extraction method was able to detect 68% to 100% genera and 42% to 95% species whereas the glycan degradation extraction method was able to detect 56% to 93% genera and 25% to 87% species. To allow a good liberation of DNA from exopolysaccharides commonly presented in stools, we recommend the mechanical lysis of stools plus glycan degradation, used here for the first time. Caution must be taken in the interpretation of current metagenomic studies, as the efficiency of DNA extraction varies widely among stool samples. PMID:27188959

  16. The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol-chloroform-isoamyl alcohol DNA extraction.

    PubMed

    Renshaw, Mark A; Olds, Brett P; Jerde, Christopher L; McVeigh, Margaret M; Lodge, David M

    2015-01-01

    Current research targeting filtered macrobial environmental DNA (eDNA) often relies upon cold ambient temperatures at various stages, including the transport of water samples from the field to the laboratory and the storage of water and/or filtered samples in the laboratory. This poses practical limitations for field collections in locations where refrigeration and frozen storage is difficult or where samples must be transported long distances for further processing and screening. This study demonstrates the successful preservation of eDNA at room temperature (20 °C) in two lysis buffers, CTAB and Longmire's, over a 2-week period of time. Moreover, the preserved eDNA samples were seamlessly integrated into a phenol-chloroform-isoamyl alcohol (PCI) DNA extraction protocol. The successful application of the eDNA extraction to multiple filter membrane types suggests the methods evaluated here may be broadly applied in future eDNA research. Our results also suggest that for many kinds of studies recently reported on macrobial eDNA, detection probabilities could have been increased, and at a lower cost, by utilizing the Longmire's preservation buffer with a PCI DNA extraction. PMID:24834966

  17. The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol–chloroform–isoamyl alcohol DNA extraction

    PubMed Central

    Renshaw, Mark A; Olds, Brett P; Jerde, Christopher L; McVeigh, Margaret M; Lodge, David M

    2015-01-01

    Current research targeting filtered macrobial environmental DNA (eDNA) often relies upon cold ambient temperatures at various stages, including the transport of water samples from the field to the laboratory and the storage of water and/or filtered samples in the laboratory. This poses practical limitations for field collections in locations where refrigeration and frozen storage is difficult or where samples must be transported long distances for further processing and screening. This study demonstrates the successful preservation of eDNA at room temperature (20 °C) in two lysis buffers, CTAB and Longmire's, over a 2-week period of time. Moreover, the preserved eDNA samples were seamlessly integrated into a phenol–chloroform–isoamyl alcohol (PCI) DNA extraction protocol. The successful application of the eDNA extraction to multiple filter membrane types suggests the methods evaluated here may be broadly applied in future eDNA research. Our results also suggest that for many kinds of studies recently reported on macrobial eDNA, detection probabilities could have been increased, and at a lower cost, by utilizing the Longmire's preservation buffer with a PCI DNA extraction. PMID:24834966

  18. Enhanced method for microbial community DNA extraction and purification from agricultural yellow loess soil.

    PubMed

    Kathiravan, Mathur Nadarajan; Gim, Geun Ho; Ryu, Jaewon; Kim, Pyung Il; Lee, Chul Won; Kim, Si Wouk

    2015-11-01

    In this study, novel DNA extraction and purification methods were developed to obtain high-quantity and reliable quality DNA from the microbial community of agricultural yellow loess soil samples. The efficiencies of five different soil DNAextraction protocols were evaluated on the basis of DNA yield, quality and DNA shearing. Our suggested extraction method, which used CTAB, EDTA and cell membrane lytic enzymes in the extraction followed by DNA precipitation using isopropanol, yielded a maximum DNA content of 42.28 ± 5.59 µg/g soil. In addition, among the five different purification protocols, the acid-treated polyvinyl polypyrrolidone (PVPP) spin column purification method yielded high-quality DNA and recovered 91% of DNA from the crude DNA. Spectrophotometry revealed that the ultraviolet A 260/A 230 and A 260/A 280 absorbance ratios of the purified DNA were 1.82 ± 0.03 and 1.94 ± 0.05, respectively. PCR-based 16S rRNA amplification showed clear bands at ~1.5 kb with acid-treated PVPP-purified DNA templates. In conclusion, our suggested extraction and purification protocols can be used to recover high concentration, high purity, and high-molecular-weight DNA from clay and silica-rich agricultural soil samples. PMID:26502961

  19. A non-invasive technique for rapid extraction of DNA from fish scales.

    PubMed

    Kumar, Ravindra; Singh, Poonam Jayant; Nagpure, N S; Kushwaha, Basdeo; Srivastava, S K; Lakra, W S

    2007-11-01

    DNA markers are being increasingly used in studies related to population genetics and conservation biology of endangered species. DNA isolation for such studies requires a source of biological material that is easy to collect, non-bulky and reliable. Further, the sampling strategies based on non-invasive procedures are desirable, especially for the endangered fish species. In view of above, a rapid DNA extraction method from fish scales has been developed with the use of a modified lysis buffer that require about 2 hr duration. This methodology is non-invasive, less expensive and reproducible with high efficiency of DNA recovery. The DNA extracted by this technique, have been found suitable for performing restriction enzyme digestion and PCR amplification. Therefore, the present DNA extraction procedure can be used as an alternative technique in population genetic studies pertaining to endangered fish species. The technique was also found equally effective for DNA isolation from fresh, dried and ethanol preserved scales. PMID:18072545

  20. Two-step adaptive extraction method for ground points and breaklines from lidar point clouds

    NASA Astrophysics Data System (ADS)

    Yang, Bisheng; Huang, Ronggang; Dong, Zhen; Zang, Yufu; Li, Jianping

    2016-09-01

    The extraction of ground points and breaklines is a crucial step during generation of high quality digital elevation models (DEMs) from airborne LiDAR point clouds. In this study, we propose a novel automated method for this task. To overcome the disadvantages of applying a single filtering method in areas with various types of terrain, the proposed method first classifies the points into a set of segments and one set of individual points, which are filtered by segment-based filtering and multi-scale morphological filtering, respectively. In the process of multi-scale morphological filtering, the proposed method removes amorphous objects from the set of individual points to decrease the effect of the maximum scale on the filtering result. The proposed method then extracts the breaklines from the ground points, which provide a good foundation for generation of a high quality DEM. Finally, the experimental results demonstrate that the proposed method extracts ground points in a robust manner while preserving the breaklines.

  1. [Rapid construction of full-length MnSOD cDNA of chickens by one-step 3'RACE].

    PubMed

    Bu, You-Quan; Luo, Xu-Gang; Liu, Bin; Li, Su-Fen

    2004-07-01

    RACE (rapid amplification of cDNA ends) is a popular technique to rapidly obtain the full-length cDNA. After obtaining the 3' cDNA and 5' cDNA fragments with a overlapped region by 3' RACE and 5' RACE, the full-length cDNA could be generated by end-to-end PCR or subcloning. In this study, 3' RACE combined with touch-down PCR was successfully used for the rapid construction of full-length MnSOD cDNA of chickens. Compared with the conventional end-to-end PCR or subcloning, this method, called one-step 3' RACE, is fast, economical and highly specific. It especially fits the rapid construction of full-length cDNA by RACE method. PMID:15640053

  2. Optimizing techniques to capture and extract environmental DNA for detection and quantification of fish.

    PubMed

    Eichmiller, Jessica J; Miller, Loren M; Sorensen, Peter W

    2016-01-01

    Few studies have examined capture and extraction methods for environmental DNA (eDNA) to identify techniques optimal for detection and quantification. In this study, precipitation, centrifugation and filtration eDNA capture methods and six commercially available DNA extraction kits were evaluated for their ability to detect and quantify common carp (Cyprinus carpio) mitochondrial DNA using quantitative PCR in a series of laboratory experiments. Filtration methods yielded the most carp eDNA, and a glass fibre (GF) filter performed better than a similar pore size polycarbonate (PC) filter. Smaller pore sized filters had higher regression slopes of biomass to eDNA, indicating that they were potentially more sensitive to changes in biomass. Comparison of DNA extraction kits showed that the MP Biomedicals FastDNA SPIN Kit yielded the most carp eDNA and was the most sensitive for detection purposes, despite minor inhibition. The MoBio PowerSoil DNA Isolation Kit had the lowest coefficient of variation in extraction efficiency between lake and well water and had no detectable inhibition, making it most suitable for comparisons across aquatic environments. Of the methods tested, we recommend using a 1.5 μm GF filter, followed by extraction with the MP Biomedicals FastDNA SPIN Kit for detection. For quantification of eDNA, filtration through a 0.2-0.6 μm pore size PC filter, followed by extraction with MoBio PowerSoil DNA Isolation Kit was optimal. These results are broadly applicable for laboratory studies on carps and potentially other cyprinids. The recommendations can also be used to inform choice of methodology for field studies. PMID:25919417

  3. The elimination of DNA from the Cry toxin-DNA complex is a necessary step in the mode of action of the Cry8 toxin.

    PubMed

    Ai, Bingjie; Li, Jie; Feng, Dongmei; Li, Feng; Guo, Shuyuan

    2013-01-01

    Several crystal (Cry) proteins are known to occur as DNA-protein complexes. However, the role of the DNA associated with the activated toxin in the mechanism of action of the Cry toxin has long been ignored. Here, we focused on the DNA-activated Cry toxin complex. Both forms of the Cry8Ca2 and Cry8Ea1 toxins, i.e., with or without bound DNA, were separately obtained. Size-exclusion chromatography analysis indicated that the Cry8Ca2 toxin-DNA complex has a tight or compact structure. The Cry8Ca2 toxin-DNA complex is more likely to move toward the air/water interface and is more hydrophobic than the toxin without DNA. Competitive binding assays indicated that the Cry8Ca2 and Cry8Ea1 toxins without DNA specifically bind to the midgut of Anomala corpulenta and Holotrichia parallela larvae, respectively. In contrast, the association of DNA with each toxin might result in the nonspecific recognition of the Cry toxin and its target receptor in the insect midgut. The association of the DNA fragment with the Cry8 toxin was shown to protect the Cry protein from digestion by proteases. Based on our results, we propose an additional step in the mechanism of action of the Cry8 toxin and elucidate the function of the associated DNA as well as the importance of the removal of this DNA for the insecticidal activity of the toxin. PMID:24324685

  4. The Elimination of DNA from the Cry Toxin-DNA Complex Is a Necessary Step in the Mode of Action of the Cry8 Toxin

    PubMed Central

    Ai, Bingjie; Li, Jie; Feng, Dongmei; Li, Feng; Guo, Shuyuan

    2013-01-01

    Several crystal (Cry) proteins are known to occur as DNA-protein complexes. However, the role of the DNA associated with the activated toxin in the mechanism of action of the Cry toxin has long been ignored. Here, we focused on the DNA-activated Cry toxin complex. Both forms of the Cry8Ca2 and Cry8Ea1 toxins, i.e., with or without bound DNA, were separately obtained. Size-exclusion chromatography analysis indicated that the Cry8Ca2 toxin-DNA complex has a tight or compact structure. The Cry8Ca2 toxin-DNA complex is more likely to move toward the air/water interface and is more hydrophobic than the toxin without DNA. Competitive binding assays indicated that the Cry8Ca2 and Cry8Ea1 toxins without DNA specifically bind to the midgut of Anomala corpulenta and Holotrichia parallela larvae, respectively. In contrast, the association of DNA with each toxin might result in the nonspecific recognition of the Cry toxin and its target receptor in the insect midgut. The association of the DNA fragment with the Cry8 toxin was shown to protect the Cry protein from digestion by proteases. Based on our results, we propose an additional step in the mechanism of action of the Cry8 toxin and elucidate the function of the associated DNA as well as the importance of the removal of this DNA for the insecticidal activity of the toxin. PMID:24324685

  5. Evaluation of DNA extraction methods for the detection of Cytomegalovirus in dried blood spots

    PubMed Central

    Koontz, D.; Baecher, K.; Amin, M.; Nikolova, S.; Gallagher, M.; Dollard, S.

    2015-01-01

    Background Dried blood spots (DBS) are collected universally from newborns and may be valuable for the diagnosis of congenital Cytomegalovirus (CMV) infection. The reported analytical sensitivity for DBS testing compared to urine or saliva varies greatly across CMV studies. The purpose of this study was to directly compare the performance of various DNA extraction methods for identification of CMV in DBS including those used most often in CMV studies. Study design Whatman® Grade 903 filter paper cards were spotted with blood samples from 25 organ transplant recipients who had confirmed CMV viremia. Six DNA extraction methods were compared for relative yield of viral and cellular DNA: 2 manual solution-based methods (Gentra Puregene, thermal shock), 2 manual silica column-based methods (QIAamp DNA Mini, QIAamp DNA Investigator), and 2 automated methods (M48 MagAttract Mini, QIAcube Investigator). DBS extractions were performed in triplicate followed by real-time quantitative PCR (qPCR). Results For extraction of both viral and cellular DNA, two methods (QIAamp DNA Investigator and thermal shock) consistently gave the highest yields, and two methods (M48 MagAttract Mini and QIAamp DNA Mini) consistently gave the lowest yields. There was an average 3-fold difference in DNA yield between the highest and lowest yield methods. Conclusion The choice of DNA extraction method is a major factor in the ability to detect low levels of CMV in DBS and can largely account for the wide range of DBS sensitivities reported in studies to date. PMID:25866346

  6. A high volume extraction and purification method for recovering DNA from human bone.

    PubMed

    Marshall, Pamela L; Stoljarova, Monika; Schmedes, Sarah E; King, Jonathan L; Budowle, Bruce

    2014-09-01

    DNA recovery, purity and overall extraction efficiency of a protocol employing a novel silica-based column, Hi-Flow(®) (Generon Ltd., Maidenhead, UK), were compared with that of a standard organic DNA extraction methodology. The quantities of DNA recovered by each method were compared by real-time PCR and quality of DNA by STR typing using the PowerPlex(®) ESI 17 Pro System (Promega Corporation, Madison, WI) on DNA from 10 human bone samples. Overall, the Hi-Flow method recovered comparable quantities of DNA ranging from 0.8ng±1 to 900ng±159 of DNA compared with the organic method ranging from 0.5ng±0.9 to 855ng±156 of DNA. Complete profiles (17/17 loci tested) were obtained for at least one of three replicates for 3/10 samples using the Hi-Flow method and from 2/10 samples with the organic method. All remaining bone samples yielded partial profiles for all replicates with both methods. Compared with a standard organic DNA isolation method, the results indicated that the Hi-Flow method provided equal or improved recovery and quality of DNA without the harmful effects of organic extraction. Moreover, larger extraction volumes (up to 20mL) can be employed with the Hi-Flow method which enabled more bone sample to be extracted at one time. PMID:24997320

  7. Using a commercial DNA extraction kit to obtain RNA from mature rice kernels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Few RNA extraction protocols or commercial kits work well with the starchy endosperm of cereal grains. Standard RNA extraction protocols are time consuming, use large amounts of expensive chemicals, and leave behind hazardous wastes. However, there are numerous commercial DNA extraction kits that ...

  8. [Method validation according to ISO 15189 and SH GTA 04: application for the extraction of DNA and its quantitative evaluation by a spectrophotometric assay].

    PubMed

    Harlé, Alexandre; Lion, Maëva; Husson, Marie; Dubois, Cindy; Merlin, Jean-Louis

    2013-01-01

    According to the French legislation on medical biology (January 16th, 2010), all biological laboratories must be accredited according to ISO 15189 for at least 50% of their activities before the end of 2016. The extraction of DNA from a sample of interest, whether solid or liquid is one of the critical steps in molecular biology and specifically in somatic or constitutional genetic. The extracted DNA must meet a number of criteria such quality and also be in sufficient concentration to allow molecular biology assays such as the detection of somatic mutations. This paper describes the validation of the extraction and purification of DNA using chromatographic column extraction and quantitative determination by spectrophotometric assay, according to ISO 15189 and the accreditation technical guide in Human Health SH-GTA-04. PMID:24113450

  9. Extracting DNA Twist Rigidity from Experimental Supercoiling Data

    NASA Astrophysics Data System (ADS)

    Neukirch, Sébastien

    2004-11-01

    We use an elastic rod model with contact to study the extension versus rotation diagrams of single supercoiled DNA molecules. We reproduce quantitatively the supercoiling response of overtwisted DNA and, using experimental data, we obtain an estimate of the effective supercoiling radius and of the twist rigidity of B-DNA. We find that the twist rigidity of DNA seems to vary widely with the nature and concentration of the salt buffer in which it is immersed.

  10. One-step synthesis of highly biocompatible multi-shaped gold nanostructures with fruit extract.

    PubMed

    Tai, Y; Tran, N T T; Tsai, Y-C; Fang, J-Y; Chang, L-W

    2011-06-01

    In this study, the authors demonstrate the synthesis of various gold nanostructures through a one-step, green and complete bio-modulation approach. Nanoparticles were successfully synthesised by the addition of gold aqueous solution to fruit extracts, including orange, papaya, peach or lemon. The particles were of various shapes and sizes with high abundance, such as sphere, marigold, triangle and hexagon. The biocompatibility of the presented gold nanostructures was examined; haemolysis tests revealed a non-toxicity result in blood cell uptake of such gold nanostructures. This study opens the exciting possibility of synthesising various multi-shaped nanoparticles through a simple and green approach, as well as paving the way for future bio-applications. PMID:21495781

  11. Effect of DNA extraction procedure, repeated extraction and ethidium monoazide (EMA)/propidium monoazide (PMA) treatment on overall DNA yield and impact on microbial fingerprints for bacteria, fungi and archaea in a reference soil

    PubMed Central

    Wagner, Andreas O.; Praeg, Nadine; Reitschuler, Christoph; Illmer, Paul

    2015-01-01

    Different DNA extraction protocols were evaluated on a reference soil. A wide difference was found in the total extractable DNA as derived from different extraction protocols. Concerning the DNA yield phenol–chloroform–isomyl alcohol extraction resulted in high DNA yield but also in a remarkable co-extraction of contaminants making PCR from undiluted DNA extracts impossible. By comparison of two different extraction kits, the Macherey&Nagel SoilExtract II kit resulted in the highest DNA yields when buffer SL1 and the enhancer solution were applied. The enhancer solution not only significantly increased the DNA yield but also the amount of co-extracted contaminates, whereas additional disintegration strategies did not. Although a three times repeated DNA extraction increased the total amount of extracted DNA, microbial fingerprints were merely affected. However, with the 5th extraction this changed. A reduction of total DGGE band numbers was observed for archaea and fungi, whereas for bacteria the diversity increased. The application of ethidium monoazide (EMA) or propidium monoazide (PMA) treatment aiming on the selective removal of soil DNA derived from cells lacking cell wall integrity resulted in a significant reduction of total extracted DNA, however, the hypothesized effect on microbial fingerprints failed to appear indicating the need for further investigations. PMID:26339125

  12. Use of magnetic beads for tissue DNA extraction and IS6110 Mycobacterium tuberculosis PCR.

    PubMed Central

    Caldarelli-Stefano, R; Vago, L; Bonetto, S; Nebuloni, M; Costanzi, G

    1999-01-01

    Polymerase chain reaction (PCR) techniques are used increasingly for the diagnosis of Mycobacterium tuberculosis infection and can be used on the DNA obtained from both frozen and formalin fixed, paraffin wax embedded tissues. However, the extraction of DNA by means of the conventional phenol/chloroform method is time consuming and requires the use of potentially dangerous chemical reagents. This paper describes a method based upon the use of magnetic beads for the extraction of M tuberculosis DNA from both routinely formalin fixed, paraffin wax embedded tissues and frozen tissues. Magnetic bead extracted DNA from brain, lymph node, and lung tissues collected from patients with human immunodeficiency virus and tuberculosis was compared with that extracted using the phenol/chloroform method. The magnetic bead extraction procedure requires less than two hours, including the time necessary to dewax the tissue sections. In all cases, the DNA extracted with both methods was amplified successfully by PCR for the M tuberculosis IS6110 sequence. Magnetic bead DNA extraction can be used on both frozen and archival tissues: the method is reliable, simple, sensitive, and rapid; in addition, it does not use hazardous procedures or specialised laboratory equipment and can be used for routine DNA isolation from various human tissues. PMID:10621838

  13. How Severely Is DNA Quantification Hampered by RNA Co-extraction?

    PubMed

    Sanchez, Ignacio; Remm, Matthieu; Frasquilho, Sonia; Betsou, Fay; Mathieson, William

    2015-10-01

    The optional RNase digest that is part of many DNA extraction protocols is often omitted, either because RNase is not provided in the kit or because users do not want to risk contaminating their laboratory. Consequently, co-eluting RNA can become a "contaminant" of unknown magnitude in a DNA extraction. We extracted DNA from liver, lung, kidney, and heart tissues and established that 28-52% of the "DNA" as assessed by spectrophotometry is actually RNA (depending on tissue type). Including an RNase digest in the extraction protocol reduced 260:280 purity ratios. Co-eluting RNA drives an overestimation of DNA yield when quantification is carried out using OD 260 nm spectrophotometry, or becomes an unquantified contaminant when spectrofluorometry is used for DNA quantification. This situation is potentially incompatible with the best practice guidelines for biobanks issued by organizations such as the International Society for Biological and Environmental Repositories, which state that biospecimens should be accurately characterized in terms of their identity, purity, concentration, and integrity. Consequently, we conclude that an RNase digest must be included in DNA extractions if pure DNA is required. We also discuss the implications of unquantified RNA contamination in DNA samples in the context of laboratory accreditation schemes. PMID:26418169

  14. An evaluation of the performance of five extraction methods: Chelex® 100, QIAamp® DNA Blood Mini Kit, QIAamp® DNA Investigator Kit, QIAsymphony® DNA Investigator® Kit and DNA IQ™.

    PubMed

    Ip, Stephen C Y; Lin, Sze-Wah; Lai, Kam-Ming

    2015-05-01

    DNA left at a crime scene was often limited in amount and far from pristine. To maximize the chance of recovering as much information as possible from such compromised samples, an appropriate extraction method using the available technologies needs to be devised. In this study, we used human blood, buffy coat and a total of 76 simulated touch DNA samples to test the effectiveness of the following five common DNA extraction methods, namely, Chelex® 100, QIAamp® DNA Blood Mini Kit, QIAamp® DNA Investigator Kit, QIAsymphony® DNA Investigator® Kit and DNA IQ™ system, in the recovery of such DNA. We demonstrated that the QIAamp® and QIAsymphony® DNA Investigator® Kits, and the DNA IQ™ system, exhibited a better effectiveness in DNA recovery amongst these methods and yielded extracts with higher success rate in subsequent DNA profiling. These extracts also generated profiles with better intra-colour signal balance. The findings in this work allowed us to propose an extraction approach as follows: 1) casework samples shall be extracted with the QIAamp®/QIAsymphony® DNA Investigator® Kits or the DNA IQ™ system, viz., QIAsymphony® DNA Investigator® Kit and DNA IQ™, due to their higher throughput, are for the touched DNA evidence from the volume crime, while QIAamp® DNA Investigator Kit is preferable for challenging bloodstain samples; and 2) control samples, such as buccal swab, with known identity can be extracted with the Chelex, due to their cheaper cost per sample. PMID:25934373

  15. Efficiency of genomic DNA extraction dependent on the size of magnetic nanoclusters

    NASA Astrophysics Data System (ADS)

    Cho, Hyun Ah; Hyun Min, Ji; Hua Wu, Jun; Woo Jang, Jin; Lim, Chae-Seung; Keun Kim, Young

    2014-05-01

    We report the efficiency of genomic DNA extraction as a function of particle size and quantity. For DNA extraction, we synthesized magnetic nanoclusters of various sizes and coated the surface of these magnetic nanoclusters with meso-2,3-dimercaptosuccinic acid. We showed that the nanoclusters had a tight particle size distribution and high crystallinity. Furthermore, we observed that the three types of magnetic nanoclusters studied exhibited ferrimagnetic behavior and that larger nanoclusters showed larger saturation magnetization values. The resultant efficiency of DNA extraction is inversely proportional to particle size in the range of nanoclusters tested, due to the fact that the surface-to-volume ratio decreases as particle size increases.

  16. DNA synthesis in yeast cell-free extracts dependent on recombinant DNA plasmids purified from Escherichia coli.

    PubMed Central

    Jong, A Y; Scott, J F

    1985-01-01

    In our attempts to establish a cell-free DNA replication system for the yeast Saccharomyces cerevisiae, we have observed that recombinant DNA plasmids purified from Escherichia coli by a common procedure (lysozyme-detergent lysis and equilibrium banding in cesium chloride ethidium bromide gradients) often serve as templates for DNA synthesis by elongation enzymes. The templates could be elongated equally well by enzymes present in the yeast cell-free extracts, by the large proteolytic fragment of E. coli DNA polymerase I or by T4 DNA polymerase. The template activity of the purified plasmids was dependent on the presence of heterologous DNA segments in the bacterial vectors. The template activity could be diminished by treatment with alkali. We propose that the ability of recombinant plasmids isolated from bacterial hosts to serve as elongation templates may lead to erroneous conclusions when these plasmids are used as templates for in vitro replication or transcription reactions. Images PMID:3889851

  17. Evaluating DNA Extraction Methods for Community Profiling of Pig Hindgut Microbial Community

    PubMed Central

    Lu, Yang; Hugenholtz, Philip; Batstone, Damien John

    2015-01-01

    Recovery of high quality PCR-amplifiable DNA has been the general minimal requirement for DNA extraction methods for bulk molecular analysis. However, modern high through-put community profiling technologies are more sensitive to representativeness and reproducibility of DNA extraction method. Here, we assess the impact of three DNA extraction methods (with different levels of extraction harshness) for assessing hindgut microbiomes from pigs fed with different diets (with different physical properties). DNA extraction from each sample was performed in three technical replicates for each extraction method and sequenced by 16S rRNA amplicon sequencing. Host was the primary driver of molecular sequencing outcomes, particularly on samples analysed by wheat based diets, but higher variability, with one failed extraction occurred on samples from a barley fed pig. Based on these results, an effective method will enable reproducible and quality outcomes on a range of samples, whereas an ineffective method will fail to generate extract, but host (rather than extraction method) remains the primary factor. PMID:26560873

  18. A simple silica-based method for metagenomic DNA extraction from soil and sediments.

    PubMed

    Rojas-Herrera, R; Narváez-Zapata, J; Zamudio-Maya, M; Mena-Martínez, M E

    2008-09-01

    A new method is described for extraction of metagenomic DNA from soil and sediments which is based on DNA adsorption to silica without the use of phenol, ethanol precipitation or a cesium chloride gradient. High-quality DNA was obtained, and PCR inhibition was overcome by adding bovine serum albumin and adjusting magnesium concentration. By using PCR-DGGE with Firmicutes and lactic acid bacteria-specific primers the extracted metagenomic DNA was shown to contain a mixture of bacterial genomes. This method can be used for screening bacterial diversity in soil and sediment samples. PMID:18373226

  19. The direct application of the polymerase chain reaction to DNA extracted from foods.

    PubMed

    Dickinson, J H; Kroll, R G; Grant, K A

    1995-04-01

    Two methods for the successful extraction of DNA from foods are described. The rapid lysis method uses a proteinase K buffer system to lyse cells and solubilize food samples. DNA is then precipitated using isopropanol. The second method achieves cell lysis using toluene and mutanolysin, and solubilization using guanidium thiocyanate. Following protein removal with organic solvents DNA is precipitated with isopropanol. Both methods enabled the polymerase chain reaction to be applied directly to DNA extracted from samples of cheese, coleslaw and raw chicken and allowed the direct rapid, sensitive and specific detection of Yersinia enterocolitica, Aerococcus viridans and Listeria monocytogenes in these foods. PMID:7766115

  20. Elimination of bioweapons agents from forensic samples during extraction of human DNA.

    PubMed

    Timbers, Jason; Wilkinson, Della; Hause, Christine C; Smith, Myron L; Zaidi, Mohsin A; Laframboise, Denis; Wright, Kathryn E

    2014-11-01

    Collection of DNA for genetic profiling is a powerful means for the identification of individuals responsible for crimes and terrorist acts. Biologic hazards, such as bacteria, endospores, toxins, and viruses, could contaminate sites of terrorist activities and thus could be present in samples collected for profiling. The fate of these hazards during DNA isolation has not been thoroughly examined. Our goals were to determine whether the DNA extraction process used by the Royal Canadian Mounted Police eliminates or neutralizes these agents and if not, to establish methods that render samples safe without compromising the human DNA. Our results show that bacteria, viruses, and toxins were reduced to undetectable levels during DNA extraction, but endospores remained viable. Filtration of samples after DNA isolation eliminated viable spores from the samples but left DNA intact. We also demonstrated that contamination of samples with some bacteria, endospores, and toxins for longer than 1 h compromised the ability to complete genetic profiling. PMID:25069670

  1. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on.

    PubMed

    Chomczynski, Piotr; Sacchi, Nicoletta

    2006-01-01

    Since its introduction, the 'single-step' method has become widely used for isolating total RNA from biological samples of different sources. The principle at the basis of the method is that RNA is separated from DNA after extraction with an acidic solution containing guanidinium thiocyanate, sodium acetate, phenol and chloroform, followed by centrifugation. Under acidic conditions, total RNA remains in the upper aqueous phase, while most of DNA and proteins remain either in the interphase or in the lower organic phase. Total RNA is then recovered by precipitation with isopropanol and can be used for several applications. The original protocol, enabling the isolation of RNA from cells and tissues in less than 4 hours, greatly advanced the analysis of gene expression in plant and animal models as well as in pathological samples, as demonstrated by the overwhelming number of citations the paper gained over 20 years. PMID:17406285

  2. Comparing DNA extraction methods for analysis of botanical materials found in anti-diabetic supplements.

    PubMed

    Llongueras, Jose P; Nair, Saraswathy; Salas-Leiva, Dayana; Schwarzbach, Andrea E

    2013-03-01

    A comparative performance evaluation of DNA extraction methods from anti-diabetic botanical supplements using various commercial kits was conducted, to determine which produces the best quality DNA suitable for PCR amplification, sequencing and species identification. All plant materials involved were of suboptimal quality showing various levels of degradation and therefore representing real conditions for testing herbal supplements. Eight different DNA extraction methods were used to isolate genomic DNA from 13 medicinal plant products. Two methods for evaluation, DNA concentration measurements that included absorbance ratios as well as PCR amplifiability, were used to determine quantity and quality of extracted DNA. We found that neither DNA concentrations nor commonly used UV absorbance ratio measurements at A(260)/A(280) between 1.7 and 1.9 are suitable for globally predicting PCR success in these plant samples, and that PCR amplifiablity itself was the best indicator of extracted product quality. However, our results suggest that A(260)/A(280) ratios below about 1.3 and above 2.3 indicated a DNA quality too poor to amplify. Therefore, A(260)/A(280) measurements are not useful to identify samples that likely will amplify but can be used to exclude samples that likely will not amplify reducing the cost for unnecessarily subjecting samples to PCR. The two Nucleospin(®) plant II kit extraction methods produced the most pure and amplifiable genomic DNA extracts. Our results suggest that there are clear, discernable differences between extraction methods for low quality plant samples in terms of producing contamination-free, high-quality genomic DNA to be used for further analysis. PMID:22403012

  3. Comparison of DNA extraction kits and modification of DNA elution procedure for the quantitation of subdominant bacteria from piggery effluents with real-time PCR

    PubMed Central

    Desneux, Jérémy; Pourcher, Anne-Marie

    2014-01-01

    Four commercial DNA extraction kits and a minor modification in the DNA elution procedure were evaluated for the quantitation of bacteria in pig manure samples. The PowerSoil®, PowerFecal®, NucleoSpin® Soil kits and QIAamp® DNA Stool Mini kit were tested on raw manure samples and on lagoon effluents for their ability to quantify total bacteria and a subdominant bacteria specific of pig manure contamination: Lactobacillus amylovorus. The NucleoSpin® Soil kit (NS kit), and to a lesser extent the PowerFecal® kit were the most efficient methods. Regardless of the kit utilized, the modified elution procedure increased DNA yield in the lagoon effluent by a factor of 1.4 to 1.8. When tested on 10 piggery effluent samples, compared to the QIAamp kit, the NS kit combined with the modified elution step, increased by a factor up to 1.7 log10 the values of the concentration of L. amylovorus. Regardless of the type of manure, the best DNA quality and the highest concentrations of bacteria were obtained using the NS kit combined with the modification of the elution procedure. The method recommended here significantly improved quantitation of subdominant bacteria in manure. PMID:24838631

  4. Comparison of DNA extraction kits and modification of DNA elution procedure for the quantitation of subdominant bacteria from piggery effluents with real-time PCR.

    PubMed

    Desneux, Jérémy; Pourcher, Anne-Marie

    2014-08-01

    Four commercial DNA extraction kits and a minor modification in the DNA elution procedure were evaluated for the quantitation of bacteria in pig manure samples. The PowerSoil(®), PowerFecal(®), NucleoSpin(®) Soil kits and QIAamp(®) DNA Stool Mini kit were tested on raw manure samples and on lagoon effluents for their ability to quantify total bacteria and a subdominant bacteria specific of pig manure contamination: Lactobacillus amylovorus. The NucleoSpin(®) Soil kit (NS kit), and to a lesser extent the PowerFecal(®) kit were the most efficient methods. Regardless of the kit utilized, the modified elution procedure increased DNA yield in the lagoon effluent by a factor of 1.4 to 1.8. When tested on 10 piggery effluent samples, compared to the QIAamp kit, the NS kit combined with the modified elution step, increased by a factor up to 1.7 log10 the values of the concentration of L. amylovorus. Regardless of the type of manure, the best DNA quality and the highest concentrations of bacteria were obtained using the NS kit combined with the modification of the elution procedure. The method recommended here significantly improved quantitation of subdominant bacteria in manure. PMID:24838631

  5. DNA extraction methods for detecting genetically modified foods: A comparative study.

    PubMed

    Elsanhoty, Rafaat M; Ramadan, Mohamed Fawzy; Jany, Klaus Dieter

    2011-06-15

    The work presented in this manuscript was achieved to compare six different methods for extracting DNA from raw maize and its derived products. The methods that gave higher yield and quality of DNA were chosen to detect the genetic modification in the samples collected from the Egyptian market. The different methods used were evaluated for extracting DNA from maize kernels (without treatment), maize flour (mechanical treatment), canned maize (sweet corn), frozen maize (sweet corn), maize starch, extruded maize, popcorn, corn flacks, maize snacks, and bread made from corn flour (mechanical and thermal treatments). The quality and quantity of the DNA extracted from the standards, containing known percentages of GMO material and from the different food products were evaluated. For qualitative detection of the GMO varieties in foods, the GMOScreen 35S/NOS test kit was used, to screen the genetic modification in the samples. The positive samples for the 35S promoter and/or the NOS terminator were identified by the standard methods adopted by EU. All of the used methods extracted yielded good DNA quality. However, we noted that the purest DNA extract were obtained using the DNA extraction kit (Roche) and this generally was the best method for extracting DNA from most of the maize-derived foods. We have noted that the yield of DNA extracted from maize-derived foods was generally lower in the processed products. The results indicated that 17 samples were positive for the presence of 35S promoter, while 34% from the samples were positive for the genetically modified maize line Bt-176. PMID:25213972

  6. Extraction of DNA from human embryos after long-term preservation in formalin and Bouin's solutions.

    PubMed

    Nagai, Momoko; Minegishi, Katsura; Komada, Munekazu; Tsuchiya, Maiko; Kameda, Tomomi; Yamada, Shigehito

    2016-05-01

    The "Kyoto Collection of Human Embryos" at Kyoto University was begun in 1961. Although morphological analyses of samples in the Kyoto Collection have been performed, these embryos have been considered difficult to genetically analyze because they have been preserved in formalin or Bouin's solution for 20-50 years. Owing to the recent advances in molecular biology, it has become possible to extract DNA from long-term fixed tissues. The purpose of this study was to extract DNA from wet preparations of human embryo samples after long-term preservation in fixing solution. We optimized the DNA extraction protocol to be suitable for tissues that have been damaged by long-term fixation, including DNA-protein crosslinking damage. Diluting Li2 CO3 with 70% ethanol effectively removed picric acid from samples fixed in Bouin's solution. Additionally, 20.0 mg/mL proteinase was valuable to lyse the long-term fixed samples. The extracted DNA was checked with PCR amplification using several sets of primers and sequence analysis. The PCR products included at least 295- and 838-bp amplicons. These results show that the extracted DNA is applicable for genetic analyses, and indicate that old embryos in the Kyoto Collection should be made available for future studies. The protocol described in this study can successfully extract DNA from old specimens and, with improvements, should be applicable in research aiming to understand the molecular mechanisms of human congenital anomalies. PMID:26662860

  7. A new DNA extraction method by controlled alkaline treatments from consolidated subsurface sediments.

    PubMed

    Kouduka, Mariko; Suko, Takeshi; Morono, Yuki; Inagaki, Fumio; Ito, Kazumasa; Suzuki, Yohey

    2012-01-01

    Microbial communities that thrive in subterranean consolidated sediments are largely unknown owing to the difficulty of extracting DNA. As this difficulty is often attributed to DNA binding onto the silica-bearing sediment matrix, we developed a DNA extraction method for consolidated sediment from the deep subsurface in which silica minerals were dissolved by being heated under alkaline conditions. NaOH concentrations (0.07 and 0.33 N), incubation temperatures (65 and 94 °C) and incubation times (30-90 min) before neutralization were evaluated based on the copy number of extracted prokaryotic DNA. Prokaryotic DNA was detected by quantitative PCR analysis after heating the sediment sample at 94 °C in 0.33 N NaOH solution for 50-80 min. Results of 16S rRNA gene sequence analysis of the extracted DNA were all consistent with regard to the dominant occurrence of the metallophilic bacterium, Cupriavidus metallidurans, and Pseudomonas spp. Mineralogical analysis revealed that the dissolution of a silica mineral (opal-CT) during alkaline treatment was maximized at 94 °C in 0.33 N NaOH solution for 50 min, which may have resulted in the release of DNA into solution. Because the optimized protocol for DNA extraction is applicable to subterranean consolidated sediments from a different locality, the method developed here has the potential to expand our understanding of the microbial community structure of the deep biosphere. PMID:22092362

  8. DNA extraction from plant food supplements: Influence of different pharmaceutical excipients.

    PubMed

    Costa, Joana; Amaral, Joana S; Fernandes, Telmo J R; Batista, Andreia; Oliveira, M Beatriz P P; Mafra, Isabel

    2015-12-01

    The consumption of plant food supplements (PFS) has been growing globally, with an increase of misleading labeling and fraudulent practices also being reported. Recently, the use of molecular biology techniques has been proposed to detect botanical adulterations, one of the possible frauds in PFS. However, difficulties in recovering DNA from some PFS samples have been described. Aiming at using DNA-based methods for the unequivocal identification of plant species in PFS, adequate DNA isolation is required. However, PFS often contain pharmaceutical excipients known to have adsorbent properties that might interfere with DNA extraction. Thus, the aim of this work was to assess the effect of different excipients (talc, silica, iron oxide and titanium dioxide) on the recovery/amplification of DNA. For that purpose, known amounts of template maize DNA were spiked either to PFS or to model mixtures of excipients and quantified by real-time PCR. The tested excipients evidenced clear adsorption phenomena that justify the hampering effect on DNA extraction from PFS. The use of either 10% talc or 0.5% dyes completely adsorbed DNA, resulting in negative PCR amplifications. For the first time, pharmaceutical excipients were shown to affect DNA extraction explaining the inability of recovering DNA from some PFS samples in previous studies. PMID:26079045

  9. Comparison of proteases in DNA extraction via quantitative polymerase chain reaction.

    PubMed

    Eychner, Alison M; Lebo, Roberta J; Elkins, Kelly M

    2015-06-01

    We compared four proteases in the QIAamp DNA Investigator Kit (Qiagen) to extract DNA for use in multiplex polymerase chain reaction (PCR) assays. The aim was to evaluate alternate proteases for improved DNA recovery as compared with proteinase K for forensic, biochemical research, genetic paternity and immigration, and molecular diagnostic purposes. The Quantifiler Kit TaqMan quantitative PCR assay was used to measure the recovery of DNA from human blood, semen, buccal cells, breastmilk, and earwax in addition to low-template samples, including diluted samples, computer keyboard swabs, chewing gum, and cigarette butts. All methods yielded amplifiable DNA from all samples. PMID:25197027

  10. Qualification Study of Two Genomic DNA Extraction Methods in Different Clinical Samples

    PubMed Central

    Javadi, Alireza; Shamaei, Masoud; Pourabdollah, Mihan; Dorudinia, Atosa; Seyedmehdi, Seyed Mohammad; Karimi, Shirin

    2014-01-01

    Introduction The purity of genomic DNA (gDNA) extracted from different clinical specimens optimizes sensitivity of polymerase chain reaction (PCR) assays. This study attempted to compare two different DNA extraction techniques namely salting-out and classic phenol-chloroform. Materials and Methods Qualification of two different DNA extraction techniques for 634 clinical specimens highly suspected of having mycobacterial infection was performed. Genomic DNA was extracted from 330 clinical samples using phenol-chloroform and 304 by non-toxic salting-out. Qualification of obtained gDNA was done through amplification of internal controls, β-actin and β-globin. Results β-actin-positive was detected in 279/330 (84%) and 272/304 (89%) samples by phenol-chloroform technique and salting-out, respectively. PCR inhibitor was found for the gDNA of 13/304 (4%) patient samples were negative by β-actin and β-globin tests via salting-out technique in comparison with gDNAs from 27/330 (8.5%) samples extracted by phenol-chloroform procedure. No statistically significant difference was found between phenol-chloroform technique and salting-out for 385 sputum, 29 bronchoalveolar lavage (BAL), 105 gastric washing, and 38 body fluid (P=0.04) samples. This illustrates that both techniques have the same quality for extracting gDNA. Conclusion This study discloses salting-out as a non-toxic DNA extraction procedure with a superior time-efficiency and cost-effectiveness in comparison with phenol-chloroform and it can be routinely used in resource-limited laboratory settings. PMID:25852760

  11. A SIMPLE VERSATILE HIGH THROUPHPUT DNA EXTRACTION METHOD SUITABLE FOR PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PCR has become the most popular technique in functional genomics. Both forward and reverse genetic projects routinely require PCR amplification of thousands of samples. Processing the samples for DNA suitable for PCR is usually the limiting step. We have developed a simple high throughput DNA extra...

