Science.gov

Sample records for dna extraction step

  1. DNA extraction from fossil eggshell.

    PubMed

    Oskam, Charlotte L; Bunce, Michael

    2012-01-01

    Avian eggshell fragments recovered from both paleontological and archaeological deposits contain a cache of well-preserved ancient DNA. Here, we describe an extraction protocol that has been optimized to maximize the recovery of ancient DNA from fossil eggshell and minimize the co-purification of PCR inhibitors. In this method, fossil eggshell fragments are powdered, then digested and heated to release DNA from the calcite matrix. The digest then undergoes a concentration step before purification and washing using silica columns. The method has been used to recover aDNA from the eggshell of many avian species including moa, elephant birds, and emu, up to 19,000 years old. PMID:22237523

  2. Phytoplasma plasmid DNA extraction.

    PubMed

    Andersen, Mark T; Liefting, Lia W

    2013-01-01

    Phytoplasma plasmids have generally been detected from DNA extracted from plants and insects using methods designed for the purification of total phytoplasma DNA. Methods include extraction from tissues that are high in phytoplasma titre, such as the phloem of plants, with the use of CsCl-bisbenzimide gradients that exploit the low G+C content of phytoplasma DNA. Many of the methods employed for phytoplasma purification have been described elsewhere in this book. Here we describe in detail two methods that are specifically aimed at isolating plasmid DNA. PMID:22987431

  3. Development of a rapid DNA extraction method and one-step nested PCR for the detection of Naegleria fowleri from the environment.

    PubMed

    Ahmad, Arine Fadzlun; Lonnen, James; Andrew, Peter W; Kilvington, Simon

    2011-10-15

    Naegleria fowleri is a small free-living amoebo-flagellate found in natural and manmade thermal aquatic habitats worldwide. The organism is pathogenic to man causing fatal primary amoebic meningoencephalitis (PAM). Infection typically results from bathing in contaminated water and is usually fatal. It is, therefore, important to identify sites containing N. fowleri in the interests of preventive public health microbiology. Culture of environmental material is the conventional method for the isolation of N. fowleri but requires several days incubation and subsequent biochemical or molecular tests to confirm identification. Here, a nested one-step PCR test, in conjunction with a direct DNA extraction from water or sediment material, was developed for the rapid and reliable detection of N. fowleri from the environment. Here, the assay detected N, fowleri in 18/109 river water samples associated with a nuclear power plant in South West France and 0/10 from a similar site in the UK. Although culture of samples yielded numerous thermophilic free-living amoebae, none were N. fowleri or other thermophilic Naegleria spp. The availability of a rapid, reliable and sensitive one-step nested PCR method for the direct detection of N. fowleri from the environment may aid ecological studies and enable intervention to prevent PAM cases. PMID:21855956

  4. 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

  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. Comparison of three DNA extraction methods for recovery of soil protist DNA.

    PubMed

    Santos, Susana S; Nielsen, Tue Kjærgaard; Hansen, Lars H; Winding, Anne

    2015-08-01

    The use of molecular methods to investigate protist communities in soil is in rapid development this decade. Molecular analysis of soil protist communities is usually dependant on direct genomic DNA extraction from soil and inefficient or differential DNA extraction of protist DNA can lead to bias in downstream community analysis. Three commonly used soil DNA extraction methods have been tested on soil samples from three European Long-Term Observatories (LTOs) with different land-use and three protist cultures belonging to different phylogenetic groups in different growth stages. The methods tested were: ISOm-11063 (a version of the ISO-11063 method modified to include a FastPrep ®-24 mechanical lysis step), GnS-GII (developed by the GenoSol platform to extract soil DNA in large-scale soil surveys) and a commercial DNA extraction kit - Power Lyzer™ PowerSoil® DNA Isolation Kit (MoBio). DNA yield and quality were evaluated along with DNA suitability for amplification of 18S rDNA fragments by PCR. On soil samples, ISOm-11063 yields significantly higher DNA for two of the three soil samples, however, MoBio extraction favors DNA quality. This method was also more effective to recover copies of 18S rDNA numbers from all soil types. In addition and despite the lower yields, higher DNA quality was observed with DNA extracted from protist cultures with the MoBio method. Likewise, a bead-beating step shows to be a good solution for DNA extraction of soil protists, since the recovery of DNA from protist cultures and from the different soil samples with the ISOm method proved to be efficient in recovering PCR-amplifiable DNA. This study showed that soil DNA extraction methods provide biased results towards the cyst stages of protist organism. PMID:25966645