  12. Plant DNA detection from grasshopper guts: A step-by-step protocol, from tissue preparation to obtaining plant DNA sequences1

    PubMed Central

    Avanesyan, Alina

    2014-01-01

    • Premise of the study: A PCR-based method of identifying ingested plant DNA in gut contents of Melanoplus grasshoppers was developed. Although previous investigations have focused on a variety of insects, there are no protocols available for plant DNA detection developed for grasshoppers, agricultural pests that significantly influence plant community composition. • Methods and Results: The developed protocol successfully used the noncoding region of the chloroplast trnL (UAA) gene and was tested in several feeding experiments. Plant DNA was obtained at seven time points post-ingestion from whole guts and separate gut sections, and was detectable up to 12 h post-ingestion in nymphs and 22 h post-ingestion in adult grasshoppers. • Conclusions: The proposed protocol is an effective, relatively quick, and low-cost method of detecting plant DNA from the grasshopper gut and its different sections. This has important applications, from exploring plant “movement” during food consumption, to detecting plant–insect interactions. PMID:25202604

  13. A Simple Method of Genomic DNA Extraction from Human Samples for PCR-RFLP Analysis

    PubMed Central

    Ghatak, Souvik; Muthukumaran, Rajendra Bose; Nachimuthu, Senthil Kumar

    2013-01-01

    Isolation of DNA from blood and buccal swabs in adequate quantities is an integral part of forensic research and analysis. The present study was performed to determine the quality and the quantity of DNA extracted from four commonly available samples and to estimate the time duration of the ensuing PCR amplification. Here, we demonstrate that hair and urine samples can also become an alternate source for reliably obtaining a small quantity of PCR-ready DNA. We developed a rapid, cost-effective, and noninvasive method of sample collection and simple DNA extraction from buccal swabs, urine, and hair using the phenol-chloroform method. Buccal samples were subjected to DNA extraction, immediately or after refrigeration (4–6°C) for 3 days. The purity and the concentration of the extracted DNA were determined spectrophotometerically, and the adequacy of DNA extracts for the PCR-based assay was assessed by amplifying a 1030-bp region of the mitochondrial D-loop. Although DNA from all the samples was suitable for PCR, the blood and hair samples provided a good quality DNA for restriction analysis of the PCR product compared with the buccal swab and urine samples. In the present study, hair samples proved to be a good source of genomic DNA for PCR-based methods. Hence, DNA of hair samples can also be used for the genomic disorder analysis in addition to the forensic analysis as a result of the ease of sample collection in a noninvasive manner, lower sample volume requirements, and good storage capability. PMID:24294115

  14. Application of magnetic hydroxyapatite nanoparticles for solid phase extraction of plasmid DNA.

    PubMed

    Shan, Zhi; Li, Xianghai; Gao, Yaying; Wang, Xianxiang; Li, Chenglei; Wu, Qi

    2012-06-15

    We developed a facile method for plasmid DNA (pDNA) extraction from crude Escherichia coli lysate using magnetic hydroxyapatite nanoparticles (MHapNPs) in the presence of polyethylene glycol (PEG)/NaCl. DNA condensation induced by PEG/NaCl is a prerequisite for achieving pronounced DNA recovery. The quality and quantity of MHapNP-purified pDNA under optimal binding buffer conditions (0.5 volume of 20% PEG 8000/2M NaCl) were comparable to those obtained using organic solvents or commercial kits. This MHapNP technique is rapid, simple, cost-effective, and environmentally friendly and has the potential to extract DNA from other cell lysates. PMID:22465330

  15. Comparative assessment of genomic DNA extraction processes for Plasmodium: Identifying the appropriate method.

    PubMed

    Mann, Riti; Sharma, Supriya; Mishra, Neelima; Valecha, Neena; Anvikar, Anupkumar R

    2015-12-01

    Plasmodium DNA, in addition to being used for molecular diagnosis of malaria, find utility in monitoring patient responses to antimalarial drugs, drug resistance studies, genotyping and sequencing purposes. Over the years, numerous protocols have been proposed for extracting Plasmodium DNA from a variety of sources. Given that DNA isolation is fundamental to successful molecular studies, here we review the most commonly used methods for Plasmodium genomic DNA isolation, emphasizing their pros and cons. A comparison of these existing methods has been made, to evaluate their appropriateness for use in different applications and identify the method suitable for a particular laboratory based study. Selection of a suitable and accessible DNA extraction method for Plasmodium requires consideration of many factors, the most important being sensitivity, cost-effectiveness and, purity and stability of isolated DNA. Need of the hour is to accentuate on the development of a method that upholds well on all these parameters. PMID:26714505

  16. Influence of DNA extraction methods on relative telomere length measurements and its impact on epidemiological studies

    PubMed Central

    Raschenberger, Julia; Lamina, Claudia; Haun, Margot; Kollerits, Barbara; Coassin, Stefan; Boes, Eva; Kedenko, Ludmilla; Köttgen, Anna; Kronenberg, Florian

    2016-01-01

    Measurement of telomere length is widely used in epidemiologic studies. Insufficient standardization of the measurements processes has, however, complicated the comparison of results between studies. We aimed to investigate whether DNA extraction methods have an influence on measured values of relative telomere length (RTL) and whether this has consequences for epidemiological studies. We performed four experiments with RTL measurement in quadruplicate by qPCR using DNA extracted with different methods: 1) a standardized validation experiment including three extraction methods (magnetic-particle-method EZ1, salting-out-method INV, phenol-chloroform-isoamyl-alcohol PCI) each in the same 20 samples demonstrated pronounced differences in RTL with lowest values with EZ1 followed by INV and PCI-isolated DNA; 2) a comparison of 307 samples from an epidemiological study showing EZ1-measurements 40% lower than INV-measurements; 3) a matching-approach of two similar non-diseased control groups including 143 pairs of subjects revealed significantly shorter RTL in EZ1 than INV-extracted DNA (0.844 ± 0.157 vs. 1.357 ± 0.242); 4) an association analysis of RTL with prevalent cardiovascular disease detected a stronger association with INV than with EZ1-extracted DNA. In summary, DNA extraction methods have a pronounced influence on the measured RTL-values. This might result in spurious or lost associations in epidemiological studies under certain circumstances. PMID:27138987

  17. Influence of DNA extraction methods on relative telomere length measurements and its impact on epidemiological studies.

    PubMed

    Raschenberger, Julia; Lamina, Claudia; Haun, Margot; Kollerits, Barbara; Coassin, Stefan; Boes, Eva; Kedenko, Ludmilla; Köttgen, Anna; Kronenberg, Florian

    2016-01-01

    Measurement of telomere length is widely used in epidemiologic studies. Insufficient standardization of the measurements processes has, however, complicated the comparison of results between studies. We aimed to investigate whether DNA extraction methods have an influence on measured values of relative telomere length (RTL) and whether this has consequences for epidemiological studies. We performed four experiments with RTL measurement in quadruplicate by qPCR using DNA extracted with different methods: 1) a standardized validation experiment including three extraction methods (magnetic-particle-method EZ1, salting-out-method INV, phenol-chloroform-isoamyl-alcohol PCI) each in the same 20 samples demonstrated pronounced differences in RTL with lowest values with EZ1 followed by INV and PCI-isolated DNA; 2) a comparison of 307 samples from an epidemiological study showing EZ1-measurements 40% lower than INV-measurements; 3) a matching-approach of two similar non-diseased control groups including 143 pairs of subjects revealed significantly shorter RTL in EZ1 than INV-extracted DNA (0.844 ± 0.157 vs. 1.357 ± 0.242); 4) an association analysis of RTL with prevalent cardiovascular disease detected a stronger association with INV than with EZ1-extracted DNA. In summary, DNA extraction methods have a pronounced influence on the measured RTL-values. This might result in spurious or lost associations in epidemiological studies under certain circumstances. PMID:27138987

  18. Rapid extraction of DNA From Escherichia coli and Cryptosporidium parvum for use in PCR.

    PubMed

    Higgins, J A; Jenkins, M C; Shelton, D R; Fayer, R; Karns, J S

    2001-11-01

    The Xtra Amp tube, Isocode paper, Instagene matrix, and PrepMan matrix methods were evaluated for their ability to rapidly extract PCR-quality DNAs from Escherichia coli O157:H7 and Cryptosporidium parvum. All methods provided satisfactory DNA from E. coli, and the Xtra Amp and Instagene reagents provided satisfactory DNA from C. parvum. PMID:11679362

  19. “Versatile toolset” for DNA or protein immobilization: Toward a single-step chemistry

    NASA Astrophysics Data System (ADS)

    Berthelot, Thomas; Garcia, Alexandre; Le, Xuan Tuan; El Morsli, Jenna; Jégou, Pascale; Palacin, Serge; Viel, Pascal

    2011-02-01

    Covalent immobilization of non-modified biological materials as proteins or nucleic acids has been performed through a single and soft method. Based on diazonium salt chemistry, this protocol leads to an ultrathin grafted film, on metallic or polymer materials, which can eventually be used as a self-adhesive primer for immobilizing biological materials from aqueous solutions through a simple dipping step. Moreover, this self-adhesive primer may be patterned by cheap and easy methods as ink or UV masking. Biological models as low molecular weight DNA from salmon sperm and glucose oxidase (GOD) were covalently immobilized by this soft procedure. In order to evaluate the consequences of this non-specific covalent immobilization method on biological activity, enzymatic activity of GOD was monitored by electrochemical detection of hydrogen peroxide (H2O2). We thus demonstrate that such a self-adhesive primer represents a new and alternative process offering a versatile toolset for immobilizing biological material for biosensor development on conductive and non-conductive materials.

  20. One-step polymer surface modification for minimizing drug, protein, and DNA adsorption in microanalytical systems.

    PubMed

    Larsen, Esben Kjær Unmack; Larsen, Niels B

    2013-02-21

    The non-specific adsorption of dissolved analytes strongly reduces the sensitivity and reliability in polymer microanalytical systems. Here, a one-step aqueous phase procedure modifies polymer material surfaces to strongly reduce their non-specific adsorption of a broad range of organic analytes including hydrophobic and hydrophilic drugs (0.23 < ClogP < 8.95), small and large proteins (insulin, albumin, IgG), and DNA. The coating is shown to limit the adsorption of even highly hydrophobic drugs (ClogP > 8) in their pharmaceutically relevant concentration range ≤100 nM. The low adsorption is mediated by photochemical conjugation, where polyethylene glycol (PEG) polymers in aqueous solution are covalently bound to the surface by UV illumination of dissolved benzophenone and a functionalized PEG. The method can coat the interior of polymer systems made from a range of materials commonly used in microanalytical systems, including polystyrene (PS), cyclic olefin copolymer (COC), liquid crystalline polymer (LCP), and polyimide (PI). PMID:23254780

  1. Characterization of the initial steps in the T7 DNA ejection process

    PubMed Central

    González-García, Verónica A; Bocanegra, Rebeca; Pulido-Cid, Mar; Martín-Benito, Jaime; Cuervo, Ana; Carrascosa, José L

    2015-01-01

    A specialized complex, the tail, is the most common strategy employed by bacterial viruses to deliver their genome without disrupting cell integrity. T7 has a short, non-contractile tail formed by a tubular structure surrounded by fibers. Recent studies showed that incubation of the virus with Escherichia coli lipopolysaccharides (LPS) resulted in complete delivery of the viral genome, demonstrating for the first time that LPS are the T7 receptor. Further screening of the bacterial envelope for proteinaceous compounds that affect T7 ejection showed that porins OmpA and OmpF affect viral particle adsorption and infection kinetics, suggesting that these proteins play a role in the first steps of virus-host interaction. Comparison of the structures before and after ejection showed the conformational changes needed in the tail for genome delivery. Structural similarities between T7 and other viruses belonging to the Podoviridae family suggests that they could also follow a similar DNA ejection mechanism. PMID:26458390

  2. Comparison of DNA extraction methodologies used for assessing fungal diversity via ITS sequencing

    PubMed Central

    Rittenour, William R.; Park, Ju-Hyeong; Cox-Ganser, Jean M.; Beezhold, Donald H.; Green, Brett J.

    2015-01-01

    Traditional methods of assessing fungal exposure have been confounded by a number of limiting variables. The recent utilization of molecular methods such as internal transcribed spacer (ITS) sequencing of ribosomal RNA genes has provided improved insight into the diversity of fungal bioaerosols in indoor, outdoor and occupational environments. However, ITS analyses may also be confounded by a number of methodological limitations. In this study, we have optimized this technology for use in occupational or environmental studies. Three commonly used DNA extraction methodologies (UltraClean Soil kit, High Pure PCR Template kit, and EluQuik/DNeasy kit) were compared in terms of sensitivity and susceptibility to PCR inhibitors in dust for three common fungal bioaerosols, Aspergillus versicolor, Rhizopus microsporus and Wallemia sebi. Environmental dust samples were then studied using each extraction methodology and results were compared to viable culture data. The extraction methods differed in terms of their ability to efficiently extract DNA from particular species of fungi (e.g. Aspergillus versicolor). In addition, the ability to remove PCR inhibitors from dust samples was most effective using the soil DNA extraction kit. The species composition varied greatly between ITS clone libraries generated with the different DNA extraction kits. However, compared to viable culture data, ITS clone libraries included additional fungal species that are incapable of growth on solid culture medium. Collectively, our data indicated that DNA extraction methodologies used in ITS sequencing studies of occupational or environmental dust samples can greatly influence the fungal species that are detected. PMID:22230933

  3. The effect of different solvents and number of extraction steps on the polyphenol content and antioxidant capacity of basil leaves (Ocimum basilicum L.) extracts.

    PubMed

    Złotek, Urszula; Mikulska, Sylwia; Nagajek, Małgorzata; Świeca, Michał

    2016-09-01

    The objectives of this study were to determine best conditions for the extraction of phenolic compounds from fresh, frozen and lyophilized basil leaves. The acetone mixtures with the highest addition of acetic acid extracted most of the phenolic compounds when fresh and freeze-dried material have been used. The three times procedure was more effective than once shaking procedure in most of the extracts obtained from fresh basil leaves - unlike the extracts derived from frozen material. Surprisingly, there were not any significant differences in the content of phenolics between the two used procedures in the case of lyophilized basil leaves used for extraction. Additionally, the positive correlation between the phenolic compounds content and antioxidant activity of the studied extracts has been noted. It is concluded that the acetone mixtures were more effective than the methanol ones for polyphenol extraction. The number of extraction steps in most of the cases was also a statistically significant factor affecting the yield of phenolic extraction as well as antioxidant potential of basil leaf extracts. PMID:27579013

  4. The validation of forensic DNA extraction systems to utilize soil contaminated biological evidence.

    PubMed

    Kasu, Mohaimin; Shires, Karen

    2015-07-01

    The production of full DNA profiles from biological evidence found in soil has a high failure rate due largely to the inhibitory substance humic acid (HA). Abundant in various natural soils, HA co-extracts with DNA during extraction and inhibits DNA profiling by binding to the molecular components of the genotyping assay. To successfully utilize traces of soil contaminated evidence, such as that found at many murder and rape crime scenes in South Africa, a reliable HA removal extraction system would often be selected based on previous validation studies. However, for many standard forensic DNA extraction systems, peer-reviewed publications detailing the efficacy on soil evidence is either lacking or is incomplete. Consequently, these sample types are often not collected or fail to yield suitable DNA material due to the use of unsuitable methodology. The aim of this study was to validate the common forensic DNA collection and extraction systems used in South Africa, namely DNA IQ, FTA elute and Nucleosave for processing blood and saliva contaminated with HA. A forensic appropriate volume of biological evidence was spiked with HA (0, 0.5, 1.5 and 2.5 mg/ml) and processed through each extraction protocol for the evaluation of HA removal using QPCR and STR-genotyping. The DNA IQ magnetic bead system effectively removed HA from highly contaminated blood and saliva, and generated consistently acceptable STR profiles from both artificially spiked samples and crude soil samples. This system is highly recommended for use on soil-contaminated evidence over the cellulose card-based systems currently being preferentially used for DNA sample collection. PMID:25690910

  5. An efficient DNA extraction method for desert Calligonum species.

    PubMed

    Abdellaoui, Raoudha; Gouja, Hassen; Sayah, Amel; Neffati, Mohamed

    2011-12-01

    Genetic conservation programs in arid environments rely on molecular methods for diversity assessments. DNA-based molecular profiling will aid in conservation and protection of species from genetic erosion. Obtaining intact genomic DNA from Calligonum species, of sufficiently high-quality that is readily amplifiable using PCR, is challenging because of the presence of the exceptionally large amount of oxidized polyphenolic compounds, polysaccharides, and other secondary metabolites. The present method involves a modification of the available CTAB method employing higher concentrations of NaCl and CTAB, and incorporating PEG 6000 (1%) and glucose. The yield of DNA was 60-670 μg g(-1) of fresh tissue. The protocol has been tested with two species from the arid region. The DNA isolated was successfully amplified by two ITS primer pairs. PCR-RFLP analysis of the ITS1-5.8S-ITS2 region among and within Calligonum species followed by sequencing is under way. PMID:21681578

  6. Antioxidant, DNA protective efficacy and HPLC analysis of Annona muricata (soursop) extracts.

    PubMed

    George, V Cijo; Kumar, D R Naveen; Suresh, P K; Kumar, R Ashok

    2015-04-01

    Annona muricata is a naturally occurring edible plant with wide array of therapeutic potentials. In India, it has a long history of traditional use in treating various ailments. The present investigation was carried out to characterize the phytochemicals present in the methanolic and aqueous leaf extracts of A. muricata, followed by validation of its radical scavenging and DNA protection activities. The extracts were also analyzed for its total phenolic contents and subjected to HPLC analysis to determine its active metabolites. The radical scavenging activities were premeditated by various complementary assays (DRSA, FRAP and HRSA). Further, its DNA protection efficacy against H2O2 induced toxicity was evaluated using pBR322 plasmid DNA. The results revealed that the extracts were highly rich in various phytochemicals including luteolin, homoorientin, tangeretin, quercetin, daidzein, epicatechin gallate, emodin and coumaric acid. Both the extracts showed significant (p < 0.05) radical scavenging activities, while methanolic extract demonstrated improved protection against H2O2-induced DNA damage when compared to aqueous extract. A strong positive correlation was observed for the estimated total phenolic contents and radical scavenging potentials of the extracts. Further HPLC analysis of the phyto-constituents of the extracts provides a sound scientific basis for compound isolation. PMID:25829616

  7. DNA stabilization and amplification from museum collections of extracts originally intended for allozyme analysis.

    PubMed

    Tan, A M; Orrego, C

    1992-10-01

    Protein extracts originally prepared for isozyme electrophoresis two decades ago contain surviving DNA sequences susceptible to amplification by the polymerase chain reaction (PCR). Amplification was also possible after stabilization of such extracts on filter paper and immersion under mineral oil without refrigeration. PMID:1344996

  8. Comparison of three DNA extraction kits to establish maximum yield and quality of coral-associated microbial DNA

    USGS Publications Warehouse

    Baker, Erin J.; Kellogg, Christina A.

    2014-01-01

    Coral microbiology is an expanding field, yet there is no standard DNA extraction protocol. Although many researchers depend on commercial extraction kits, no specific kit has been optimized for use with coral samples. Both soil and plant DNA extraction kits from MO BIO Laboratories, Inc., have been used by many research groups for this purpose. MO BIO recently replaced their PowerPlant® kit with an improved PowerPlantPro kit, but it was unclear how these changes would affect the kit’s use with coral samples. In order to determine which kit produced the best results, we conducted a comparison between the original PowerPlant kit, the new PowerPlantPro kit, and an alternative kit, PowerSoil, using samples from several different coral genera. The PowerPlantPro kit had the highest DNA yields, but the lack of 16S rRNA gene amplification in many samples suggests that much of the yield may be coral DNA rather than microbial DNA. The most consistent positive amplifications came from the PowerSoil kit.

  9. Two-steps extraction of essential oil, polysaccharides and biphenyl cyclooctene lignans from Schisandra chinensis Baill fruits.

    PubMed

    Cheng, Zhenyu; Yang, Yingjie; Liu, Yan; Liu, Zhigang; Zhou, Hongli; Hu, Haobin

    2014-08-01

    A method for two-steps extraction of essential oil, polysaccharides and lignans from Schisandra chinensis Baill had been established. Firstly, S. chinensis was extracted by hydro-distillation, the extracted solution was separated from the water-insoluble residue and precipitated by adding dehydrated alcohol after the essential oil was collected, and then the precipitate as polysaccharide was collected. Finally, second extraction was performed to obtained lignans from the water-insoluble residue with ultrasonic-microwave assisted extraction (UMAE) method. Response surface methodology was employed to optimize the UMAE parameters, the optimal conditions were as follows: microwave power 430W, ethanol concentration 84%, particle size of sample 120-mesh sieves, ratio of water to raw material 15 and extraction time 2.1min. Under these optimized conditions, the total extraction yields of five lignans (Schisandrol A, Schisantherin A, Deoxyschisandrin, Schisandrin B and Schisandrin C) had reached 14.22±0.135mg/g. Compared with the traditional method of direct extraction of different bioactive components in respective procedure, the extraction yields of polysaccharides and the five lignans had reached 99% and 95%, respectively. The mean recoveries of the 5 lignan compounds and polysaccharides were 97.75-101.08% and their RSD value was less than 3.88%.The approach proposed in this study not only improved the extraction yield of lignans, but also elevated the utilization of Schisandra resources. PMID:24755113

  10. A universal DNA extraction and PCR amplification method for fungal rDNA sequence-based identification.

    PubMed

    Romanelli, A M; Fu, J; Herrera, M L; Wickes, B L

    2014-10-01

    Accurate identification of fungal pathogens using a sequence-based approach requires an extraction method that yields template DNA pure enough for polymerase chain reaction (PCR) or other types of amplification. Therefore, the objective of this study was to develop and standardise a rapid, inexpensive DNA extraction protocol applicable to the major fungal phyla, which would yield sufficient template DNA pure enough for PCR and sequencing. A total of 519 clinical and culture collection strains, comprised of both yeast and filamentous fungi, were prepared using our extraction method to determine its applicability for PCR, which targeted the ITS and D1/D2 regions in a single PCR amplicon. All templates were successfully amplified and found to yield the correct strain identification when sequenced. This protocol could be completed in approximately 30 min and utilised a combination of physical and chemical extraction methods but did not require organic solvents nor ethanol precipitation. The method reduces the number of tube manipulations and yielded suitable template DNA for PCR amplification from all phyla that were tested. PMID:24865530

  11. DNA unwinding step-size of E. coli RecBCD helicase determined from single turnover chemical quenched-flow kinetic studies.

    PubMed

    Lucius, Aaron L; Vindigni, Alessandro; Gregorian, Razmic; Ali, Janid A; Taylor, Andrew F; Smith, Gerald R; Lohman, Timothy M

    2002-11-29

    The mechanism by which Escherichia coli RecBCD DNA helicase unwinds duplex DNA was examined in vitro using pre-steady-state chemical quenched-flow kinetic methods. Single turnover DNA unwinding experiments were performed by addition of ATP to RecBCD that was pre-bound to a series of DNA substrates containing duplex DNA regions ranging from 24 bp to 60 bp. In each case, the time-course for formation of completely unwound DNA displayed a distinct lag phase that increased with duplex length, reflecting the transient formation of partially unwound DNA intermediates during unwinding catalyzed by RecBCD. Quantitative analysis of five independent sets of DNA unwinding time courses indicates that RecBCD unwinds duplex DNA in discrete steps, with an average unwinding "step-size", m=3.9(+/-1.3)bp step(-1), with an average unwinding rate of k(U)=196(+/-77)steps s(-1) (mk(U)=790(+/-23)bps(-1)) at 25.0 degrees C (10mM MgCl(2), 30 mM NaCl (pH 7.0), 5% (v/v) glycerol). However, additional steps, not linked directly to DNA unwinding are also detected. This kinetic DNA unwinding step-size is similar to that determined for the E.coli UvrD helicase, suggesting that these two SF1 superfamily helicases may share similar mechanisms of DNA unwinding. PMID:12445778

  12. Stereospecific removal of methyl phosphotriesters from DNA by an Escherichia coli ada+ extract.

    PubMed

    Weinfeld, M; Drake, A F; Saunders, J K; Paterson, M C

    1985-10-11

    The ada+ gene product, a DNA methyltransferase present in extracts from an Escherichia coli strain constitutive for the adaptive response, removes only half of the methyl phosphotriesters from alkylated DNA. Since DNA phosphotriesters occur in two isomeric configurations (denoted Rp and Sp), we examined whether this reflects a stereospecific mode of repair by the methyltransferase. Analysis by reverse-phase HPLC, phosphorus NMR and circular dichroism established that only triesters in the Sp configuration are acted upon by the E. coli extract. PMID:3903661

  13. Optimisation of DNA and RNA extraction from archival formalin-fixed tissue.

    PubMed Central

    Coombs, N J; Gough, A C; Primrose, J N

    1999-01-01

    Archival, formalin-fixed, paraffin-embedded tissue is an invaluable resource for molecular genetic studies but the extraction of high quality nucleic acid may be problematic. We have optimised DNA extraction by comparing 10 protocols, including a commercially available kit and a novel method that utilises a thermal cycler. The thermal cycler and Chelex-100 extraction method yielded DNA capable of amplification by PCR from every block and 61% of sections versus 54% using microwave and Chelex-100, 15% with classical xylene-based extraction and 60% of sections using the kit. Successful RNA extraction was observed, by beta-actin amplification, in 83.7% sections for samples treated by the thermal cycler and Chelex-100 method. Thermal cycler and Chelex-100 extraction of nucleic acid is reliable, quick and inexpensive. PMID:10454649

  14. Global analysis of ion dependence unveils hidden steps in DNA binding and bending by integration host factor

    NASA Astrophysics Data System (ADS)

    Vivas, Paula; Velmurugu, Yogambigai; Kuznetsov, Serguei V.; Rice, Phoebe A.; Ansari, Anjum

    2013-09-01

    Proteins that recognize and bind to specific sites on DNA often distort the DNA at these sites. The rates at which these DNA distortions occur are considered to be important in the ability of these proteins to discriminate between specific and nonspecific sites. These rates have proven difficult to measure for most protein-DNA complexes in part because of the difficulty in separating the kinetics of unimolecular conformational rearrangements (DNA bending and kinking) from the kinetics of bimolecular complex association and dissociation. A notable exception is the Integration Host Factor (IHF), a eubacterial architectural protein involved in chromosomal compaction and DNA recombination, which binds with subnanomolar affinity to specific DNA sites and bends them into sharp U-turns. The unimolecular DNA bending kinetics has been resolved using both stopped-flow and laser temperature-jump perturbation. Here we expand our investigation by presenting a global analysis of the ionic strength dependence of specific binding affinity and relaxation kinetics of an IHF-DNA complex. This analysis enables us to obtain each of the underlying elementary rates (DNA bending/unbending and protein-DNA association/dissociation), and their ionic strength dependence, even under conditions where the two processes are coupled. Our analysis indicates interesting differences in the ionic strength dependence of the bi- versus unimolecular steps. At moderate [KCl] (100-500 mM), nearly all the ionic strength dependence to the overall equilibrium binding affinity appears in the bimolecular association/dissociation of an initial, presumably weakly bent, encounter complex, with a slope SKbi ≈ 8 describing the loglog-dependence of the equilibrium constant to form this complex on [KCl]. In contrast, the unimolecular equilibrium constant to form the fully wrapped specific complex from the initial complex is nearly independent of [KCl], with SKuni < 0.5. This result is counterintuitive because there

  15. Non-destructive high-throughput DNA extraction and genotyping methods for cotton seeds and seedlings.

    PubMed

    Zheng, Xiuting; Hoegenauer, Kevin A; Maeda, Andrea B V; Wang, Fei; Stelly, David M; Nichols, Robert L; Jones, Don C

    2015-05-01

    Extensive use of targeted PCR-based genotyping is precluded for many plant research laboratories by the cost and time required for DNA extraction. Using cotton (Gossypium hirsutum) as a model for plants with medium-sized seeds, we report here manual procedures for inexpensive non-destructive high-throughput extraction of DNA suitable for PCR-based genotyping of large numbers of individual seeds and seedlings. By sampling only small amounts of cotyledon tissue of ungerminated seed or young seedlings, damage is minimized, and viability is not discernibly affected. The yield of DNA from each seed or seedling is typically sufficient for 1000 or 500 PCR reactions, respectively. For seeds, the tissue sampling procedure relies on a modified 96-well plate that is used subsequently for seed storage. For seeds and seedlings, the DNA is extracted in a strongly basic DNA buffer that is later neutralized and diluted. Extracts can be used directly for high-throughput PCR-based genotyping. Any laboratory can thus extract DNA from thousands of individual seeds/seedlings per person-day at a very modest cost for consumables (~$0.05 per sample). Being non-destructive, our approach enables a wide variety of time- and resource-saving applications, such as marker-assisted selection (MAS), before planting, transplanting, and flowering. PMID:25967902

  16. Attempted DNA extraction from a Rancho La Brea Columbian mammoth (Mammuthus columbi): prospects for ancient DNA from asphalt deposits

    PubMed Central

    Gold, David A; Robinson, Jacqueline; Farrell, Aisling B; Harris, John M; Thalmann, Olaf; Jacobs, David K

    2014-01-01

    Fossil-bearing asphalt deposits are an understudied and potentially significant source of ancient DNA. Previous attempts to extract DNA from skeletons preserved at the Rancho La Brea tar pits in Los Angeles, California, have proven unsuccessful, but it is unclear whether this is due to a lack of endogenous DNA, or if the problem is caused by asphalt-mediated inhibition. In an attempt to test these hypotheses, a recently recovered Columbian mammoth (Mammuthus columbi) skeleton with an unusual pattern of asphalt impregnation was studied. Ultimately, none of the bone samples tested successfully amplified M. columbi DNA. Our work suggests that reagents typically used to remove asphalt from ancient samples also inhibit DNA extraction. Ultimately, we conclude that the probability of recovering ancient DNA from fossils in asphalt deposits is strongly (perhaps fatally) hindered by the organic compounds that permeate the bones and that at the Rancho La Brea tar pits, environmental conditions might not have been ideal for the general preservation of genetic material. PMID:24634719

  17. Complementation of DNA repair in xeroderma pigmentosum group A cell extracts by a protein with affinity for damaged DNA.

    PubMed

    Robins, P; Jones, C J; Biggerstaff, M; Lindahl, T; Wood, R D

    1991-12-01

    Complementation group A of xeroderma pigmentosum (XP) represents one of the most prevalent and serious forms of this cancer-prone disorder. Because of a marked defect in DNA excision repair, cells from individuals with XP-A are hypersensitive to the toxic and mutagenic effects of ultraviolet light and many chemical agents. We report here the isolation of the XP-A DNA repair protein by complementation of cell extracts from a repair-defective human XP-A cell line. XP-A protein purified from calf thymus migrates on denaturing gel electrophoresis as a doublet of 40 and 42 kilodaltons. The XP-A protein binds preferentially to ultraviolet light-irradiated DNA, with a preference for damaged over nondamaged nucleotides of approximately 10(3). This strongly suggests that the XP-A protein plays a direct role in the recognition of and incision at lesions in DNA. We further show that this protein corresponds to the product encoded by a recently isolated gene that can restore excision repair to XP-A cells. Thus, excision repair of plasmid DNA by cell extracts sufficiently resembles genomic repair in cells to reveal accurately the repair defect in an inherited disease. The general approach described here can be extended to the identification and isolation of other human DNA repair proteins. PMID:1935910

  18. Complementation of DNA repair in xeroderma pigmentosum group A cell extracts by a protein with affinity for damaged DNA.

    PubMed Central

    Robins, P; Jones, C J; Biggerstaff, M; Lindahl, T; Wood, R D

    1991-01-01

    Complementation group A of xeroderma pigmentosum (XP) represents one of the most prevalent and serious forms of this cancer-prone disorder. Because of a marked defect in DNA excision repair, cells from individuals with XP-A are hypersensitive to the toxic and mutagenic effects of ultraviolet light and many chemical agents. We report here the isolation of the XP-A DNA repair protein by complementation of cell extracts from a repair-defective human XP-A cell line. XP-A protein purified from calf thymus migrates on denaturing gel electrophoresis as a doublet of 40 and 42 kilodaltons. The XP-A protein binds preferentially to ultraviolet light-irradiated DNA, with a preference for damaged over nondamaged nucleotides of approximately 10(3). This strongly suggests that the XP-A protein plays a direct role in the recognition of and incision at lesions in DNA. We further show that this protein corresponds to the product encoded by a recently isolated gene that can restore excision repair to XP-A cells. Thus, excision repair of plasmid DNA by cell extracts sufficiently resembles genomic repair in cells to reveal accurately the repair defect in an inherited disease. The general approach described here can be extended to the identification and isolation of other human DNA repair proteins. Images PMID:1935910

  19. A pressure cooking-based DNA extraction from archival formalin fixed, paraffin embedded tissue

    PubMed Central

    Chung, Joon-Yong; Yi, Joo Mi; Xie, Ran; Brown, Victoria; Lee, Olivia; Ahuja, Nita; Braunschweig, Till; Hewitt, Stephen M.

    2012-01-01

    As emerging novel DNA-based methodologies are adopted, nucleic acid-based assays depend critically on the quality and quantity of extracted DNA. Formalin fixed, paraffin embedded (FFPE) tissue samples provide an invaluable resource for subsequent molecular studies of clinical phenotypes, but high quality DNA extraction from archival FFPE tissue specimen remains complex and time consuming. To address this challenge, we have developed a reliable rapid DNA extraction method for FFPE tissue specimens. It is based on deparaffinization at high temperature coupled with relieving crosslink in a pressure cooker. The DNA yield by this rapid method resulted in an average 1.8-fold increase in comparison with the commercial kit; O.D 260/280 ratios between 1.87 and 1.95. The DNA obtained by the rapid method was suitable for methylation analyses in colon cancer patients. These data suggest that this new DNA extraction method coupled with MSP can be used for epigenetic studies with the advantages of rapidity and high quality, and may contribute to the development of biomarkers in clinical studies. PMID:22449494

  20. An optimized DNA extraction and purification method from dairy manure compost for genetic diversity analysis.

    PubMed

    Tian, Wei; Zhang, Zhenhua; Liu, Dongyang; Zhou, Tiantian; Shen, Qirong; Shen, Biao

    2013-05-01

    An unbiased DNA extraction protocol is necessary for analysis of genetic diversity, particularly, of genes in complex environmental samples by nucleic acid techniques. In the present study, three manual extraction methods and two commonly used commercial kits, which were accompanied by two DNA purification strategies, were compared based on cell lysis efficiency, DNA and humic acid yields, PCR amplification and denaturing gradient gel electrophoresis (DGGE) analysis. The results show that in spite of higher cell lysis efficiencies of the two commercial kits, the purified DNA yields were only one-third of that obtained by the two manual methods of FTSP (Freeze-thaw-SDS-Protein K) and FTSPP (Freeze-thaw-SDS-Protein K-Polyvinylpolypyrrolidone). The purified DNA from all five methods was pure enough for successful PCR and real-time PCR amplifications in the presence of 1 μg μL(-1) BSA. However, the FTSPP extraction method with DNA purification by a Wizard(®) kit yielded the largest number of 16S rRNA gene copies and ribotypes or bands in DGGE profiles, which indicated a superiority over the other four methods. The development of this optimized DNA extraction and purification method may provide a valuable tool for further molecular analysis of compost. PMID:23239373

  1. A Rapid and Economical Method for Efficient DNA Extraction from Diverse Soils Suitable for Metagenomic Applications

    PubMed Central

    Devi, Selvaraju Gayathri; Fathima, Anwar Aliya; Radha, Sudhakar; Arunraj, Rex; Curtis, Wayne R.; Ramya, Mohandass

    2015-01-01

    A rapid, cost effective method of metagenomic DNA extraction from soil is a useful tool for environmental microbiology. The present work describes an improved method of DNA extraction namely “powdered glass method” from diverse soils. The method involves the use of sterile glass powder for cell lysis followed by addition of 1% powdered activated charcoal (PAC) as purifying agent to remove humic substances. The method yielded substantial DNA (5.87 ± 0.04 μg/g of soil) with high purity (A260/280: 1.76 ± 0.05) and reduced humic substances (A340: 0.047 ± 0.03). The quality of the extracted DNA was compared against five different methods based on 16S rDNA PCR amplification, BamHI digestion and validated using quantitative PCR. The digested DNA was used for a metagenomic library construction with the transformation efficiency of 4 X 106 CFU mL-1. Besides providing rapid, efficient and economical extraction of metgenomic DNA from diverse soils, this method’s applicability is also demonstrated for cultivated organisms (Gram positive B. subtilis NRRL-B-201, Gram negative E. coli MTCC40, and a microalgae C. sorokiniana UTEX#1666). PMID:26167854

  2. Automated serial extraction of DNA and RNA from biobanked tissue specimens

    PubMed Central

    2013-01-01

    Background With increasing biobanking of biological samples, methods for large scale extraction of nucleic acids are in demand. The lack of such techniques designed for extraction from tissues results in a bottleneck in downstream genetic analyses, particularly in the field of cancer research. We have developed an automated procedure for tissue homogenization and extraction of DNA and RNA into separate fractions from the same frozen tissue specimen. A purpose developed magnetic bead based technology to serially extract both DNA and RNA from tissues was automated on a Tecan Freedom Evo robotic workstation. Results 864 fresh-frozen human normal and tumor tissue samples from breast and colon were serially extracted in batches of 96 samples. Yields and quality of DNA and RNA were determined. The DNA was evaluated in several downstream analyses, and the stability of RNA was determined after 9 months of storage. The extracted DNA performed consistently well in processes including PCR-based STR analysis, HaloPlex selection and deep sequencing on an Illumina platform, and gene copy number analysis using microarrays. The RNA has performed well in RT-PCR analyses and maintains integrity upon storage. Conclusions The technology described here enables the processing of many tissue samples simultaneously with a high quality product and a time and cost reduction for the user. This reduces the sample preparation bottleneck in cancer research. The open automation format also enables integration with upstream and downstream devices for automated sample quantitation or storage. PMID:23957867

  3. Comparison of two silica-based extraction methods for DNA isolation from bones.

    PubMed

    Rothe, Jessica; Nagy, Marion

    2016-09-01

    One of the most demanding DNA extractions is from bones and teeth due to the robustness of the material and the relatively low DNA content. The greatest challenge is due to the manifold nature of the material, which is defined by various factors, including age, storage, environmental conditions, and contamination with inhibitors. However, most published protocols do not distinguish between different types or qualities of bone material, but are described as being generally applicable. Our laboratory works with two different extraction methods based on silica membranes or the use of silica beads. We compared the amplification success of the two methods from bone samples with different qualities and in the presence of inhibitors. We found that the DNA extraction using the silica membrane method results an in higher DNA yield but also in a higher risk of co-extracting impurities, which can act as inhibitors. In contrast the silica beads method shows decreased co-extraction of inhibitors but also less DNA yield. Related to our own experiences it has to be considered that each bone material should be reviewed independently regarding the analysis and extraction method. Therefore, the most ambitious task is determining the quality of the bone material, which requires substantial experience. PMID:27591537

  4. High-quality genomic DNA extraction from formalin-fixed and paraffin-embedded samples deparaffinized using mineral oil.

    PubMed

    Lin, Jianghai; Kennedy, Stephen H; Svarovsky, Therese; Rogers, Jeffrey; Kemnitz, Joseph W; Xu, Anlong; Zondervan, Krina T

    2009-12-15

    Extracting DNA from formalin-fixed and paraffin-embedded (FFPE) tissue remains a challenge, despite numerous attempts to develop a more effective method. Polymerase chain reaction (PCR) success rates with DNA extracted using current methods remain low. We extracted DNA from 140 long-term archived FFPE samples using a simple but effective deparaffinization method, removing the wax with mineral oil, and a commercially available DNA extraction kit. DNA quality was subsequently tested in a genotyping experiment with 14 microsatellite markers. High-quality DNA was obtained with a mean PCR success rate of 97% (range: 88-100%) across markers. The results suggested that DNA extracted using this novel method is likely to be suitable for genetic studies involving DNA fragments <200 bp. PMID:19698695

  5. [Extraction and purification method of rice DNA from rice powder containing Konjak flour].