  7. 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…

  8. 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

  9. 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

  10. Simplified buccal DNA extraction with FTA Elute Cards.

    PubMed

    Wolfgramm, Eldamária de Vargas; de Carvalho, Fernanda Magri; Aguiar, Vitor Rezende da Costa; Sartori, Mariana Penha De Nadai; Hirschfeld-Campolongo, Gabriela C R; Tsutsumida, Weslley M; Louro, Iúri Drumond

    2009-03-01

    DNA isolation is the initial step of most genetic studies, and ideally it should use a reliable and non-invasive method. Buccal samples are adequate for such purposes, being painless, easy to collect and a very reliable DNA source. FTA Elute Cards are relatively new on the market and are designed for rapid blood DNA extraction, in which DNA is solubilized in water, instead of attached to the paper matrix. In this study, we sought to test if FTA Elute Cards are suitable for buccal DNA extraction. Furthermore, several time and temperature conditions were analyzed in order to determine the best concentration/time ratio. Twenty-five different conditions were tested in quadruplicate and extracted DNA was quantified by real-time PCR. PMID:19215882

  11. 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

  12. Rapid extraction and preservation of genomic DNA from human samples

    PubMed Central

    Kalyanasundaram, D.; Kim, J.-H.; Yeo, W.-H.; Oh, K.; Lee, K.-H.; Kim, M.-H.; Ryew, S.-M.; Ahn, S.-G.; Gao, D.; Cangelosi, G. A.; Chung, J.-H.

    2013-01-01

    Simple and rapid extraction of human genomic DNA remains a bottle neck for genome analysis and disease diagnosis. Current methods using microfilters require cumbersome, multiple handling steps in part because salt conditions must be controlled for attraction and elution of DNA in porous silica. We report a novel extraction method of human genomic DNA from buccal swab- and saliva samples. DNA is attracted on to a gold-coated microchip by an electric field and capillary action while the captured DNA is eluted by thermal heating at 70 °C. A prototype device was designed to handle 4 microchips, and a compatible protocol was developed. The extracted DNA using microchips was characterized by qPCR for different sample volumes, using different lengths of PCR amplicon, and nuclear and mitochondrial genes. In comparison with a commercial kit, an equivalent yield of DNA extraction was achieved with fewer steps. Room-temperature preservation for one month was demonstrated for captured DNA, facilitating straightforward collection, delivery and handling of genomic DNA in an environment-friendly protocol. PMID:23307121

  13. 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

  14. 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

  15. Experiments in DNA extraction and PCR amplification from bighorn sheep feces: the importance of DNA extraction method.

    PubMed

    Wehausen, J D; Ramey, R R; Epps, C W

    2004-01-01

    Reliability of genotyping is an issue for studies using non-invasive sources of DNA. We emphasize the importance of refining DNA extraction methods to maximize reliability and efficiency of genotyping for such DNA sources. We present a simple and general method to quantitatively compare genotyping reliability of various DNA extraction techniques and sample materials used. For bighorn sheep (Ovis canadensis) fecal samples we compare different fecal pellet materials, different amounts of fecal pellet material, and the effects of eliminating two DNA extraction steps for four microsatellite loci and four samples heterozygous at each locus. We evaluated 192 PCR outcomes for each treatment using indices of PCR success and peak height (signal strength) developed from analysis output of sequencer chromatograms. Outermost pellet material produced PCR results almost equivalent to DNA extracted from blood. Where any inner pellet material was used for DNA extraction, PCR results were poorer and inconsistent among samples. PCR success was not sensitive to amount of pellet material used until it was decreased to 15 mg from 60 mg. Our PCR index provides considerably more information relative to potential genotyping errors than simply comparing genotypes derived from paired fecal and blood or tissue samples. Our DNA extraction method probably has wide applicability to herbivores that produce pelleted feces where samples dry rapidly after deposition. PMID:15475396