    PubMed

    Minematsu, Kazuhiko; Nakamura, Kosuke; Akiyama, Hiroshi; Harikai, Naoki; Nakajima, Osamu; Kitta, Kazumi; Teshima, Reiko; Iizuka, Tayoshi

    2010-01-01

    Rice powder containing Konjak flour made with tuberous roots of Amorphophallus konjac is imported as a rice-processed product from China to Japan. An improved DNA purification method for the polymerase chain reaction (PCR) analysis of rice in such products is necessary, since Konjak flour constituents absorb the DNA purification buffer to form a gel, and cause problems in the subsequent purification steps. Here, we present a simple preparative system for isolation of the rice and a purification method of the rice DNA from the product. The purified DNA was confirmed to be a good template for both PCR and real-time PCR. PMID:21071909

  6. Antigenotoxic Effect of Trametes spp. Extracts against DNA Damage on Human Peripheral White Blood Cells

    PubMed Central

    Knežević, Aleksandar; Živković, Lada; Stajić, Mirjana; Vukojević, Jelena; Milovanović, Ivan; Spremo-Potparević, Biljana

    2015-01-01

    Trametes species have been used for thousands of years in traditional and conventional medicine for the treatment of various types of diseases. The goal was to evaluate possible antigenotoxic effects of mycelium and basidiocarp extracts of selected Trametes species and to assess dependence on their antioxidant potential. Trametes versicolor, T. hirsuta, and T. gibbosa were the species studied. Antigenotoxic potentials of extracts were assessed on human peripheral white blood cells with basidiocarp and mycelium extracts of the species. The alkaline comet test was used for detection of DNA strand breaks and alkali-labile sites, as well as the extent of DNA migration. DPPH assay was used to estimate antioxidative properties of extracts. Fruiting body extracts of T. versicolor and T. gibbosa as well as T. hirsuta extracts, except that at 20.0 mg/mL, were not genotoxic agents. T. versicolor extract had at 5.0 mg/mL the greatest antigenotoxic effect in both pre- and posttreatment of leukocytes. The mycelium extracts of the three species had no genotoxic activity and significant antigenotoxic effect against H2O2-induced DNA damage, both in pre- and posttreatment. The results suggest that extracts of these three species could be considered as strong antigenotoxic agents able to stimulate genoprotective response of cells. PMID:26258163

  7. DNA in ancient bone - where is it located and how should we extract it?

    PubMed

    Campos, Paula F; Craig, Oliver E; Turner-Walker, Gordon; Peacock, Elizabeth; Willerslev, Eske; Gilbert, M Thomas P

    2012-01-20

    Despite the widespread use of bones in ancient DNA (aDNA) studies, relatively little concrete information exists in regard to how the DNA in mineralised collagen degrades, or where it survives in the material's architecture. While, at the macrostructural level, physical exclusion of microbes and other external contaminants may be an important feature, and, at the ultrastructural level, the adsorption of DNA to hydroxyapatite and/or binding of DNA to Type I collagen may stabilise the DNA, the relative contribution of each, and what other factors may be relevant, are unclear. There is considerable variation in the quality of DNA retrieved from bones and teeth. This is in part due to various environmental factors such as temperature, proximity to free water or oxygen, pH, salt content, and exposure to radiation, all of which increase the rate of DNA decay. For example, bone specimens from sites at high latitudes usually yield better quality DNA than samples from temperate regions, which in turn yield better results than samples from tropical regions. However, this is not always the case, and rates of success of DNA recovery from apparently similar sites are often strikingly different. The question arises as to whether this may be due to post-collection preservation or just an artefact of the extraction methods used in these different studies? In an attempt to resolve these questions, we examine the efficacy of DNA extraction methods, and the quality and quantity of DNA recovered from both artificially degraded, and genuinely ancient, but well preserved, bones. In doing so we offer hypotheses relevant to the DNA degradation process itself, and to where and how the DNA is actually preserved in ancient bone. PMID:21855309

  8. Comparison and optimization of methods for the simultaneous extraction of DNA, RNA, proteins, and metabolites.

    PubMed

    Vorreiter, Fränze; Richter, Silke; Peter, Michel; Baumann, Sven; von Bergen, Martin; Tomm, Janina M

    2016-09-01

    The challenge of performing a time-resolved comprehensive analysis of molecular systems has led to the quest to optimize extraction methods. When the size of a biological sample is limited, there is demand for the simultaneous extraction of molecules representing the four areas of "omics": genomics, transcriptomics, proteomics, and metabolomics. Here we optimized a protocol for the simultaneous extraction of DNA, RNA, proteins, and metabolites and compared it with two existing protocols. Our optimization comprised the addition of a methanol/chloroform metabolite purification before the separation of DNA/RNA and proteins. Extracted DNA, RNA, proteins, and metabolites were quantitatively and/or qualitatively analyzed. Of the three methods, only the newly developed protocol yielded all biomolecule classes of adequate quantity and quality. PMID:27237373

  9. Tracing tree nut allergens in chocolate: A comparison of DNA extraction protocols.

    PubMed

    Costa, Joana; Melo, Vítor S; Santos, Cristina G; Oliveira, M Beatriz P P; Mafra, Isabel

    2015-11-15

    The present work aimed at comparing different DNA extraction methods, from chocolate matrices, for the effective application in molecular techniques to detect tree nut allergens. For this study, DNA from almond or hazelnut model chocolates was extracted using seven selected protocols: the in-house methods of CTAB-PVP (cetyltrimethylammonium bromide-polyvinylpyrrolidone), Wizard with and without RNase, Wizard-PVP with and without RNase, and the Wizard Magnetic and Nucleospin kits. The extracts were assessed for their suitability for amplification by qualitative PCR and real-time PCR. From the evaluated protocols, Nucleospin presented the best results for almond and hazelnut amplification, achieving a limit of detection of 0.005% (w/w) with high PCR efficiency, linearity and range of amplification. These results highlight the importance of the DNA extraction protocol in the case of food allergens from complex matrices, such as chocolate, in which sensitivity is a key parameter. PMID:25977052

  10. Enzymatic Treatment of Specimens before DNA Extraction Directly Influences Molecular Detection of Infectious Agents

    PubMed Central

    Goldschmidt, Pablo; Degorge, Sandrine; Merabet, Lilia; Chaumeil, Christine

    2014-01-01

    Introduction Biological samples, pharmaceuticals or food contain proteins, lipids, polymers, ammoniums and macromolecules that alter the detection of infectious agents by DNA amplification techniques (PCR). Moreover the targeted DNA has to be released from the complex cell walls and the compact nucleoprotein matrixes and cleared from potential inhibitors. The goal of the present work was to assess the efficiency of enzymatic pretreatments on infectious agents to make DNA available for further extraction and amplification. Methods Staphylococcus epidermidis, Streptococcus mitis, Propionibacterium acnes, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger and Fusarium solani were mixed with an internal control virus and treated with: 1) proteinase K; 2) lyticase and 3) lyticase followed by proteinase K. DNAs was manually extracted using the QIAmp DNA Mini kit or the MagNA Pure Compact automate. DNA extraction yields and the inhibitors were assessed with a phocid Herpesvirus. Bacterial detection was performed using TaqMan real-time PCR and yeasts and filamentous Fungi with HRM (real-time PCR followed by high-resolution melting analysis). Results Viral DNA was released, extracted and detected using manual and automatic methods without pre enzymatic treatments. Either the manual or the automatic DNA extraction systems did not meet the sensitivity expectations if enzymatic treatments were not performed before: lyticase for Fungi and Proteinase K for Bacteria. The addition of lyticase and proteinase K did not improve results. For Fungi the detection after lyticase was higher than for Proteinase K, for which melting analysis did not allow fungal specification. Discussion Columns and magnetic beads allowed collecting DNA and separate PCR inhibitors. Detection rates cannot be related to DNA-avidity of beads or to elution but to the lack of proteolysis. PMID:24936792

  11. Preservation of and DNA Extraction from Muscle Tissue.

    PubMed

    McNevin, Dennis

    2016-01-01

    As well as protecting DNA for subsequent analysis, tissue preservation methods ideally should be safe, readily available, and easy to transport at relatively low cost. Formalin (formaldehyde solution), used extensively to preserve medical and museum specimens, irreparably damages DNA. We have found four tissue preservatives (solid salt, salt-saturated dimethyl sulfoxide (DMSO)-EDTA solution, ethanol solution, and ethanol-EDTA solution) that preserved muscle tissue at 35 °C for up to 1 month: full short tandem repeat (STR) profiles were obtained after preservation. In addition, salt-saturated DMSO-EDTA solution yielded full STR profiles from aliquots of the liquid preservative surrounding muscle tissue. PMID:27259730

  12. A Non-uniform Stepping Mechanism for E. coli UvrD Monomer Translocation along Single Stranded DNA

    PubMed Central

    Tomko, Eric J.; Fischer, Christopher J.; Niedziela-Majka, Anita; Lohman, Timothy M.

    2007-01-01

    Summary E. coli UvrD is an SF1 helicase involved in several DNA metabolic processes. Although a UvrD dimer is needed for helicase activity, a monomer can translocate with 3′ to 5′ directionality along single stranded DNA and this ATP-dependent translocation is likely involved in RecA displacement. In order to understand how the monomeric translocase functions, we have combined fluorescence stopped-flow kinetic methods with novel analysis methods to determine the kinetic mechanism, including ATP coupling stoichiometry, for UvrD monomer translocation along ssDNA. Our results suggest that the macroscopic rate of UvrD monomer translocation is not limited by each ATPase cycle, but rather by a slow step (pause) in each translocation cycle that occurs after four to five rapid one nucleotide translocation steps, with each rapid step coupled to hydrolysis of one ATP. These results suggest a non-uniform stepping mechanism that differs from either a Brownian motor or previous structure based inch-worm mechanisms. PMID:17499041

  13. A nonuniform stepping mechanism for E. coli UvrD monomer translocation along single-stranded DNA.

    PubMed

    Tomko, Eric J; Fischer, Christopher J; Niedziela-Majka, Anita; Lohman, Timothy M

    2007-05-11

    E. coli UvrD is an SF1 helicase involved in several DNA metabolic processes. Although a UvrD dimer is needed for helicase activity, a monomer can translocate with 3' to 5' directionality along single-stranded DNA, and this ATP-dependent translocation is likely involved in RecA displacement. In order to understand how the monomeric translocase functions, we have combined fluorescence stopped-flow kinetic methods with recently developed analysis methods to determine the kinetic mechanism, including ATP coupling stoichiometry, for UvrD monomer translocation along ssDNA. Our results suggest that the macroscopic rate of UvrD monomer translocation is not limited by each ATPase cycle but rather by a slow step (pause) in each translocation cycle that occurs after four to five rapid 1 nt translocation steps, with each rapid step coupled to hydrolysis of one ATP. These results suggest a nonuniform stepping mechanism that differs from either a Brownian motor or previous structure-based inchworm mechanisms. PMID:17499041

  14. A RAPID DNA EXTRACTION METHOD IS SUCCESSFULLY APPLIED TO ITS-RFLP ANALYSIS OF MYCORRHIZAL ROOT TIPS

    EPA Science Inventory

    A rapid method for extracting DNA from intact, single root tips using a Xanthine solution was developed to handle very large numbers of analyses of ectomycorrhizas. By using an extraction without grinding we have attempted to bias the extraction towards the fungal DNA in the man...

  15. Trapping of DNA topoisomerase I on nick-containing DNA in cell free extracts of Saccharomyces cerevisiae.

    PubMed

    Lebedeva, Natalia; Auffret Vander Kemp, Patricia; Bjornsti, Mary-Ann; Lavrik, Olga; Boiteux, Serge

    2006-07-13

    The aim of the present study was to identify proteins that bind nicked DNA intermediates formed in the course of base excision repair (BER) in cell free extracts of Saccharomyces cerevisiae. In mammalian cells, nicks in DNA are targets of proteins such as PARP-1 or XRCC1 that have no homologues in yeast. One of the most promising methodologies to trap proteins that interact with damaged DNA lies in using a photocrosslinking technique with photoactivable dNTP analogues such as exo-N-{2-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]-aminoethyl}-2'-deoxycytidine-5'-triphosphate (FAP-dCTP) for enzymatic synthesis of DNA probes with a photoreactive dNMP residue at the 3'-margin of a nick. Using this approach, we identified a major covalent DNA-protein adduct between a nick-containing 34-mer DNA duplex and a protein of a molecular mass of around 100-kDa. Unexpectedly, the formation of the 100-kDa adduct did not require the incorporation of the photoreactive dNMP residue at the 3'-margin of the nick nor exposure to near UV-light. However, the formation of the 100-kDa adduct strictly required a nick or a short gap in the DNA probe. Furthermore, the 100-kDa adduct was not detected in yeast extracts lacking DNA topoisomerase I (Top1). To further establish the nature of crosslinked protein, yeast Top1 was tagged with a Myc-epitope. In this case, the mobility of the Top1-DNA adduct increased by 7- kDa. Therefore, our data speak in favor of Top1 trapping by nicked DNA. In support of this hypothesis, purified yeast Top1 was also crosslinked to nicked DNA structures. Undamaged, uracil- and abasic (AP) site-containing DNAs were unable to trap Top1 under the same assay conditions. Since nicked DNA structures are frequently formed in the course of BER, their covalent linkage to Top1 has the potential to interfere with BER in vivo. PMID:16713756

  16. Comparison of two methods for the small-scale extraction of DNA from subgingival microorganisms.

    PubMed

    Smith, G L; Sansone, C; Socransky, S S

    1989-09-01

    Two methods were compared for the extraction of DNA from small numbers of bacterial cells. The first method involved lysis of cells with SDS in the presence of proteinase K, treatment with hexadecyltrimethyl ammonium bromide (CTAB) and precipitation of DNA with isopropanol. In the second method, DNA was extracted by treatment of the cells with guanidine hydrochloride (GHCl) and precipitated with ethanol. Thirty strains of representative gram positive and gram negative species were included in the study. Preparations derived from confluent growth on one-quarter of the surface of agar plates and from 10(8) cells were subjected to each extraction procedure and analyzed for their content of DNA, RNA and protein. The suitabilities of the resultant DNA for restriction enzyme digestion and biotin-labelling by a random primer technique were also assessed. In general, the CTAB method yielded greater amounts of DNA than the GHCl procedure. RNA was present in most preparations of both types, but in amounts detectable only by agarose gel electrophoresis. The latter technique also revealed that DNA was not excessively sheared by either procedure. Protein was detected in some CTAB and GHCl preparations, but was not consistently associated with one or the other method. DNA obtained by both methods could be digested by the restriction enzyme EcoR I. In addition, biotin-labelled DNA probes prepared from CTAB and GHCl preparations were capable of hybridizing with homologous target DNA fixed to nitrocellulose. Since the CTAB method was consistently successful in recovering DNA from preparations containing 10(8) cells, it may be more suitable for the direct treatment of single colonies taken from primary isolation plates or plaque samples. PMID:2639297

  17. Protective effect of enzymatic extracts from microalgae against DNA damage induced by H2O2.

    PubMed

    Karawita, Rohan; Senevirathne, Mahinda; Athukorala, Yasantha; Affan, Abu; Lee, Young-Jae; Kim, Se-Kwon; Lee, Joon-Baek; Jeon, You-Jin

    2007-01-01

    The enzymatic extracts from seven species of microalgae (Pediastrum duplex, Dactylococcopsis fascicularis, Halochlorococcum porphyrae, Oltmannsiellopsis unicellularis, Achnanthes longipes, Navicula sp. and Amphora coffeaeformis) collected from three habitats (freshwater, tidal pool, and coastal benthic) at Jeju Island in Korea were investigated for their antioxidant activity. Of the extracts tested, the AMG 300 L (an exo 1, 4-alpha-D-glucosidase) extract of P. duplex, the Viscozyme extract of Navicula sp., and the Celluclast extract of A. longipes provided the most potential as antioxidants. Meanwhile, the Termamyl extract of P. duplex in an H(2)O(2) scavenging assay exhibited an approximate 60% scavenging effect. In this study, we report that the DNA damage inhibitory effects of P. duplex (Termamyl extract) and D. fascicularis (Kojizyme extract) were nearly 80% and 69% respectively at a concentration of 100 microg/ml. Thus, it is suggested that the microalgae tested in this study yield promising DNA damage inhibitory properties on mouse lymphoma L 5178 cells that are treated with H(2)O(2). Therefore, microalgae such as P. duplex may be an excellent source of naturally occurring antioxidant compounds with potent DNA damage inhibition potential. PMID:17520314

  18. DNA extraction from paraffin embedded material for genetic and epigenetic analyses.

    PubMed

    Pikor, Larissa A; Enfield, Katey S S; Cameron, Heryet; Lam, Wan L

    2011-01-01

    Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures (1). To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion (2). Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions

  19. Exploring microbial diversity in volcanic environments: a review of methods in DNA extraction.

    PubMed

    Herrera, Aude; Cockell, Charles S

    2007-07-01

    The last decade has been marked by a large number of studies focused on understanding the distribution of microorganisms in volcanic environments. These studies are motivated by the desire to elucidate how the geochemically extreme conditions of such environments can influence microbial diversity both on the surface and in the subsurface of the Earth. The exploration of microbial community diversity has generally not relied on culture-dependent methods, but has been carried out using environmental DNA extraction. Because of the large diversity of chemically and physically complex samples, extracting DNA from volcanic environments is technically challenging. In view of the emerging literature, and our own experience in the optimisation of methods for DNA extraction from volcanic materials, it is timely to provide a methodological comparison. This review highlights and discusses new insights and methods published on DNA extraction methods from volcanic samples, considering the different volcanic environments. A description of a recent method for DNA extraction from basalt and obsidian glass rock samples from Iceland is included. Finally, we discuss these approaches in the wider context of modern work to understand the microbial diversity of volcanic environments. PMID:17540467

  20. Comparison of customized spin-column and salt-precipitation finger-prick blood DNA extraction

    PubMed Central

    Poh, Jun-Jie; Gan, Samuel Ken-En

    2014-01-01

    gDNA (genomic DNA extraction from blood is a fundamental process in many diagnostic, identification and research applications. Numerous extraction methods have been reported and are available commercially. However, there is insufficient understanding of the impact of chemical buffers on DNA yield from either whole or nucleated blood. Moreover, these commercial kits are often costly, constraining less well-funded laboratories to traditional and more cost-effective salt-precipitation methods. Towards this, we compared a salt-precipitation and a customized cost-effective spin-column-based method, studying the impact of different chemical constituents on the yields. This customized method resulted in a shortening of the extraction process, higher gDNA yields, and more successful PCR amplification of gDNA genes compared with the salt-precipitation method. Optimizing different chemical buffers on whole- and nucleated blood materials further revealed that certain chemicals boosted extractions from whole- but not nucleated blood. These findings may be useful to laboratories that do not have ready access to commercial kits, and improve their nucleic acid extractions from blood economically. PMID:25222694

  1. Ginkgo biloba leaf extract induces DNA damage by inhibiting topoisomerase II activity in human hepatic cells

    PubMed Central

    Zhang, Zhuhong; Chen, Si; Mei, Hu; Xuan, Jiekun; Guo, Xiaoqing; Couch, Letha; Dobrovolsky, Vasily N.; Guo, Lei; Mei, Nan

    2015-01-01

    Ginkgo biloba leaf extract has been shown to increase the incidence in liver tumors in mice in a 2-year bioassay conducted by the National Toxicology Program. In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined. A molecular docking study revealed that quercetin, a flavonoid constituent of Ginkgo biloba, showed a higher potential to interact with topoisomerase II (Topo II) than did the other Ginkgo biloba constituents; this in silico prediction was confirmed by using a biochemical assay to study Topo II enzyme inhibition. Moreover, as measured by the Comet assay and the induction of γ-H2A.X, quercetin, followed by keampferol and isorhamnetin, appeared to be the most potent DNA damage inducer in HepG2 cells. In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage is directly associated with Topo II. DNA damage was also observed when cells were treated with commercially available Ginkgo biloba extract product. Our findings suggest that Ginkgo biloba leaf extract- and quercetin-induced in vitro genotoxicity may be the result of Topo II inhibition. PMID:26419945

  2. Ginkgo biloba leaf extract induces DNA damage by inhibiting topoisomerase II activity in human hepatic cells.

    PubMed

    Zhang, Zhuhong; Chen, Si; Mei, Hu; Xuan, Jiekun; Guo, Xiaoqing; Couch, Letha; Dobrovolsky, Vasily N; Guo, Lei; Mei, Nan

    2015-01-01

    Ginkgo biloba leaf extract has been shown to increase the incidence in liver tumors in mice in a 2-year bioassay conducted by the National Toxicology Program. In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined. A molecular docking study revealed that quercetin, a flavonoid constituent of Ginkgo biloba, showed a higher potential to interact with topoisomerase II (Topo II) than did the other Ginkgo biloba constituents; this in silico prediction was confirmed by using a biochemical assay to study Topo II enzyme inhibition. Moreover, as measured by the Comet assay and the induction of γ-H2A.X, quercetin, followed by keampferol and isorhamnetin, appeared to be the most potent DNA damage inducer in HepG2 cells. In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage is directly associated with Topo II. DNA damage was also observed when cells were treated with commercially available Ginkgo biloba extract product. Our findings suggest that Ginkgo biloba leaf extract- and quercetin-induced in vitro genotoxicity may be the result of Topo II inhibition. PMID:26419945

  3. Moricandia arvensis extracts protect against DNA damage, mutagenesis in bacteria system and scavenge the superoxide anion.

    PubMed

    Skandrani, Ines; Bouhlel, Ines; Limem, Ilef; Boubaker, Jihed; Bhouri, Wissem; Neffati, Aicha; Ben Sghaier, Mohamed; Kilani, Soumaya; Ghedira, Kamel; Ghedira-Chekir, Leila

    2009-02-01

    The mutagenic potential of total aqueous, total oligomers flavonoids (TOF), ethyl acetate (EA), chloroform (Chl), petroleum ether (PE) and methanol (MeOH) extracts from aerial parts of Moricandia arvensis was assessed using Ames Salmonella tester strains TA100 and TA1535 with and without metabolic activation (S9), and using plasmid pBluescript DNA assay. None of the different extracts produced a mutagenic effect, except aqueous extract when incubated with Salmonella typhimurium TA100 after metabolic activation. Likewise, the antimutagenicity of the same extracts was tested using the "Ames test". Our results showed that M. arvensis extracts possess antimutagenic effects against sodium azide (SA) in the two tested Salmonella assay systems, except metabolized aqueous and PE extracts when tested with S. typhimurium TA100 assay system. Different extracts were also found to be effective in protecting plasmid DNA against the strand breakage induced by hydroxyl radicals, except PE and aqueous extracts. Antioxidant capacity of the tested extracts was evaluated using the enzymatic (xanthine/xanthine oxidase assay) (X/XOD) and the non enzymatic (NBT/Riboflavine assay) systems. TOF extract was the more effective one in inhibiting both xanthine oxidase activity and NBT reduction. PMID:19015021

  4. Optimisation of DNA extraction and validation of PCR assays to detect Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Timms, Verlaine J; Mitchell, Hazel M; Neilan, Brett A

    2015-05-01

    The aim of this study was to investigate DNA extraction methods and PCR assays suitable for the detection of Mycobacterium paratuberculosis in bovine tissue. The majority of methods currently used to detect M. paratuberculosis have been developed using bovine samples, such as faeces, blood or tissue and, in many cases, have been based on detection from pooled samples from a herd. However most studies have not compared PCR results to culture results. In order to address this problem, four DNA extraction protocols and three PCR assays were employed to detect M. paratuberculosis in bovine tissue. Given that culture is reliable from cows, the results were then compared with the known M. paratuberculosis culture status. The following DNA extractions were included, two commercial kits, a boiling method, an in house extraction based on a published method and enrichment by sonication. The three PCR assays used included single round IS900 and f57 assays and a nested IS900 assay. In addition, another PCR assay was validated for the detection of any Mycobacterial species and a universal bacterial 16S rRNA gene assay was used to detect sample inhibition. The in-house DNA extraction was the most consistent in extracting good quality DNA compared to all other methods. The use of two PCR markers, IS900 and f57, and a universal PCR enabled the correct samples to be identified as M. paratuberculosis positive. In addition, when compared to the culture result, false-positives did not occur and PCR inhibition was readily identified. Using an in house DNA extraction coupled with the IS900 and f57 PCR markers, this study provides a reliable and simple method to detect M. paratuberculosis in both veterinary and spill over infections. PMID:25797305

  5. Ancient DNA in historical parchments - identifying a procedure for extraction and amplification of genetic material.

    PubMed

    Lech, T

    2016-01-01

    Historical parchments in the form of documents, manuscripts, books, or letters, make up a large portion of cultural heritage collections. Their priceless historical value is associated with not only their content, but also the information hidden in the DNA deposited on them. Analyses of ancient DNA (aDNA) retrieved from parchments can be used in various investigations, including, but not limited to, studying their authentication, tracing the development of the culture, diplomacy, and technology, as well as obtaining information on the usage and domestication of animals. This article proposes and verifies a procedure for aDNA recovery from historical parchments and its appropriate preparation for further analyses. This study involved experimental selection of an aDNA extraction method with the highest efficiency and quality of extracted genetic material, from among the multi-stage phenol-chloroform extraction methods, and the modern, column-based techniques that use selective DNA-binding membranes. Moreover, current techniques to amplify entire genetic material were questioned, and the possibility of using mitochondrial DNA for species identification was analyzed. The usefulness of the proposed procedure was successfully confirmed in identification tests of historical parchments dating back to the 13-16th century AD. PMID:27173330

  6. Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins

    PubMed Central

    Gillespie, Peter J.; Gambus, Agnieszka; Blow, J. Julian

    2012-01-01

    The use of cell-free extracts prepared from eggs of the South African clawed toad, Xenopus laevis, has led to many important discoveries in cell cycle research. These egg extracts recapitulate the key nuclear transitions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. DNA added to the extract is first assembled into a nucleus and is then efficiently replicated. Progression of the extract into mitosis then allows the separation of paired sister chromatids. The Xenopus cell-free system is therefore uniquely suited to the study of the mechanisms, dynamics and integration of cell cycle regulated processes at a biochemical level. In this article we describe methods currently in use in our laboratory for the preparation of Xenopus egg extracts and demembranated sperm nuclei for the study of DNA replication in vitro. We also detail how DNA replication can be quantified in this system. In addition, we describe methods for isolating chromatin and chromatin-bound protein complexes from egg extracts. These recently developed and revised techniques provide a practical starting point for investigating the function of proteins involved in DNA replication. PMID:22521908

  7. Properties of damage-dependent DNA incision by nucleotide excision repair in human cell-free extracts.

    PubMed Central

    Calsou, P; Salles, B

    1994-01-01

    Nucleotide excision repair (NER) is the primary mechanism for the removal of many lesions from DNA. This repair process can be broadly divided in two stages: first, incision at damaged sites and second, synthesis of new DNA to replace the oligonucleotide removed by excision. In order to dissect the repair mechanism, we have recently devised a method to analyze the incision reaction in vitro in the absence of repair synthesis (1). Damage-specific incisions take place in a repair reaction in which mammalian cell-free extracts are mixed with undamaged and damaged plasmids. Most of the incision events are accompanied by excision. Using this assay, we investigated here various parameters that specifically affect the level of damage-dependent incision activity by cell-free extracts in vitro. We have defined optimal conditions for the reaction and determined the kinetics of the incision with cell-free extracts from human cells. We present direct evidence that the incision step of NER is ATP-dependent. In addition, we observe that Mn2+ but no other divalent cation can substitute for Mg2+ in the incision reaction. Images PMID:7800483

  8. Two-step polymerase chain reactions and restriction endonuclease analyses detect and differentiate ompA DNA of Chlamydia spp.

    PubMed Central

    Kaltenboeck, B; Kousoulas, K G; Storz, J

    1992-01-01

    Specific and sensitive amplification of major outer membrane protein (MOMP) gene (ompA) DNA sequences of Chlamydia species with various MOMP genotypes was achieved by a two-step polymerase chain reaction (PCR). Degenerate, inosine-containing oligonucleotide primers homologous to the 5' and 3' ends of the translated regions of all chlamydial MOMP genes were used in a PCR to amplify a DNA fragment of approximately 1,120 bp. A portion of this DNA fragment was amplified in a second genus-specific reaction that yielded a DNA fragment of approximately 930 bp. A pair of degenerate oligonucleotide primers homologous to internal sequences of the primary DNA fragment was used in this PCR. This method detected three cognate chlamydial genomes in a background of 1 microgram of unrelated DNA. MOMP genes of 13 representative chlamydial MOMP genotypes of the species C. trachomatis, C. pneumoniae, and C. psittaci were amplified. In a secondary PCR, group-specific detection was achieved by the simultaneous use of one genus-specific primer and three primers derived from different fingerprint regions of three major groups of chlamydiae. This multiplex PCR differentiated the groups by the length of the amplified DNA fragments and detected the simultaneous presence of DNA sequences of the Chlamydia spp. with different MOMP genotypes. Further differentiation as ompA restriction fragment length polymorphism types among all chlamydial strains with the various MOMP genotypes analyzed here was achieved by restriction endonuclease analysis of the secondary PCR products. DNA sequences corresponding to the ompA restriction fragment length polymorphism type B577 of C. psittaci were detected in two of seven milk samples from cases of bovine mastitis. Images PMID:1349899

  9. DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure

    DOE PAGESBeta

    None

    2014-12-01

    The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illuminamore » 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied.« less

  10. DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure

    SciTech Connect

    2014-12-01

    The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illumina 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied.

  11. DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure

    PubMed Central

    Rubin, Benjamin E R; Sanders, Jon G; Hampton-Marcell, Jarrad; Owens, Sarah M; Gilbert, Jack A; Moreau, Corrie S

    2014-01-01

    The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illumina 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied. PMID:25257543

  12. Extraction of PCR-amplifiable genomic DNA from Bacillus anthracisspores

    SciTech Connect

    Torok, Tamas

    2003-05-19

    Bacterial endospore disruption and nucleic acid extractionresulting in DNA of PCR-amplifiable quality and quantity are not trivial.Responding to the needs of the Hazardous Materials Response Unit (HMRU),Laboratory Division, Federal Bureau of Investigation, protocols weredeveloped to close these gaps. Effectiveness and reproducibility of thetechniques were validated with laboratory grown pure spores of Bacillusanthracis and its close phylogenetic neighbors, and with spiked soils anddamaged samples.

  13. Novel extraction method of genomic DNA suitable for long-fragment amplification from small amounts of milk.

    PubMed

    Liu, Y F; Gao, J L; Yang, Y F; Ku, T; Zan, L S

    2014-11-01

    Isolation of genomic DNA is a prerequisite for assessment of milk quality. As a source of genomic DNA, milk somatic cells from milking ruminants are practical, animal friendly, and cost-effective sources. Extracting DNA from milk can avoid the stress response caused by blood and tissue sampling of cows. In this study, we optimized a novel DNA extraction method for amplifying long (>1,000 bp) DNA fragments and used it to evaluate the isolation of DNA from small amounts of milk. The techniques used for the separation of milk somatic cell were explored and combined with a sodium dodecyl sulfate (SDS)-phenol method for optimizing DNA extraction from milk. Spectrophotometry was used to determine the concentration and purity of the extracted DNA. Gel electrophoresis and DNA amplification technologies were used for to determine DNA size and quality. The DNA of 112 cows was obtained from milk (samples of 13 ± 1 mL) and the corresponding optical density ratios at 260:280 nm were between 1.65 and 1.75. Concentrations were between 12 and 45 μg/μL and DNA size and quality were acceptable. The specific PCR amplification of 1,019- and 729-bp bovine DNA fragments was successfully carried out. This novel method can be used as a practical, fast, and economical mean for long genomic DNA extraction from a small amount of milk. PMID:25218756

  14. Optimization of DNA extraction from fresh leaf tissues of Melanoxylon brauna (Fabaceae).

    PubMed

    Borges, D B; Amorim, M B; Waldschmidt, A M; Mariano-Neto, E; Vivas, C V; Pereira, D G

    2012-01-01

    Melanoxylon brauna (Fabaceae - Caesalpinioideae) is an endemic and valuable hardwood tree species in the Brazilian Atlantic rainforest; it is comparable to African ebony wood. We tested three protocols of DNA extraction based on the citrimonium bromide (CTAB) method and evaluated the quantity, purity and integrity of the DNA. We also determined whether these procedures interfere with PCR amplification in order to develop a protocol for M. brauna. We found that the quality and integrity of DNA were improved with the use of proteinase K in the extraction buffer and by modifications in the centrifugation speed. The lowest concentration of DNA was obtained with Doyle and Doyle's protocol (5.42 ng/μL). Ferreira and Grattapaglia's protocol modified for M. brauna provided the most DNA (36.89 ng/μL) and the highest quality DNA (purity ratio of 1.80 nm). The original Ferreira and Grattapaglia protocol provided 13.42 ng/μL DNA; however, the purity ratio (1.44 nm) indicates protein contamination. PCR results showed that Ferreira and Grattapaglia's protocol modified for M. brauna gave satisfactory quantity and purity of DNA for molecular studies. PMID:22653632

  15. An evaluation of the effect of microwave irradiation on bone decalcification aimed to DNA extraction.

    PubMed

    Imaizumi, Kazuhiko; Taniguchi, Kei; Ogawa, Yoshinori

    2013-09-01

    An effect of intermittent microwave irradiation on decalcification of compact bone followed by DNA extraction was verified. In order to perform quantitative analysis regarding the degree of decalcification, Cubic bone specimens were prepared from bovine metacarpal bone and micro-focus X-ray CT imaging was applied to measure precise volume of decalcified area in the cubes. Microwave irradiation was performed under strict control of temperature using commercially available experimental device which is designed for advancing tissue fixation, decalcification, and antigen-antibody reaction by intermittent microwave. The integrity of the DNA obtained from irradiated specimen was also examined by PCR analysis. The results of morphological analysis with CT imaging showed that microwave irradiation has a positive effect on decalcification though that effect is not so drastic. The results obtained from PCR analysis showed that microwave irradiation decrease amplifiable DNA, suggesting that we should be careful to use microwave for the purpose of bone DNA extraction. PMID:23838266

  16. GC Preps: Fast and Easy Extraction of Stable Yeast Genomic DNA

    PubMed Central

    Blount, Benjamin A.; Driessen, Maureen R. M.; Ellis, Tom

    2016-01-01

    Existing yeast genomic DNA extraction methods are not ideally suited to extensive screening of colonies by PCR, due to being too lengthy, too laborious or yielding poor quality DNA and inconsistent results. We developed the GC prep method as a solution to this problem. Yeast cells from colonies or liquid cultures are lysed by vortex mixing with glass beads and then boiled in the presence of a metal chelating resin. In around 12 minutes, multiple samples can be processed to extract high yields of genomic DNA. These preparations perform as effectively in PCR screening as DNA purified by organic solvent methods, are stable for up to 1 year at room temperature and can be used as the template for PCR amplification of fragments of at least 8 kb. PMID:27240644

  17. GC Preps: Fast and Easy Extraction of Stable Yeast Genomic DNA.

    PubMed

    Blount, Benjamin A; Driessen, Maureen R M; Ellis, Tom

    2016-01-01

    Existing yeast genomic DNA extraction methods are not ideally suited to extensive screening of colonies by PCR, due to being too lengthy, too laborious or yielding poor quality DNA and inconsistent results. We developed the GC prep method as a solution to this problem. Yeast cells from colonies or liquid cultures are lysed by vortex mixing with glass beads and then boiled in the presence of a metal chelating resin. In around 12 minutes, multiple samples can be processed to extract high yields of genomic DNA. These preparations perform as effectively in PCR screening as DNA purified by organic solvent methods, are stable for up to 1 year at room temperature and can be used as the template for PCR amplification of fragments of at least 8 kb. PMID:27240644

  18. Single-step extraction followed by LC for determination of (fluoro)quinolone drug residues in muscle, eggs, and milk.

    PubMed

    Cho, Hee-Jung; Yi, Hee; Cho, Soo Min; Lee, Dong Goo; Cho, Kyul; Abd el-Aty, A M; Shim, Jae-Han; Lee, Soon-Ho; Jeong, Ji-Yoon; Shin, Ho-Chul

    2010-04-01

    In this study, a simplified method for the extraction and determination of seven fluoroquinolone residues (danofloxacin, difloxacin, enrofloxacin, marbofloxacin, orbifloxacin, ofloxacin, and sarafloxacin) and three quinolones (oxolinic acid, flumequine, and nalidixic acid), in porcine muscle, table eggs, and commercial whole milk, which required no cleanup step, was devised. This procedure involves the extraction of analytes from the samples via liquid-phase extraction, and the subsequent quantitative determination was accomplished via LC-fluorescence detection. Analyte separation was successfully conducted on an XBridge-C(18) column, with a linear gradient mobile phase composed of acetonitrile and 0.01 M oxalic acid buffer at pH=3.5. The one-step liquid-liquid extraction method evidenced good selectivity, precision (RSDs=0.26-15.07%), and recovery of the extractable analytes, ranging from 61.12 to 115.93% in matrices. The LOQs ranged from 0.3 to 25 microg/kg. A survey of ten samples purchased from local markets was conducted, and none of the samples harbored fluoroquinolone residues. This method is an improvement over existing methodologies, since no additional cleanup was necessary. PMID:20175091

  19. Comparison of five protocols to extract DNA from paraffin-embedded tissues for the detection of human papillomavirus.

    PubMed

    Alvarez-Aldana, Adalucy; Martínez, José William; Sepúlveda-Arias, Juan C

    2015-02-01

    Formalin-fixed paraffin-embedded (FFPE) tissues are a valuable source of DNA with which to perform large retrospective studies on the epidemiology of HPV infection. Five different DNA extraction protocols were carried out to evaluate the DNA obtained from FFPE samples with polymerase chain reaction (PCR) using two primer sets to amplify a constitutive human gene, β-globin, and two primer sets to detect the L1 and E6 HPV genes. From the five DNA extraction protocols evaluated, the best results were obtained with protocol A, corresponding to a crude extract from the sample. With the procedures described herein, we were able to amplify DNA extracted from archival paraffin blocks stored for six years. However, the amplification products were more efficiently obtained with primers that amplified shorter fragments. This result indicates that a major factor limiting the extraction process in these samples is DNA fragmentation, a factor that will naturally vary between the different specimens evaluated. Also, depending upon the extraction method, PCR amplification of a human gene does not necessarily guarantee the successful extraction of viral DNA. In conclusion, different DNA and HPV detection methods can significantly influence the results. Therefore, the DNA extraction methods and primers used for DNA amplification in fixed tissues need to be chosen carefully, depending on the specific requirements of the study being carried out. PMID:25444238

  20. Effect of nucleic acid binding dyes on DNA extraction, amplification, and STR typing.

    PubMed

    Haines, Alicia M; Tobe, Shanan S; Kobus, Hilton J; Linacre, Adrian

    2015-10-01

    We report on the effects of six dyes used in the detection of DNA on the process of DNA extraction, amplification, and detection of STR loci. While dyes can be used to detect the presence of DNA, their use is restricted if they adversely affect subsequent DNA typing processes. Diamond™ Nucleic Acid Dye, GelGreen™, GelRed™, RedSafe™, SYBR(®) Green I, and EvaGreen™ were evaluated in this study. The percentage of dye removed during the extraction process was determined to be: 70.3% for SYBR(®) Green I; 99.6% for RedSafe™; 99.4% for EvaGreen™; 52.7% for Diamond™ Dye; 50.6% for GelRed™, and; could not be determined for GelGreen™. It was then assumed that the amount of dye in the fluorescent quantification assay had no effect on the DNA signal. The presence of all six dyes was then reviewed for their effect on DNA extraction. The t-test showed no significant difference between the dyes and the control. These extracts were then STR profiled and all dyes and control produced full DNA profiles. STR loci in the presence of GelGreen(TM) at 1X concentration showed increased amplification products in comparison to the control samples. Full STR profiles were detected in the presence of EvaGreen™ (1X), although with reduced amplification products. RedSafe™ (1X), Diamond™ Dye (1X), and SYBR(®) Green I (1X) all exhibited varying degrees of locus drop-out with GelRed™ generating no loci at all. We provide recommendations for the best dye to visualize the presence of DNA profile as a biological stain and its subsequent amplification and detection. PMID:26202628

  1. A hyper-step DNA computing system based on surface plasmon resonance

    NASA Astrophysics Data System (ADS)

    Chang, Tsung-Yao; Lin, Che-Hsin; Yang, Chia-Ning; Lin, Chii-Wann

    2007-01-01

    We reported a reusable DNA computing platform for solving satisfiability (SAT) problem based on surface plamon resonance (SPR) technology in this paper. Three different sequences of 18-mer ssDNAs with thiol terminal were first immobilized on the gold surface and then hybridized with their complementary sequences at specific sites via microfluidic channels under room temperature. We also conjugated monoclonal antibody (human IgG) to these complementary pairs chemically to amplify the hybridization signal and thus enhance the noise margin to distinguish Boolean value of true and false. In order to keep the reaction temperature and SPR measurement stable, repeated DNA annealing and denaturing is doned by varying salt concentration (by adding NaOH to denature DNA) of reaction solution rather than changing reaction temperature. The experimental results successfully demonstrated a multi-channel microfluidic DNA computation system to solve a three variables (X, Y, Z) Boolean SAT problem (formula) with reusability and specificity using protein-ssDNA conjugates to link to complementary ssDNA SAM surface under room temperature within one hour. This technique provide a feasible solution to miniaturize the DNA computation platform for possible iterated hyperstep computing processes.