  16. Nondestructive DNA extraction from museum specimens.

    PubMed

    Hofreiter, Michael

    2012-01-01

    Natural history museums around the world hold millions of animal and plant specimens that are potentially amenable to genetic analyses. With more and more populations and species becoming extinct, the importance of these specimens for phylogenetic and phylogeographic analyses is rapidly increasing. However, as most DNA extraction methods damage the specimens, nondestructive extraction methods are useful to balance the demands of molecular biologists, morphologists, and museum curators. Here, I describe a method for nondestructive DNA extraction from bony specimens (i.e., bones and teeth). In this method, the specimens are soaked in extraction buffer, and DNA is then purified from the soaking solution using adsorption to silica. The method reliably yields mitochondrial and often also nuclear DNA. The method has been adapted to DNA extraction from other types of specimens such as arthropods. PMID:22237527

  17. DNA nanoarchitectures: steps towards biological applications.

    PubMed

    Tintoré, Maria; Eritja, Ramon; Fábrega, Carmen

    2014-07-01

    DNA's remarkable molecular recognition properties, flexibility, and structural features make it one of the most promising scaffolds to design a variety of nanostructures. During recent decades, two major methods have been developed for the construction of DNA nanomaterials in a programmable way; both generate nanostructures in one, two, and three dimensions. The tile-based assembly process is a useful tool to construct large and simple structures; the DNA origami method is suitable for the production of smaller, more sophisticated and well-defined structures. Proteins, nanoparticles and other functional elements have been specifically positioned into designed patterns on these structures. They can also act as templates to study chemical reactions, help in the structural determination of proteins, and be used as platform for genomic and drug delivery applications. In this review we examine recent progresses towards the potential use of DNA nanostructures in molecular and cellular biology. PMID:24953971

  18. 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

  19. 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

  20. 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

  1. 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)

  2. An Efficient Method for Genomic DNA Extraction from Different Molluscs Species

    PubMed Central

    Pereira, Jorge C.; Chaves, Raquel; Bastos, Estela; Leitão, Alexandra; Guedes-Pinto, Henrique

    2011-01-01

    The selection of a DNA extraction method is a critical step when subsequent analysis depends on the DNA quality and quantity. Unlike mammals, for which several capable DNA extraction methods have been developed, for molluscs the availability of optimized genomic DNA extraction protocols is clearly insufficient. Several aspects such as animal physiology, the type (e.g., adductor muscle or gills) or quantity of tissue, can explain the lack of efficiency (quality and yield) in molluscs genomic DNA extraction procedure. In an attempt to overcome these aspects, this work describes an efficient method for molluscs genomic DNA extraction that was tested in several species from different orders: Veneridae, Ostreidae, Anomiidae, Cardiidae (Bivalvia) and Muricidae (Gastropoda), with different weight sample tissues. The isolated DNA was of high molecular weight with high yield and purity, even with reduced quantities of tissue. Moreover, the genomic DNA isolated, demonstrated to be suitable for several downstream molecular techniques, such as PCR sequencing among others. PMID:22174651

  3. A simple method to extract DNA from hair shafts using enzymatic laundry powder.

    PubMed

    Guan, Zheng; Zhou, Yu; Liu, Jinchuan; Jiang, Xiaoling; Li, Sicong; Yang, Shuming; Chen, Ailiang

    2013-01-01

    A simple method to extract DNA from hair shafts was developed by using enzymatic laundry powder at the first step of the process. The whole extraction can be finished in less than 2 hours. The simple extraction reagent proposed here contains only two cheap components: ordinary enzymatic laundry powder and PCR buffer. After extraction, an ultra sensitive fluorescent nucleic acid stain, PicoGreen, was used for quantifying trace amount of double-stranded DNA in the solution extracted. For further validation of DNA extraction, four primers were employed to amplify DNA microsatellite loci. Both fluorescence spectroscopy and PCR results suggested that this method can extract DNA from hair shafts with good efficiency and repeatability. The study will greatly facilitate the use of hair shafts in future for DNA analyses on genome-wide scale. PMID:23922747

  4. A Simple Method to Extract DNA from Hair Shafts Using Enzymatic Laundry Powder

    PubMed Central

    Liu, Jinchuan; Jiang, Xiaoling; Li, Sicong; Yang, Shuming; Chen, Ailiang

    2013-01-01

    A simple method to extract DNA from hair shafts was developed by using enzymatic laundry powder at the first step of the process. The whole extraction can be finished in less than 2 hours. The simple extraction reagent proposed here contains only two cheap components: ordinary enzymatic laundry powder and PCR buffer. After extraction, an ultra sensitive fluorescent nucleic acid stain, PicoGreen, was used for quantifying trace amount of double-stranded DNA in the solution extracted. For further validation of DNA extraction, four primers were employed to amplify DNA microsatellite loci. Both fluorescence spectroscopy and PCR results suggested that this method can extract DNA from hair shafts with good efficiency and repeatability. The study will greatly facilitate the use of hair shafts in future for DNA analyses on genome-wide scale. PMID:23922747

  5. Plant and metagenomic DNA extraction of mucilaginous seeds

    PubMed Central

    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

  6. 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

  7. Determination of an efficient and reliable method for DNA extraction from ticks.

    PubMed

    Halos, Lénaïg; Jamal, Taoufik; Vial, Laurence; Maillard, Renaud; Suau, Antonia; Le Menach, Arnaud; Boulouis, Henri-Jean; Vayssier-Taussat, Muriel

    2004-01-01

    Molecular detection of pathogenic microorganisms in ticks is based on DNA amplification of the target pathogen; therefore, extraction of DNA from the tick is a major step. In this study, we compared three different tick DNA extraction protocols based on an enzymatic digestion by proteinase K followed by DNA extraction by a commercial kit (method 1), or on mortar crushing, proteinase K digestion and phenol/chloroform DNA extraction (method 2) and fine crushing with a beads beater, proteinase K digestion and DNA extraction using a commercial kit (method 3). The absence of PCR inhibitors and the DNA quality were evaluated by PCR amplification of the tick mitochondrial 16S rRNA gene using tick-specific primers. With method 1, 23/30 (77%) of the samples were extracted; with method 2, 30/31 (97%) of the samples were extracted and with method 3, 30/30 (100%) of the samples were extracted. DNA extraction efficiency using method 3 is significantly higher than DNA extraction efficiency using method 1 (100% versus 77%, P < 0.05). There was no significant difference between methods 2 and 3. Method 3 was however more adapted to cohort studies than method 2. This technique was validated for cohort tick DNA extraction and applicable to the treatment of small samples such as nymphs and soft ticks with 100% efficiency. PMID:15535960

  8. 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.

  9. Single-step intercalating dye strategies for DNA damage studies.

    PubMed

    Paidipalli, Manasa; Pjescic, Ilija; Hindmarsh, Patrick L; Crews, Niel D

    2013-08-01

    Many analytical protocols exist for the quantification of varied types of DNA damage, which span a range of complexity and sensitivity. As an alternative or companion to existing procedures, this article demonstrates the application of quantitative PCR (qPCR) and high-resolution DNA melting analysis (HRMA) to the detection and quantification of intramolecular DNA damage and/or strand breaks. These proven molecular biology methods are essentially single-step processes. When implemented with a third-generation saturating DNA dye, high sensitivity can be obtained. The experiments presented here demonstrate how DNA damage can inhibit amplification of the affected molecules. This corresponding decrease in the initial concentration of amplifiable DNA can be measured with qPCR. In addition, damage in the form of intramolecular dimerization and strand breaks alters the stored energy in the hydrogen bonds between the two strands in the dsDNA molecule. This significantly affects the thermal stability, which can be measured with extreme precision using HRMA. Simplified damage models were used in these experiments: UV-C irradiation to produce photoproducts, and restriction enzyme digestion to simulate double-strand breaks. The findings of this work, however, can be intuitively applied to the broad scope of DNA damage mechanisms. PMID:23770014

  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. Single-strand DNA translation initiation step analyzed by Isothermal Titration Calorimetry