  2. DNA extraction from bristles and quills of Chaetomys subspinosus (Rodentia: Erethizontidae) using a novel protocol.

    PubMed

    Oliveira, C G; Martinez, R A; Gaiotto, F A

    2007-01-01

    DNA extraction protocols are as varied as DNA sources. When it comes to endangered species, it is especially important to pay attention to all details that ensure the completion of the study goals and effectiveness in attaining useful data for conservation. Chaetomys subspinosus (Rodentia: Erethizontidae) is a secretive arboreal porcupine endemic to certain ecosystems of the Brazilian Atlantic Forest. A multidisciplinary study (including genetic data) was performed to create a management plan for the conservation of this species. Individuals from natural populations of the states of Bahia, Espírito Santo and Sergipe were sampled. To obtain a reliable and abundant amount of starting material, non-destructive methods were tested, extracting DNA from the bristles and quills that comprise most of this animal's hide. This method has also been innovative in adapting a DNA extraction protocol traditionally used for plants. Digestion using proteinase K was followed by protein precipitation with CTAB, a chloroform-isoamyl alcohol cleaning and DNA precipitation with isopropyl alcohol. This protocol supplies good-quality DNA for genetic analysis with molecular markers based on PCR. PMID:18050086

  3. Method for RNA extraction and cDNA library construction from microbes in crop rhizosphere soil.

    PubMed

    Fang, Changxun; Xu, Tiecheng; Ye, Changliang; Huang, Likun; Wang, Qingshui; Lin, Wenxiong

    2014-02-01

    Techniques to analyze the transcriptome of the soil rhizosphere are essential to reveal the interactions and communications between plants and microorganisms in the soil ecosystem. In this study, different volumes of Al₂(SO₄)₃ were added to rhizosphere soil samples to precipitate humic substances, which interfere with most procedures of RNA and DNA analyses. After humic substances were precipitated, cells of soil microorganisms were broken by vortexing with glass beads, and then DNA and RNA were recovered using Tris-HCl buffer with LiCl, SDS, and EDTA. The crude extract was precipitated and dissolved in RNAse-free water, and then separated by agarose gel electrophoresis. We determined the optimum volume of Al₂(SO₄)₃ for treating rhizosphere soil of rice, tobacco, sugarcane, Rehmannia glutinosa, and Pseudostellaria heterophylla. The crude nucleic acids extract from rice soil was treated with DNase I and then RNA was purified using a gel filtration column. The purified RNA was reverse-transcribed into single-strand cDNA and then ligated with an adaptor at each end before amplifying ds cDNA. The ds cDNA was sub-cloned for subsequent gene sequence analysis. We conducted qPCR to amplify 16S ribosomal DNA and observed highly efficient amplification. These results show that the extraction method can be optimized to isolate and obtain high-quality nucleic acids from microbes in different rhizosphere soils, suitable for genomic and post-genomic analyses. PMID:24078111

  4. An Improved Method for High Quality Metagenomics DNA Extraction from Human and Environmental Samples

    PubMed Central

    Bag, Satyabrata; Saha, Bipasa; Mehta, Ojasvi; Anbumani, D.; Kumar, Naveen; Dayal, Mayanka; Pant, Archana; Kumar, Pawan; Saxena, Shruti; Allin, Kristine H.; Hansen, Torben; Arumugam, Manimozhiyan; Vestergaard, Henrik; Pedersen, Oluf; Pereira, Verima; Abraham, Philip; Tripathi, Reva; Wadhwa, Nitya; Bhatnagar, Shinjini; Prakash, Visvanathan Gnana; Radha, Venkatesan; Anjana, R. M.; Mohan, V.; Takeda, Kiyoshi; Kurakawa, Takashi; Nair, G. Balakrish; Das, Bhabatosh

    2016-01-01

    To explore the natural microbial community of any ecosystems by high-resolution molecular approaches including next generation sequencing, it is extremely important to develop a sensitive and reproducible DNA extraction method that facilitate isolation of microbial DNA of sufficient purity and quantity from culturable and uncultured microbial species living in that environment. Proper lysis of heterogeneous community microbial cells without damaging their genomes is a major challenge. In this study, we have developed an improved method for extraction of community DNA from different environmental and human origin samples. We introduced a combination of physical, chemical and mechanical lysis methods for proper lysis of microbial inhabitants. The community microbial DNA was precipitated by using salt and organic solvent. Both the quality and quantity of isolated DNA was compared with the existing methodologies and the supremacy of our method was confirmed. Maximum recovery of genomic DNA in the absence of substantial amount of impurities made the method convenient for nucleic acid extraction. The nucleic acids obtained using this method are suitable for different downstream applications. This improved method has been named as the THSTI method to depict the Institute where the method was developed. PMID:27240745

  5. Chaga mushroom extract inhibits oxidative DNA damage in human lymphocytes as assessed by comet assay.

    PubMed

    Park, Yoo Kyoung; Lee, Hyang Burm; Jeon, Eun-Jae; Jung, Hack Sung; Kang, Myung-Hee

    2004-01-01

    The Chaga mushroom (Inonotus obliquus) is claimed to have beneficial properties for human health, such as anti-bacterial, anti-allergic, anti-inflammatory and antioxidant activities. The antioxidant effects of the mushroom may be partly explained by protection of cell components against free radicals. We evaluated the effect of aqueous Chaga mushroom extracts for their potential for protecting against oxidative damage to DNA in human lymphocytes. Cells were pretreated with various concentrations (10, 50, 100 and 500 microg/mL) of the extract for 1 h at 37 degrees C. Cells were then treated with 100 microM of H2O2 for 5 min as an oxidative stress. Evaluation of oxidative damage was performed using single-cell gel electrophoresis for DNA fragmentation (Comet assay). Using image analysis, the degree of DNA damage was evaluated as the DNA tail moment. Cells pretreated with Chaga extract showed over 40% reduction in DNA fragmentation compared with the positive control (100 micromol H2O2 treatment). Thus, Chaga mushroom treatment affords cellular protection against endogenous DNA damage produced by H2O2. PMID:15630179

  6. An Improved Method for High Quality Metagenomics DNA Extraction from Human and Environmental Samples.

    PubMed

    Bag, Satyabrata; Saha, Bipasa; Mehta, Ojasvi; Anbumani, D; Kumar, Naveen; Dayal, Mayanka; Pant, Archana; Kumar, Pawan; Saxena, Shruti; Allin, Kristine H; Hansen, Torben; Arumugam, Manimozhiyan; Vestergaard, Henrik; Pedersen, Oluf; Pereira, Verima; Abraham, Philip; Tripathi, Reva; Wadhwa, Nitya; Bhatnagar, Shinjini; Prakash, Visvanathan Gnana; Radha, Venkatesan; Anjana, R M; Mohan, V; Takeda, Kiyoshi; Kurakawa, Takashi; Nair, G Balakrish; Das, Bhabatosh

    2016-01-01

    To explore the natural microbial community of any ecosystems by high-resolution molecular approaches including next generation sequencing, it is extremely important to develop a sensitive and reproducible DNA extraction method that facilitate isolation of microbial DNA of sufficient purity and quantity from culturable and uncultured microbial species living in that environment. Proper lysis of heterogeneous community microbial cells without damaging their genomes is a major challenge. In this study, we have developed an improved method for extraction of community DNA from different environmental and human origin samples. We introduced a combination of physical, chemical and mechanical lysis methods for proper lysis of microbial inhabitants. The community microbial DNA was precipitated by using salt and organic solvent. Both the quality and quantity of isolated DNA was compared with the existing methodologies and the supremacy of our method was confirmed. Maximum recovery of genomic DNA in the absence of substantial amount of impurities made the method convenient for nucleic acid extraction. The nucleic acids obtained using this method are suitable for different downstream applications. This improved method has been named as the THSTI method to depict the Institute where the method was developed. PMID:27240745

  7. A comparison of methods for total community DNA preservation and extraction from various thermal environments.

    PubMed

    Mitchell, Kendra R; Takacs-Vesbach, Cristina D

    2008-10-01

    The widespread use of molecular techniques in studying microbial communities has greatly enhanced our understanding of microbial diversity and function in the natural environment and contributed to an explosion of novel commercially viable enzymes. One of the most promising environments for detecting novel processes, enzymes, and microbial diversity is hot springs. We examined potential biases introduced by DNA preservation and extraction methods by comparing the quality, quantity, and diversity of environmental DNA samples preserved and extracted by commonly used methods. We included samples from sites representing the spectrum of environmental conditions that are found in Yellowstone National Park thermal features. Samples preserved in a non-toxic sucrose lysis buffer (SLB), along with a variation of a standard DNA extraction method using CTAB resulted in higher quality and quantity DNA than the other preservation and extraction methods tested here. Richness determined using DGGE revealed that there was some variation within replicates of a sample, but no statistical difference among the methods. However, the sucrose lysis buffer preserved samples extracted by the CTAB method were 15-43% more diverse than the other treatments. PMID:18633656

  8. Multiple DNA Extractions Coupled with Stable-Isotope Probing of Anthracene-Degrading Bacteria in Contaminated Soil▿†

    PubMed Central

    Jones, Maiysha D.; Singleton, David R.; Sun, Wei; Aitken, Michael D.

    2011-01-01

    In many of the DNA-based stable-isotope probing (SIP) studies published to date in which soil communities were investigated, a single DNA extraction was performed on the soil sample, usually using a commercial DNA extraction kit, prior to recovering the 13C-labeled (heavy) DNA by density-gradient ultracentrifugation. Recent evidence suggests, however, that a single extraction of a soil sample may not lead to representative recovery of DNA from all of the organisms in the sample. To determine whether multiple DNA extractions would affect the DNA yield, the eubacterial 16S rRNA gene copy number, or the identification of anthracene-degrading bacteria, we performed seven successive DNA extractions on the same aliquot of contaminated soil either untreated or enriched with [U-13C]anthracene. Multiple extractions were necessary to maximize the DNA yield and 16S rRNA gene copy number from both untreated and anthracene-enriched soil samples. Sequences within the order Sphingomonadales, but unrelated to any previously described genus, dominated the 16S rRNA gene clone libraries derived from 13C-enriched DNA and were designated “anthracene group 1.” Sequences clustering with Variovorax spp., which were also highly represented, and sequences related to the genus Pigmentiphaga were newly associated with anthracene degradation. The bacterial groups collectively identified across all seven extracts were all recovered in the first extract, although quantitative PCR analysis of SIP-identified groups revealed quantitative differences in extraction patterns. These results suggest that performing multiple DNA extractions on soil samples improves the extractable DNA yield and the number of quantifiable eubacterial 16S rRNA gene copies but have little qualitative effect on the identification of the bacterial groups associated with the degradation of a given carbon source by SIP. PMID:21398486

  9. Unraveling the sequence-dependent polymorphic behavior of d(CpG) steps in B-DNA

    PubMed Central

    Dans, Pablo Daniel; Faustino, Ignacio; Battistini, Federica; Zakrzewska, Krystyna; Lavery, Richard; Orozco, Modesto

    2014-01-01

    We have made a detailed study of one of the most surprising sources of polymorphism in B-DNA: the high twist/low twist (HT/LT) conformational change in the d(CpG) base pair step. Using extensive computations, complemented with database analysis, we were able to characterize the twist polymorphism in the d(CpG) step in all the possible tetranucleotide environment. We found that twist polymorphism is coupled with BI/BII transitions, and, quite surprisingly, with slide polymorphism in the neighboring step. Unexpectedly, the penetration of cations into the minor groove of the d(CpG) step seems to be the key element in promoting twist transitions. The tetranucleotide environment also plays an important role in the sequence-dependent d(CpG) polymorphism. In this connection, we have detected a previously unexplored intramolecular C-H···O hydrogen bond interaction that stabilizes the low twist state when 3′-purines flank the d(CpG) step. This work explains a coupled mechanism involving several apparently uncorrelated conformational transitions that has only been partially inferred by earlier experimental or theoretical studies. Our results provide a complete description of twist polymorphism in d(CpG) steps and a detailed picture of the molecular choreography associated with this conformational change. PMID:25223784

  10. The Effect of a Grape Seed Extract on Radiation-Induced DNA Damage in Human Lymphocytes

    NASA Astrophysics Data System (ADS)

    Dicu, Tiberius; Postescu, Ion D.; Foriş, Vasile; Brie, Ioana; Fischer-Fodor, Eva; Cernea, Valentin; Moldovan, Mircea; Cosma, Constantin

    2009-05-01

    Plant-derived antioxidants due to their phenolic compounds content are reported as potential candidates for reducing the levels of oxidative stress in living organisms. Grape seed extracts are very potent antioxidants and exhibit numerous interesting pharmacologic activities. Hydroethanolic (50/50, v/v) standardized extract was obtained from red grape seed (Vitis vinifera, variety Burgund Mare—BM). The total polyphenols content was evaluated by Folin-Ciocalteu procedure and expressed as μEq Gallic Acid/ml. The aim of this study was to evaluate the potential antioxidant effects of different concentrations of BM extract against 60Co γ-rays induced DNA damage in human lymphocytes. Samples of human lymphocytes were incubated with BM extract (12.5, 25.0 and 37.5 μEq GA/ml, respectively) administered at 30 minutes before in vitro irradiation with γ-rays (2 Gy). The DNA damage and repair in lymphocytes were evaluated using alkaline comet assay. Using the lesion score, the radiation-induced DNA damage was found to be significantly different (p<0.05) from control, both in the absence and presence of BM extract (except the lymphocytes treated with 37.5 μEq GA/ml BM extract). DNA repair analyzed by incubating the irradiated cells at 37° C and 5% CO2 atmosphere for 2 h, indicated a significant difference (p<0.05) in the lymphocytes group treated with 25.0 μEq GA/ml BM extract, immediately and two hours after irradiation. These results suggest radioprotective effects after treatment with BM extract in human lymphocytes.

  11. Comparative Evaluation of DNA Extraction Methods from Feces of Multiple Host Species for Downstream Next-Generation Sequencing

    PubMed Central

    Hart, Marcia L.; Meyer, Alexandra; Johnson, Philip J.; Ericsson, Aaron C.

    2015-01-01

    The gastrointestinal tract contains a vast community of microbes that to this day remain largely unculturable, making studies in this area challenging. With the newly affordable advanced sequencing technology, important breakthroughs in this exciting field are now possible. However, standardized methods of sample collection, handling, and DNA extraction have yet to be determined. To help address this, we investigated the use of 5 common DNA extraction methods on fecal samples from 5 different species. Our data show that the method of DNA extraction impacts DNA concentration and purity, successful NGS amplification, and influences microbial communities seen in NGS output dependent on the species of fecal sample and the DNA extraction method used. These data highlight the importance of careful consideration of DNA extraction method used when designing and interpreting data from cross species studies. PMID:26599606

  12. Probabilistically determining the cellular source of DNA derived from differential extractions in sexual assault scenarios.

    PubMed

    Taylor, Duncan

    2016-09-01

    Sexual assault cases are the type of case that often produces questions about the cellular source of DNA. In these cases multiple findings of microscopy, DNA profiling and presumptive testing need to be considered when addressing source level propositions. In this work, I consider a line of questioning that has been raised a number of times in the recent past, where in court it was disputed that low levels of sperm seen on a microscope slide were the cellular source of the male DNA profile component generated from the sperm fraction of a differential DNA extraction. I demonstrate how the cell scoring results and DNA profiling results can be considered together, in helping address this source level question through the use of Bayesian Networks. PMID:27388428

  13. Standardization of DNA extraction from methanol acetic acid fixed cytogenetic cells of cattle and buffalo.

    PubMed

    Kotikalapudi, Rosaiah; Patel, Rajesh K; Katragadda, Sanghamitra

    2013-12-01

    The aim of the study is to standardize the simple method for extracting DNA from cells fixed in fixative (3:1 ratio of methanol and acetic acid glacial) mostly used for chromosomal studies in cattle and buffaloes. These fixed cells were stored for more than 6 months at refrigerated temperature. The fixed cells were washed 2-3 times by the ice cold 1x Phosphate Buffer Saline (PBS) with pH 7.4, so that effect of fixative may be eliminated. The genomic DNA was extracted by adding cell lysis and nucleus lysis buffers. The quality and quantity of DNA were estimated. The readings of nano drop and agarose gel electrophoresis indicate good quality DNA isolated with a rapid and simple protocol routinely using in our laboratory. The method enables us to study the DNA of a cattle and buffaloes after completing cytogenetic investigation or in cases where DNA samples are otherwise not available. This protocol may be useful for molecular analysis of DNA from fixed cells palettes. PMID:24506057

  14. Extraction and fractionation of RNA and DNA from single cells using selective lysing and isotachophoresis

    NASA Astrophysics Data System (ADS)

    Shintaku, Hirofumi; Santiago, Juan G.

    2015-03-01

    Single cell analyses of RNA and DNA are crucial to understanding the heterogeneity of cell populations. The numbers of approaches to single cells analyses are expanding, but sequence specific measurements of nucleic acids have been mostly limited to studies of either DNA or RNA, and not both. This remains a challenge as RNA and DNA have very similar physical and biochemical properties, and cross-contamination with each other can introduce false positive results. We present an electrokinetic technique which creates the opportunity to fractionate and deliver cytoplasmic RNA and genomic DNA to independent downstream analyses. Our technique uses an on-chip system that enables selective lysing of cytoplasmic membrane, extraction of RNA (away from genomic DNA and nucleus), focusing, absolute quantification of cytoplasmic RNA mass. The absolute RNA mass quantification is performed using fluorescence observation without enzymatic amplification in < 5 min. The cell nucleus is left intact and the relative genomic DNA amount in the nucleus can be measured. We demonstrate the technique using single mouse B lymphocyte cells, for which we extracted an average of 14.1 pg total cytoplasmic RNA per cell. We also demonstrate correlation analysis between the absolute amount of cytoplasmic RNA and relative amount of genomic DNA, showing heterogeneity associated with cell cycle.

  15. DNA repair enhancement of aqueous extracts of Uncaria tomentosa in a human volunteer study.

    PubMed

    Sheng, Y; Li, L; Holmgren, K; Pero, R W

    2001-07-01

    The Uncaria tomentosa water extracts (C-Med-100) have been shown to enhance DNA repair, mitogenic response and leukocyte recovery after chemotherapy-induced DNA damage in vivo. In this study, the effect of C-Med-100 supplement was evaluated in a human volunteer study. Twelve apparently healthy adults working in the same environment were randomly assigned into 3 groups with age and gender matched. One group was daily supplemented with a 250 mg tablet containing an aqueous extract of Uncaria tomentosa of C-Med-100, and another group with a 350 mg tablet, for 8 consecutive weeks. DNA repair after induction of DNA damage by a standard dose of hydrogen peroxide was measured 3 times before supplement and 3 times after the supplement for the last 3 weeks of the 8 week-supplement period. There were no drug-related toxic responses to C-Med-100 supplement when judged in terms of clinical symptoms, serum clinical chemistry, whole blood analysis and leukocyte differential counts. There was a statistically significant decrease of DNA damage and a concomitant increase of DNA repair in the supplement groups (250 and 350 mg/day) when compared with non-supplemented controls (p < 0.05). There was also an increased tendency of PHA induced lymphocyte proliferation in the treatment groups. Taken together, this trial has confirmed the earlier results obtained in the rat model when estimating DNA repair enhancement by C-Med-100. PMID:11515717

  16. Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis.

    PubMed

    Huang, Shichu; Do, Jaephil; Mahalanabis, Madhumita; Fan, Andy; Zhao, Lei; Jepeal, Lisa; Singh, Satish K; Klapperich, Catherine M

    2013-01-01

    In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile) DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA) to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65°C±2°C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.25×10(-2) pg of C. difficile DNA) while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health. PMID:23555883

  17. Imaging and spectroscopic comparison of multi-step methods to form DNA arrays based on the biotin-streptavidin system.

    PubMed

    Gajos, Katarzyna; Petrou, Panagiota; Budkowski, Andrzej; Awsiuk, Kamil; Bernasik, Andrzej; Misiakos, Konstantinos; Rysz, Jakub; Raptis, Ioannis; Kakabakos, Sotirios

    2015-02-21

    Three multi-step multi-molecular approaches using the biotin-streptavidin system to contact-print DNA arrays on SiO2 surfaces modified with (3-glycidoxypropyl)trimethoxysilane are examined after each deposition/reaction step by atomic force microscopy, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry. Surface modification involves the spotting of preformed conjugates of biotinylated oligonucleotides with streptavidin onto surfaces coated with biotinylated bovine serum albumin b-BSA (approach I) or the spotting of biotinylated oligonucleotides onto a streptavidin coating, the latter prepared through a reaction with immobilized b-BSA (approach II) or direct adsorption (approach III). AFM micrographs, quantified by autocorrelation and height histogram parameters (e.g. roughness), reveal uniform coverage after each modification step with distinct nanostructures after the reaction of biotinylated BSA with streptavidin or of a streptavidin conjugate with biotinylated oligonucleotides. XPS relates the immobilization of biomolecules with covalent binding to the epoxy-silanized surface. Protein coverage, estimated from photoelectron attenuation, shows that regarding streptavidin the highest and the lowest immobilization efficiency is achieved by following approaches I and III, respectively, as confirmed by TOF-SIMS microanalysis. The size of the DNA spot reflects the contact radius of the printed droplet and increases with protein coverage (and roughness) prior to the spotting, as epoxy-silanized surfaces are hardly hydrophilic. Representative TOF-SIMS images show sub-millimeter spots: uniform for approach I, doughnut-like (with a small non-zero minimum) for approach II, both with coffee-rings or peak-shaped for approach III. Spot features, originating from pinned contact lines and DNA surface binding and revealed by complementary molecular distributions (all material, DNA, streptavidin, BSA, epoxy, SiO2), indicate two modes of droplet

  18. Efficient extraction of canthaxanthin from Escherichia coli by a 2-step process with organic solvents.

    PubMed

    Scaife, Mark A; Ma, Cynthia A; Armenta, Roberto E

    2012-05-01

    Canthaxanthin has a substantial commercial market in aquaculture, poultry production, and cosmetic and nutraceutical industries. Commercial production is dominated by chemical synthesis; however, changing consumer demands fuel research into the development of biotechnology processes. Highly productive microbial systems to produce carotenoids can be limited by the efficiency of extraction methods. Extraction with hexane, acetone, methanol, 2-propanol, ethanol, 1-butanol, tetrahydrofuran and ethyl acetate was carried out with each solvent separately, and subsequently the most efficient solvents were tested in combination, both as mixtures and sequentially. Sequential application of methanol followed by acetone proved most efficient. Extraction efficiency remained stable over a solvent to biomass range of 100:1 to 55:1, but declined significantly at a ratio of 25:1. Application of this method to a canthaxanthin-producing Escherichia coli production system enabled efficient canthaxanthin extraction of up to 8.5 mg g(-1) dry biomass. PMID:22353211

  19. A robust two-step PCR method of template DNA production for high-throughput cell-free protein synthesis.

    PubMed

    Yabuki, Takashi; Motoda, Yoko; Hanada, Kazuharu; Nunokawa, Emi; Saito, Miyuki; Seki, Eiko; Inoue, Makoto; Kigawa, Takanori; Yokoyama, Shigeyuki

    2007-12-01

    A two-step PCR method has been developed for the robust, high-throughput production of linear templates ready for cell-free protein synthesis. The construct made from the cDNA expresses a target protein region with N- and/or C-terminal tags. The procedure consists only of mixing, dilution, and PCR steps, and is free from cloning and purification steps. In the first step of the two-step PCR, a target region within the coding sequence is amplified using two gene-specific forward and reverse primers, which contain the linker sequences and the terminal sequences of the target region. The second PCR concatenates the first PCR product with the N- and C-terminal double-stranded fragments, which contain the linker sequences as well as the sequences for the tag(s) and the initiation and termination, respectively, for T7 transcription and ribosomal translation, and amplifies it with the universal primer. Proteins can be fused with a variety of tags, such as natural poly-histidine, glutathione-S-transferase, maltose-binding protein, and/or streptavidin-binding peptide. The two-step PCR method was successfully applied to 42 human target protein regions with various GC contents (38-77%). The robustness of the two-step PCR method against possible fluctuations of experimental conditions in practical use was explored. The second PCR product was obtained at 60-120 microg/ml, and was used without purification as a template at a concentration of 2-4 microg/ml in an Escherichia coli coupled transcription-translation system. This combination of two-step PCR with cell-free protein synthesis is suitable for the rapid production of proteins in milligram quantities for genome-scale studies. PMID:18167031

  20. Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR

    NASA Astrophysics Data System (ADS)

    Ansari, Aseem Z.; Chael, Mark L.; O'Halloran, Thomas V.

    1992-01-01

    POSITIVE control of transcription often involves stimulatory protein-protein interactions between regulatory factors and RNA polymerase1. Critical steps in the activation process itself are seldom ascribed to protein-DNA distortions. Activator-induced DNA bending is typically assigned a role in binding-site recognition2, alterations in DNA loop structures3 or optimal positioning of the activator for interaction with polymerase4. Here we present a transcriptional activation mechanism that does not require a signal-induced DNA bend but rather a receptor-induced untwisting of duplex DNA. The allosterically modulated transcription factor MerR is a represser and an Hg(II)-responsive activator of bacterial mercury-resistance genes5-7.Escherichia coliRNA polymerase binds to the MerR-promoter complex but cannot proceed to a transcriptionally active open complex until Hg(II) binds to MerR (ref. 6). Chemical nuclease studies show that the activator form, but not the represser, induces a unique alteration of the helical structure localized at the centre of the DNA-binding site6. Data presented here indicate that this Hg-MerR-induced DNA distortion corresponds to a local underwinding of the spacer region of the promoter by about 33° relative to the MerR-operator complex. The magnitude and the direction of the Hg-MerR-induced change in twist angle are consistent with a positive control mechanism involving reorientation of conserved, but suboptimally phased, promoter elements and are consistent with a role for torsional stress in formation of an open complex.

  1. Evaluation of antioxidant activity and preventing DNA damage effect of pomegranate extracts by chemiluminescence method.

    PubMed

    Guo, Shanshan; Deng, Qianchun; Xiao, Junsong; Xie, Bijun; Sun, Zhida

    2007-04-18

    The antioxidant activities of three parts (peel, juice, and seed) and extracts of three pomegranate varieties in China were investigated by using a chemiluminescence (CL) method in vitro. The scavenging ability of pomegranate extracts (PEs) on superoxide anion, hydroxide radical, and hydrogen peroxide was determined by the pyrogallol-luminol system, the CuSO4-Phen-Vc-H2O2 system, and the luminol-H2O2 system, respectively. DNA damage preventing the effect of PE was determined by the CuSO4-Phen-Vc-H2O2-DNA CL system. The results showed that the peel extract of red pomegranate had the best effect on the scavenging ability of superoxide anion because its IC50 value (4.01 +/- 0.09 microg/mL) was the lowest in all PEs. The seed extract of white pomegranate could scavenge hydroxide radical most effectively of the nine extracts (the IC50 value was 1.69 +/- 0.03 microg/mL). The peel extract of white pomegranate had the best scavenging ability on hydrogen peroxide, which had the lowest IC50 value (0.032 +/- 0.003 microg/mL) in the nine extracts. The seed extract of white pomegranate (the IC50 value was 3.67 +/- 0.03 microg/mL) was the most powerful on the DNA damage-preventing effect in all of the PEs. Also, the statistical analysis indicated that there were significant differences (at P < 0.05) among the extracts of the different varieties and parts in each system. PMID:17381116

  2. Automated DNA extraction platforms offer solutions to challenges of assessing microbial biofouling in oil production facilities.

    PubMed

    Oldham, Athenia L; Drilling, Heather S; Stamps, Blake W; Stevenson, Bradley S; Duncan, Kathleen E

    2012-01-01

    The analysis of microbial assemblages in industrial, marine, and medical systems can inform decisions regarding quality control or mitigation. Modern molecular approaches to detect, characterize, and quantify microorganisms provide rapid and thorough measures unbiased by the need for cultivation. The requirement of timely extraction of high quality nucleic acids for molecular analysis is faced with specific challenges when used to study the influence of microorganisms on oil production. Production facilities are often ill equipped for nucleic acid extraction techniques, making the preservation and transportation of samples off-site a priority. As a potential solution, the possibility of extracting nucleic acids on-site using automated platforms was tested. The performance of two such platforms, the Fujifilm QuickGene-Mini80™ and Promega Maxwell®16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm™ DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition. Three pipeline biofilm samples were chosen for these comparisons; two contained crude oil and corrosion products and the third transported seawater. Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach. DNA quality was evaluated for amplification by quantitative PCR (qPCR) and end-point PCR to generate 454 pyrosequencing libraries for 16S rRNA microbial community analysis. Microbial community structure, as assessed by DGGE analysis and pyrosequencing, was comparable among the three extraction methods. Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources. PMID:23168231

  3. Automated DNA extraction platforms offer solutions to challenges of assessing microbial biofouling in oil production facilities

    PubMed Central

    2012-01-01

    The analysis of microbial assemblages in industrial, marine, and medical systems can inform decisions regarding quality control or mitigation. Modern molecular approaches to detect, characterize, and quantify microorganisms provide rapid and thorough measures unbiased by the need for cultivation. The requirement of timely extraction of high quality nucleic acids for molecular analysis is faced with specific challenges when used to study the influence of microorganisms on oil production. Production facilities are often ill equipped for nucleic acid extraction techniques, making the preservation and transportation of samples off-site a priority. As a potential solution, the possibility of extracting nucleic acids on-site using automated platforms was tested. The performance of two such platforms, the Fujifilm QuickGene-Mini80™ and Promega Maxwell®16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm™ DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition. Three pipeline biofilm samples were chosen for these comparisons; two contained crude oil and corrosion products and the third transported seawater. Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach. DNA quality was evaluated for amplification by quantitative PCR (qPCR) and end-point PCR to generate 454 pyrosequencing libraries for 16S rRNA microbial community analysis. Microbial community structure, as assessed by DGGE analysis and pyrosequencing, was comparable among the three extraction methods. Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources. PMID:23168231

  4. A comparison of different pre-lysis methods and extraction kits for recovery of Streptococcus agalacticae (Lancefield group B Streptococcus) DNA from whole blood.

    PubMed

    Burke, Rachael M; McKenna, James P; Cox, Ciara; Coyle, Peter V; Shields, Michael D; Fairley, Derek J

    2016-10-01

    Sub-optimal recovery of bacterial DNA from whole blood samples can limit the sensitivity of molecular assays to detect pathogenic bacteria. We compared 3 different pre-lysis protocols (none, mechanical pre-lysis and achromopeptidase pre-lysis) and 5 commercially available DNA extraction platforms for direct detection of Group B Streptococcus (GBS) in spiked whole blood samples, without enrichment culture. DNA was extracted using the QIAamp Blood Mini kit (Qiagen), UCP Pathogen Mini kit (Qiagen), QuickGene DNA Whole Blood kit S (Fuji), Speed Xtract Nucleic Acid Kit 200 (Qiagen) and MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche Diagnostics Corp). Mechanical pre-lysis increased yields of bacterial genomic DNA by 51.3 fold (95% confidence interval; 31.6-85.1, p<0.001) and pre-lysis with achromopeptidase by 6.1 fold (95% CI; 4.2-8.9, p<0.001), compared with no pre-lysis. Differences in yield due to pre-lysis were 2-3 fold larger than differences in yield between extraction methods. Including a pre-lysis step can improve the limits of detection of GBS using PCR or other molecular methods without need for culture. PMID:27546716

  5. DNA Barcoding Marine Biodiversity: Steps from Mere Cataloguing to Giving Reasons for Biological Differences.

    PubMed

    Nikinmaa, Mikko; Götting, Miriam

    2016-01-01

    DNA barcoding has become a useful tool in many contexts and has opened up a completely new avenue for taxonomy. DNA barcoding has its widest application in biodiversity and ecological research to detect and describe diversity whenever morphological discrimination is difficult or impossible (e.g., in the case of species lacking diagnostic characters, early life stages, or cryptic species). In this chapter, we outline the utility of including physiological parameters as part of species description in publicly available databases that catalog taxonomic information resulting from barcoding projects. Cryptic species or different life stages of a species often differ in their physiological traits. Thus, if physiological aspects were included in species definitions, the presently cryptic species could be distinguished. We furthermore give suggestions for physiological information that should be included in a species description and describe potential applications of DNA barcoding for research with physiological components. PMID:27460377

  6. Comparison of DNA extraction protocols for the analysis of gut microbiota in fishes.

    PubMed

    Larsen, Andrea M; Mohammed, Haitham H; Arias, Covadonga R

    2015-03-01

    This study investigated the impacts of bacterial DNA extraction methodology on downstream analysis of fish gut microbiota. Feces and intestine samples were taken from three sympatric freshwater fish species with varying diets. Samples were processed immediately (approximately 4 h after capture; fresh), stored at -20 °C for 15 days or preserved in RNAlater® reagent for 15 days. DNA was then extracted using two commercial kits: one designed for animal tissues and one specifically formulated for stool samples. Microbial community fingerprints were generated using ribosomal intergenic spacer analysis. Factors including diversity as depicted by band number, band intensity, repeatability and practicalities such as cost and time were considered. Despite significant differences in microbiota structure, results were similar between feces and intestine samples. Frozen samples were consistently outperformed by other storage methods and the stool kit typically outperformed the tissue kit. Overall, we recommend extraction of bacterial DNA from fresh samples using the stool kit for both sample types. If samples cannot be processed immediately, preservation in RNAlater® is preferred to freezing. Choice of DNA extraction method significantly influences the results of downstream microbial community analysis and thus should be taken into consideration for metadata analysis. PMID:25757730

  7. DNA adducts induced by in vitro activation of diesel and biodiesel exhaust extracts

    EPA Science Inventory

    The abstract reports the results of studies assessing the relative DNA damage potential of extracts of exhaust particles resulting from the combustion of petroleum diesel, biodiesel, and petroleum diesel-biodiesel blends. Results indicate that the commercially available B20 petr...

  8. Large scale sex typing of ostriches using DNA extracted from feathers

    PubMed Central

    Malagó, Wilson; Franco, Heitor M; Matheucci, Euclides; Medaglia, Adriana; Henrique-Silva, Flavio

    2002-01-01

    Background Ostrich (Struthio camelus) breeds have been gaining increasing significance around the world. The large-scale sex determination of chicks is an important task in the development of these breeds. To date, two PCR-based methods have been established for ostrich sex typing, neither of them intended for large-scale analyses. Here, we report on a protocol adapted to carry out large-scale gender scoring using DNA obtained from chick feathers. Results The DNA was extracted using a fast and simple alkaline extraction protocol that provided sufficient template for an early diagnosis. Tests with several primer sets enabled us to determine the best internal control primers associated with the sex-specific primers, avoiding spurious bands. Using DNA extracted from a single bulb and the best set of primers, we applied this protocol to simultaneously sex-type 96 individuals accurately. Conclusion We have established a fast, safe, accurate and inexpensive procedure for large-scale sex typing of ostriches using DNA extracted from feathers. This procedure is useful for the gender identification of chicks in the first days of nestling life. PMID:12359044

  9. Chaga mushroom extract inhibits oxidative DNA damage in lymphocytes of patients with inflammatory bowel disease.

    PubMed

    Najafzadeh, Mojgan; Reynolds, P Dominic; Baumgartner, Adolf; Jerwood, David; Anderson, Diana

    2007-01-01

    Inflammatory Bowel Disease (IBD) is partly caused by oxidative stress from free radicals and reduced antioxidant levels. Using hydrogen peroxide to induce oxidative stress in vitro in peripheral lymphocytes we investigated the induction of DNA damage supplemented with ethanolic extract of Chaga mushroom as a protective antioxidant. Lymphocytes were obtained from 20 IBD patients and 20 healthy volunteers. For treatment, a constant H_{2}O_{2 } dose (50 microg/ml) was used with variable doses of Chaga extract (10-500 microg/ml). DNA damage was evaluated in 50 cells per individual and dose using the Comet assay (making 1000 observations per experimental point ensuring appropriate statistical power). Chaga supplementation resulted in a 54.9% (p < 0.001) reduction of H_{2}O_{2 } induced DNA damage within the patient group and 34.9% (p < 0.001) within the control group. Lymphocytes from Crohn's disease (CD) patients had a greater basic DNA damage than Ulcerative Colitis (UC) patients (p < 0.001). Conclusively, Chaga extract reduces oxidative stress in lymphocytes from IBD patients and also healthy individuals when challenged in vitro. Thus, Chaga extract could be a possible and valuable supplement to inhibit oxidative stress in general. PMID:18997282

  10. A versatile bacterial expression vector designed for single-step cloning of multiple DNA fragments using homologous recombination.

    PubMed

    Holmberg, Mats A; Gowda, Naveen Kumar Chandappa; Andréasson, Claes

    2014-06-01

    Production of recombinant proteins is the starting point for biochemical and biophysical analyses and requires methodology to efficiently proceed from gene sequence to purified protein. While optimized strategies for the efficient cloning of single-gene fragments for bacterial expression is available, efficient multiple DNA fragment cloning still presents a challenge. To facilitate this step, we have developed an efficient cloning strategy based on yeast homologous recombination cloning (YHRC) into the new pET-based bacterial expression vector pSUMO-YHRC. The vector supports cloning for untagged expression as well as fusions to His6-SUMO or His6 tags. We demonstrate that YHRC from single PCR products of 6 independent genes into the vector results in virtually no background. Importantly, in a quantitative assay for functional expression we find that single-step YHRC of 7 DNA fragments can be performed with very high cloning efficiencies. The method and reagents described in this paper significantly simplifies the construction of expression plasmids from multiple DNA fragments, including complex gene fusions, chimeric genes and polycistronic constructs. PMID:24631626

  11. PicoNewton-Millisecond Force Steps Reveal the Transition Kinetics and Mechanism of the Double-Stranded DNA Elongation

    PubMed Central

    Bianco, Pasquale; Bongini, Lorenzo; Melli, Luca; Dolfi, Mario; Lombardi, Vincenzo

    2011-01-01

    We study the kinetics of the overstretching transition in λ-phage double-stranded (ds) DNA from the basic conformation (B state) to the 1.7-times longer and partially unwound conformation (S state), using the dual-laser optical tweezers under force-clamp conditions at 25°C. The unprecedented resolution of our piezo servo-system, which can impose millisecond force steps of 0.5–2 pN, reveals the exponential character of the elongation kinetics and allows us to test the two-state nature of the B-S transition mechanism. By analyzing the load-dependence of the rate constant of the elongation, we find that the elementary elongation step is 5.85 nm, indicating a cooperativity of ∼25 basepairs. This mechanism increases the free energy for the elementary reaction to ∼94 kBT, accounting for the stability of the basic conformation of DNA, and explains why ds-DNA can remain in equilibrium as it overstretches. PMID:21843477

  12. Cdc7–Drf1 kinase links chromosome cohesion to the initiation of DNA replication in Xenopus egg extracts

    PubMed Central

    Takahashi, Tatsuro S.; Basu, Abhijit; Bermudez, Vladimir; Hurwitz, Jerard; Walter, Johannes C.

    2008-01-01

    To establish functional cohesion between replicated sister chromatids, cohesin is recruited to chromatin before S phase. Cohesin is loaded onto chromosomes in the G1 phase by the Scc2–Scc4 complex, but little is known about how Scc2–Scc4 itself is recruited to chromatin. Using Xenopus egg extracts as a vertebrate model system, we showed previously that the chromatin association of Scc2 and cohesin is dependent on the prior establishment of prereplication complexes (pre-RCs) at origins of replication. Here, we report that Scc2–Scc4 exists in a stable complex with the Cdc7–Drf1 protein kinase (DDK), which is known to bind pre-RCs and activate them for DNA replication. Immunodepletion of DDK from Xenopus egg extracts impairs chromatin association of Scc2–Scc4, a defect that is reversed by wild-type, but not catalytically inactive DDK. A complex of Scc4 and the N terminus of Scc2 is sufficient for chromatin loading of Scc2–Scc4, but not for cohesin recruitment. These results show that DDK is required to tether Scc2–Scc4 to pre-RCs, and they underscore the intimate link between early steps in DNA replication and cohesion. PMID:18628396

  13. Optimized DNA extraction methods for encysted embryos of the endangered fairy shrimp, Branchinecta sandiegonensis

    USGS Publications Warehouse

    Steele, A.N.; Simovich, M.A.; Pepino, D.; Schroeder, K.M.; Vandergast, A.G.; Bohonak, A.J.

    2009-01-01

    The San Diego fairy shrimp Branchinecta sandiegonensis is a federally endangered species endemic to vernal pools in southern California, USA. Filling events in these habitats are highly variable, with some pools failing to hold water long enough for reproduction over many successive years. Studies of this species are thus hindered by the relatively rare appearance of aquatically active life history phases. Because diapausing cysts are abundant and present at all times, they provide an underutilized opportunity for both species identification and genetic studies. However, methods for extracting DNA from cysts are technically challenging because of their structure and size. Here we present a protocol for extracting DNA from B. sandiegonensis cysts in sufficient quantities for "DNA Barcoding", microsatellite analysis and other genotyping and sequencing applications. The technique will aid in population genetic studies and species identification (since taxonomic keys only distinguish among adults), and will be applicable to other crustaceans with similar diapausing cysts. ?? Springer Science+Business Media B.V. 2008.

  14. Determination of ABO genotypes with DNA extracted from formalin-fixed, paraffin-embedded tissues.

    PubMed

    Yamada, M; Yamamoto, Y; Tanegashima, A; Kane, M; Ikehara, Y; Fukunaga, T; Nishi, K

    1994-01-01

    The gene encoding the specific glycosyltransferases which catalyze the conversion of the H antigen to A or B antigens shows a slight but distinct variation in its allelic nucleotide sequence and can be divided into 6 genotypes when digested with specific restriction enzymes. We extracted DNA from formalin-fixed, paraffin-embedded tissues using SDS/proteinase K treatment followed by phenol/chloroform extraction. The sequence of nucleotides for the A, B and O genes was amplified by the polymerase chain reaction (PCR). DNA fragments of 128 bp and 200 bp could be amplified in the second round of PCR, using an aliquot of the first round PCR product as template. Degraded DNA from paraffin blocks stored for up to 10.7 years could be successfully typed. The ABO genotype was deduced from the digestion patterns with an appropriate combination of restriction enzymes and was compatible with the phenotype obtained from the blood sample. PMID:7947334

  15. A New Method to Extract Dental Pulp DNA: Application to Universal Detection of Bacteria

    PubMed Central

    Tran-Hung, Lam; Tran-Thi, Ny; Aboudharam, Gérard; Raoult, Didier; Drancourt, Michel

    2007-01-01

    Background Dental pulp is used for PCR-based detection of DNA derived from host and bacteremic microorganims. Current protocols require odontology expertise for proper recovery of the dental pulp. Dental pulp specimen exposed to laboratory environment yields contaminants detected using universal 16S rDNA-based detection of bacteria. Methodology/Principal Findings We developed a new protocol by encasing decontaminated tooth into sterile resin, extracting DNA into the dental pulp chamber itself and decontaminating PCR reagents by filtration and double restriction enzyme digestion. Application to 16S rDNA-based detection of bacteria in 144 teeth collected in 86 healthy people yielded a unique sequence in only 14 teeth (9.7%) from 12 individuals (14%). Each individual yielded a unique 16S rDNA sequence in 1–2 teeth per individual. Negative controls remained negative. Bacterial identifications were all confirmed by amplification and sequencing of specific rpoB sequence. Conclusions/Significance The new protocol prevented laboratory contamination of the dental pulp. It allowed the detection of bacteria responsible for dental pulp colonization from blood and periodontal tissue. Only 10% such samples contained 16S rDNA. It provides a new tool for the retrospective diagnostic of bacteremia by allowing the universal detection of bacterial DNA in animal and human, contemporary or ancient tooth. It could be further applied to identification of host DNA in forensic medicine and anthropology. PMID:17957246

  16. Evaluation of extraction methods from paraffin wax embedded tissues for PCR amplification of human and viral DNA

    PubMed Central

    Chan, P; Chan, D; To, K; Yu, M; Cheung, J; Cheng, A

    2001-01-01

    Aim—To evaluate the efficiency of phenol/chloroform, microwave, and Qiagen spin column based DNA extractions from paraffin wax embedded tissue for use in the polymerase chain reaction (PCR). In addition, to assess the reliability of amplifying a housekeeping gene to indicate successful viral DNA extraction. Methods—DNA samples extracted from 20 blocks of cervical carcinoma tissues using the three methods were subjected to PCRs targeting 509 bp and 355 bp of the ß globin gene, and 450 bp and 150 bp of human papillomavirus (HPV) DNA. Results—Microwave extraction showed the highest positive rate for ß globin PCR, whereas the spin column method was the most efficient for HPV DNA extraction. When the 509 bp ß globin and 450 bp HPV PCR results were correlated, two of 10, eight of 12, and nine of 10 ß globin positive extractions prepared by means of the phenol/chloroform, microwave, and spin column methods, respectively, yielded HPV DNA of the expected size. For the ß globin negative samples, HPV was detected in three of 10, two of eight, and four of 10 samples. Conclusions—HPV DNA extraction was most efficient using the Qiagen spin column and had the highest positive predictive value when a housekeeping gene was used as an indicator of successful viral DNA extraction; the phenol/chloroform method was the least efficient. The potential drawbacks of some extraction methods when using a human housekeeping gene to assess the quality of viral DNA extraction need to be considered. Key Words: cervical cancer • DNA extraction • polymerase chain reaction PMID:11328843

  17. An economical and effective high-throughput DNA extraction protocol for molecular marker analysis in honey bees

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Extraction of DNA from tissue samples can be expensive both in time and monetary resources and can often require handling and disposal of hazardous chemicals. We have developed a high throughput protocol for extracting DNA from honey bees that is of a high enough quality and quantity to enable hundr...

  18. An Improved Method for Soil DNA Extraction to Study the Microbial Assortment within Rhizospheric Region

    PubMed Central

    Fatima, Faria

    2014-01-01

    The need for identification of soil microbial community mainly depends on direct extraction of DNA from soil, a multifaceted environment that is a major pool for microbial genetic diversity. The soil DNA extraction procedures usually suffer from two major problems, namely, inappropriate rupturing of cells and contamination with humic substances. In the present study, five protocols for single type of rhizospheric soil were investigated and their comparison indicated that the inclusion of 120 mM phosphate buffered saline (PBS) for washing and mannitol in the lysis buffer allowed the processing of soil sample in minimal time with no specific equipment requirement. Furthermore, DNA purity and yield were also improved, which allowed the exploitation of genetic potential of soil microbes within soil sample thereby facilitating the amplification of metagenomic DNA. The effectiveness of methods was analyzed using random amplification of polymorphic DNA. The banding patterns revealed that both the abundance and the composition of indigenous microbial community depend on the DNA recovery method. PMID:25302120

  19. An Improved DNA Extraction Method for Efficient and Quantitative Recovery of Phytoplankton Diversity in Natural Assemblages

    PubMed Central

    Yuan, Jian; Li, Meizhen; Lin, Senjie

    2015-01-01

    Marine phytoplankton are highly diverse with different species possessing different cell coverings, posing challenges for thoroughly breaking the cells in DNA extraction yet preserving DNA integrity. While quantitative molecular techniques have been increasingly used in phytoplankton research, an effective and simple method broadly applicable to different lineages and natural assemblages is still lacking. In this study, we developed a bead-beating protocol based on our previous experience and tested it against 9 species of phytoplankton representing different lineages and different cell covering rigidities. We found the bead-beating method enhanced the final yield of DNA (highest as 2 folds) in comparison with the non-bead-beating method, while also preserving the DNA integrity. When our method was applied to a field sample collected at a subtropical bay located in Xiamen, China, the resultant ITS clone library revealed a highly diverse assemblage of phytoplankton and other micro-eukaryotes, including Archaea, Amoebozoa, Chlorophyta, Ciliphora, Bacillariophyta, Dinophyta, Fungi, Metazoa, etc. The appearance of thecate dinoflagellates, thin-walled phytoplankton and “naked” unicellular organisms indicates that our method could obtain the intact DNA of organisms with different cell coverings. All the results demonstrate that our method is useful for DNA extraction of phytoplankton and environmental surveys of their diversity and abundance. PMID:26218575

  20. One-step extraction for gas chromatography with flame photometric detection of 18 organophosphorus pesticides in Chinese medicine health wines.

    PubMed

    Liu, Qianzhen; Kong, Weijun; Qiu, Feng; Wei, Jianhe; Yang, Shihai; Zheng, Yuguo; Yang, Meihua

    2012-02-15

    An easy, rapid and selective gas chromatography with flame photometric detection (GC-FPD) method was established for simultaneously determining 18 organophosphorus pesticides (OPPs) in 80 Chinese medicine (CM) health wines. This method was based on a simple one-step extraction procedure using a little solvent without any further cleanup steps. The optimized extraction solvent for the pesticides is acetone:dichloromethane (1:1, V/V) with extraction recovery of 79.0-109.1% and relative standard deviation (RSD) of 0.36-12.68%, respectively. The limits of detection (LODs) of the established GC-FPD method for all investigated pesticides ranged from 1 to 15ngmL(-1) and limits of quantification (LOQs) from 4 to 50ngmL(-1). Out of all 80 CM health wines, 18 OPPs were found in 8 samples at low concentrations of 8.2-37.9ngmL(-1). These pesticides were successfully confirmed by GC-MS. This is the first report of determining OPPs in CM health wines, providing references for monitoring the quality of CM health wine in routine analysis. PMID:22285000

  1. Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor (L. Moench).

    PubMed

    Maropola, Mapula Kgomotso Annah; Ramond, Jean-Baptiste; Trindade, Marla

    2015-05-01

    Culture-independent studies rely on the quantity and quality of the extracted environmental metagenomic DNA (mDNA). To fully access the plant tissue microbiome, the extracted plant mDNA should allow optimal PCR applications and the genetic content must be representative of the total microbial diversity. In this study, we evaluated the endophytic bacterial diversity retrieved using different mDNA extraction procedures. Metagenomic DNA from sorghum (Sorghum bicolor L. Moench) stem and root tissues were extracted using two classical DNA extraction protocols (CTAB- and SDS-based) and five commercial kits. The mDNA yields and quality as well as the reproducibility were compared. 16S rRNA gene terminal restriction fragment length polymorphism (t-RFLP) was used to assess the impact on endophytic bacterial community structures observed. Generally, the classical protocols obtained high mDNA yields from sorghum tissues; however, they were less reproducible than the commercial kits. Commercial kits retrieved higher quality mDNA, but with lower endophytic bacterial diversities compared to classical protocols. The SDS-based protocol enabled access to the highest sorghum endophytic diversities. Therefore, "SDS-extracted" sorghum root and stem microbiome diversities were analysed via 454 pyrosequencing, and this revealed that the two tissues harbour significantly different endophytic communities. Nevertheless, both communities are dominated by agriculturally important genera such as Microbacterium, Agrobacterium, Sphingobacterium, Herbaspirillum, Erwinia, Pseudomonas and Stenotrophomonas; which have previously been shown to play a role in plant growth promotion. This study shows that DNA extraction protocols introduce biases in culture-independent studies of environmental microbial communities by influencing the mDNA quality, which impacts the microbial diversity analyses and evaluation. Using the broad-spectrum SDS-based DNA extraction protocol allows the recovery of the most

  2. Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

    PubMed Central

    Tatti, Enrico; McKew, Boyd A.; Whitby, Corrine; Smith, Cindy J.

    2016-01-01

    Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included. PMID:27341629

  3. High-Capacity Conductive Nanocellulose Paper Sheets for Electrochemically Controlled Extraction of DNA Oligomers

    PubMed Central

    Razaq, Aamir; Nyström, Gustav; Strømme, Maria; Mihranyan, Albert; Nyholm, Leif

    2011-01-01

    Highly porous polypyrrole (PPy)-nanocellulose paper sheets have been evaluated as inexpensive and disposable electrochemically controlled three-dimensional solid phase extraction materials. The composites, which had a total anion exchange capacity of about 1.1 mol kg−1, were used for extraction and subsequent release of negatively charged fluorophore tagged DNA oligomers via galvanostatic oxidation and reduction of a 30–50 nm conformal PPy layer on the cellulose substrate. The ion exchange capacity, which was, at least, two orders of magnitude higher than those previously reached in electrochemically controlled extraction, originated from the high surface area (i.e. 80 m2 g−1) of the porous composites and the thin PPy layer which ensured excellent access to the ion exchange material. This enabled the extractions to be carried out faster and with better control of the PPy charge than with previously employed approaches. Experiments in equimolar mixtures of (dT)6, (dT)20, and (dT)40 DNA oligomers showed that all oligomers could be extracted, and that the smallest oligomer was preferentially released with an efficiency of up to 40% during the reduction of the PPy layer. These results indicate that the present material is very promising for the development of inexpensive and efficient electrochemically controlled ion-exchange membranes for batch-wise extraction of biomolecules. PMID:22195031

  4. Differential efficiency among DNA extraction methods influences detection of the amphibian pathogen Batrachochytrium dendrobatidis.

    PubMed

    Bletz, M C; Rebollar, E A; Harris, R N

    2015-02-10

    Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is responsible for massive declines and extinctions of amphibians worldwide. The most common method for detecting Bd is quantitative polymerase chain reaction (qPCR). qPCR is a highly sensitive detection technique, but its ability to determine the presence and accurately quantify the amount of Bd is also contingent on the efficiency of the DNA extraction method used prior to PCR. Using qPCR, we compared the extraction efficiency of 3 different extraction methods commonly used for Bd detection across a range of zoospore quantities: PrepMan Ultra Reagent, Qiagen DNeasy Blood and Tissue Kit, and Mobio PowerSoil DNA Isolation Kit. We show that not all extraction methods led to successful detection of Bd for the low zoospore quantities and that there was variation in the estimated zoospore equivalents among the methods, which demonstrates that these methods have different extraction efficiencies. These results highlight the importance of considering the extraction method when comparing across studies. The Qiagen DNeasy kit had the highest efficiency. We also show that replicated estimates of less than 1 zoospore can result from known zoospore concentrations; therefore, such results should be considered when obtained from field data. Additionally, we discuss the implications of our findings for interpreting previous studies and for conducting future Bd surveys. It is imperative to use the most efficient DNA extraction method in tandem with the highly sensitive qPCR technique in order to accurately diagnose the presence of Bd as well as other pathogens. PMID:25667331

  5. One-step DNA-programmed growth of luminescent and biofunctionalized nanocrystals

    NASA Astrophysics Data System (ADS)

    Ma, Nan; Sargent, Edward H.; Kelley, Shana O.

    2009-02-01

    Colloidal semiconductor nanocrystals are widely used as lumiphores in biological imaging because their luminescence is both strong and stable, and because they can be biofunctionalized. During synthesis, nanocrystals are typically passivated with hydrophobic organic ligands, so it is then necessary either to replace these ligands or encapsulate the nanocrystals with hydrophilic moieties to make the lumiphores soluble in water. Finally, biological labels must be added to allow the detection of nucleic acids, proteins and specific cell types. This multistep process is time- and labour-intensive and thus out of reach of many researchers who want to use luminescent nanocrystals as customized lumiphores. Here, we show that a single designer ligand-a chimeric DNA molecule-can controllably program both the growth and the biofunctionalization of the nanocrystals. One part of the DNA sequence controls the nanocrystal passivation and serves as a ligand, while another part controls the biorecognition. The synthetic protocol reported here is straightforward and produces a homogeneous dispersion of nanocrystal lumiphores functionalized with a single biomolecular receptor. The nanocrystals exhibit strong optical emission in the visible region, minimal toxicity and have hydrodynamic diameters of ~6 nm, which makes them suitable for bioimaging. We show that the nanocrystals can specifically bind DNA, proteins or cells that have unique surface recognition markers.

  6. To beat or not to beat a tick: comparison of DNA extraction methods for ticks (Ixodes scapularis).

    PubMed

    Ammazzalorso, Alyssa D; Zolnik, Christine P; Daniels, Thomas J; Kolokotronis, Sergios-Orestis

    2015-01-01

    Background. Blacklegged ticks (Ixodes scapularis) are important disease vectors in the United States, known to transmit a variety of pathogens to humans, including bacteria, protozoa, and viruses. Their importance as a disease vector necessitates reliable and comparable methods for extracting microbial DNA from ticks. Furthermore, to explore the population genetics or genomics of this tick, appropriate DNA extraction techniques are needed for both the vector and its microbes. Although a few studies have investigated different methods of DNA isolation from ticks, they are limited in the number and types of DNA extraction and lack species-specific quantification of DNA yield. Methods. Here we determined the most efficient and consistent method of DNA extraction from two different developmental stages of I. scapularis-nymph and adult-that are the most important for disease transmission. We used various methods of physical disruption of the hard, chitinous exoskeleton, as well as commercial and non-commercial DNA isolation kits. To gauge the effectiveness of these methods, we quantified the DNA yield and confirmed the DNA quality via PCR of both tick and microbial genetic material. Results. DNA extraction using the Thermo GeneJET Genomic DNA Purification Kit resulted in the highest DNA yields and the most consistent PCR amplification when combined with either cutting or bead beating with select matrices across life stages. DNA isolation methods using ammonium hydroxide as well as the MoBio PowerSoil kit also produced strong and successful PCR amplification, but only for females. Discussion. We contrasted a variety of readily available methods of DNA extraction from single individual blacklegged ticks and presented the results through a quantitative and qualitative assessment. PMID:26290800

  7. Validation of a DNA IQ-based extraction method for TECAN robotic liquid handling workstations for processing casework.

    PubMed

    Frégeau, Chantal J; Lett, C Marc; Fourney, Ron M

    2010-10-01

    A semi-automated DNA extraction process for casework samples based on the Promega DNA IQ™ system was optimized and validated on TECAN Genesis 150/8 and Freedom EVO robotic liquid handling stations configured with fixed tips and a TECAN TE-Shake™ unit. The use of an orbital shaker during the extraction process promoted efficiency with respect to DNA capture, magnetic bead/DNA complex washes and DNA elution. Validation studies determined the reliability and limitations of this shaker-based process. Reproducibility with regards to DNA yields for the tested robotic workstations proved to be excellent and not significantly different than that offered by the manual phenol/chloroform extraction. DNA extraction of animal:human blood mixtures contaminated with soil demonstrated that a human profile was detectable even in the presence of abundant animal blood. For exhibits containing small amounts of biological material, concordance studies confirmed that DNA yields for this shaker-based extraction process are equivalent or greater to those observed with phenol/chloroform extraction as well as our original validated automated magnetic bead percolation-based extraction process. Our data further supports the increasing use of robotics for the processing of casework samples. PMID:20457033

  8. New sorbent in the dispersive solid phase extraction step of quick, easy, cheap, effective, rugged, and safe for the extraction of organic contaminants in drinking water treatment sludge.

    PubMed

    Cerqueira, Maristela B R; Caldas, Sergiane S; Primel, Ednei G

    2014-04-01

    Recent studies have shown a decrease in the concentration of pesticides, pharmaceuticals and personal care products (PCPs) in water after treatment. A possible explanation for this phenomenon is that these compounds may adhere to the sludge; however, investigation of these compounds in drinking water treatment sludge has been scarce. The sludge generated by drinking water treatment plants during flocculation and decantation steps should get some special attention not only because it has been classified as non-inert waste but also because it is a very complex matrix, consisting essentially of inorganic (sand, argil and silt) and organic (humic substances) compounds. In the first step of this study, three QuEChERS methods were used, and then compared, for the extraction of pesticides (atrazine, simazine, clomazone and tebuconazole), pharmaceuticals (amitriptyline, caffeine, diclofenac and ibuprofen) and PCPs (methylparaben, propylparaben, triclocarban and bisphenol A) from drinking water treatment sludge. Afterwards, the study of different sorbents in the dispersive solid phase extraction (d-SPE) step was evaluated. Finally, a new QuEChERS method employing chitin, obtained from shrimp shell waste, was performed in the d-SPE step. After having been optimized, the method showed limits of quantification (LOQ) between 1 and 50 μg kg(-1) and the analytical curves showed r values higher than 0.98, when liquid chromatography tandem mass spectrometry was employed. Recoveries ranged between 50 and 120% with RSD≤15%. The matrix effect was evaluated and compensated with matrix-matched calibration. The method was applied to drinking water treatment sludge samples and methylparaben and tebuconazole were found in concentration

  9. Genomic DNA extraction methods using formalin-fixed paraffin-embedded tissue.

    PubMed

    Potluri, Keerti; Mahas, Ahmed; Kent, Michael N; Naik, Sameep; Markey, Michael

    2015-10-01

    As new technologies come within reach for the average cytogenetic laboratory, the study of chromosome structure has become increasingly more sophisticated. Resolution has improved from karyotyping (in which whole chromosomes are discernible) to fluorescence in situ hybridization and comparative genomic hybridization (CGH, with which specific megabase regions are visualized), array-based CGH (aCGH, examining hundreds of base pairs), and next-generation sequencing (providing single base pair resolution). Whole genome next-generation sequencing remains a cost-prohibitive method for many investigators. Meanwhile, the cost of aCGH has been reduced during recent years, even as resolution has increased and protocols have simplified. However, aCGH presents its own set of unique challenges. DNA of sufficient quantity and quality to hybridize to arrays and provide meaningful results is required. This is especially difficult for DNA from formalin-fixed paraffin-embedded (FFPE) tissues. Here, we compare three different methods for acquiring DNA of sufficient length, purity, and "amplifiability" for aCGH and other downstream applications. Phenol-chloroform extraction and column-based commercial kits were compared with adaptive focused acoustics (AFA). Of the three extraction methods, AFA samples showed increased amplicon length and decreased polymerase chain reaction (PCR) failure rate. These findings support AFA as an improvement over previous DNA extraction methods for FFPE tissues. PMID:26126956

  10. Robust and effective methodologies for cryopreservation and DNA extraction from anaerobic gut fungi.

    PubMed

    Solomon, Kevin V; Henske, John K; Theodorou, Michael K; O'Malley, Michelle A

    2016-04-01

    Cell storage and DNA isolation are essential to developing an expanded suite of microorganisms for biotechnology. However, many features of non-model microbes, such as an anaerobic lifestyle and rigid cell wall, present formidable challenges to creating strain repositories and extracting high quality genomic DNA. Here, we establish accessible, high efficiency, and robust techniques to store lignocellulolytic anaerobic gut fungi long term without specialized equipment. Using glycerol as a cryoprotectant, gut fungal isolates were preserved for a minimum of 23 months at -80 °C. Unlike previously reported approaches, this improved protocol is non-toxic and rapid, with samples surviving twice as long with negligible growth impact. Genomic DNA extraction for these isolates was optimized to yield samples compatible with next generation sequencing platforms (e.g. Illumina, PacBio). Popular DNA isolation kits and precipitation protocols yielded preps that were unsuitable for sequencing due to carbohydrate contaminants from the chitin-rich cell wall and extensive energy reserves of gut fungi. To address this, we identified a proprietary method optimized for hardy plant samples that rapidly yielded DNA fragments in excess of 10 kb with minimal RNA, protein or carbohydrate contamination. Collectively, these techniques serve as fundamental tools to manipulate powerful biomass-degrading gut fungi and improve their accessibility among researchers. PMID:26620247

  11. Extraction of single-copy nuclear DNA from forensic specimens with a variety of postmortem histories.

    PubMed

    Evison, M P; Smillie, D M; Chamberlain, A T

    1997-11-01

    Specimens of human bone, teeth and dried blood spots from 3 months to 91 years old, with a variety of postmortem histories, were used in a comparative study of recovery of single-copy nuclear DNA sequences from forensic material. Sequences of the amelogenin and HLA-DPB1 genes were chosen for their value in sexing and identification. Sequences of the mitochondrial non-coding region V were also amplified to compare the recovery of mitochondrial and single-copy nuclear DNA. A variation of the silica method for DNA extraction was refined for application to the forensic specimens in this sample. Single-copy nuclear DNA was amplified from 100% of recent postoperative bone specimens (n = 6), 80% of forensic teeth and bone specimens (n = 10), 78% of recently extracted teeth (n = 18), 78% of exhumed bone up to 91 years old (n = 37) and 69% of 15 year old bone specimens fixed in 10% formalin (n = 20). Amelogenin sexing was correct in 85% of cases (n = 74) in which the sex of the donor had been recorded. There was no correlation between the age of the specimen and the extent of DNA preservation. PMID:9397544

  12. DNA Extraction Protocols for Whole-Genome Sequencing in Marine Organisms.

    PubMed

    Panova, Marina; Aronsson, Henrik; Cameron, R Andrew; Dahl, Peter; Godhe, Anna; Lind, Ulrika; Ortega-Martinez, Olga; Pereyra, Ricardo; Tesson, Sylvie V M; Wrange, Anna-Lisa; Blomberg, Anders; Johannesson, Kerstin

    2016-01-01

    The marine environment harbors a large proportion of the total biodiversity on this planet, including the majority of the earths' different phyla and classes. Studying the genomes of marine organisms can bring interesting insights into genome evolution. Today, almost all marine organismal groups are understudied with respect to their genomes. One potential reason is that extraction of high-quality DNA in sufficient amounts is challenging for many marine species. This is due to high polysaccharide content, polyphenols and other secondary metabolites that will inhibit downstream DNA library preparations. Consequently, protocols developed for vertebrates and plants do not always perform well for invertebrates and algae. In addition, many marine species have large population sizes and, as a consequence, highly variable genomes. Thus, to facilitate the sequence read assembly process during genome sequencing, it is desirable to obtain enough DNA from a single individual, which is a challenge in many species of invertebrates and algae. Here, we present DNA extraction protocols for seven marine species (four invertebrates, two algae, and a marine yeast), optimized to provide sufficient DNA quality and yield for de novo genome sequencing projects. PMID:27460368

  13. Methods for optimizing DNA extraction before quantifying oral bacterial numbers by real-time PCR.

    PubMed

    Nadkarni, Mangala A; Martin, F Elizabeth; Hunter, Neil; Jacques, Nicholas A

    2009-07-01

    Methods for the optimal extraction of genomic DNA for real-time PCR enumeration of oral bacteria using the muramidase, mutanolysin, were developed using a simple in vitro oral flora model comprised of the facultative anaerobic gram-positive bacteria, Lactobacillus acidophilus and Streptococcus mutans, the gram-positive anaerobe, Parvimonas micra, and the gram-negative anaerobes, Porphyromonas gingivalis, Prevotella melaninogenica and Fusobacterium nucleatum. Traditional, as well as more elaborate, methods of quantifying bacterial numbers, including colony counting and estimation of DNA content using 4',6-diamino-2-phenylindole were compared in order to validate the real-time PCR approach. Evidence was obtained that P. gingivalis nuclease activity adversely affected the extraction of double-stranded DNA from this bacterium either alone or when it formed part of a consortium with the other bacteria. This nuclease activity could be overcome by treatment of the bacteria with either 20 mM diethyl pyrocarbonate or 70% ethanol at 4 degrees C overnight. A final purification of the DNA to remove any potential PCR inhibitors was added to the protocol in order to accurately quantify the amount of DNA by real-time PCR and hence the number of bacteria in a sample. PMID:19459962

  14. A simple method to determine bioethanol content in gasoline using two-step extraction and liquid scintillation counting.

    PubMed

    Yunoki, Shunji; Saito, Masaaki

    2009-12-01

    A simple method for determining bioethanol content in gasoline containing bioethanol (denoted as E-gasoline in this study) is urgently required. Liquid scintillation counting (LSC) was employed based on the principle that (14)C exists in bioethanol but not in synthetic ethanol. Bioethanol was extracted in two steps by water from E-gasoline containing 3% (E3) or 10% (E10) bioethanol. The (14)C radioactivity was measured by LSC and converted to the amount of bioethanol. The bioethanol content in E-gasoline was determined precisely from the partition coefficient in the extraction and the amount of bioethanol in the water phases: 2.98+/-0.10% for E3 and 10.0+/-0.1% for E10 (means+/-SD; n=3). It appears that this method can be used to determine bioethanol content in E-gasoline quickly and easily. PMID:19577920

  15. Usefulness of FTA® cards as a Pneumocystis-DNA extraction method in bronchoalveolar lavage samples.

    PubMed

    Rodiño, Jenniffer M; Aguilar, Yudy A; Rueda, Zulma Vanessa; Vélez, Lázaro A

    2016-05-01

    Background FTA® cards (Fast Technology for Analysis of Nucleic Acids) are an alternative DNA extraction method in bronchoalveolar lavage (BAL) samples for Pneumocystis jirovecii molecular analyses. The goal was to evaluate the usefulness of FTA® cards to detect P. jirovecii-DNA by PCR in BAL samples compared to silica adsorption chromatography (SAC). Methods This study used 134 BAL samples from immunocompromised patients previously studied to establish microbiological aetiology of pneumonia, among them 15 cases of Pneumocystis pneumonia (PCP) documented by staining and 119 with other alternative diagnoses. The FTA® system and SAC were used for DNA extraction and then amplified by nested PCR to detect P. jirovecii. Performance and concordance of the two DNA extraction methods compared to P. jirovecii microscopy were calculated. The influence of the macroscopic characteristics, transportation of samples and the duration of the FTA® card storage (1, 7, 10 or 12 months) were also evaluated. Results Among 134 BAL samples, 56% were positive for P. jirovecii-DNA by SAC and 27% by FTA®. All 15 diagnosed by microscopy were detected by FTA® and SAC. Specificity of the FTA® system and SAC were 82.4% and 49.6%, respectively. Compared to SAC, positivity by FTA® decreased with the presence of blood in BAL (62% vs 13.5%). The agreement between samples at 7, 10 and 12 months was 92.5% for FTA®. Positive cases by FTA® remained the same after shipment by mail. Conclusions Results suggest that FTA® is a practical, safe and economical method to preserve P. jirovecii-DNA in BAL samples for molecular studies. PMID:26950684

  16. Comparison of eight commercially available kits for DNA extraction from formalin-fixed paraffin-embedded tissues.

    PubMed

    Janecka, Anna; Adamczyk, Agnieszka; Gasińska, Anna

    2015-05-01

    A proper extraction method from formalin-fixed paraffin-embedded (FFPE) blocks is essential to obtain DNA of satisfactory quality/quantity. We compared the effectiveness of eight commercially available kits for DNA extraction based on 10 FFPE tissues. Kits differed significantly in terms of DNA yield, purity, and quality. Using the QIAamp DNA FFPE Tissue Kit (Qiagen) and the ReliaPrep FFPE gDNA Miniprep System (Promega), we obtained DNA of the highest quality and acceptable quantity. We also demonstrated that overnight digestion of samples usually improved DNA yield and/or purity. For precious or limited material, double elution is recommended for obtaining up to 42% higher amount of DNA. PMID:25640584

  17. Novel enzyme immunoassay and optimized DNA extraction for the detection of polymerase-chain-reaction-amplified viral DNA from paraffin-embedded tissue.

    PubMed Central

    Merkelbach, S.; Gehlen, J.; Handt, S.; Füzesi, L.

    1997-01-01

    Four different DNA extraction methods were compared to determine their ability to provide DNA for amplification of viral sequences from paraffin-embedded human tissue samples by polymerase chain reaction (PCR). The suitability of extraction methods was assessed using parameters like DNA yield, length of recovered DNA fragments, and duration. Furthermore, the efficiency of amplifying a human single-copy gene, the beta-globin gene, from DNA samples was tested. The best preservation of DNA molecules could be achieved by binding the DNA onto a silica column before further purification. Viral DNA sequences could be amplified by PCR in DNA extracted from routinely processed paraffin blocks from cases with clinically or morphologically suspected cytomegalovirus or Epstein-Barr virus infections. The PCR products were specified by a novel liquid hybridization assay called PCR-enzyme-linked immunosorbent assay. Using this assay, the time-consuming Southern hybridization could be replaced and the time requirement for the detection of PCR products could be reduced from 1 day to 4 hours. The assay system described here represents a reliable, sensitive, and specific method for the detection of viral DNA from paraffin-embedded tissue samples. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:9137080

  18. Extraction of DNA from small sections of frozen tissue with simultaneous histological examination.

    PubMed Central

    Cotter, F E; Hall, P A; Young, B D

    1988-01-01

    Though analysis of small sections of biopsy material by molecular techniques permits increased sensitivity, it also requires accurate histological examination of the tissue in order to reduce sampling error. A technique for the extraction of DNA from small sections of frozen biopsy material with simultaneous histological examination from adjacent sections is described that may enhance the accuracy of characterisation of the tissue, particularly where there is focal variation. The quality of the DNA obtained enables a full range of molecular studies to be carried out. Images Fig 1 Fig 2 PMID:3192737

  19. A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle.

    PubMed

    Majumdar, Gipsy; Vera, Santiago; Elam, Marshall B; Raghow, Rajendra

    2015-04-01

    We combined the TRIzol method of nucleic acid extraction with QIAamp columns to achieve coextraction of RNA and genomic DNA from peripheral blood mononuclear cells (PBMCs) and biopsied skeletal muscle, both stored at -80 °C for many months. Total RNA was recovered from the upper aqueous phase of TRIzol. The interphase and organic phases were precipitated with ethanol, digested with proteinase K, and filtered through QIAamp MinElute columns to recover DNA. The combined protocol yielded excellent quality and quantity of nucleic acids from archived human PBMCs and muscle and may be easily adapted for other tissues. PMID:25579785

  20. Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits.

    PubMed

    Demeke, Tigst; Jenkins, G Ronald

    2010-03-01

    Biotechnology-derived varieties of canola, cotton, corn and soybean are being grown in the USA, Canada and other predominantly grain exporting countries. Although the amount of farmland devoted to production of biotechnology-derived crops continues to increase, lingering concerns that unintended consequences may occur provide the EU and most grain-importing countries with justification to regulate these crops. Legislation in the EU requires traceability of grains/oilseeds, food and feed products, and labelling, when a threshold level of 0.9% w/w of genetically engineered trait is demonstrated to be present in an analytical sample. The GE content is routinely determined by quantitative PCR (qPCR) and plant genomic DNA provides the template for the initial steps in this process. A plethora of DNA extraction methods exist for qPCR applications. Implementing standardized methods for detection of genetically engineered traits is necessary to facilitate grain marketing. The International Organization for Standardization draft standard 21571 identifies detergent-based methods and commercially available kits that are widely used for DNA extraction, but also indicates that adaptations may be necessary depending upon the sample matrix. This review assesses advantages and disadvantages of various commercially available DNA extraction kits, as well as modifications to published cetyltrimethylammonium bromide methods. Inhibitors are a major obstacle for efficient amplification in qPCR. The types of PCR inhibitors and techniques to minimize inhibition are discussed. Finally, accurate quantification of DNA for applications in qPCR is not trivial. Many confounders contribute to differences in analytical measurements when a particular DNA quantification method is applied and different methods do not always provide concordant results on the same DNA sample. How these differences impact measurement uncertainty in qPCR is considered. PMID:19789856

  1. DNA analysis of soil extracts can be used to investigate fine root depth distribution of trees.

    PubMed

    Bithell, Sean L; Tran-Nguyen, Lucy T T; Hearnden, Mark N; Hartley, Diana M

    2014-01-01

    Understanding the root distribution of trees by soil coring is time -: consuming as it requires the separation of roots from soil and classification of roots into particular size classes. This labour-intensive process can limit sample throughput and therefore sampling intensity. We investigated the use of quantitative polymerase chain reaction (qPCR) on soil DNA extractions to determine live fine root DNA density (RDD, mg DNA m(-2)) for mango (Mangifera indica) trees. The specificity of the qPCR was tested against DNA extracted from 10 mango cultivars and 14 weed species. All mango cultivars and no weeds were detected. Mango DNA was successfully quantified from control soil spiked with mango roots and weed species. The DNA yield of mango root sections stored in moist soil at 23-28 °C declined after 15 days to low concentrations as roots decayed, indicating that dead root materials in moist soil would not cause false-positive results. To separate large roots from samples, a root separation method for field samples was used to target the root fragments remaining in sieved (minimum 2 mm aperture) soil for RDD comparisons. Using this method we compared the seasonal RDD values of fine roots for five mango rootstock cultivars in a field trial. The mean cultivar DNA yields by depth from root fragments in the sieved soil samples had the strongest relationship (adjusted multiple R(2) = 0.9307, P < 0.001) with the dry matter (g m(-2)) of fine (diameter <0.64 mm) roots removed from the soil by sieving. This method provides a species-specific and rapid means of comparing the distribution and concentration of live fine roots of trees in orchards using soil samples up to 500 g. PMID:25552675

  2. DNA analysis of soil extracts can be used to investigate fine root depth distribution of trees

    PubMed Central

    Bithell, Sean L.; Tran-Nguyen, Lucy T. T.; Hearnden, Mark N.; Hartley, Diana M.

    2015-01-01

    Understanding the root distribution of trees by soil coring is time-consuming as it requires the separation of roots from soil and classification of roots into particular size classes. This labour-intensive process can limit sample throughput and therefore sampling intensity. We investigated the use of quantitative polymerase chain reaction (qPCR) on soil DNA extractions to determine live fine root DNA density (RDD, mg DNA m−2) for mango (Mangifera indica) trees. The specificity of the qPCR was tested against DNA extracted from 10 mango cultivars and 14 weed species. All mango cultivars and no weeds were detected. Mango DNA was successfully quantified from control soil spiked with mango roots and weed species. The DNA yield of mango root sections stored in moist soil at 23–28 °C declined after 15 days to low concentrations as roots decayed, indicating that dead root materials in moist soil would not cause false-positive results. To separate large roots from samples, a root separation method for field samples was used to target the root fragments remaining in sieved (minimum 2 mm aperture) soil for RDD comparisons. Using this method we compared the seasonal RDD values of fine roots for five mango rootstock cultivars in a field trial. The mean cultivar DNA yields by depth from root fragments in the sieved soil samples had the strongest relationship (adjusted multiple R2 = 0.9307, P < 0.001) with the dry matter (g m−2) of fine (diameter <0.64 mm) roots removed from the soil by sieving. This method provides a species-specific and rapid means of comparing the distribution and concentration of live fine roots of trees in orchards using soil samples up to 500 g. PMID:25552675

  3. Initial steps towards a production platform for DNA sequence analysis on the grid

    PubMed Central

    2010-01-01

    Background Bioinformatics is confronted with a new data explosion due to the availability of high throughput DNA sequencers. Data storage and analysis becomes a problem on local servers, and therefore it is needed to switch to other IT infrastructures. Grid and workflow technology can help to handle the data more efficiently, as well as facilitate collaborations. However, interfaces to grids are often unfriendly to novice users. Results In this study we reused a platform that was developed in the VL-e project for the analysis of medical images. Data transfer, workflow execution and job monitoring are operated from one graphical interface. We developed workflows for two sequence alignment tools (BLAST and BLAT) as a proof of concept. The analysis time was significantly reduced. All workflows and executables are available for the members of the Dutch Life Science Grid and the VL-e Medical virtual organizations All components are open source and can be transported to other grid infrastructures. Conclusions The availability of in-house expertise and tools facilitates the usage of grid resources by new users. Our first results indicate that this is a practical, powerful and scalable solution to address the capacity and collaboration issues raised by the deployment of next generation sequencers. We currently adopt this methodology on a daily basis for DNA sequencing and other applications. More information and source code is available via http://www.bioinformaticslaboratory.nl/ PMID:21156038

  4. Next-generation DNA sequencing of HEXA: a step in the right direction for carrier screening.

    PubMed

    Hoffman, Jodi D; Greger, Valerie; Strovel, Erin T; Blitzer, Miriam G; Umbarger, Mark A; Kennedy, Caleb; Bishop, Brian; Saunders, Patrick; Porreca, Gregory J; Schienda, Jaclyn; Davie, Jocelyn; Hallam, Stephanie; Towne, Charles

    2013-11-01

    Tay-Sachs disease (TSD) is the prototype for ethnic-based carrier screening, with a carrier rate of ∼1/27 in Ashkenazi Jews and French Canadians. HexA enzyme analysis is the current gold standard for TSD carrier screening (detection rate ∼98%), but has technical limitations. We compared DNA analysis by next-generation DNA sequencing (NGS) plus an assay for the 7.6 kb deletion to enzyme analysis for TSD carrier screening using 74 samples collected from participants at a TSD family conference. Fifty-one of 74 participants had positive enzyme results (46 carriers, five late-onset Tay-Sachs [LOTS]), 16 had negative, and seven had inconclusive results. NGS + 7.6 kb del screening of HEXA found a pathogenic mutation, pseudoallele, or variant of unknown significance (VUS) in 100% of the enzyme-positive or obligate carrier/enzyme-inconclusive samples. NGS detected the B1 allele in two enzyme-negative obligate carriers. Our data indicate that NGS can be used as a TSD clinical carrier screening tool. We demonstrate that NGS can be superior in detecting TSD carriers compared to traditional enzyme and genotyping methodologies, which are limited by false-positive and false-negative results and ethnically focused, limited mutation panels, respectively, but is not ready for sole use due to lack of information regarding some VUS. PMID:24498621

  5. Automated microfluidic DNA/RNA extraction with both disposable and reusable components

    NASA Astrophysics Data System (ADS)

    Kim, Jungkyu; Johnson, Michael; Hill, Parker; Sonkul, Rahul S.; Kim, Jongwon; Gale, Bruce K.

    2012-01-01

    An automated microfluidic nucleic extraction system was fabricated with a multilayer polydimethylsiloxane (PDMS) structure that consists of sample wells, microvalves, a micropump and a disposable microfluidic silica cartridge. Both the microvalves and micropump structures were fabricated in a single layer and are operated pneumatically using a 100 µm PDMS membrane. To fabricate the disposable microfluidic silica cartridge, two-cavity structures were made in a PDMS replica to fit the stacked silica membranes. A handheld controller for the microvalves and pumps was developed to enable system automation. With purified ribonucleic acid (RNA), whole blood and E. coli samples, the automated microfluidic nucleic acid extraction system was validated with a guanidine-based solid phase extraction procedure. An extraction efficiency of ~90% for deoxyribonucleic acid (DNA) and ~54% for RNA was obtained in 12 min from whole blood and E. coli samples, respectively. In addition, the same quantity and quality of extracted DNA was confirmed by polymerase chain reaction (PCR) amplification. The PCR also presented the appropriate amplification and melting profiles. Automated, programmable fluid control and physical separation of the reusable components and the disposable components significantly decrease the assay time and manufacturing cost and increase the flexibility and compatibility of the system with downstream components.

  6. DNA topoisomerase IIα controls replication origin cluster licensing and firing time in Xenopus egg extracts

    PubMed Central

    Gaggioli, Vincent; Le Viet, Barbara; Germe, Thomas; Hyrien, Olivier

    2013-01-01

    Sperm chromatin incubated in Xenopus egg extracts undergoes origin licensing and nuclear assembly before DNA replication. We found that depletion of DNA topoisomerase IIα (topo IIα), the sole topo II isozyme of eggs and its inhibition by ICRF-193, which clamps topo IIα around DNA have opposite effects on these processes. ICRF-193 slowed down replication origin cluster activation and fork progression in a checkpoint-independent manner, without altering replicon size. In contrast, topo IIα depletion accelerated origin cluster activation, and topo IIα add-back negated overinitiation. Therefore, topo IIα is not required for DNA replication, but topo IIα clamps slow replication, probably by forming roadblocks. ICRF-193 had no effect on DNA synthesis when added after nuclear assembly, confirming that topo IIα activity is dispensable for replication and revealing that topo IIα clamps formed on replicating DNA do not block replication, presumably because topo IIα acts behind and not in front of forks. Topo IIα depletion increased, and topo IIα addition reduced, chromatin loading of MCM2-7 replicative helicase, whereas ICRF-193 did not affect MCM2-7 loading. Therefore, topo IIα restrains MCM2-7 loading in an ICRF-193-resistant manner during origin licensing, suggesting a model for establishing the sequential firing of origin clusters. PMID:23757188

  7. A magnetic nanoparticles-based method for DNA extraction from the saliva of stroke patients

    PubMed Central

    Yi, Li; Huang, Ying; Wu, Ting; Wu, Jun

    2013-01-01

    C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene is a risk factor for stroke, suggesting that widespread detection could help to prevent stroke. DNA from 70 stroke patients and 70 healthy controls was extracted from saliva using a magnetic nanoparticles-based method and from blood using conventional methods. Real-time PCR results revealed that the C677T polymorphism was genotyped by PCR using DNA extracted from both saliva and blood samples. The genotype results were confirmed by gene sequencing, and results for saliva and blood samples were consistent. The mutation TT genotype frequency was significantly higher in the stroke group than in controls. Homocysteine levels were significantly higher than controls in both TT genotype groups. Therefore, this noninvasive magnetic nanoparticles-based method using saliva samples could be used to screen for the MTHFR C677T polymorphism in target populations. PMID:25206624

  8. Single-step procedure for labeling DNA strand breaks with fluorescein- or BODIPY-conjugated deoxynucleotides: Detection of apoptosis and bromodeoxyuridine incorporation

    SciTech Connect

    Xun Li; Traganos, F.; Melamed, M.R.; Darzynkiewicz, Z.

    1995-06-01

    The methods of in situ labeling of DNA strand breaks have been used to identify apoptotic cells and/or DNA replicating cells. While discrimination of apoptotic cells is based on the inherent presence of numerous DNA strand breaks in their chromatin, DNA proliferating cells can be discriminated by the selective DNA strand break induction by photolysis (SBIP) methodology at the sites that contain incorporated bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd). In both instances, DNA strand breaks are labeled with biotin- or digoxygenin-conjugated deoxynucleotides, preferably in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase; fluorescein tagged avidin (streptavidin) or digoxygenin antibody is used in the second step of the reaction. In the present study, DNA strand break labeling was simplified by using directly labeled deoxynucleotides, in a single-step reaction. Apoptotic cells in HL-60 cultures treated with camptothecin or in primary cultures of non-Hodgkin`s lymphoma cells treated with prednisolone were easily identified utilizing BODIPY-conjugated dUTP (B-dUTP). The single-step procedure, requiring fewer centrifugation steps, resulted in less cell loss compared to the two-step cell labeling technique. The morphology of cells subjected to SBIP was excellent, allowing visualization of distinct DNA replication points. Because, unlike the immunocytochemical methods used to detect BrdUrd incorporation, the SBIP methodology does not require DNA denaturation by heat or acid, nuclear proteins are expected to remain undenatured in situ, allowing one to study colocalization of various constituents, detected immunocytochemically, at the DNA replication points. 30 refs., 7 figs.

  9. Computer-based knowledge extraction tool: a step in the development of a cognitive skills tutor

    SciTech Connect

    Stoddard, M.L.; Kern, R.P.; Emerson, J.D.

    1986-01-01

    Los Alamos National Laboratory, under the sponsorship of the Army Research Institute, is developing an experimental computer-tutor to be used as part of the Armor Officer's Basic Course at Fort Knox, Kentucky. The tutor's objective is to train students to apply the types of cognitive processing strategies needed to more effectively organize their knowledge for application in planning and conducting tactical operations. The tutor is being developed through an iterative process with the first phase being a knowledge extraction computer-based tool. Student knowledge organization in this domain will be obtained through collection of online and offline performance data. The tool is designed to obtain the knowledge organization through a motivating, realistic tactical operations exercise. 18 refs.

  10. Rapid techniques for DNA extraction from routinely processed archival tissue for use in PCR.

    PubMed Central

    Sepp, R; Szabó, I; Uda, H; Sakamoto, H

    1994-01-01

    AIMS--To evaluate the ability of four rapid DNA extraction methods to provide DNA for the polymerase chain reaction (PCR) from routinely fixed, paraffin wax embedded archival tissues. METHODS--Eighteen blocks of various tissues, 18 blocks of cervical cancer specimens, and nine blocks of B cell lymphomas were investigated. Both normal and biopsy specimen sized tissues were studied. DNA was extracted using four methods: boiling for 20 minutes in distilled water; boiling for 20 minutes in 5% Chelex-100 resin solution; 3-hour proteinase K digestion; and 3-hour proteinase K digestion, followed by boiling in 5% Chelex-100. Different exons of the p53 gene, human papillomavirus type 16 (HPV 16) sequence, and immunoglobulin heavy chain (IgH) gene rearrangement were amplified from the extracts. RESULTS--The Chelex boiling, proteinase K digestion, and proteinase K digestion-Chelex boiling methods produced DNA suitable for amplification in all of the 45 samples. Boiling in water yielded insufficient template for the PCR in three of the 45 cases (7%), and in six of 42 positive cases (14%) much fainter bands were observed, mostly when the processed material was either biopsy specimen sized or a B cell lymphoma sample. Fragments of the p53 gene were successfully amplified up to 408 base pairs in water boiled extracts, up to 647 in Chelex boiled preparates, and up to 984 in proteinase K digested and proteinase K digested-Chelex boiled samples, although with decreased sensitivity in the last case. All of the templates were reusable after 3 months of storage at -20 degrees C. CONCLUSIONS--Chelex boiling, proteinase K digestion, and proteinase K digestion followed by Chelex boiling produce suitable templates for the PCR from a large variety of paraffin wax embedded tissues. As the simple 20 minute boiling method in 5% Chelex-100 solution requires minimal manipulation and time, it could be useful, especially in the routine processing of large amounts of material. Images PMID:8027368

  11. Near-quantitative extraction of genomic DNA from various food-borne eubacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this work we have tested a dozen commercial bacterial genomic DNA extraction methodologies on an average of 7.70E6 (± 9.05%), 4.77E8 (± 31.0%), and 5.93E8 (± 4.69%) colony forming units (CFU) associated with 3 cultures (n = 3) each of Brochothrix thermosphacta (Bt), Shigella sonnei (Ss), and Esch...