    SciTech Connect

    Damian, Luminita; Universite de Toulouse, UPS, IPBS, F-31077 Toulouse; IUB, School of Engineering and Science, D-28727 Bremen ; Marty-Detraves, Claire; Universite de Toulouse, UPS, IPBS, F-31077 Toulouse ; Winterhalter, Mathias; Fournier, Didier; Universite de Toulouse, UPS, IPBS, F-31077 Toulouse ; Paquereau, Laurent; Universite de Toulouse, UPS, IPBS, F-31077 Toulouse

    2009-07-31

    Is single-strand DNA translatable? Since the 60s, the question still remains whether or not DNA could be directly translated into protein. Some discrepancies in the results were reported about functional translation of single-strand DNA but all results converged on a similar behavior of RNA and ssDNA in the initiation step. Isothermal Titration Calorimetry method was used to determine thermodynamic constants of interaction between single-strand DNA and S30 extract of Escherichia coli. Our results showed that the binding was not affected by the nature of the template tested and the dissociation constants were in the same range when ssDNA (K{sub d} = 3.62 {+-} 2.1 x 10{sup -8} M) or the RNA corresponding sequence (K{sub d} = 2.7 {+-} 0.82 x 10{sup -8} M) bearing SD/ATG sequences were used. The binding specificity was confirmed by antibiotic interferences which block the initiation complex formation. These results suggest that the limiting step in translation of ssDNA is the elongation process.

  12. 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

  13. 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.

  14. 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

  15. 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

    2016-02-01

    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.

  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. Optimized microbial DNA extraction from diarrheic stools

    PubMed Central

    2012-01-01

    Background The detection of enteropathogens in stool specimens increasingly relies on the detection of specific nucleic acid sequences. We observed that such detection was hampered in diarrheic stool specimens and we set-up an improved protocol combining lyophilization of stools prior to a semi-automated DNA extraction. Findings A total of 41 human diarrheic stool specimens comprising of 35 specimens negative for enteropathogens and six specimens positive for Salmonella enterica in culture, were prospectively studied. One 1-mL aliquot of each specimen was lyophilised and total DNA was extracted from lyophilised and non-lyophilised aliquots by combining automatic and phenol-chloroform DNA extraction. DNA was incorporated into real-time PCRs targeting the 16S rRNA gene of Bacteria and the archaea Methanobrevibacter smithii and the chorismate synthase gene of S. enterica. Whereas negative controls consisting in DNA-free water remained negative, M. smithii was detected in 26/41 (63.4%) non-lyophilised (Ct value 28.78 ± 9.1) versus 39/41 (95.1%) lyophilised aliquots (Ct value 22.04 ± 5.5); bacterial 16S rRNA was detected in 33/41 (80.5%) non-lyophilised (Ct value 28.11 ± 5.9) versus 40/41 (97.6%) lyophilised aliquots (Ct value 24.94 ± 6.6); and S. enterica was detected in 6/6 (100%) non-lyophilized and lyophilized aliquots (Ct value 26.98 ± 4.55 and 26.16 ± 4.97, respectively). S. enterica was not detected in the 35 remaining diarrheal-stool specimens. The proportion of positive specimens was significantly higher after lyophilization for the detection of M. smithii (p = 0.00043) and Bacteria (p = 0.015). Conclusion Lyophilization of diarrheic stool specimens significantly increases the PCR-based detection of microorganisms. The semi-automated protocol described here could be routinely used for the molecular diagnosis of infectious diarrhea. PMID:23273000

  19. 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,…

  20. 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,

  1. Bacterial and fungal DNA extraction from positive blood culture bottles: a manual and an automated protocol.

    PubMed

    Mäki, Minna

    2015-01-01

    When adapting a gene amplification-based method in a routine sepsis diagnostics using a blood culture sample as a specimen type, a prerequisite for a successful and sensitive downstream analysis is the efficient DNA extraction step. In recent years, a number of in-house and commercial DNA extraction solutions have become available. Careful evaluation in respect to cell wall disruption of various microbes and subsequent recovery of microbial DNA without putative gene amplification inhibitors should be conducted prior selecting the most feasible DNA extraction solution for the downstream analysis used. Since gene amplification technologies have been developed to be highly sensitive for a broad range of microbial species, it is also important to confirm that the used sample preparation reagents and materials are bioburden-free to avoid any risks for false-positive result reporting or interference of the diagnostic process. Here, one manual and one automated DNA extraction system feasible for blood culture samples are described. PMID:25319779

  2. 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

  3. 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

  4. Direct DNA Extraction from Mycobacterium tuberculosis Frozen Stocks as a Reculture-Independent Approach to Whole-Genome Sequencing

    PubMed Central

    Zallet, J.; Lillebaek, T.; Andersen, A. B.; Niemann, S.; Rasmussen, E. M.; Kohl, T. A.