  12. A spin cartridge system for DNA extraction from paraffin wax embedded tissues.

    PubMed Central

    Pinto, A P; Villa, L L

    1998-01-01

    A simple and efficient method of DNA extraction from paraffin wax embedded tissues using a spin cartridge system is described. Such DNAs were shown to be suitable for amplification by the polymerase chain reaction, which targeted two human papillomavirus genes and one globin fragment giving rise to products of 450, 150, and 110 base pairs, respectively. Different human tissues, stored for up to 20 years, were successfully amplified, demonstrating the usefulness of this very simple procedure for retrospective studies. PMID:9624421

  13. Comparative Study of Seven Commercial Kits for Human DNA Extraction from Urine Samples Suitable for DNA Biomarker-Based Public Health Studies

    PubMed Central

    El Bali, Latifa; Diman, Aurélie; Bernard, Alfred; Roosens, Nancy H. C.; De Keersmaecker, Sigrid C. J.

    2014-01-01

    Human genomic DNA extracted from urine could be an interesting tool for large-scale public health studies involving characterization of genetic variations or DNA biomarkers as a result of the simple and noninvasive collection method. These studies, involving many samples, require a rapid, easy, and standardized extraction protocol. Moreover, for practicability, there is a necessity to collect urine at a moment different from the first void and to store it appropriately until analysis. The present study compared seven commercial kits to select the most appropriate urinary human DNA extraction procedure for epidemiological studies. DNA yield has been determined using different quantification methods: two classical, i.e., NanoDrop and PicoGreen, and two species-specific real-time quantitative (q)PCR assays, as DNA extracted from urine contains, besides human, microbial DNA also, which largely contributes to the total DNA yield. In addition, the kits giving a good yield were also tested for the presence of PCR inhibitors. Further comparisons were performed regarding the sampling time and the storage conditions. Finally, as a proof-of-concept, an important gene related to smoking has been genotyped using the developed tools. We could select one well-performing kit for the human DNA extraction from urine suitable for molecular diagnostic real-time qPCR-based assays targeting genetic variations, applicable to large-scale studies. In addition, successful genotyping was possible using DNA extracted from urine stored at −20°C for several months, and an acceptable yield could also be obtained from urine collected at different moments during the day, which is particularly important for public health studies. PMID:25365790

  14. Optimization design for selective extraction of size-fractioned DNA sample in microfabricated electrophoresis devices

    NASA Astrophysics Data System (ADS)

    Lin, Rongsheng; Burke, David T.; Burns, Mark A.

    2004-03-01

    In recent years, there has been tremendous interest in developing a highly integrated DNA analysis system using microfabrication techniques. With the success of incorporating sample injection, reaction, separation and detection onto a monolithic silicon device, addition of otherwise time-consuming components in macroworld such as sample preparation is gaining more and more attention. In this paper, we designed and fabricated a miniaturized device, capable of separating size-fractioned DNA sample and extracting the band of interest. In order to obtain pure target band, a novel technique utilizing shaping electric field is demonstrated. Both theoretical analysis and experimental data shows significant agreement in designing appropriate electrode structures to achieve the desired electric field distribution. This technique has a very simple fabrication procedure and can be readily added with other existing components to realize a highly integrated "lab-on-a-chip" system for DNA analysis.

  15. Sarcoptes mite from collection to DNA extraction: the lost realm of the neglected parasite.

    PubMed

    Alasaad, S; Rossi, L; Soriguer, R C; Rambozzi, L; Soglia, D; Pérez, J M; Zhu, X Q

    2009-03-01

    Sarcoptes mite from collection to DNA extraction forms the cornerstone for studies on Sarcoptes scabiei. Whilst the new science era took a shy leap into the different facets of mite studies, the cornerstone was almost entirely neglected. Mite collection, cleaning, storage and DNA extraction were, basically, humble attempts to extrapolate, adapt, modify or 'pirate' those existing methods to the peculiarities of Sarcoptes research. These aspects usually constituted few lines, bashfully mentioned, in the materials and methods section of some papers, which arose in unique problems concerning cost-effectiveness, time profitability, safety and even worse, the credibility of the results, creating contradictory conclusions in some cases. This 'noisy' situation encouraged us to collect, classify and review, for the first time to our knowledge, some aspects relating to studies on Sarcoptes mite from collection to DNA extraction, which will be useful for further studies on Sarcoptes, and have implications for the effective control of the diseases Sarcoptes mite causes. Further studies are needed, especially to compare the profitability, safety, sensibility and specificity of the different methods of this neglected realm of the ubiquitous ectoparasite. PMID:19159955

  16. Evaluation of simplified DNA extraction methods for emm typing of group A streptococci.

    PubMed

    Jose, J J M; Brahmadathan, K N

    2006-04-01

    Simplified methods of DNA extraction for amplification and sequencing for emm typing of group A streptococci (GAS) can save valuable time and cost in resource crunch situations. To evaluate this, we compared two methods of DNA extraction directly from colonies with the standard CDC cell lysate method for emm typing of 50 GAS strains isolated from children with pharyngitis and impetigo. For this, GAS colonies were transferred into two sets of PCR tubes. One set was preheated at 94 degrees C for two minutes in the thermal cycler and cooled while the other set was frozen overnight at -20 degrees C and then thawed before adding the PCR mix. For the cell lysate method, cells were treated with mutanolysin and hyaluronidase before heating at 100 degrees C for 10 minutes and cooling immediately as recommended in the CDC method. All 50 strains could be typed by sequencing the hyper variable region of the emm gene after amplification. The quality of sequences and the emm types identified were also identical. Our study shows that the two simplified DNA extraction methods directly from colonies can conveniently be used for typing a large number of GAS strains easily in relatively short time. PMID:16687865

  17. DNA extraction-free quantification of Dehalococcoides spp. in groundwater using a hand-held device.

    PubMed

    Stedtfeld, Robert D; Stedtfeld, Tiffany M; Kronlein, Maggie; Seyrig, Gregoire; Steffan, Robert J; Cupples, Alison M; Hashsham, Syed A

    2014-12-01

    Nucleic acid amplification of biomarkers is increasingly used to measure microbial activity and predict remedial performance in sites with trichloroethene (TCE) contamination. Field-based genetic quantification of microorganisms associated with bioremediation may help increase accuracy that is diminished through transport and processing of groundwater samples. Sterivex cartridges and a previously undescribed mechanism for eluting biomass was used to concentrate cells. DNA extraction-free loop mediated isothermal amplification (LAMP) was monitored in real-time with a point of use device (termed Gene-Z). A detection limit of 10(5) cells L(–1) was obtained, corresponding to sensitivity between 10 to 100 genomic copies per reaction for assays targeting the Dehalococcoides spp. specific 16S rRNA gene and vcrA gene, respectively. The quantity of Dehalococcoides spp. genomic copies measured from two TCE contaminated groundwater samples with conventional means of quantification including filtration, DNA extraction, purification, and qPCR was comparable to the field ready technique. Overall, this method of measuring Dehalococcoides spp. and vcrA genes in groundwater via direct amplification without intentional DNA extraction and purification is demonstrated, which may provide a more accurate mechanism of predicting remediation rates. PMID:25360694

  18. DNA Fingerprinting in a Forensic Teaching Experiment

    ERIC Educational Resources Information Center

    Wagoner, Stacy A.; Carlson, Kimberly A.

    2008-01-01

    This article presents an experiment designed to provide students, in a classroom laboratory setting, a hands-on demonstration of the steps used in DNA forensic analysis by performing DNA extraction, DNA fingerprinting, and statistical analysis of the data. This experiment demonstrates how DNA fingerprinting is performed and how long it takes. It…

  19. A simple and efficient method for extraction of PCR-amplifiable DNA from chicken eggshells.

    PubMed

    Rikimaru, Kazuhiro; Takahashi, Hideaki

    2009-04-01

    Recently, we reported a method for discriminating a Japanese brand of chicken, the Hinai-jidori. As an application of this method for discriminating Hinai-jidori eggs, we here report an efficient method for extracting maternal DNA from eggshells. Eggshell powder was completely decalcified with EDTA solution, and then DNA was isolated by conventional phenol-chloroform extraction and ethanol precipitation. The efficiency of DNA recovery from eggshells was 50-fold higher than that of a previously reported method. The recovered DNA could be used for PCR, and 10 markers for identifying the Hinai-jidori chicken were detected. The genotypes of the Hinai-jidori exactly matched those of the Hinai-dori breed. Using this method, Hinai-jidori and Hinai-dori eggs could be distinguished from the eggs of Rhode Island Reds. This is the first report of a technique that can be used to discriminate the eggs of Hinai-jidori from those of other chickens, and it can also be utilized to validate the labeling of Hinai-jidori eggs in the market. PMID:20163594

  20. The beyond 12/23 restriction is imposed at the nicking and pairing steps of DNA cleavage during V(D)J recombination.

    PubMed

    Drejer-Teel, Anna H; Fugmann, Sebastian D; Schatz, David G

    2007-09-01

    The beyond 12/23 (B12/23) rule ensures inclusion of a Dbeta gene segment in the assembled T-cell receptor (TCR) beta variable region exon and is manifest by a failure of direct Vbeta-to-Jbeta gene segment joining. The restriction is enforced during the DNA cleavage step of V(D)J recombination by the recombination-activating gene 1 and 2 (RAG1/2) proteins and the recombination signal sequences (RSSs) flanking the TCRbeta gene segments. Nothing is known about the step(s) at which DNA cleavage is defective or how TCRbeta locus sequences contribute to these defects. To address this, we examined the steps of DNA cleavage by the RAG proteins using TCRbeta locus V, D, and J RSS oligonucleotide substrates. The results demonstrate that the B12/23 rule is enforced through slow nicking of Jbeta substrates and to some extent through poor synapsis of Vbeta and Jbeta substrates. Nicking is controlled largely by the coding flank and, unexpectedly, the RSS spacer, while synapsis is controlled primarily by the RSS nonamer. The results demonstrate that different Jbeta substrates are crippled at different steps of cleavage by distinct combinations of defects in the various DNA elements and strongly suggest that the DNA nicking step of V(D)J recombination can be rate limiting in vivo. PMID:17636023

  1. The Beyond 12/23 Restriction Is Imposed at the Nicking and Pairing Steps of DNA Cleavage during V(D)J Recombination▿

    PubMed Central

    Drejer-Teel, Anna H.; Fugmann, Sebastian D.; Schatz, David G.

    2007-01-01

    The beyond 12/23 (B12/23) rule ensures inclusion of a Dβ gene segment in the assembled T-cell receptor (TCR) β variable region exon and is manifest by a failure of direct Vβ-to-Jβ gene segment joining. The restriction is enforced during the DNA cleavage step of V(D)J recombination by the recombination-activating gene 1 and 2 (RAG1/2) proteins and the recombination signal sequences (RSSs) flanking the TCRβ gene segments. Nothing is known about the step(s) at which DNA cleavage is defective or how TCRβ locus sequences contribute to these defects. To address this, we examined the steps of DNA cleavage by the RAG proteins using TCRβ locus V, D, and J RSS oligonucleotide substrates. The results demonstrate that the B12/23 rule is enforced through slow nicking of Jβ substrates and to some extent through poor synapsis of Vβ and Jβ substrates. Nicking is controlled largely by the coding flank and, unexpectedly, the RSS spacer, while synapsis is controlled primarily by the RSS nonamer. The results demonstrate that different Jβ substrates are crippled at different steps of cleavage by distinct combinations of defects in the various DNA elements and strongly suggest that the DNA nicking step of V(D)J recombination can be rate limiting in vivo. PMID:17636023

  2. Qualitative and quantitative evaluation of the genomic DNA extracted from GMO and non-GMO foodstuffs with four different extraction methods.

    PubMed

    Peano, Clelia; Samson, Maria Cristina; Palmieri, Luisa; Gulli, Mariolina; Marmiroli, Nelson

    2004-11-17

    The presence of DNA in foodstuffs derived from or containing genetically modified organisms (GMO) is the basic requirement for labeling of GMO foods in Council Directive 2001/18/CE (Off. J. Eur. Communities 2001, L1 06/2). In this work, four different methods for DNA extraction were evaluated and compared. To rank the different methods, the quality and quantity of DNA extracted from standards, containing known percentages of GMO material and from different food products, were considered. The food products analyzed derived from both soybean and maize and were chosen on the basis of the mechanical, technological, and chemical treatment they had been subjected to during processing. Degree of DNA degradation at various stages of food production was evaluated through the amplification of different DNA fragments belonging to the endogenous genes of both maize and soybean. Genomic DNA was extracted from Roundup Ready soybean and maize MON810 standard flours, according to four different methods, and quantified by real-time Polymerase Chain Reaction (PCR), with the aim of determining the influence of the extraction methods on the DNA quantification through real-time PCR. PMID:15537304

  3. RecA-dependent programmable endonuclease Ref cleaves DNA in two distinct steps

    PubMed Central

    Ronayne, Erin A.; Cox, Michael M.

    2014-01-01

    The bacteriophage P1 recombination enhancement function (Ref) protein is a RecA-dependent programmable endonuclease. Ref targets displacement loops formed when an oligonucleotide is bound by a RecA filament and invades homologous double-stranded DNA sequences. Mechanistic details of this reaction have been explored, revealing that (i) Ref is nickase, cleaving the two target strands of a displacement loop sequentially, (ii) the two strands are cleaved in a prescribed order, with the paired strand cut first and (iii) the two cleavage events have different requirements. Cutting the paired strand is rapid, does not require RecA-mediated ATP hydrolysis and is promoted even by Ref active site variant H153A. The displaced strand is cleaved much more slowly, requires RecA-mediated ATP hydrolysis and does not occur with Ref H153A. The two cleavage events are also affected differently by solution conditions. We postulate that the second cleavage (displaced strand) is limited by some activity of RecA protein. PMID:24371286

  4. DNA Extraction Protocol for Plants with High Levels of Secondary Metabolites and Polysaccharides without Using Liquid Nitrogen and Phenol

    PubMed Central

    Sahu, Sunil Kumar; Thangaraj, Muthusamy; Kathiresan, Kandasamy

    2012-01-01

    Mangroves and salt marsh species are known to synthesize a wide spectrum of polysaccharides and polyphenols including flavonoids and other secondary metabolites which interfere with the extraction of pure genomic DNA. Although a plethora of plant DNA isolation protocols exist, extracting DNA from mangroves and salt marsh species is a challenging task. This study describes a rapid and reliable cetyl trimethylammonium bromide (CTAB) protocol suited specifically for extracting DNA from plants which are rich in polysaccharides and secondary metabolites, and the protocol also excludes the use of expensive liquid nitrogen and toxic phenols. Purity of extracted DNA was excellent as evident by A260/A280 ratio ranging from 1.78 to 1.84 and A260/A230 ratio was >2, which also suggested that the preparations were sufficiently free of proteins and polyphenolics/polysaccharide compounds. DNA concentration ranged from 8.8 to 9.9 μg μL−1. The extracted DNA was amenable to RAPD, restriction digestion, and PCR amplification of plant barcode genes (matK and rbcl). The optimized method is suitable for both dry and fresh leaves. The success of this method in obtaining high-quality genomic DNA demonstrated the broad applicability of this method. PMID:27335662

  5. Modified salting-out method: high-yield, high-quality genomic DNA extraction from whole blood using laundry detergent.

    PubMed

    Nasiri, H; Forouzandeh, M; Rasaee, M J; Rahbarizadeh, F

    2005-01-01

    Different approaches have been used to extract DNA from whole blood. In most of these methods enzymes (such as proteinase K and RNAse A) or toxic organic solvents (such as phenol or guanidine isothiocyanate) are used. Since these enzymes are expensive, and most of the materials that are used routinely are toxic, it is desirable to apply an efficient DNA extraction procedure that does not require the use of such materials. In this study, genomic DNA was extracted by the salting-out method, but instead of using an analytical-grade enzyme and chemical detergents, as normally used for DNA isolation, a common laundry powder was used. Different concentrations of the powder were tested, and proteins were precipitated by NaCl-saturated distilled water. Finally, DNA precipitation was performed with the use of 96% ethanol. From the results, we conclude that the optimum concentration of laundry powder for the highest yield and purity of isolated DNA is 30 mg/mL. The procedure was optimized, and a final protocol is suggested. Following the same protocol, DNA was extracted from 100 blood samples, and their amounts were found to be >50 microg/mL of whole blood. The integrity of the DNA fragments was confirmed by agarose gel electrophoresis. Furthermore, the extracted DNA was used as a template for PCR reaction. The results obtained from PCR showed that the final solutions of extracted DNA did not contain any inhibitory material for the enzyme used in the PCR reaction, and indicated that the isolated DNA was of good quality. These results show that this method is simple, fast, safe, and cost-effective, and can be used in medical laboratories and research centers. PMID:16302208

  6. Extraction of helium from individual interplanetary dust particles by step-heating

    NASA Astrophysics Data System (ADS)

    Nier, A. O.; Schlutter, D. J.

    1992-06-01

    Fragments from 20 individual particles, collected in the earth's stratosphere and believed to be interplanetary dust particles (IDPs), were subjected to step-heating to see if differences in the release pattern for He-4 could be observed which might provide clues to the origin of the particles. Comparisons were made to the release pattern for 18 individual lunar surface grains heated in the same manner. Twelve of the IDP fragments contained an appreciable amount of He-4, 50 percent of which was released by the time the particles were heated to approximately 630 C. For the 18 individual lunar grains, the corresponding average temperature was 660 C. The He-3/He-4 ratios found for these fragments agreed well with those found for deep Pacific magnetic fines believed to be of extraterrestrial origin, and were comparable to those which have been observed for the solar wind and lunar surface soil grains. Four of the IDP fragments contained appreciably less He-4, and this was released at a higher temperature. The remaining four fragments had too little He-4 to permit a determination. From Flynn's analyses of the problem of the heating of IDPs in their descent in the atmosphere, the present results suggest that the parent IDPs of the 12 particles which contained an appreciable amount of He-4 suffered very little heating in their descent and are likely of asteroidal origin, although one cannot rule out the possibility that at least some of them had a cometary origin and entered the earth's atmosphere at a grazing angle.

  7. Improving Molecular Detection of Fungal DNA in Formalin-Fixed Paraffin-Embedded Tissues: Comparison of Five Tissue DNA Extraction Methods Using Panfungal PCR▿

    PubMed Central

    Muñoz-Cadavid, C.; Rudd, S.; Zaki, S. R.; Patel, M.; Moser, S. A.; Brandt, M. E.; Gómez, B. L.

    2010-01-01

    DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues is difficult and requires special protocols in order to extract small amounts of DNA suitable for amplification. Most described methods report an amplification success rate between 60 and 80%; therefore, there is a need to improve molecular detection and identification of fungi in FFPE tissue. Eighty-one archived FFPE tissues with a positive Gomori methenamine silver (GMS) stain were evaluated using five different commercial DNA extraction kits with some modifications. Three different panfungal PCR assays were used to detect fungal DNA, and two housekeeping genes were used to assess the presence of amplifiable DNA and to detect PCR inhibitors. The sensitivities of the five extraction protocols were compared, and the quality of DNA detection (calculated for each kit as the number of housekeeping gene PCR-positive samples divided by the total number of samples) was 60 to 91% among the five protocols. The efficiencies of the three different panfungals used (calculated as the number of panfungal-PCR-positive samples divided by the number of housekeeping gene PCR-positive samples) were 58 to 93%. The panfungal PCR using internal transcribed spacer 3 (ITS3) and ITS4 primers yielded a product in most FFPE tissues. Two of the five DNA extraction kits (from TaKaRa and Qiagen) showed similar and promising results. However, one method (TaKaRa) could extract fungal DNA from 69 of the 74 FFPE tissues from which a housekeeping gene could be amplified and was also cost-effective, with a nonlaborious protocol. Factors such as sensitivity, cost, and labor will help guide the selection of the most appropriate method for the needs of each laboratory. PMID:20392915

  8. Protective effects of black tea extract against oxidative DNA damage in human lymphocytes.

    PubMed

    Ježovičová, Miriam; Koňariková, Katarína; Ďuračková, Zdeňka; Keresteš, Ján; Králik, Gabriel; Žitňanová, Ingrid

    2016-02-01

    The aim of the present study was to examine the genoprotective and radioprotective effects of black tea extract (BTE) against the induction of single strand DNA breaks in human lymphocytes subjected to hydrogen peroxide (H2O2) or gamma-rays (2 Gy dose). Lymphocytes were incubated with or without different concentrations of BTE (0.005-500 µg/ml) for 30 min, followed by treatment with or without H2O2 (0.088 µmol/l) for 5 min. To examine the radioprotective effect of BTE, the lymphocytes were incubated with or without BTE for 30 and 60 min prior to and following in vitro irradiation. Oxidative damage to DNA was monitored using a comet assay. BTE at lower concentrations prevented H2O2-induced DNA damage. An increase in BTE concentrations resulted in increased formation of single strand DNA breaks. BTE also exerted significant protective effects against gamma radiation-induced total DNA damage in healthy lymphocytes during their 30 or 60 min incubation with BTE prior to or following irradiation. Therefore, the protective effect of BTE against irradiation was time-dependent. The results contribute to the research on potential beneficial effects of natural compounds, such as BTE, in cancer and its protective effects of normal tissue during radiation therapy. PMID:26718244

  9. Sources of Pre-Analytical Variations in Yield of DNA Extracted from Blood Samples: Analysis of 50,000 DNA Samples in EPIC

    PubMed Central

    Caboux, Elodie; Lallemand, Christophe; Ferro, Gilles; Hémon, Bertrand; Mendy, Maimuna; Biessy, Carine; Sims, Matt; Wareham, Nick; Britten, Abigail; Boland, Anne; Hutchinson, Amy; Siddiq, Afshan; Vineis, Paolo; Riboli, Elio; Romieu, Isabelle; Rinaldi, Sabina; Gunter, Marc J.; Peeters, Petra H. M.; van der Schouw, Yvonne T.; Travis, Ruth; Bueno-de-Mesquita, H. Bas; Canzian, Federico; Sánchez, Maria-José; Skeie, Guri; Olsen, Karina Standahl; Lund, Eiliv; Bilbao, Roberto; Sala, Núria; Barricarte, Aurelio; Palli, Domenico; Navarro, Carmen; Panico, Salvatore; Redondo, Maria Luisa; Polidoro, Silvia; Dossus, Laure; Boutron-Ruault, Marie Christine; Clavel-Chapelon, Françoise; Trichopoulou, Antonia; Trichopoulos, Dimitrios; Lagiou, Pagona; Boeing, Heiner; Fisher, Eva; Tumino, Rosario; Agnoli, Claudia; Hainaut, Pierre

    2012-01-01

    The European Prospective Investigation into Cancer and nutrition (EPIC) is a long-term, multi-centric prospective study in Europe investigating the relationships between cancer and nutrition. This study has served as a basis for a number of Genome-Wide Association Studies (GWAS) and other types of genetic analyses. Over a period of 5 years, 52,256 EPIC DNA samples have been extracted using an automated DNA extraction platform. Here we have evaluated the pre-analytical factors affecting DNA yield, including anthropometric, epidemiological and technical factors such as center of subject recruitment, age, gender, body-mass index, disease case or control status, tobacco consumption, number of aliquots of buffy coat used for DNA extraction, extraction machine or procedure, DNA quantification method, degree of haemolysis and variations in the timing of sample processing. We show that the largest significant variations in DNA yield were observed with degree of haemolysis and with center of subject recruitment. Age, gender, body-mass index, cancer case or control status and tobacco consumption also significantly impacted DNA yield. Feedback from laboratories which have analyzed DNA with different SNP genotyping technologies demonstrate that the vast majority of samples (approximately 88%) performed adequately in different types of assays. To our knowledge this study is the largest to date to evaluate the sources of pre-analytical variations in DNA extracted from peripheral leucocytes. The results provide a strong evidence-based rationale for standardized recommendations on blood collection and processing protocols for large-scale genetic studies. PMID:22808065

  10. EVALUATION OF DIFFERENT METHODS FOR THE EXTRACTION OF DNA FROM FUNGAL CONIDIA BY QUANTITATIVE COMPETITIVE PCR ANALYSIS

    EPA Science Inventory

    Five different DNA extraction methods were evaluated for their effectiveness in recovering PCR templates from the conidia of a series of fungal species often encountered in indoor air. The test organisms were Aspergillus versicolor, Penicillium chrysogenum, Stachybotrys chartaru...

  11. Trace DNA from insect skins: a comparison of five extraction protocols and direct PCR on chironomid pupal exuviae.

    PubMed

    Kranzfelder, Petra; Ekrem, Torbjørn; Stur, Elisabeth

    2016-01-01

    Insect skins (exuviae) are of extracellular origin and shed during moulting. The skins do not contain cells or DNA themselves, but epithelial cells and other cell-based structures might accidentally attach as they are shed. This source of trace DNA can be sufficient for PCR amplification and sequencing of target genes and aid in species identification through DNA barcoding or association of unknown life stages. Species identification is essential for biomonitoring programs, as species vary in sensitivities to environmental factors. However, it requires a DNA isolation protocol that optimizes the output of target DNA. Here, we compare the relative effectiveness of five different DNA extraction protocols and direct PCR in isolation of DNA from chironomid pupal exuviae. Chironomidae (Diptera) is a species-rich group of aquatic macroinvertebrates widely distributed in freshwater environments and considered a valuable bioindicator of water quality. Genomic DNA was extracted from 61.2% of 570 sampled pupal exuviae. There were significant differences in the methods with regard to cost, handling time, DNA quantity, PCR success, sequence success and the ability to sequence target taxa. The NucleoSpin(®) Tissue XS Kit, DNeasy(®) Blood and Tissue kit, and QuickExtract(™) DNA Extraction Solution provided the best results in isolating DNA from single pupal exuviae. Direct PCR and DTAB/CTAB methods gave poor results. While the observed differences in DNA isolation methods on trace DNA will be relevant to research that focuses on aquatic macroinvertebrate ecology, taxonomy and systematics, they should also be of interest for studies using environmental barcoding and metabarcoding of aquatic environments. PMID:26186122

  12. Transient kinetics measured with force steps discriminate between double-stranded DNA elongation and melting and define the reaction energetics.

    PubMed

    Bongini, Lorenzo; Melli, Luca; Lombardi, Vincenzo; Bianco, Pasquale

    2014-03-01

    Under a tension of ∼65 pN, double-stranded DNA undergoes an overstretching transition from its basic (B-form) conformation to a 1.7 times longer conformation whose nature is only recently starting to be understood. Here we provide a structural and thermodynamic characterization of the transition by recording the length transient following force steps imposed on the λ-phage DNA with different melting degrees and temperatures (10-25°C). The shortening transient following a 20-35 pN force drop from the overstretching force shows a sequence of fast shortenings of double-stranded extended (S-form) segments and pauses owing to reannealing of melted segments. The lengthening transients following a 2-35 pN stretch to the overstretching force show the kinetics of a two-state reaction and indicate that the whole 70% extension is a B-S transition that precedes and is independent of melting. The temperature dependence of the lengthening transient shows that the entropic contribution to the B-S transition is one-third of the entropy change of thermal melting, reinforcing the evidence for a double-stranded S-form that maintains a significant fraction of the interstrand bonds. The cooperativity of the unitary elongation (22 bp) is independent of temperature, suggesting that structural factors, such as the nucleic acid sequence, control the transition. PMID:24353317

  13. An easy, rapid, and cost-effective method for DNA extraction from various lichen taxa and specimens suitable for analysis of fungal and algal strains.

    PubMed

    Park, Sook-Young; Jang, Seol-Hwa; Oh, Soon-Ok; Kim, Jung A; Hur, Jae-Seoun

    2014-12-01

    Lichen studies, including biodiversity, phylogenetic relationships, and conservation concerns require definitive species identification, however many lichens can be challenging to identify at the species level. Molecular techniques have shown efficacy in discriminating among lichen taxa, however, obtaining genomic DNA from herbarium and fresh lichen thalli by conventional methods has been difficult, because lichens contain high proteins, polysaccharides, and other complex compounds in their cell walls. Here we report a rapid, easy, and inexpensive protocol for extracting PCR-quality DNA from various lichen species. This method involves the following two steps: first, cell breakage using a beadbeater; and second, extraction, isolation, and precipitation of genomic DNA. The procedure requires approximately 10 mg of lichen thalli and can be completed within 20 min. The obtained DNAs were of sufficient quality and quantity to amplify the internal transcribed spacer region from the fungal and algal lichen components, as well as to sequence the amplified products. In addition, 26 different lichen taxa were tested, resulting in successful PCR products. The results of this study validated the experimental protocols, and clearly demonstrated the efficacy and value of our KCl extraction method applied in the fungal and algal samples. PMID:25606001

  14. An Easy, Rapid, and Cost-Effective Method for DNA Extraction from Various Lichen Taxa and Specimens Suitable for Analysis of Fungal and Algal Strains

    PubMed Central

    Park, Sook-Young; Jang, Seol-Hwa; Oh, Soon-Ok; Kim, Jung A

    2014-01-01

    Lichen studies, including biodiversity, phylogenetic relationships, and conservation concerns require definitive species identification, however many lichens can be challenging to identify at the species level. Molecular techniques have shown efficacy in discriminating among lichen taxa, however, obtaining genomic DNA from herbarium and fresh lichen thalli by conventional methods has been difficult, because lichens contain high proteins, polysaccharides, and other complex compounds in their cell walls. Here we report a rapid, easy, and inexpensive protocol for extracting PCR-quality DNA from various lichen species. This method involves the following two steps: first, cell breakage using a beadbeater; and second, extraction, isolation, and precipitation of genomic DNA. The procedure requires approximately 10 mg of lichen thalli and can be completed within 20 min. The obtained DNAs were of sufficient quality and quantity to amplify the internal transcribed spacer region from the fungal and algal lichen components, as well as to sequence the amplified products. In addition, 26 different lichen taxa were tested, resulting in successful PCR products. The results of this study validated the experimental protocols, and clearly demonstrated the efficacy and value of our KCl extraction method applied in the fungal and algal samples. PMID:25606001

  15. Extraction of DNA from orange juice, and detection of bacterium Candidatus Liberibacter asiaticus by real-time PCR.

    PubMed

    Bai, Jinhe; Baldwin, Elizabeth; Liao, Hui-Ling; Zhao, Wei; Kostenyuk, Igor; Burns, Jacqueline; Irey, Mike

    2013-10-01

    Orange juice processed from Huanglongbing (HLB) affected fruit is often associated with bitter taste and/or off-flavor. HLB disease in Florida is associated with Candidatus Liberibacter asiaticus (CLas), a phloem-limited bacterium. The current standard to confirm CLas for citrus trees is to take samples from midribs of leaves, which are rich in phloem tissues, and use a quantitative real-time polymerase chain reaction (qPCR) test to detect the 16S rDNA gene of CLas. It is extremely difficult to detect CLas in orange juice because of the low CLas population, high sugar and pectin concentration, low pH, and possible existence of an inhibitor to DNA amplification. The objective of this research was to improve extraction of DNA from orange juice and detection of CLas by qPCR. Homogenization using a sonicator increased DNA yield by 86% in comparison to mortar and pestle extraction. It is difficult to separate DNA from pectin; however, DNA was successfully extracted by treating the juice with pectinase. Application of an elution column successfully removed the unidentified inhibitor to DNA amplification. This work provided a protocol to extract DNA from whole orange juice and detect CLas in HLB-affected fruit. PMID:24047134

  16. COMPARISON OF RAPID METHODS FOR THE EXTRACTION OF BACTERIAL DNA FROM COLONIC AND CECAL LUMEN CONTENTS OF THE PIG

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The increasing use of DNA methodologies to study the microflora of the pig gastrointestinal tract requires an efficient recovery of bacterial DNA from intestinal samples. Thus, the objective of this study was to determine which extraction methods are most effective for colonic and cecal lumen sampl...

  17. The Extraction and Partial Purification of Bacterial DNA as a Practical Exercise for GCE Advanced Level Students.

    ERIC Educational Resources Information Center

    Falconer, A. C.; Hayes, L. J.

    1986-01-01

    Describes a relatively simple method of extraction and purification of bacterial DNA. This technique permits advanced secondary-level science students to obtain adequate amounts of DNA from very small pellets of bacteria and to observe some of its polymer properties. (ML)

  18. Single step aqueous two-phase extraction for downstream processing of C-phycocyanin from Spirulina platensis.

    PubMed

    Chethana, S; Nayak, Chetan A; Madhusudhan, M C; Raghavarao, K S M S

    2015-04-01

    C-phycocyanin, a natural food colorant, is gaining importance worldwide due to its several medical and pharmaceutical applications. In the present study, aqueous two-phase extraction was shown to be an attractive alternative for the downstream processing of C-phycocyanin from Spirulina platensis. By employing differential partitioning, C-phycocyanin selectively partitioned to the polymer rich (top) phase in concentrated form and contaminant proteins to the salt rich (bottom) phase. This resulted in an increase in the product purity (without losing much of the yield) in a single step without the need of multiple processing steps. Effect of process parameters such as molecular weight, tie line length, phase volume ratio, concentration of phase components on the partitioning behavior of C-phycocyanin was studied. The results were explained based on relative free volume of the phase systems. C-phycocyanin with a purity of 4.32 and yield of about 79 % was obtained at the standardized conditions. PMID:25829627

  19. Non-destructive genetic sampling in fish. An improved method for DNA extraction from fish fins and scales.

    PubMed

    Wasko, Adriane P; Martins, Cesar; Oliveira, Claudio; Foresti, Fausto

    2003-01-01

    DNA-based studies have been one of the major interests in conservation biology of endangered species and in population genetics. As species and population genetic assessment requires a source of biological material, the sampling strategy can be overcome by non-destructive procedures for DNA isolation. An improved method for obtaining DNA from fish fins and scales with the use of an extraction buffer containing urea and further DNA purification with phenol-chloroform is described. The methodology combines the benefits of a non-destructive DNA sampling and its high efficiency. In addition, comparisons with other methodologies for isolating DNA from fish demonstrated that the present procedure also becomes a very attractive alternative to obtain large amounts of high-quality DNA for use in different molecular analyses. The DNA samples, isolated from different fish species, have been successfully used on random amplified polymorphic DNA (RAPD) experiments, as well as on amplification of specific ribosomal and mitochondrial DNA sequences. The present DNA extraction procedure represents an alternative for population approaches and genetic studies on rare or endangered taxa. PMID:14641478

  20. A Two-Step Nanofiltration Process for the Production of Phenolic-Rich Fractions from Artichoke Aqueous Extracts

    PubMed Central

    Cassano, Alfredo; Conidi, Carmela; Ruby Figueroa, René; Castro Muñoz, Roberto

    2015-01-01

    Commercial nanofiltration (NF) membranes in spiral-wound configuration (NP030 from Microdyn Nadir and Desal DK from GE Water & Process Technologies) were used in a sequential design in order to produce a separated fraction of phenolic and sugar compounds from an aqueous artichoke extract. For both membranes, the effect of transmembrane pressure (TMP) on the permeation flux was evaluated. In optimized conditions of TMP, the NP030 membrane exhibited high rejections of apigenin, cynarin and chlorogenic acid (higher than 85%); on the other hand, very low rejections of fructose, glucose and sucrose (lower than 4%) were measured. Starting from an extract with a total antioxidant activity (TAA) of 5.28 mM trolox a retentate fraction with a TAA of 47.75 mM trolox was obtained. The NF permeate from the NP030 membrane was processed with the Desal DK membrane in optimized conditions of TMP producing a permeate stream free of phenolic and sugar compounds. Accordingly, as most part of phenolic compounds was removed in the first NF step, the concentration of sugar compounds in the NF retentate had much higher results than that of phenolic compounds. PMID:25913377

  1. A two-step nanofiltration process for the production of phenolic-rich fractions from artichoke aqueous extracts.

    PubMed

    Cassano, Alfredo; Conidi, Carmela; Figueroa, René Ruby; Muñoz, Roberto Castro

    2015-01-01

    Commercial nanofiltration (NF) membranes in spiral-wound configuration (NP030 from Microdyn Nadir and Desal DK from GE Water & Process Technologies) were used in a sequential design in order to produce a separated fraction of phenolic and sugar compounds from an aqueous artichoke extract. For both membranes, the effect of transmembrane pressure (TMP) on the permeation flux was evaluated. In optimized conditions of TMP, the NP030 membrane exhibited high rejections of apigenin, cynarin and chlorogenic acid (higher than 85%); on the other hand, very low rejections of fructose, glucose and sucrose (lower than 4%) were measured. Starting from an extract with a total antioxidant activity (TAA) of 5.28 mM trolox a retentate fraction with a TAA of 47.75 mM trolox was obtained. The NF permeate from the NP030 membrane was processed with the Desal DK membrane in optimized conditions of TMP producing a permeate stream free of phenolic and sugar compounds. Accordingly, as most part of phenolic compounds was removed in the first NF step, the concentration of sugar compounds in the NF retentate had much higher results than that of phenolic compounds. PMID:25913377

  2. Inhibitory Effects of Bangladeshi Medicinal Plant Extracts on Interactions between Transcription Factors and Target DNA Sequences

    PubMed Central

    Lampronti, Ilaria; Khan, Mahmud T.H.; Borgatti, Monica; Bianchi, Nicoletta

    2008-01-01

    Several transcription factors (TFs) play crucial roles in governing the expression of different genes involved in the immune response, embryo or cell lineage development, cell apoptosis, cell cycle progression, oncogenesis, repair and fibrosis processes and inflammation. As far as inflammation, TFs playing pivotal roles are nuclear factor kappa B (NF-kB), activator protein (AP-1), signal transducer and activator of transcription (STATs), cAMP response element binding protein (CREB) and GATA-1 factors. All these TFs regulate the expression of pro-inflammatory cytokines and are involved in the pathogenesis of a number of human disorders, particularly those with an inflammatory component. Since several medicinal plants can be employed to produce extracts exhibiting biological effects and because alteration of gene transcription represents a very interesting approach to control the expression of selected genes, this study sought to verify the ability of several extracts derived from Bangladeshi medicinal plants in interfering with molecular interactions between different TFs and specific DNA sequences. We first analyzed the antiproliferative activity of 19 medicinal plants on different human cell lines, including erythroleukemia K562, B lymphoid Raji and T lymphoid Jurkat cell lines. Secondly, we employed the electrophoretic mobility shift assay as a suitable technique for a fast screening of plant extracts altering the binding between NF-kB, AP-1, GATA-1, STAT-3, CREB and the relative target DNA elements. PMID:18830455

  3. A one-step extraction procedure for the screening of cocaine, amphetamines and cannabinoids in postmortem blood samples.

    PubMed

    Pelição, Fabrício Souza; Peres, Mariana Dadalto; Pissinate, Jauber Fornaciari; De Martinis, Bruno Spinosa

    2014-01-01

    A gas chromatography-mass spectrometric (GC-MS) method was developed and validated for the simultaneous detection and quantification in postmortem whole blood samples of cocaine (COC), amphetamines (AMPs) and cannabis; the main drugs involved in cases of impaired driving in Brazil. The analytes were extracted by solid-phase extraction by means of Bond-Elute Certify cartridges, derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide at 80°C for 30 min and analyzed by GC-MS. Linearity ranged from 10 to 500 ng/mL, except for ecgonine methyl ester, for which linearity ranged from 10 to 100 ng/mL. Inter- and intra-day imprecision ranged from 2.8 to 18.4% and from 1.5 to 14.9%, respectively. Accuracy values lay between 86.9 and 104.4%. The limit of quantitation for all drugs was 10 ng/mL and recoveries were >74% for all analytes, except for cannabinoids, which showed poor recovery (∼30%). The developed method was applied to real samples collected from deceased victims due to traffic accidents. These samples were selected according to the results obtained in immunoassay screening on collected urine samples. Five samples were positive for the presence of COC and metabolites, four samples were positive for cannabinoids, six samples were positive for AMPs and two samples were drug negative. Some samples were positive for more than one class of drug. Results obtained from whole blood samples showed good agreement with urine screening. The developed method proved capable of quantifying all three classes of drugs of abuse proposed in this study, through a one-step extraction procedure. PMID:24782143

  4. Nanofluidic laboratory-on-chip device for mapping of single molecule DNA extracted from single cells

    NASA Astrophysics Data System (ADS)

    Mahshid, Sara; Berard, Daniel; Sladek, Robert; Leslie, Sabrina; Reisner, Walter

    2014-03-01

    The aim of this project is to create a nanofluidic platform to provide comprehensive maps of single-cell genomes at 1 kbp resolution based on the direct analysis of single 1-10 Mbp extended DNA molecules extracted from individual cells on-chip. We have developed a nanodevice in which all biochemical processing of single cells (cell lysis, DNA purification and fragmentation) is performed in situ. The platform has the following three components: (1) a micro-cavity (50 ×20 micron in dimension) for trapping and biochemical processing of single cells; (2) post arrays (1 micron depth) for untangling the released genomic contents and (3) parallel nanochannel arrays (100 nm) for extension of ~ 1-10 Mbp DNA for high-throughput optical mapping. Moreover, we use ``Convex Lense-Induced Nanoconfinement'' (CLIC) technique for trapping of single cell and dragging DNA into nanochannels. The principle is that a convex lens is pushed down to deform a flexible coverslip lid above the aforesaid platform containing nano/micro patterns, creating a locally confined region that pins molecules in the embedded nano/micro features. CLIC is used to lower the device lid over a cell isolated in the microcavity with an adjustable gap for buffer exchange. The released DNA is untangled using 1 micron-deep post arrays and driven into nanochannel array where its genomic content is revealed. In particular, using CLIC we were able to successfully trap 20 micron lymphoblast cells inside microcavity and lyse the trapped cell to drive out DNA.

  5. Optimization of DNA extraction and PCR protocols for phylogenetic analysis in Schinopsis spp. and related Anacardiaceae.

    PubMed

    Mogni, Virginia Y; Kahan, Mariano A; de Queiroz, Luciano Paganucci; Vesprini, José L; Ortiz, Juan Pablo A; Prado, Darién E

    2016-01-01

    The Anacardiaceae is an important and worldwide distributed family of ecological and socio-economic relevance. Notwithstanding that, molecular studies in this family are scarce and problematic because of the particularly high concentration of secondary metabolites-i.e. tannins and oleoresins-that are present in almost all tissues of the many members of the group, which complicate the purification and amplification of the DNA. The objective of this work was to improve an available DNA isolation method for Schinopsis spp. and other related Anacardiaceae, as well as the PCR protocols for DNA amplification of the chloroplast trnL-F, rps16 and ndhF and nuclear ITS-ETS fragments. The modifications proposed allowed the extraction of 70-120 µg of non-degraded genomic DNA per gram of dry tissue that resulted useful for PCR amplification. PCR reactions produced the expected fragments that could be directly sequenced. Sequence analyses of amplicons showed similarity with the corresponding Schinopsis accessions available at GenBank. The methodology presented here can be routinely applied for molecular studies of the group aimed to clarify not only aspects on the molecular biology but also the taxonomy and phylogeny of this fascinating group of vascular plants. PMID:27217992

  6. Protective Effects of Extracts from Fructus rhodomyrti against Oxidative DNA Damage In Vitro and In Vivo

    PubMed Central

    Ke, Yuebin; Xu, Xinyun; Wu, Shuang; Huang, Juan; Misra, Hara; Li, Yunbo

    2013-01-01

    Objective. To evaluate the potential protective effects of extracts from Fructus rhodomyrti (FR) against oxidative DNA damage using a cellular system and the antioxidant ability on potassium bromate- (KBrO3-) mediated oxidative stress in rats. Methods. The effects of FR on DNA damage induced by hydrogen peroxide (H2O2) were evaluated by comet assay in primary spleen lymphocytes cultures. The effects of FR on the activities of SOD, CAT, and GPx and the levels of GSH, hydroperoxides, and 8-OHdG were determined in the plasma and tissues of rats treated with KBrO3. Results. FR was shown to effectively protect against DNA damage induced by H2O2  in vitro, and the maximum protective effect was observed when FR was diluted 20 times. Endogenous antioxidant status, namely, the activities of SOD, CAT, and GPx and the levels of GSH were significantly decreased in the plasma, the liver, and the kidney of the KBrO3-treated rats, while the pretreatment of FR prevented the decreases of these parameters. In addition, the pretreatment of FR was also able to prevent KBrO3-induced increases in the levels of hydroperoxides and 8-OHdG in the plasma, the liver, and the kidney in rats. Conclusions. Our findings suggested that FR might act as a chemopreventive agent with antioxidant properties offering effective protection against oxidative DNA damage in a concentration-dependent manner in vitro and in vivo. PMID:24089629

  7. Ancient microbes from halite fluid inclusions: optimized surface sterilization and DNA extraction.

    PubMed

    Sankaranarayanan, Krithivasan; Timofeeff, Michael N; Spathis, Rita; Lowenstein, Tim K; Lum, J Koji

    2011-01-01

    Fluid inclusions in evaporite minerals (halite, gypsum, etc.) potentially preserve genetic records of microbial diversity and changing environmental conditions of Earth's hydrosphere for nearly one billion years. Here we describe a robust protocol for surface sterilization and retrieval of DNA from fluid inclusions in halite that, unlike previously published methods, guarantees removal of potentially contaminating surface-bound DNA. The protocol involves microscopic visualization of cell structures, deliberate surface contamination followed by surface sterilization with acid and bleach washes, and DNA extraction using Amicon centrifugal filters. Methods were verified on halite crystals of four different ages from Saline Valley, California (modern, 36 ka, 64 ka, and 150 ka), with retrieval of algal and archaeal DNA, and characterization of the algal community using ITS1 sequences. The protocol we developed opens up new avenues for study of ancient microbial ecosystems in fluid inclusions, understanding microbial evolution across geological time, and investigating the antiquity of life on earth and other parts of the solar system. PMID:21694765

  8. A novel reliable method of DNA extraction from olive oil suitable for molecular traceability.

    PubMed

    Raieta, Katia; Muccillo, Livio; Colantuoni, Vittorio

    2015-04-01

    Extra virgin olive oil production has a worldwide economic impact. The use of this brand, however, is of great concern to Institutions and private industries because of the increasing number of fraud and adulteration attempts to the market products. Here, we present a novel, reliable and not expensive method for extracting the DNA from commercial virgin and extra virgin olive oils. The DNA is stable overtime and amenable for molecular analyses; in fact, by carrying out simple sequence repeats (SSRs) markers analysis, we characterise the genetic profile of monovarietal olive oils. By comparing the oil-derived pattern with that of the corresponding tree, we can unambiguously identify four cultivars from Samnium, a region of Southern Italy, and distinguish them from reference and more widely used varieties. Through a parentage statistical analysis, we also identify the putative pollinators, establishing an unprecedented and powerful tool for olive oil traceability. PMID:25442596

  9. A novel approach on fluid dispensing for a DNA/RNA extraction chip package

    NASA Astrophysics Data System (ADS)

    Xie, Ling; Premachandran, C. S.; Chew, Michelle; Yao, Qiang; Xu, Diao; Pinjala, D.

    2008-02-01

    Micro fluidic package with integrated reservoirs has been developed for DNA /RNA extraction application. A membrane based pump which consists of a reservoir to store reagents and a pin valve to control the fluid is developed to dispense the reagents into the chip. A programmable external actuator is fabricated to dispense the fluid from the membrane pump into the DNA chip. An elastic and high elongation thin rubber membrane is used to seal the membrane pump and at the same time prevent actuator from mixing with different reagents in the micro fluidic package. Break displacement during actuation of membrane pump sealing material is studied with different ratios of PDMS and other types of rubber materials. The fluid flow from the reservoir to the chip is controlled by a pin valve which is activated during the external actuation. A CFD simulation is performed to study the pumping action dusting the external actuation and is validated with experimental results.

  10. Evaluation of antibacterial, antioxidant and DNA protective capacity of Chenopodium album's ethanolic leaf extract.

    PubMed

    Elif Korcan, S; Aksoy, Onur; Erdoğmuş, S Feyza; Ciğerci, İ Hakkı; Konuk, Muhsin

    2013-01-01

    We have investigated the antibacterial effects of Chenopodium album's ethanolic leaf extract (CAE) on all the Gram (+) and Gram (-) microorganisms and evaluated the protective effects of CAE on both yeast and human mononuclear leukocytes' genomic DNA upon oxidative shock. Antibacterial activity was recorded on Bacillus subtilis with 13 mm of inhibition zone. Total oxidative status (TOS) and the total antioxidative status (TAS) levels were determined to evaluate the antioxidant activity of CAE. Results indicated that there was a good correlation between dose of CAE and TAS levels. We also observed that CAE protect the DNA of both yeast and mononuclear leukocytes against the damaging effect of hydrogen peroxide. The comet assay, applied on both Saccharomyces cerevisiae BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and human leukocytes, results suggested that there was statistically significant correlation between CAE dilutions and antigenotoxic activity. PMID:22897836

  11. High quality DNA obtained with an automated DNA extraction method with 70+ year old formalin-fixed celloidin-embedded (FFCE) blocks from the indiana medical history museum

    PubMed Central

    Niland, Erin E; McGuire, Audrey; Cox, Mary H; Sandusky, George E

    2012-01-01

    DNA and RNA have been used as markers of tissue quality and integrity throughout the last few decades. In this research study, genomic quality DNA of kidney, liver, heart, lung, spleen, and brain were analyzed in tissues from post-mortem patients and surgical cancer cases spanning the past century. DNA extraction was performed on over 180 samples from: 70+ year old formalin-fixed celloidin-embedded (FFCE) tissues, formalin-fixed paraffin-embedded (FFPE) tissue samples from surgical cases and post-mortem cases from the 1970’s, 1980’s, 1990’s, and 2000’s, tissues fixed in 10% neutral buffered formalin/stored in 70% ethanol from the 1990’s, 70+ year old tissues fixed in unbuffered formalin of various concentrations, and fresh tissue as a control. To extract DNA from FFCE samples and ethanol-soaked samples, a modified standard operating procedure was used in which all tissues were homogenized, digested with a proteinase K solution for a long period of time (24-48 hours), and DNA was extracted using the Autogen Flexstar automated extraction machine. To extract DNA from FFPE, all tissues were soaked in xylene to remove the paraffin from the tissue prior to digestion, and FFPE tissues were not homogenized. The results were as follows: celloidin-embedded and paraffin-embedded tissues yielded the highest DNA concentration and greatest DNA quality, while the formalin in various concentrations, and long term formalin/ethanol-stored tissue yielded both the lowest DNA concentration and quality of the tissues tested. The average DNA yield for the various fixatives was: 367.77 μg/ mL FFCE, 590.7 μg/mL FFPE, 53.74 μg/mL formalin-fixed/70% ethanol-stored and 33.2 μg/mL unbuffered formalin tissues. The average OD readings for FFCE, FFPE, formalin-fixed/70% ethanol-stored tissues, and tissues fixed in unbuffered formalin were 1.86, 1.87, 1.43, and 1.48 respectively. The results show that usable DNA can be extracted from tissue fixed in formalin and embedded in celloidin

  12. Homologous pairing between single-stranded DNA immobilized on a nitrocellulose membrane and duplex DNA is specific for RecA activity in bacterial crude extract.

    PubMed Central

    Bertrand, P; Corteggiani, E; Dutreix, M; Coppey, J; Lopez, B S

    1993-01-01

    Reaction between a circular single stranded and a linear double stranded DNA molecule (ssDNA and dsDNA) provides an efficient system to study recombination mediated by RecA protein. However, classical assays using reaction in solution require highly purified enzymes. This limits biochemical studies of mutant RecA proteins from Escherichia coli or of RecA proteins from other organisms. We describe here an assay that is specific for RecA activity even in bacterial crude extracts. In this assay, the ssDNA is bound to a nitrocellulose membrane, proteins are loaded on this membrane and it is then incubated with a labeled homologous dsDNA. Joint molecules are visualized by autoradiography. We have shown that, despite the reduced mobility of the DNA due to its binding to the membrane, RecA protein is able to promote formation of stable plectonemic joints, in a homology dependent manner. Fourteen other proteins involved in DNA metabolism were checked and did not produce a signal in our assay. Moreover, in Dot blot analysis as well as after native electrophoresis and electrotransfer on a ssDNA coated membrane, production of a signal was strictly dependent on the presence of active RecA protein in the bacterial crude extracts used. We named this assay Pairing On Membrane blot (POM blot). Images PMID:8367282

  13. 5-Methylation of Cytosine in CG:CG Base-pair Steps: A Physicochemical Mechanism for the Epigenetic Control of DNA Nanomechanics

    PubMed Central

    Yusufaly, Tahir I.; Li, Yun; Olson, Wilma K.

    2014-01-01

    Van der Waals density functional theory is integrated with analysis of a non-redundant set of protein-DNA crystal structures from the Nucleic Acid Database to study the stacking energetics of CG:CG base-pair steps, specifically the role of cytosine 5-methylation. Principal component analysis of the steps reveals the dominant collective motions to correspond to a tensile ‘opening’ mode and two shear ‘sliding’ and ‘tearing’ modes in the orthogonal plane. The stacking interactions of the methyl groups globally inhibit CG:CG step overtwisting while simultaneously softening the modes locally via potential energy modulations that create metastable states. Additionally, the indirect effects of the methyl groups on possible base-pair steps neighboring CG:CG are observed to be of comparable importance to their direct effects on CG:CG. The results have implications for the epigenetic control of DNA mechanics. PMID:24313757

  14. Antioxidant and DNA Repair Stimulating Effect of Extracts from Transformed and Normal Roots of Rhaponticum carthamoides against Induced Oxidative Stress and DNA Damage in CHO Cells

    PubMed Central

    Skała, Ewa; Sitarek, Przemysław; Różalski, Marek; Krajewska, Urszula; Szemraj, Janusz; Wysokińska, Halina; Śliwiński, Tomasz

    2016-01-01

    Rhaponticum carthamoides has a long tradition of use in Siberian folk medicine. The roots and rhizomes of this species are used in various dietary supplements or nutraceutical preparations to increase energy level or eliminate physical weakness. This is the first report to reveal the protective and DNA repair stimulating abilities of R. carthamoides root extracts in Chinese hamster ovary (CHO) cells exposed to an oxidative agent. Both transformed root extract (TR extract) and extract of soil-grown plant roots (NR extract) may be responsible for stimulating CHO cells to repair oxidatively induced DNA damage, but CHO cells stimulated with extract from the transformed roots demonstrated significantly stronger properties than cells treated with the soil-grown plant root extract. These differences in biological activity may be attributed to the differences in the content of phenolic compounds in these root extracts. Preincubation of the CHO cells with TR and NR extracts showed an increase in gene expression and protein levels of catalase (CAT) and superoxide dismutase (SOD2). R. carthamoides may possess antioxidant properties that protect CHO cells against oxidative stress. PMID:27034736

  15. Antioxidant and DNA Repair Stimulating Effect of Extracts from Transformed and Normal Roots of Rhaponticum carthamoides against Induced Oxidative Stress and DNA Damage in CHO Cells.

    PubMed

    Skała, Ewa; Sitarek, Przemysław; Różalski, Marek; Krajewska, Urszula; Szemraj, Janusz; Wysokińska, Halina; Śliwiński, Tomasz

    2016-01-01

    Rhaponticum carthamoides has a long tradition of use in Siberian folk medicine. The roots and rhizomes of this species are used in various dietary supplements or nutraceutical preparations to increase energy level or eliminate physical weakness. This is the first report to reveal the protective and DNA repair stimulating abilities of R. carthamoides root extracts in Chinese hamster ovary (CHO) cells exposed to an oxidative agent. Both transformed root extract (TR extract) and extract of soil-grown plant roots (NR extract) may be responsible for stimulating CHO cells to repair oxidatively induced DNA damage, but CHO cells stimulated with extract from the transformed roots demonstrated significantly stronger properties than cells treated with the soil-grown plant root extract. These differences in biological activity may be attributed to the differences in the content of phenolic compounds in these root extracts. Preincubation of the CHO cells with TR and NR extracts showed an increase in gene expression and protein levels of catalase (CAT) and superoxide dismutase (SOD2). R. carthamoides may possess antioxidant properties that protect CHO cells against oxidative stress. PMID:27034736

  16. A SYBR green-based real-time polymerase chain reaction protocol and novel DNA extraction technique to detect Xylella fastidiosa in Homalodisca coagulata.

    PubMed

    Bextine, Blake; Blua, Matthew; Harshman, Dave; Miller, Thomas A

    2005-06-01

    Homalodisca coagulata Say (Hemiptera: Cicadellidae) is a major agronomic pest because it transmits Xylella fastidiosa (Wells), the bacterium that causes Pierce's disease of grapevine. The ability to easily detect X. fastidiosa in populations of H. coagulata facilitates epidemiological studies and development of a monitoring program supporting disease management. Such a program depends on a detection protocol that is rapid, reproducible, and amenable to large sample sizes, while remaining sensitive enough to detect low amounts of pathogen DNA. In this study, we developed an improved method to speed DNA extraction by implementing a simple vacuum step that replaces labor- and time-intensive maceration of tissue samples and that is compatible with manufactured DNA extraction kits. Additionally, we have developed a SYBR Green-based real-time (RT)-polymerase chain reaction (PCR) system, which uses traditional PCR primers that are relatively inexpensive and effective. Using this extraction/RT-PCR system, we found no statistically significant differences in the detection of X. fastidiosa among samples that were either immediately extracted or stored dry or in mineral oil for 10 d at -4 degrees C. In further testing, we found no significant reduction in detection capabilities for X. fastidiosa-fed H. coagulata left in the sun on yellow sticky cards for up to 6 d. Therefore, we recommend a field-based detection system that includes recovery of H. coagulata from sticky traps for up to 6 d after trapping, subsequent freezing of samples for as long as 10 d before vacuum extraction is performed, and detection of the bacterium by SYBR Green-based RT-PCR. PMID:16022291

  17. Influence of amount of starting material for DNA extraction on detection of low-level presence of genetically engineered traits.

    PubMed

    Demeke, Tigst; Phan, Anh; Ratnayaka, Indira; Holigroski, Michelle; Jenkins, G Ronald

    2014-05-14

    Two laboratories independently examined how the amount of starting material influences DNA extraction efficiency and, ultimately, the detection of low-level presence of genetically engineered (GE) traits in commercialized grains. GE traits from one maize, two canola, and two soybean samples were used as prototypical models in the study design as well as two commonly used DNA extraction methods, a small scale (0.1 and 0.2 g samples) and a large scale (1.0 and 2.0 g samples). The DNA samples were fortified (spiked) at 0.1 and 0.01% (w/w) levels. The amount of DNA recovery varied between the two laboratories, although a sufficient amount of DNA was obtained to perform replicate PCR analysis by both laboratories. Reliable detection of all five events was achieved by both laboratories at 0.1% level using either small-scale or large-scale DNA extractions. Reliable detection of the GE events was achieved at 0.01% level for soybean and canola but not for maize. Variability was observed among the two laboratories in terms of the Ct values generated. There was no difference between small-scale and large-scale DNA extraction methods for qualitative PCR detections of all five GE events. PMID:24745691

  18. Comparison of six commercial kits to extract bacterial chromosome and plasmid DNA for MiSeq sequencing.

    PubMed

    Becker, Laura; Steglich, Matthias; Fuchs, Stephan; Werner, Guido; Nübel, Ulrich

    2016-01-01

    We compared commercial kits for extraction of genomic DNA from the Gram-negative bacterium Klebsiella pneumoniae for subsequent Miseq sequencing. Purification of DNA was based on matrix binding (silica or anion exchange resin) or differential precipitation (salting out), respectively. The choice of extraction kit had little effect on sequencing quality and coverage across drastically different replicons, except for an apparent depletion of small plasmids (<5 kb) during precipitation-based extractions. Sequencing coverage provided copy-number estimates for small plasmids that were consistently higher than those from quantitative real-time PCR. PMID:27312200

  19. Comparison of six commercial kits to extract bacterial chromosome and plasmid DNA for MiSeq sequencing

    PubMed Central

    Becker, Laura; Steglich, Matthias; Fuchs, Stephan; Werner, Guido; Nübel, Ulrich

    2016-01-01

    We compared commercial kits for extraction of genomic DNA from the Gram-negative bacterium Klebsiella pneumoniae for subsequent Miseq sequencing. Purification of DNA was based on matrix binding (silica or anion exchange resin) or differential precipitation (salting out), respectively. The choice of extraction kit had little effect on sequencing quality and coverage across drastically different replicons, except for an apparent depletion of small plasmids (<5 kb) during precipitation-based extractions. Sequencing coverage provided copy-number estimates for small plasmids that were consistently higher than those from quantitative real-time PCR. PMID:27312200

  20. Quantitative and qualitative validations of a sonication-based DNA extraction approach for PCR-based molecular biological analyses.

    PubMed

    Dai, Xiaohu; Chen, Sisi; Li, Ning; Yan, Han

    2016-05-15

    The aim of this study was to comprehensively validate the sonication-based DNA extraction method, in hope of the replacement of the so-called 'standard DNA extraction method' - the commercial kit method. Microbial cells in the digested sludge sample, containing relatively high amount of PCR-inhibitory substances, such as humic acid and protein, were applied as the experimental alternatives. The procedure involving solid/liquid separation of sludge sample and dilution of both DNA templates and inhibitors, the minimum templates for PCR-based analyses, and the in-depth understanding from the bias analysis by pyrosequencing technology were obtained and confirmed the availability of the sonication-based DNA extraction method. PMID:26774955

  1. Two separable functions of Ctp1 in the early steps of meiotic DNA double-strand break repair

    PubMed Central

    Ma, Lijuan; Milman, Neta; Nambiar, Mridula; Smith, Gerald R.

    2015-01-01

    Meiotic programmed DNA double-strand break (DSB) repair is essential for crossing-over and viable gamete formation and requires removal of Spo11-oligonucleotide complexes from 5′ ends (clipping) and their resection to generate invasive 3′-end single-stranded DNA (resection). Ctp1 (Com1, Sae2, CtIP homolog) acting with the Mre11-Rad50-Nbs1 (MRN) complex is required in both steps. We isolated multiple S. pombe ctp1 mutants deficient in clipping but proficient in resection during meiosis. Remarkably, all of the mutations clustered in or near the conserved CxxC or RHR motif in the C-terminal portion. The mutants tested, like ctp1Δ, were clipping-deficient by both genetic and physical assays­. But, unlike ctp1Δ, these mutants were recombination-proficient for Rec12 (Spo11 homolog)-independent break-repair and resection-proficient by physical assay. We conclude that the intracellular Ctp1 C-terminal portion is essential for clipping, while the N-terminal portion is sufficient for DSB end-resection. This conclusion agrees with purified human CtIP resection and endonuclease activities being independent. Our mutants provide intracellular evidence for separable functions of Ctp1. Some mutations truncate Ctp1 in the same region as one of the CtIP mutations linked to the Seckel and Jawad severe developmental syndromes, suggesting that these syndromes are caused by a lack of clipping at DSB ends that require repair. PMID:26130711

  2. Reconstitution of the cellular response to DNA damage in vitro using damage-activated extracts from mammalian cells

    SciTech Connect

    Roper, Katherine; Coverley, Dawn

    2012-03-10

    In proliferating mammalian cells, DNA damage is detected by sensors that elicit a cellular response which arrests the cell cycle and repairs the damage. As part of the DNA damage response, DNA replication is inhibited and, within seconds, histone H2AX is phosphorylated. Here we describe a cell-free system that reconstitutes the cellular response to DNA double strand breaks using damage-activated cell extracts and naieve nuclei. Using this system the effect of damage signalling on nuclei that do not contain DNA lesions can be studied, thereby uncoupling signalling and repair. Soluble extracts from G1/S phase cells that were treated with etoposide before isolation, or pre-incubated with nuclei from etoposide-treated cells during an in vitro activation reaction, restrain both initiation and elongation of DNA replication in naieve nuclei. At the same time, H2AX is phosphorylated in naieve nuclei in a manner that is dependent upon the phosphatidylinositol 3-kinase-like protein kinases. Notably, phosphorylated H2AX is not focal in naieve nuclei, but is evident throughout the nucleus suggesting that in the absence of DNA lesions the signal is not amplified such that discrete foci can be detected. This system offers a novel screening approach for inhibitors of DNA damage response kinases, which we demonstrate using the inhibitors wortmannin and LY294002. -- Highlights: Black-Right-Pointing-Pointer A cell free system that reconstitutes the response to DNA damage in the absence of DNA lesions. Black-Right-Pointing-Pointer Damage-activated extracts impose the cellular response to DNA damage on naieve nuclei. Black-Right-Pointing-Pointer PIKK-dependent response impacts positively and negatively on two separate fluorescent outputs. Black-Right-Pointing-Pointer Can be used to screen for inhibitors that impact on the response to damage but not on DNA repair. Black-Right-Pointing-Pointer LY294002 and wortmannin demonstrate the system's potential as a pathway focused screening

  3. Improved Methods for Capture, Extraction, and Quantitative Assay of Environmental DNA from Asian Bigheaded Carp (Hypophthalmichthys spp.)

    PubMed Central

    Turner, Cameron R.; Miller, Derryl J.; Coyne, Kathryn J.; Corush, Joel

    2014-01-01

    Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA) is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp.), an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR) assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species. PMID:25474207

  4. Comparison of DNA Extraction Kits for Detection of Burkholderia pseudomallei in Spiked Human Whole Blood Using Real-Time PCR

    PubMed Central

    Podnecky, Nicole L.; Elrod, Mindy G.; Newton, Bruce R.; Dauphin, Leslie A.; Shi, Jianrong; Chawalchitiporn, Sutthinan; Baggett, Henry C.; Hoffmaster, Alex R.; Gee, Jay E.

    2013-01-01

    Burkholderia pseudomallei, the etiologic agent of melioidosis, is endemic in northern Australia and Southeast Asia and can cause severe septicemia that may lead to death in 20% to 50% of cases. Rapid detection of B. pseudomallei infection is crucial for timely treatment of septic patients. This study evaluated seven commercially available DNA extraction kits to determine the relative recovery of B. pseudomallei DNA from spiked EDTA-containing human whole blood. The evaluation included three manual kits: the QIAamp DNA Mini kit, the QIAamp DNA Blood Mini kit, and the High Pure PCR Template Preparation kit; and four automated systems: the MagNAPure LC using the DNA Isolation Kit I, the MagNAPure Compact using the Nucleic Acid Isolation Kit I, and the QIAcube using the QIAamp DNA Mini kit and the QIAamp DNA Blood Mini kit. Detection of B. pseudomallei DNA extracted by each kit was performed using the B. pseudomallei specific type III secretion real-time PCR (TTS1) assay. Crossing threshold (CT) values were used to compare the limit of detection and reproducibility of each kit. This study also compared the DNA concentrations and DNA purity yielded for each kit. The following kits consistently yielded DNA that produced a detectable signal from blood spiked with 5.5×104 colony forming units per mL: the High Pure PCR Template Preparation, QIAamp DNA Mini, MagNA Pure Compact, and the QIAcube running the QIAamp DNA Mini and QIAamp DNA Blood Mini kits. The High Pure PCR Template Preparation kit yielded the lowest limit of detection with spiked blood, but when this kit was used with blood from patients with confirmed cases of melioidosis, the bacteria was not reliably detected indicating blood may not be an optimal specimen. PMID:23460920

  5. Evaluation of DNA extraction techniques for detecting Mycobacterium tuberculosis complex organisms in Asian elephant trunk wash samples.

    PubMed

    Kay, Meagan K; Linke, Lyndsey; Triantis, Joni; Salman, M D; Larsen, R Scott

    2011-02-01

    Rapid and sensitive diagnostic assays for the detection of tuberculous mycobacteria in elephants are lacking. DNA extraction with PCR analysis is useful for tuberculosis screening in many species but has not been validated on elephant trunk wash samples. We estimated the analytical sensitivity and specificity of three DNA extraction methods to detect Mycobacterium tuberculosis complex organisms in trunk wash specimens. A ZR soil microbe DNA kit (ZR) and a traditional salt and ethanol precipitation (TSEP) approach were evaluated under three different treatment conditions: heat treatment, phenol treatment, and contamination with Mycobacterium avium. A third approach, using a column filtration method, was evaluated for samples contaminated with soil. Trunk wash samples from uninfected elephants were spiked with various concentrations of M. bovis cells and subjected to the described treatment conditions prior to DNA extraction. Extracted DNA was amplified using IS6110-targeted PCR analysis. The ZR and TSEP methods detected as low as 1 to 5 M. bovis cells and 10 M. bovis cells, respectively, per 1.5 ml of trunk wash under all three conditions. Depending on the amount of soil present, the column filtration method detected as low as 5 to 50 M. bovis cells per 1.5 ml of trunk wash. Analytical specificity was assessed by DNA extraction from species of nontuberculous mycobacteria and amplification using the same PCR technique. Only M. bovis DNA was amplified, indicating 100% analytical specificity of this PCR technique. Our results indicate that these DNA extraction techniques offer promise as useful tests for detection of M. tuberculosis complex organisms in elephant trunk wash specimens. PMID:21159933

  6. The impact of different methods of DNA extraction on microbial community measures of BALF samples based on metagenomic data

    PubMed Central

    Wen, Yan; Xiao, Fei; Wang, Chen; Wang, Zhen

    2016-01-01

    Purpose: It is a challenge to find a better microorganisms DNA extraction method for samples taken from the lower airways for metagenomic sequencing, as the concentrations of bacteria in the alveoli and small airways are likely considerably less than that of the mouth or lower digestive tract. Background DNA from the host, and extraction biases can significantly interfere with microbiota assessment and increase the cost of sequencing. This study aimed to develop an optimized DNA extraction method, which would enable a higher concentration of microbial DNA to be extracted from the samples. Methods: We compared the microbiota profiles of the lower airway communities in twelve individuals with IIP. DNA was extracted using three different extraction methods: QIAamp UCP PurePathogen Blood Kit named kit3 in this study, QIAamp UCP Pathogen Mini Kit named kit2, and QIAamp DNA Microbiome Kit named kit1. DNA libraries were constructed according to the manufacturer’s instructions (Illumina). The same workflows from Illumina were used to perform cluster generation, template hybridization, isothermal amplification, linearization, blocking, denaturing, and hybridization of the sequencing primers. Raw data was uploaded to MG-RAST v3 and analyzed. Results: A great number of bacterium inhabits the lower airways of patients with IIP, though there is no airway infection. More bacterium was found in mouth or upper airway. DNA concentrations of DNA samples isolated with kit1 with Benzonase were significantly lower than those isolated with the other two kits for BALF and mouthwash samples. Moreover, the ratio of human genome in clean reads of samples isolated with kit1 with Benzonase was remarkably smaller than those isolated with kit2 and kit3. The relative abundance of total bacteria, the total number of taxa, and the relative abundance of taxa in BALF samples as opposed to mouthwash samples with kit1 were significantly higher than for those extracted the other kits. Conclusion: A

  7. Optimization of DNA extraction from seeds and fresh leaf tissues of wild marigold (Tagetes minuta) for polymerase chain reaction analysis.

    PubMed

    Shahzadi, I; Ahmed, R; Hassan, A; Shah, M M

    2010-01-01

    Tagetes, a genus of flowering marigolds in the family Asteraceae (Compositeae), is reported to be a medicinal plant with hypotensive, spasmolytic, anti-inflammatory, antimicrobial, and antifungal properties. Tagetes minuta characteristically contains high concentrations of essential oils, flavonoids, polyphenols, and polysaccharides that interfere with DNA, causing erroneous or no PCR products. We tested and modified various standard protocols in an effort to isolate high-quality DNA from different plant tissues of T. minuta. We used sun-dried, shade-dried and fresh-leaf tissues, as well as seeds for DNA analysis. The DNA obtained from seeds and fresh-leaf tissues with a modified cetyltrimethylammonium bromide buffer protocol was of good quality, with no colored pigments and contaminants. We were able to obtain good quality DNA from fresh leaf tissues without using liquid nitrogen. A relatively large amount of DNA was also extracted from the sun- and shade-dried tissues, but its quality was not as good as that from seeds. The DNA extracted from seeds and fresh leaves was successfully amplified by PCR using arbitrary RAPD primers. The same protocol will probably be useful for extracting high-molecular weight DNA from other plant materials containing large amounts of secondary metabolites and essential oils. PMID:20309824

  8. In vitro free radical scavenging and DNA damage protective property of Coriandrum sativum L. leaves extract.

    PubMed

    Harsha, S N; Anilakumar, K R

    2014-08-01

    Coriandrum sativum L. (coriander), an everyday spice in the Indian kitchen is known to add flavor to the cuisine. It is an annual herb belonging to the Apiaceae (Umbellifera) family. The hydro-alcohol extract of Coriandrum sativum L. at the dose of 1 mg/ml was subjected to a series of in vitro assays viz. 2, 2'- diphenyl-1-picrylhydrazyl, lipid peroxidation by thiobarbituric acid, reducing power and nitric oxide (NO) radical scavenging in order to study its antioxidant efficacy in detail. The amount of flavonoids in 70% ethanol extract was found to be 44.5 μg and that of the total phenols was 133.74 μg gallic acid equivalents per mg extract. The extracts of the leaves showed metal chelating power, with IC50 values, 368.12 μg/ml where as that of standard EDTA was 26.7 μg/ml. The IC50 values for 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid radical scavenging was 222 μg/ml where as that of standard ascorbic acid was 22.6 μg/ml. The NO scavenging activity of the extract of the leaves showed IC50 value of 815.6 μg/ml; at the same time the standard BHA had 49.1 μg/ml. All the plant extracts provided DNA damage protection; however, the protection provided at the dose of 8 μg/ml was comparable to that of standard gallic acid. The Coriandrum sativum leaf extract was able to prevent in vitro lipid peroxidation with IC50 values; 589.6 μg/ml where as that of standard BHA was 16.3 μg/ml. Our results also showed significant ferric reducing power indicating the hydrogen donating ability of the extract. This study indicated the potential of the leaf extract as a source of natural antioxidants or nutraceuticals that could be of use in food industry with potential application to reduce oxidative stress in living system. PMID:25114344

  9. Rapid method for DNA extraction from the honey bee Apis mellifera and the parasitic bee mite Varroa destructor using lysis buffer and proteinase K.

    PubMed

    Issa, M R C; Figueiredo, V L C; De Jong, D; Sakamoto, C H; Simões, Z L P

    2013-01-01

    We developed a rapid method for extraction of DNA from honey bees, Apis mellifera, and from the parasitic bee mite, Varroa destructor. The advantages include fast processing and low toxicity of the substances that are utilized. We used lysis buffer with nonionic detergents to lyse cell walls and proteinase K to digest proteins. We tested whole thorax, thoracic muscle mass, legs, and antennae from individual bees; the mites were processed whole (1 mite/sample). Each thorax was incubated whole, without cutting, because exocuticle color pigment darkened the extraction solution, interfering with PCR results. The procedure was performed with autoclaved equipment and laboratory gloves. For each sample, we used 100 µL lysis buffer (2 mL stock solution of 0.5 M Tris/HCl, pH 8.5, 10 mL stock solution of 2 M KCl, 500 µL solution of 1 M MgCl2, 2 mL NP40, and 27.6 g sucrose, completed to 200 mL with bidistilled water and autoclaved) and 2 µL proteinase K (10 mg/mL in bidistilled water previously autoclaved, as proteinase K cannot be autoclaved). Tissues were incubated in the solutions for 1-2 h in a water bath (62°-68 °C) or overnight at 37 °C. After incubation, the tissues were removed from the extraction solution (lysis buffer + proteinase K) and the solution heated to 92 °C for 10 min, for proteinase K inactivation. Then, the solution with the extracted DNA was stored in a refrigerator (4°-8 °C) or a freezer (-20 °C). This method does not require centrifugation or phenol/chloroform extraction. The reduced number of steps allowed us to sample many individuals/day. Whole mites and bee antennae were the most rapidly processed. All bee tissues gave the same quality DNA. This method, even using a single bee antenna or a single mite, was adequate for extraction and analysis of bee genomic and mitochondrial DNA and mite genomic DNA. PMID:24301746

  10. Comparison of different DNA-extraction techniques to investigate the bacterial community of marine copepods

    NASA Astrophysics Data System (ADS)

    Brandt, Petra; Gerdts, Gunnar; Boersma, Maarten; Wiltshire, Karen H.; Wichels, Antje

    2010-12-01

    Marine zooplanktic organisms, such as copepods, are usually associated with large numbers of bacteria. Some of these bacteria live attached to copepods’ exoskeleton, while others prevail in their intestine and faecal pellets. Until now, general conclusions concerning the identity of these bacteria are problematic since the majority of previous studies focused on cultivable bacteria only. Hence, to date little is known on whether copepod genera or species harbour distinct bacterial populations and about the nature of this association. To shed more light on these copepod/bacteria consortia, the focus of this study was the development and evaluation of a suitable approach to extract bacterial DNA from different North Sea copepod genera. Furthermore, the bacterial DNA was analysed by PCR-DGGE and subsequent sequencing of excised bands. The result of this work was an appropriate extraction method for batches of ten to one copepod specimens and offered first insights as to which bacteria are attached to the copepods Acartia sp . and Temora sp . from Helgoland Roads (German Bight) and a laboratory-grown Acartia tonsa culture. It revealed the prevalence of Alphaproteobacteria.

  11. DNA Protection against Oxidative Damage Using the Hydroalcoholic Extract of Garcinia mangostana and Alpha-Mangostin.

    PubMed

    Carvalho-Silva, Ronaldo; Pereira, Alanna Cibelle Fernandes; Dos Santos Alves, Rúbens Prince; Guecheva, Temenouga N; Henriques, João A P; Brendel, Martin; Pungartnik, Cristina; Rios-Santos, Fabrício

    2016-01-01

    Garcinia mangostana, popularly known as "mangosteen fruit," originates from Southeast Asia and came to Brazil about 80 years ago where it mainly grows in the states of Pará and Bahia. Although mangosteen or its extracts have been used for ages in Asian folk medicine, data on its potential genotoxicity is missing. We, therefore, evaluated genotoxicity/mutagenicity of hydroethanolic mangosteen extract [HEGM, 10 to 640 μg/mL] in established test assays (Comet assay, micronucleus test, and Salmonella/microsome test). In the Comet assay, HEGM-exposed human leukocytes showed no DNA damage. No significant HEGM-induced mutation in TA98 and TA100 strains of Salmonella typhimurium (with or without metabolic activation) was observed and HEGM-exposed human lymphocytes had no increase of micronuclei. However, HEGM suggested exposure concentration-dependent antigenotoxic potential in leukocytes and antioxidant potential in the yeast Saccharomyces cerevisiae. HEGM preloading effectively protected against H2O2-induced DNA damage in leukocytes (Comet assay). Preloading of yeast with HEGM for up to 4 h significantly protected the cells from lethality of chronic H2O2-exposure, as expressed in better survival. Absence of genotoxicity and demonstration of an antigenotoxic and antioxidant potential suggest that HEGM or some substances contained in it may hold promise for pharmaceutical or nutraceutical application. PMID:27042187

  12. Evaluation and optimisation of bacterial genomic DNA extraction for no-culture techniques applied to vinegars.

    PubMed

    Mamlouk, Dhouha; Hidalgo, Claudio; Torija, María-Jesús; Gullo, Maria

    2011-10-01

    Direct genomic DNA extraction from vinegars was set up and suitability for PCR assays performed by PCR/DGGE and sequencing of 16S rRNA gene. The method was tested on 12 intermediary products of special vinegars, fruit vinegars and condiments produced from different raw materials and procedures. DNAs extraction was performed on pellets by chemical, enzymatic, resin mediated methods and their modifications. Suitable yield and DNA purity were obtained by modification of a method based on the use of PVP/CTAB to remove polyphenolic components and esopolysaccharides. By sequencing of bands from DGGE gel, Gluconacetobacter europaeus, Acetobacter malorum/cerevisiae and Acetobacter orleanensis were detected as main species in samples having more than 4% of acetic acid content. From samples having no acetic acid content, sequences retrieved from excised bands revealed high similarity with prokaryotes with no function on vinegar fermentation: Burkholderia spp., Cupriavidus spp., Lactococcus lactis and Leuconostoc mesenteroides. The method was suitable to be applied for no-culture study of vinegars containing polyphenols and esopolysaccharides allowing a more complete assessment of vinegar bacteria. PMID:21839388

  13. DNA Protection against Oxidative Damage Using the Hydroalcoholic Extract of Garcinia mangostana and Alpha-Mangostin

    PubMed Central

    Carvalho-Silva, Ronaldo; Pereira, Alanna Cibelle Fernandes; dos Santos Alves, Rúbens Prince; Guecheva, Temenouga N.; Henriques, João A. P.; Brendel, Martin; Rios-Santos, Fabrício

    2016-01-01

    Garcinia mangostana, popularly known as “mangosteen fruit,” originates from Southeast Asia and came to Brazil about 80 years ago where it mainly grows in the states of Pará and Bahia. Although mangosteen or its extracts have been used for ages in Asian folk medicine, data on its potential genotoxicity is missing. We, therefore, evaluated genotoxicity/mutagenicity of hydroethanolic mangosteen extract [HEGM, 10 to 640 μg/mL] in established test assays (Comet assay, micronucleus test, and Salmonella/microsome test). In the Comet assay, HEGM-exposed human leukocytes showed no DNA damage. No significant HEGM-induced mutation in TA98 and TA100 strains of Salmonella typhimurium (with or without metabolic activation) was observed and HEGM-exposed human lymphocytes had no increase of micronuclei. However, HEGM suggested exposure concentration-dependent antigenotoxic potential in leukocytes and antioxidant potential in the yeast Saccharomyces cerevisiae. HEGM preloading effectively protected against H2O2-induced DNA damage in leukocytes (Comet assay). Preloading of yeast with HEGM for up to 4 h significantly protected the cells from lethality of chronic H2O2-exposure, as expressed in better survival. Absence of genotoxicity and demonstration of an antigenotoxic and antioxidant potential suggest that HEGM or some substances contained in it may hold promise for pharmaceutical or nutraceutical application. PMID:27042187

  14. No Evidence for Interference of Hematoxylin and Eosin (HE) Staining in DNA Testing: Utility of DNA Extraction from HE-Stained Archival Tissue Sections

    PubMed Central

    Morikawa, Teppei; Shima, Kaori; Kuchiba, Aya; Yamauchi, Mai; Tanaka, Noriko; Imamura, Yu; Liao, Xiaoyun; Qian, Zhi Rong; Brahmandam, Mohan; Longtine, Janina A.; Lindeman, Neal I.; Fuchs, Charles S.; Ogino, Shuji

    2012-01-01

    Although histochemical staining has been believed to inhibit DNA amplification reaction, no previous study has systematically evaluated the influence of histochemical staining on downstream molecular assays. To evaluate an influence of hematoxylin and eosin (HE) staining on DNA testing, we isolated DNA from 10 unstained, 10 hematoxylin-stained, 10 eosin-stained or 10 HE-stained tissue sections (ie, 4 groups), from each of 5 colon cancers. Among those 4 groups, we did not observe any significant or appreciable difference in DNA fragmentation by agarose gel electrophoresis; in DNA amplification by real-time PCR; in microsatellite PCR fragment analyses; or in PCR-Pyrosequencing. As a proof-of-principle study, we successfully performed microsatellite instability analysis and sequencing of KRAS and BRAF on over 1300 colorectal cancers using DNA extracted from HE stained tissue sections. Our data provide no evidence for interfering effect of HE staining on DNA testing, suggesting that DNA from HE-stained sections can be effectively used for routine DNA testing. PMID:22706867

  15. Determination of gold, indium, tellurium and thallium in the same sample digest of geological materials by atomic-absorption spectroscopy and two-step solvent extraction

    USGS Publications Warehouse

    Hubert, A.E.; Chao, T.T.

    1985-01-01

    A rock, soil, or stream-sediment sample is decomposed with hydrofluoric acid, aqua regia, and hydrobromic acid-bromine solution. Gold, thallium, indium and tellurium are separated and concentrated from the sample digest by a two-step MIBK extraction at two concentrations of hydrobromic add. Gold and thallium are first extracted from 0.1M hydrobromic acid medium, then indium and tellurium are extracted from 3M hydrobromic acid in the presence of ascorbic acid to eliminate iron interference. The elements are then determined by flame atomic-absorption spectrophotometry. The two-step solvent extraction can also be used in conjunction with electrothermal atomic-absorption methods to lower the detection limits for all four metals in geological materials. ?? 1985.

  16. Enhanced fungal DNA-Extraction from Formalin fixed, paraffin embedded tissue specimens by application of thermal energy

    PubMed Central

    Rickerts, V.; Khot, P.D.; Ko, D.L.; Fredricks, D.N.

    2014-01-01

    Summary Determining the etiology of invasive fungal infections (IFI) is critical for patient management as fungi vary in their susceptibility to antifungals. However, the etiology remains obscure in many cases due to negative culture results. The identification of fungal DNA from pathology blocks by PCR and sequencing is an alternative approach to determine the etiology of IFI. Previous studies identified fungal DNA in only 50% of samples with positive histopathology, probably due to DNA damage by the tissue fixation. We used realtime PCR to quantify human and fungal DNA from Formalin-fixed, paraffin embedded tissue specimens in order to study the effect of thermal energy during extraction on the yield of amplifiable DNA and subsequent identification of fungal DNA. Tissue sections from eight patients with proven IFI were subjected to DNA extraction with varying exposure to thermal energy. Amplifiable DNA increased up to 76-fold by increasing incubation temperature from 65°C to 90°C. An additional increase was documented by incubation for up to 6 hours at 90°C. The augmented amplification of fungal DNA was associated with improved species identification by sequencing of the PCR amplicons. This may help illuminate the etiology of IFI and thereby improve patient management by guiding antifungal therapy. PMID:22414380

  17. In-vitro Evaluation of Protective Effects on DNA Damage and Antioxidative Activities of Ilex Spinigera Loes. Extracts

    PubMed Central

    Mohadjerani, Maryam; Vosoghi Roodgar, Mina

    2016-01-01

    Ilex spinigera (Aquifoliaceae) plant is an evergreen tree or shrub with thick glossy dark green leaves and red fruits. This plant has medicinal properties and has been used traditionally in northern Iran for malaria treatment. The aim of this work is to evaluate the antioxidative activities and the inhibitory effect of I. spinigera on the oxidation of DNA. We have found no reports about the popular use of I. spinigera in terms of its chemistry and biology. In this study we report the antioxidant activity of I. spinigera extracts for the first time. Water, ethanol and methanol were used as extraction solvents. Various experimental models including iron (III) reducing power, total antioxidant capacity, DPPH radical scavenging activity, PAB assay and in-vitro inhibition of AAPH-induced oxidation of DNA were used for characterization of antioxidant activity of the extracts. The three extracts showed varying degrees of efficacy in each assay in a dose-dependent manner. The aqueous extract with the highest content of total phenolics, was the most potent antioxidant in all assays except in DPPH assay. The methanol extract with the highest amount of total flavonoids was the potent scavenger of DPPH radical with an IC50 value of 102.22 ± 0.001 μg/mL. Aqueous extract of I. spinigera also showed the protective effect on DNA damage-induced by AAPH. According to our results, I. spinigera leaves extract have the potential for chemoprotective studies. PMID:27610169

  18. In-vitro Evaluation of Protective Effects on DNA Damage and Antioxidative Activities of Ilex Spinigera Loes. Extracts.

    PubMed

    Mohadjerani, Maryam; Vosoghi Roodgar, Mina

    2016-01-01

    Ilex spinigera (Aquifoliaceae) plant is an evergreen tree or shrub with thick glossy dark green leaves and red fruits. This plant has medicinal properties and has been used traditionally in northern Iran for malaria treatment. The aim of this work is to evaluate the antioxidative activities and the inhibitory effect of I. spinigera on the oxidation of DNA. We have found no reports about the popular use of I. spinigera in terms of its chemistry and biology. In this study we report the antioxidant activity of I. spinigera extracts for the first time. Water, ethanol and methanol were used as extraction solvents. Various experimental models including iron (III) reducing power, total antioxidant capacity, DPPH radical scavenging activity, PAB assay and in-vitro inhibition of AAPH-induced oxidation of DNA were used for characterization of antioxidant activity of the extracts. The three extracts showed varying degrees of efficacy in each assay in a dose-dependent manner. The aqueous extract with the highest content of total phenolics, was the most potent antioxidant in all assays except in DPPH assay. The methanol extract with the highest amount of total flavonoids was the potent scavenger of DPPH radical with an IC50 value of 102.22 ± 0.001 μg/mL. Aqueous extract of I. spinigera also showed the protective effect on DNA damage-induced by AAPH. According to our results, I. spinigera leaves extract have the potential for chemoprotective studies. PMID:27610169

  19. Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen suppressive soil

    SciTech Connect

    Hjort, K.; Bergstrom, M.; Adesina, M.F.; Jansson, J.K.; Smalla, K.; Sjoling, S.

    2009-09-01

    Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal-restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal-restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF{sup 103} of the isolate, Streptomyces mutomycini and/or Streptomyces clavifer. There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.

  20. Characterization and quantification of diacylglycerol species in biological extracts after one-step derivatization: a shotgun lipidomics approach.

    PubMed

    Wang, Miao; Hayakawa, Jun; Yang, Kui; Han, Xianlin

    2014-02-18

    Diacylglycerols (DAGs) are important intermediates of lipid metabolism and cellular signaling. It is well-known that the mass levels of DAG are altered under disease states. Therefore, quantitative analysis of DAGs in biological samples can provide critical information to uncover underlying mechanisms of various cellular functional disorders. Although great efforts on the analysis of individual DAG species have recently been made by utilizing mass spectrometry with or without derivatization, cost-effective and high throughput methodologies for identification and quantification of all DAG species including regioisomers, particularly in an approach of shotgun lipidomics, are still missing. Herein, we described a novel method for directly identifying and quantifying DAG species including regioisomers present in lipid extracts of biological samples after facile one-step derivatization with dimethylglycine based on the principles of multidimensional mass spectrometry-based shotgun lipidomics. The established method provided substantial sensitivity (low limit of quantification at amol/μL), high specificity, and broad linear dynamics range (2500-fold) without matrix effects. By exploiting this novel method, we revealed a 16-fold increase of total DAG mass in the livers of ob/ob mice compared to their wild type controls at 4 months of age (an insulin-resistant state) versus a 5-fold difference between 3 month old mice (with normal insulin). These results demonstrated the importance and power of the method for studying biochemical mechanisms underpinning disease states. PMID:24432906

  1. MPF amplification in Xenopus oocyte extracts depends on a two-step activation of cdc25 phosphatase.

    PubMed

    Karaïskou, A; Cayla, X; Haccard, O; Jessus, C; Ozon, R

    1998-11-01

    The activation of Cdc2 kinase induces the entry into M-phase of all eukaryotic cells. We have developed a cell-free system prepared from prophase-arrested Xenopus oocytes to analyze the mechanism initiating the all-or-none activation of Cdc2 kinase. Inhibition of phosphatase 2A, the major okadaic acid-sensitive Ser/Thr phosphatase, in these extracts, provokes Cdc2 kinase amplification and concomitant hyperphosphorylation of Cdc25 phosphatase, with a lag of about 1 h. Polo-like kinase (Plx1 kinase) is activated slightly after Cdc2. All these events are totally inhibited by the cdk inhibitor p21(Cip1), demonstrating that Plx1 kinase activation depends on Cdc2 kinase activity. Addition of a threshold level of recombinant Cdc25 induces a linear activation of Cdc2 and Plx1 kinases and a partial phosphorylation of Cdc25. We propose that the Cdc2 positive feedback loop involves two successive phosphorylation steps of Cdc25 phosphatase: the first one is catalyzed by Cdc2 kinase and/or Plx1 kinase but it does not modify Cdc25 enzymatic activity, the second one requires a new kinase counteracted by phosphatase 2A. Furthermore we demonstrate that, under our conditions, Cdc2 amplification and MAP kinase activation are two independent events. PMID:9806800

  2. Characterization and Quantification of Diacylglycerol Species in Biological Extracts after One-step Derivatization: A Shotgun Lipidomics Approach

    PubMed Central

    Wang, Miao; Hayakawa, Jun; Yang, Kui; Han, Xianlin

    2014-01-01

    Diacylglycerols (DAGs) are important intermediates of lipid metabolism and cellular signaling. It is well known that the mass levels of DAG are altered under disease states. Therefore, quantitative analysis of DAGs in biological samples can provide critical information to uncover underlying mechanisms of various cellular functional disorders. Although great efforts on the analysis of individual DAG species have recently been made by utilizing mass spectrometry with or without derivatization, cost effective and high throughput methodology for identification and quantification of all DAG species including regioisomers, particularly in an approach of shotgun lipidomics, are still missing. Herein, we described a novel method for directly identifying and quantifying DAG species including regioisomers present in lipid extracts of biological samples after facile one-step derivatization with dimethylglycine based on the principles of multi-dimensional mass spectrometry-based shotgun lipidomics. The established method provided substantial sensitivity (low limit of quantification at amol/µl), high specificity, and broad linear dynamics range (2,500 folds) without matrix effects. By exploiting this novel method, we revealed a 16-fold increase of total DAG mass in the livers of ob/ob mice compared to their wild type controls at 4 months of age (an insulin-resistant state) vs. a 5-fold difference between 3-month old mice (with normal insulin). These results demonstrated the importance and power of the method for studying biochemical mechanisms underpinning disease states. PMID:24432906

  3. Technical Reproducibility of Single-Nucleotide and Size-Based DNA Biomarker Assessment Using DNA Extracted from Formalin-Fixed, Paraffin-Embedded Tissues

    PubMed Central

    Zhang, Shenli; Tan, Iain B.; Sapari, Nur S.; Grabsch, Heike I.; Okines, Alicia; Smyth, Elizabeth C.; Aoyama, Toru; Hewitt, Lindsay C.; Inam, Imran; Bottomley, Dan; Nankivell, Matthew; Stenning, Sally P.; Cunningham, David; Wotherspoon, Andrew; Tsuburaya, Akira; Yoshikawa, Takaki; Soong, Richie; Tan, Patrick

    2015-01-01

    DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues has been used in the past to analyze genetic polymorphisms. We evaluated the technical reproducibility of different types of assays for gene polymorphisms using DNA extracted from FFPE material. By using the MassARRAY iPLEX system, we investigated polymorphisms in DPYD (rs1801159 and rs3918290), UMPS (rs1801019), ERCC1 (rs11615), ERCC1 (rs3212986), and ERCC2 (rs13181) in 56 FFPE DNA samples. By using PCR, followed by size-based gel electrophoresis, we also examined TYMS 5′ untranslated region 2R/3R repeats and GSTT1 deletions in 50 FFPE DNA samples and 34 DNAs extracted from fresh-frozen tissues and cell lines. Each polymorphism was analyzed by two independent runs. We found that iPLEX biomarker assays measuring single-nucleotide polymorphisms provided consistent concordant results. However, by using FFPE DNA, size-based PCR biomarkers (GSTT1 and TYMS 5′ untranslated region) were discrepant in 32.7% (16/49, with exact 95% CI, 19.9%–47.5%; exact binomial confidence limit test) and 4.2% (2/48, with exact 95% CI, 0.5%–14.3%) of cases, respectively, whereas no discrepancies were observed using intact genomic DNA. Our findings suggest that DNA from FFPE material can be used to reliably test single-nucleotide polymorphisms. However, results based on size-based PCR biomarkers, and particularly GSTT1 deletions, using FFPE DNA need to be interpreted with caution. Independent repeated assays should be performed on all cases to assess potential discrepancies. PMID:25746798

  4. Droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling for simpler and faster PCR assay using wire-guided manipulations.

    PubMed

    You, David J; Yoon, Jeong-Yeol

    2012-01-01

    A computer numerical control (CNC) apparatus was used to perform droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling on a single superhydrophobic surface and a multi-chambered PCB heater. Droplets were manipulated using "wire-guided" method (a pipette tip was used in this study). This methodology can be easily adapted to existing commercial robotic pipetting system, while demonstrated added capabilities such as vibrational mixing, high-speed centrifuging of droplets, simple DNA extraction utilizing the hydrophobicity difference between the tip and the superhydrophobic surface, and rapid thermocycling with a moving droplet, all with wire-guided droplet manipulations on a superhydrophobic surface and a multi-chambered PCB heater (i.e., not on a 96-well plate). Serial dilutions were demonstrated for diluting sample matrix. Centrifuging was demonstrated by rotating a 10 μL droplet at 2300 round per minute, concentrating E. coli by more than 3-fold within 3 min. DNA extraction was demonstrated from E. coli sample utilizing the disposable pipette tip to cleverly attract the extracted DNA from the droplet residing on a superhydrophobic surface, which took less than 10 min. Following extraction, the 1500 bp sequence of Peptidase D from E. coli was amplified using rapid droplet thermocycling, which took 10 min for 30 cycles. The total assay time was 23 min, including droplet centrifugation, droplet DNA extraction and rapid droplet thermocycling. Evaporation from of 10 μL droplets was not significant during these procedures, since the longest time exposure to air and the vibrations was less than 5 min (during DNA extraction). The results of these sequentially executed processes were analyzed using gel electrophoresis. Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of

  5. Droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling for simpler and faster PCR assay using wire-guided manipulations

    PubMed Central

    2012-01-01

    A computer numerical control (CNC) apparatus was used to perform droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling on a single superhydrophobic surface and a multi-chambered PCB heater. Droplets were manipulated using “wire-guided” method (a pipette tip was used in this study). This methodology can be easily adapted to existing commercial robotic pipetting system, while demonstrated added capabilities such as vibrational mixing, high-speed centrifuging of droplets, simple DNA extraction utilizing the hydrophobicity difference between the tip and the superhydrophobic surface, and rapid thermocycling with a moving droplet, all with wire-guided droplet manipulations on a superhydrophobic surface and a multi-chambered PCB heater (i.e., not on a 96-well plate). Serial dilutions were demonstrated for diluting sample matrix. Centrifuging was demonstrated by rotating a 10 μL droplet at 2300 round per minute, concentrating E. coli by more than 3-fold within 3 min. DNA extraction was demonstrated from E. coli sample utilizing the disposable pipette tip to cleverly attract the extracted DNA from the droplet residing on a superhydrophobic surface, which took less than 10 min. Following extraction, the 1500 bp sequence of Peptidase D from E. coli was amplified using rapid droplet thermocycling, which took 10 min for 30 cycles. The total assay time was 23 min, including droplet centrifugation, droplet DNA extraction and rapid droplet thermocycling. Evaporation from of 10 μL droplets was not significant during these procedures, since the longest time exposure to air and the vibrations was less than 5 min (during DNA extraction). The results of these sequentially executed processes were analyzed using gel electrophoresis. Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of

  6. Evaluating variation in human gut microbiota profiles due to DNA extraction method and inter-subject differences

    PubMed Central

    Wagner Mackenzie, Brett; Waite, David W.; Taylor, Michael W.

    2015-01-01

    The human gut contains dense and diverse microbial communities which have profound influences on human health. Gaining meaningful insights into these communities requires provision of high quality microbial nucleic acids from human fecal samples, as well as an understanding of the sources of variation and their impacts on the experimental model. We present here a systematic analysis of commonly used microbial DNA extraction methods, and identify significant sources of variation. Five extraction methods (Human Microbiome Project protocol, MoBio PowerSoil DNA Isolation Kit, QIAamp DNA Stool Mini Kit, ZR Fecal DNA MiniPrep, phenol:chloroform-based DNA isolation) were evaluated based on the following criteria: DNA yield, quality and integrity, and microbial community structure based on Illumina amplicon sequencing of the V4 region of bacterial and archaeal 16S rRNA genes. Our results indicate that the largest portion of variation within the model was attributed to differences between subjects (biological variation), with a smaller proportion of variation associated with DNA extraction method (technical variation) and intra-subject variation. A comprehensive understanding of the potential impact of technical variation on the human gut microbiota will help limit preventable bias, enabling more accurate diversity estimates. PMID:25741335

  7. Mechanistic pathway for controlled extraction of guest molecule bound to herring sperm DNA using α-cyclodextrin

    NASA Astrophysics Data System (ADS)

    Jaffer, S. Syed; Ghosh, Prasun; Purkayastha, Pradipta

    2011-05-01

    trans-2-[4-(Dimethylamino)styryl]benzothiazole (DMASBT) is known to have dual emitting states where the locally excited (LE) state is responsible for fluorescence in less polar environment and in polar milieu fluorescence is from the twisted intramolecular charge transfer (TICT) state. This compound also undergoes minor groove binding to herring sperm DNA (hsDNA) evidenced by the absorption spectra before and after the binding process and an effect on DMASBT fluorescence by an anionic quencher. The binding occurs efficiently in a 1:1 manner, i.e. one guest molecule binds to one site on the hsDNA. Instead of following the DNA twist, the aromatic part seems to project outward. Thus, the bound molecule can be successfully extracted out from the DNA in a controlled way by the hydrophobic cavity of α-cyclodextrin (α-CD). The extraction starts even with a low concentration of α-CD and increases as the concentration is increased. Absorption, steady-state and time resolved fluorescence spectroscopic methods have been employed to explore the mechanistic pathway of binding of DMASBT to hsDNA. The mechanistic approach toward controlled extraction of the guest molecules from hsDNA by α-CD is reported and is expected to serve a significant purpose in treatment of drug overdose.

  8. Evaluating variation in human gut microbiota profiles due to DNA extraction method and inter-subject differences.

    PubMed

    Wagner Mackenzie, Brett; Waite, David W; Taylor, Michael W

    2015-01-01

    The human gut contains dense and diverse microbial communities which have profound influences on human health. Gaining meaningful insights into these communities requires provision of high quality microbial nucleic acids from human fecal samples, as well as an understanding of the sources of variation and their impacts on the experimental model. We present here a systematic analysis of commonly used microbial DNA extraction methods, and identify significant sources of variation. Five extraction methods (Human Microbiome Project protocol, MoBio PowerSoil DNA Isolation Kit, QIAamp DNA Stool Mini Kit, ZR Fecal DNA MiniPrep, phenol:chloroform-based DNA isolation) were evaluated based on the following criteria: DNA yield, quality and integrity, and microbial community structure based on Illumina amplicon sequencing of the V4 region of bacterial and archaeal 16S rRNA genes. Our results indicate that the largest portion of variation within the model was attributed to differences between subjects (biological variation), with a smaller proportion of variation associated with DNA extraction method (technical variation) and intra-subject variation. A comprehensive understanding of the potential impact of technical variation on the human gut microbiota will help limit preventable bias, enabling more accurate diversity estimates. PMID:25741335

  9. A new rapid method for Clostridium difficile DNA extraction and detection in stool: toward point-of-care diagnostic testing.

    PubMed

    Freifeld, Alison G; Simonsen, Kari A; Booth, Christine S; Zhao, Xing; Whitney, Scott E; Karre, Teresa; Iwen, Peter C; Viljoen, Hendrik J

    2012-01-01

    We describe a new method for the rapid diagnosis of Clostridium difficile infection, with stool sample preparation and DNA extraction by heat and physical disruption in a single-use lysis microreactor (LMR), followed by a rapid PCR amplification step. All steps can be accomplished in <20 minutes overall. Gel electrophoresis is currently used to detect the amplification product, pending real-time availability with an ultra-rapid thermocycler. Compared with the dual enzyme immunoassay (EIA) screening test (C. diff Quik Chek Complete; Techlab, Blacksburg, VA), the novel LMR/PCR assay showed complete concordance with all glutamate dehydrogenase (GDH) results (GDH(+)/toxin(+), n = 48; GDH(-)/toxin(-), n = 81). All 69 stool samples with discordant EIA results (GDH(+)/toxin(-)) were tested by both the LMR/PCR assay and the loop-mediated isothermal amplification test (LAMP) (Illumigene C. difficile; Meridian Bioscience, Cincinnati, OH). In 64/69 EIA-discordant samples, LAMP and LMR/PCR results matched (both positive in 29 sample and both negative in 35 samples); in the remaining 5 samples, results were discrepant between the LAMP assay (all five negative) and the LMR/PCR assay (all 5 positive). Overall, LMR/PCR testing matched the current algorithm of EIA and/or LAMP reflex testing in 193/198 (97.5%) samples. The present proof-of-concept study suggests that the novel LMR/PCR technique described here may be developed as an inexpensive, rapid, and reliable point-of-care diagnostic test for C. difficile infection and other infectious diseases. PMID:22402170

  10. Meta-barcoded evaluation of the ISO standard 11063 DNA extraction procedure to characterize soil bacterial and fungal community diversity and composition

    PubMed Central

    Terrat, Sebastien; Plassart, Pierre; Bourgeois, Emilie; Ferreira, Stéphanie; Dequiedt, Samuel; Adele-Dit-De-Renseville, Nathalie; Lemanceau, Philippe; Bispo, Antonio; Chabbi, Abad; Maron, Pierre-Alain; Ranjard, Lionel

    2015-01-01

    This study was designed to assess the influence of three soil DNA extraction procedures, namely the International Organization for Standardization (ISO-11063, GnS-GII and modified ISO procedure (ISOm), on the taxonomic diversity and composition of soil bacterial and fungal communities. The efficacy of each soil DNA extraction method was assessed on five soils, differing in their physico-chemical characteristics and land use. A meta-barcoded pyrosequencing approach targeting 16S and 18S rRNA genes was applied to characterize soil microbial communities. We first observed that the GnS-GII introduced some heterogeneity in bacterial composition between replicates. Then, although no major difference was observed between extraction procedures for soil bacterial diversity, we saw that the number of fungal genera could be underestimated by the ISO-11063. In particular, this procedure underestimated the detection in several soils of the genera Cryptococcus, Pseudallescheria, Hypocrea and Plectosphaerella, which are of ecological interest. Based on these results, we recommend using the ISOm method for studies focusing on both the bacterial and fungal communities. Indeed, the ISOm procedure provides a better evaluation of bacterial and fungal communities and is limited to the modification of the mechanical lysis step of the existing ISO-11063 standard. PMID:25195809

  11. Analysis of several methods for the extraction of high quality DNA from acetic acid bacteria in wine and vinegar for characterization by PCR-based methods.

    PubMed

    Jara, C; Mateo, E; Guillamón, J M; Torija, M J; Mas, A

    2008-12-10

    Acetic acid bacteria (AAB) are fastidious microorganisms with poor recovery in culture. Culture-independent methods are currently under examination. Good DNA extraction is a strict requirement of these methods. We compared five methods for extracting the DNA of AAB directly from wine and vinegar samples. Four matrices (white wine, red wine, superficial vinegar and submerged vinegar) contaminated with two AAB strains belonging to Acetobacter pasteurianus and Gluconacetobacter hansenii were assayed. To improve the yield and quality of the extracted DNA, a sample treatment (washing with polyvinyl pyrrolidone or NaCl) was also tested. DNA quality was measured by amplification of the 16S rRNA gene with conventional PCR. DNA recovery rate was assessed by real-time PCR. DNA amplification was always successful with the Wizard method though DNA recovery was poor. A CTAB-based method and NucleoSpin protocol extracted the highest DNA recoveries from wine and vinegar samples. Both of these methods require treatment to recover suitable DNA for amplification with maximum recovery. Both may therefore be good solutions for DNA extraction in wine and vinegar samples. DNA extraction of Ga hansenii was more effective than that of A. pasteurianus. The fastest and cheapest method we evaluated (the Thermal shock protocol) produced the worst results both for DNA amplification and DNA recovery. PMID:18950887

  12. Performance verification of the Maxwell 16 Instrument and DNA IQ Reference Sample Kit for automated DNA extraction of known reference samples.

    PubMed

    Krnajski, Z; Geering, S; Steadman, S

    2007-12-01

    Advances in automation have been made for a number of processes conducted in the forensic DNA laboratory. However, because most robotic systems are designed for high-throughput laboratories batching large numbers of samples, smaller laboratories are left with a limited number of cost-effective options for employing automation. The Maxwell 16 Instrument and DNA IQ Reference Sample Kit marketed by Promega are designed for rapid, automated purification of DNA extracts from sample sets consisting of sixteen or fewer samples. Because the system is based on DNA capture by paramagnetic particles with maximum binding capacity, it is designed to generate extracts with yield consistency. The studies herein enabled evaluation of STR profile concordance, consistency of yield, and cross-contamination performance for the Maxwell 16 Instrument. Results indicate that the system performs suitably for streamlining the process of extracting known reference samples generally used for forensic DNA analysis and has many advantages in a small or moderate-sized laboratory environment. PMID:25869266

  13. Antioxidant and DNA damage protecting potentials of polysaccharide extracted from Phellinus baumii using a delignification method.

    PubMed

    Jin, Qun-Li; Zhang, Zuo-Fa; Lv, Guo-Ying; Cai, Wei-Ming; Cheng, Jun-Wen; Wang, Jian-Gong; Fan, Lei-Fa

    2016-11-01

    A delignification method was employed to extract the polysaccharide from the fruiting body of Phellinus baumii. The three parameters, processing temperature, ratio of water to raw material and amount of acetic acid every time were optimized using the Box-Behnken design. As a result, the optimal extraction conditions were: processing temperature 70.3°C, ratio of water to raw material of 34.7mL/g and amount of acetic acid of 0.32mL every time. Under these conditions, the highest yield of polysaccharide (10.28%) was obtained. The main fraction (PPB-2) purified from PPB was composed of fucose, arabinose, galactose, glucose, xylose and mannose, while glucose was the predominant monosaccharide. PPB-2 exhibited noticeable antioxidant activity and strong protection against oxidative DNA damage. These findings implied that acid-chlorite delignification was a superior method to extract the polysaccharide from P. baumii and PPB-2 may be useful for cancer chemoprevention. PMID:27516306

  14. 5-Methylation of Cytosine in CG:CG Base-Pair Steps: A Physicochemical Mechanism for the Epigenetic Control of DNA Nanomechanics

    NASA Astrophysics Data System (ADS)

    Yusufaly, Tahir; Olson, Wilma; Li, Yun

    2014-03-01

    Van der Waals density functional theory is integrated with analysis of a non-redundant set of protein-DNA crystal structures from the Nucleic Acid Database to study the stacking energetics of CG:CG base-pair steps, specifically the role of cytosine 5-methylation. Principal component analysis of the steps reveals the dominant collective motions to correspond to a tensile ``opening'' mode and two shear ``sliding'' and ``tearing'' modes in the orthogonal plane. The stacking interactions of the methyl groups are observed to globally inhibit CG:CG step overtwisting while simultaneously softening the modes locally via potential energy modulations that create metastable states. The results have implications for the epigenetic control of DNA mechanics.

  15. Ethanolic extract of the Goldenseal, Hydrastis canadensis, has demonstrable chemopreventive effects on HeLa cells in vitro: Drug-DNA interaction with calf thymus DNA as target.

    PubMed

    Saha, Santu Kumar; Sikdar, Sourav; Mukherjee, Avinaba; Bhadra, Kakali; Boujedaini, Naoual; Khuda-Bukhsh, Anisur Rahman

    2013-07-01

    This study tested chemotherapeutic potential of Hydrastis canadensis (HC) extract in HeLa cells in vitro, with emphasis on its drug-DNA interaction and apoptosis induction ability. Nuclear uptake of HC by DAPI, Ao/Eb staining and internucleosomal DNA damage by comet assay was studied through fluorescence microscopy. Possible changes in MMP and apoptotic signalling events were critically analyzed. Cell cycle progression studied through FACS and fragmented DNA through "TUNEL" assay were critically analyzed. RT-PCR studies were conducted for analyzing Cyt-C and Bax translocation in mitochondrial and cytosolic extracts, and Caspase 3 in whole cell lysate. Role of p53-mediated regulation of NF-κβ and TNF-α was elucidated by Western blot analysis. Data of CD and Tm profile of CT-DNA were analyzed. Overall results indicated anti-cancer potential of HC through its ability to induce apoptosis, and interaction with CT-DNA that changed structural conformation of DNA, proving HC to be a promising candidate for chemoprevention. PMID:23628949

  16. Evaluation of five DNA extraction methods for purification of DNA from atherosclerotic tissue and estimation of prevalence of Chlamydia pneumoniae in tissue from a Danish population undergoing vascular repair

    PubMed Central

    Mygind, Tina; Østergaard, Lars; Birkelund, Svend; Lindholt, Jes S; Christiansen, Gunna

    2003-01-01

    Background To date PCR detection of Chlamydia pneumoniae DNA in atherosclerotic lesions from Danish patients has been unsuccessful. To establish whether non-detection was caused by a suboptimal DNA extraction method, we tested five different DNA extraction methods for purification of DNA from atherosclerotic tissue. Results The five different DNA extraction methods were tested on homogenate of atherosclerotic tissue spiked with C. pneumoniae DNA or EB, on pure C. pneumoniae DNA samples and on whole C. pneumoniae EB. Recovery of DNA was measured with a C. pneumoniae-specific quantitative real-time PCR. A DNA extraction method based on DNA-binding to spin columns with a silica-gel membrane (DNeasy Tissue kit) showed the highest recovery rate for the tissue samples and pure DNA samples. However, an automated extraction method based on magnetic glass particles (MagNA Pure) performed best on intact EB and atherosclerotic tissue spiked with EB. The DNeasy Tissue kit and MagNA Pure methods and the highly sensitive real-time PCR were subsequently used on 78 atherosclerotic tissue samples from Danish patients undergoing vascular repair. None of the samples were positive for C. pneumoniae DNA. The atherosclerotic samples were tested for inhibition by spiking with two different, known amounts of C. pneumoniae DNA and no samples showed inhibition. Conclusion As a highly sensitive PCR method and an optimised DNA extraction method were used, non-detection in atherosclerotic tissue from the Danish population was probably not caused by use of inappropriate methods. However, more samples may need to be analysed per patient to be completely certain on this. Possible methodological and epidemiological reasons for non-detection of C. pneumoniae DNA in atherosclerotic tissue from the Danish population are discussed. Further testing of DNA extraction methods is needed as this study has shown considerable intra- and inter-method variation in DNA recovery. PMID:12952556

  17. A two-step method for extraction of lipopolysaccharide from Shigella dysenteriae serotype 1 and Salmonella typhimurium: An improved method for enhanced yield and purity.

    PubMed

    Ahamad, Nadim; Katti, Dhirendra S

    2016-08-01

    Bacterial lipopolysaccharide (LPS) has been widely used as an antigen and adjuvant in immunological applications. Amongst the methods developed for extraction of LPS, hot phenol extraction (HPE) method is the gold standard. However, the HPE method provides poor yield of LPS (~4.5% by weight), is associated with relatively higher impurities of proteins and nucleic acids, and the acidic hot phenol can cause a degradative effect on LPS. In this work a two-step extraction (TSE) method was developed using a non-capsulated, [Shigella dysenteriae serotype-1] (Sd1) and capsulated [Salmonella typhimurium type B (StB)] species as model pathogens. The TSE method takes advantage of growth kinetics of bacteria wherein a two-step sequential approach for LPS extraction was employed. In step-1, culture supplemented with CaCl2 during early log phase of growth was induced to release LPS by the effect of EDTA at their late exponential phase of growth. In step-II, cells with left over LPS were subjected to modified HPE method that reduced both the degradative effect of acidic hot phenol and associated impurities. The LPS produced using TSE method enabled not only enhanced yield (~2.78 and ~2.91 fold higher for Sd1 and StB respectively) requiring nearly similar duration of extraction, but also was structurally and functionally comparable with LPS produced using HPE method and commercially procured LPS. Overall, the developed TSE method is relatively more efficient (enhanced yield), clean (healthy extraction with reduced impurities), safe (reduced handling of larger pathogenic culture) and cost-effective for LPS extraction with potential for scale up. PMID:27208662

  18. Simple DNA extraction protocol for a 16S rDNA study of bacterial diversity in tropical landfarm soil used for bioremediation of oil waste.

    PubMed

    Maciel, B M; Santos, A C F; Dias, J C T; Vidal, R O; Dias, R J C; Gross, E; Cascardo, J C M; Rezende, R P

    2009-01-01

    Landfarm soil is used to bioremediate oil wastes from petrochemical industries. We developed a simplified protocol for microbial DNA extraction of tropical landfarm soil using only direct lysis of macerated material. Two samples of tropical landfarm soil from a Brazilian refinery were analyzed by this protocol (one consisted of crude oil-contaminated soil; the other was continuously enriched for nine months with petroleum). The soil samples were lysed by maceration with liquid nitrogen, eliminating the need for detergents, organic solvents and enzymatic cell lysis. Then, the DNA from the lysed soil sample was extracted using phenol-chloroform-isoamyl alcohol or guanidium isothiocyanate, giving high DNA yields (more than 1 micro g DNA/g soil) from both soil types. This protocol compared favorably with an established method of DNA template preparation that included mechanical, chemical and enzymatic treatment for cell lysis. The efficiency of this extraction protocol was confirmed by polymerase chain reaction amplification of the 16S rRNA gene, denaturing gradient gel electrophoresis and cloning assays. Fifty-one different clones were obtained; their sequences were classified into at least seven different phyla of the Eubacteria group (Proteobacteria - alpha, gamma and delta, Chloroflexi, Actinobacteria, Acidobac teria, Planctomycetes, Bacteroidetes, and Firmicutes). Forty percent of the sequences could not be classified into these phyla, demonstrating the genetic diversity of this microbial community. Only eight isolates had sequences similar to known sequences of 16S rRNA of cultivable organisms or of known environmental isolates and therefore could be identified to the genus level. This method of DNA extraction is a useful tool for analysis of the bacteria responsible for petroleum degradation in contaminated environments. PMID:19440973

  19. Mild Two-Step Method to Construct DNA-Conjugated Silicon Nanoparticles: Scaffolds for the Detection of MicroRNA-21

    PubMed Central

    2015-01-01

    We describe a novel two-step method, starting from bulk silicon wafers, to construct DNA conjugated silicon nanoparticles (SiNPs). This method first utilizes reactive high-energy ball milling (RHEBM) to obtain alkene grafted SiNPs. The alkene moieties are subsequently reacted with commercially available thiol-functionalized DNA via thiol–ene click chemistry to produce SiNP DNA conjugates wherein the DNA is attached through a covalent thioether bond. Further, to show the utility of this synthetic strategy, we illustrate how these SiNP ODN conjugates can detect cancer-associated miR-21 via a fluorescence ON strategy. Given that an array of biological molecules can be prepared with thiol termini and that SiNPs are biocompatible and biodegradable, we envision that this synthetic protocol will find utility in salient SiNP systems for potential therapeutic and diagnostic applications. PMID:25243490

  20. Combined DNA extraction and antibody elution from filter papers for the assessment of malaria transmission intensity in epidemiological studies

    PubMed Central

    2013-01-01

    Background Informing and evaluating malaria control efforts relies on knowledge of local transmission dynamics. Serological and molecular tools have demonstrated great sensitivity to quantify transmission intensity in low endemic settings where the sensitivity of traditional methods is limited. Filter paper blood spots are commonly used a source of both DNA and antibodies. To enhance the operational practicability of malaria surveys, a method is presented for combined DNA extraction and antibody elution. Methods Filter paper blood spots were collected as part of a large cross-sectional survey in the Kenyan highlands. DNA was extracted using a saponin/chelex method. The eluate of the first wash during the DNA extraction process was used for antibody detection and compared with previously validated antibody elution procedures. Antibody elution efficiency was assessed by total IgG ELISA for malaria antigens apical membrane antigen-1 (AMA-1) and merozoite-surface protein-1 (MSP-142). The sensitivity of nested 18S rRNA and cytochrome b PCR assays and the impact of doubling filter paper material for PCR sensitivity were determined. The distribution of cell material and antibodies throughout filter paper blood spots were examined using luminescent and fluorescent reporter assays. Results Antibody levels measured after the combined antibody/DNA extraction technique were strongly correlated to those measured after standard antibody elution (p < 0.0001). Antibody levels for both AMA-1 and MSP-142 were generally slightly lower (11.3-21.4%) but age-seroprevalence patterns were indistinguishable. The proportion of parasite positive samples ranged from 12.9% to 19.2% in the different PCR assays. Despite strong agreement between outcomes of different PCR assays, none of the assays detected all parasite-positive individuals. For all assays doubling filter paper material for DNA extraction increased sensitivity. The concentration of cell and antibody material was not

  1. Sensing cocaine in saliva with attenuated total reflection infrared (ATR-IR) spectroscopy combined with a one-step extraction method

    NASA Astrophysics Data System (ADS)

    Hans, Kerstin M.-C.; Gianella, Michele; Sigrist, Markus W.

    2012-03-01

    On-site drug tests have gained importance, e.g., for protecting the society from impaired drivers. Since today's drug tests are majorly only positive/negative, there is a great need for a reliable, portable and preferentially quantitative drug test. In the project IrSens we aim to bridge this gap with the development of an optical sensor platform based on infrared spectroscopy and focus on cocaine detection in saliva. We combine a one-step extraction method, a sample drying technique and infrared attenuated total reflection (ATR) spectroscopy. As a first step we have developed an extraction technique that allows us to extract cocaine from saliva to an almost infrared-transparent solvent and to record ATR spectra with a commercially available Fourier Transform-infrared spectrometer. To the best of our knowledge this is the first time that such a simple and easy-to-use one-step extraction method is used to transfer cocaine from saliva into an organic solvent and detect it quantitatively. With this new method we are able to reach a current limit of detection around 10 μg/ml. This new extraction method could also be applied to waste water monitoring and controlling caffeine content in beverages.

  2. Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

    PubMed Central

    Cankar, Katarina; Štebih, Dejan; Dreo, Tanja; Žel, Jana; Gruden, Kristina

    2006-01-01

    Background Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Results Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary

  3. High abundance of ammonia-oxidizing Archaea in coastal waters, determined using a modified DNA extraction method.

    PubMed

    Urakawa, Hidetoshi; Martens-Habbena, Willm; Stahl, David A

    2010-04-01

    Molecular characterizations of environmental microbial populations based on recovery and analysis of DNA generally assume efficient or unbiased extraction of DNA from different sample matrices and microbial groups. Appropriate controls to verify this basic assumption are rarely included. Here three different DNA extractions, performed with two commercial kits (FastDNA and UltraClean) and a standard phenol-chloroform method, and two alternative filtration methods (Sterivex and 25-mm-diameter polycarbonate filters) were evaluated, using the addition of Nitrosopumilus maritimus cells to track the recovery of DNA from marine Archaea. After the comparison, a simplified phenol-chloroform extraction method was developed and shown to be significantly superior, in terms of both the recovery and the purity of DNA, to other protocols now generally applied to environmental studies. The simplified and optimized method was used to quantify ammonia-oxidizing Archaea at different depth intervals in a fjord (Hood Canal) by quantitative PCR. The numbers of Archaea increased with depth, often constituting as much as 20% of the total bacterial community. PMID:20118363

  4. Fast and simple DNA extraction from saliva and sperm cells obtained from the skin or isolated from swabs.

    PubMed

    von Wurmb-Schwark, Nicole; Mályusz, Victoria; Fremdt, Heike; Koch, Christine; Simeoni, Eva; Schwark, Thorsten

    2006-05-01

    The forensic scientist often has to cope with problematic samples from the crime scene due to their minute size and thus the low amount of extractable DNA. The retrieval of DNA from swabs taken from the surface of the skin, for example, in cases of strangulation, can be especially difficult. We systematically investigated swabs taken from the skin (to obtain a genetic profile from the victim and also from a possible offender) and from sperm cell containing swabs using two extraction kits: the Invisorb forensic and the Invisorb spin swab kit (both Invitek, Germany). DNA quality and quantity were tested on ethidium bromide containing agarose gels and in a highly sensitive duplex-PCR, which amplifies fragments specific for mitochondrial and nuclear DNA. Absolute quantification was done using real time PCR. Samples, which were positive in the duplex-PCR, were also employed to genetic fingerprinting using the Powerplex ES and the AmpFlSTRIdentifiler(TM) kits. Our study shows that the easy-to-use Invisorb spin swab kit is very suitable for DNA isolation from swabs taken from the skin and also from sperm cells. Retrieval of cells from the skin with swabs moistened in extraction buffer, not in distilled water, led to a significant higher DNA yield. PMID:16516526

  5. Evaluation of four DNA extraction methods for the detection of Tritrichomonas foetus in feline stool specimens by polymerase chain reaction.

    PubMed

    Stauffer, Stephen H; Birkenheuer, Adam J; Levy, Michael G; Marr, Henry; Gookin, Jody L

    2008-09-01

    Feces are increasingly valued as practical samples for molecular diagnosis of infectious disease. However, extraction of polymerase chain reaction (PCR) quality DNA from fecal samples can be challenging because of coextraction of PCR inhibitors. Because the type and quantity of PCR inhibitors is influenced by diet, endogenous flora, and concurrent disease, it is unlikely that extraction method performance with human feces can be directly extrapolated to that of domestic cats. In the present study, 4 commercially available DNA extraction methods were examined for their influence on the sensitivity of PCR for the detection of Tritrichomonas foetus in feline stool. DNA was extracted from serially diluted feline-origin T. foetus trophozoites in the absence or presence of feline feces. The ZR Fecal DNA kit was identified as affording the greatest analytical sensitivity and reproducibility and was able to detect >or=10 T. foetus organisms per 100 mg feces in 100% of PCR reactions. Further, the identified extraction method could be completed in the shortest time of all kits tested. PMID:18776100

  6. In Vitro Effect of Aqueous Extract and Fraction IV Portion of Ximenia americana Stem Bark on Trypanosoma congolense DNA

    PubMed Central

    Maikai, Victor Ambrose; Maikai, Beatty Viv; Kobo, Patricia Ishyaku

    2014-01-01

    Trypanosomosis is a debilitating disease affecting mainly livestock and humans in tropical Africa. Chemically synthesized drugs and medicinal plants have been used in the treatment and control of this disease. In this study, the in vitro effect of aqueous extracts and fraction IV extract of Ximenia americana stem bark on Trypanosoma congolense DNA was investigated. The extracts were incubated with the parasites in vitro at 300 mg/mL aqueous extract and 25 mg/mL fraction IV portion for 30, 60, and 120 mins. The DNA of the trypanosomes was isolated and digested using ECOR1 enzyme and subsequently PCR was carried out. Results showed that aqueous extract and fraction IV portion immobilized 55% and 90% of the trypanosomes after 30-minute incubation. Subsequent isolation of the parasite DNA and agarose gel electrophoresis did not reveal that cell death was as a result of DNA fragmentation. This suggests that cell death was by another mechanism of action. PMID:24587898

  7. Enhanced DPPH radical scavenging activity and DNA protection effect of litchi pericarp extract by Aspergillus awamori bioconversion

    PubMed Central

    2012-01-01

    Background Litchi (Litchi chinensis Sonn.) pericarp is a major byproduct which contains a significant amount of polyphenol. This study was designed to biotransformation litchi pericarp extract (LPE) by Aspergillus awamori to produce more bioactive compounds with stronger antioxidant activities. Results The study exhibited that the 2,2-diphenyl-1-picrylhydrazyl radical scavenging activities significantly (p < 0.05) increased from 15.53% to 18.23% in the water-extracted fraction and from 25.41% to 36.82% in the ethyl acetate-extracted fraction. Application of DNA cleavage assay further demonstrated the enhanced protection effect of the fermented phenolics on DNA damage. It is also noted that the water-extracted fraction of the fermented LPE possessed a much stronger capacity than the ethyl acetate-extracted fraction to prevent from damage of supercoiled DNA. Interestingly, it was found that some new compounds such as catechin and quercetin appeared after of A. awamori fermentation of LPE, which could account for the enhanced antioxidant activity. Conclusion The DPPH radical scavenging activity and DNA protection effect of LPE were increased by Aspergillus awamori bioconversion while some compounds responsible for the enhanced antioxidant activity were identified. This study provided an effective way of utilizing fruit pericarp as a readily accessible source of the natural antioxidants in food industry and, thus, extended the application area such as fruit by-products. PMID:23016522

  8. Aqueous extract of Crataegus azarolus protects against DNA damage in human lymphoblast Cell K562 and enhances antioxidant activity.