    2015-01-01

    Culturing before DNA extraction represents a major time-consuming step in whole-genome sequencing of slow-growing bacteria, such as Mycobacterium tuberculosis. We report a workflow to extract DNA from frozen isolates without reculturing. Prepared libraries and sequence data were comparable with results from recultured aliquots of the same stocks. PMID:26019203

  5. 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 translocation of DNA temporally segregated into two distinct phases of the mechanochemical cycle of the entire ring. Because this ring-ATPase is a member of the ASCE superfamily of ATPases, these results may have implications for a broad and diverse family of cellular motors.

  6. 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

  7. Selective Extraction of Bacterial DNA from the Surfaces of Macroalgae▿

    PubMed Central

    Burke, Catherine; Kjelleberg, Staffan; Thomas, Torsten

    2009-01-01

    A novel method has been developed for the selective extraction of DNA from surface-associated bacterial communities from the two model marine benthic algae Ulva australis and Delisea pulchra. The extracted DNA had no detectable contamination with host DNA, was recovered in high yield and quality, and was representative of the bacterial community on the algal surfaces. The DNA is suitable for a variety of subsequent applications, including the construction of large-insert clone libraries and metagenomic sequencing. PMID:18978081

  8. STR typing of ancient DNA extracted from hair shafts of Siberian mummies.

    PubMed

    Amory, S; Keyser, C; Crubézy, E; Ludes, B

    2007-03-01

    The aim of this study was to determine if ancient hair shafts could be suitable for nuclear DNA analysis and to develop an efficient and straightforward protocol for DNA extraction and STR typing of ancient specimens. The developed method was validated on modern and forensic samples and then successfully applied on ancient hairs collected from Siberian mummies dating from the 16th to the early 19th centuries. In parallel extractions including or excluding a washing step were performed at least two times for each sample in order to evaluate the influence on the quantity of nuclear DNA yielded and on the typing efficiency. Twelve ancient individuals were analyzed through our approach and full and reliable profiles were obtained for four of them. These profiles were validated by comparison with those obtained from bone and teeth DNA extracted from the same ancient specimens. The present study demonstrates that the washing step cannot be considered as deleterious for DNA retrieval since the same results were obtained by the two approaches. This finding challenges the hypothesis that recoverable nuclear DNA is only found on the outer surface of hair shafts and provides evidence that nuclear DNA can be successfully extracted from ancient hair shafts. The method described here constitutes a promising way for non-invasive investigations in ancient DNA analysis for precious or historical samples as well as forensic casework analyses. PMID:16839727

  9. 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

  10. 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

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

    PubMed Central

    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 peoples own DNA extraction protocols and not as an advertisement of a commercially available product.

  12. 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. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain. PMID:23328684

  13. 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.

  14. 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 510 minutes.

  15. 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

  16. 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...

  17. A SIMPLE VERSATILE HIGH THROUGHPUT DNA EXTRACTION METHOD SUITABLE FOR PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PCR has become one of the most popular techniques in functional genomics. Projects in both forward and reverse genetics routinely require PCR amplification of thousands of samples. Processing samples to extract DNA of sufficient purity for PCR is often a limiting step. We have developed a simple ...

  18. A simple DNA extraction method for marijuana samples used in amplified fragment length polymorphism (AFLP) analysis.

    PubMed

    Miller Coyle, Heather; Shutler, Gary; Abrams, Sharon; Hanniman, Janet; Neylon, Suzanne; Ladd, Carll; Palmbach, Timothy; Lee, Henry C

    2003-03-01

    As a first step in developing a molecular method for the individualization of marijuana samples, we evaluated a plant DNA extraction kit. The QIAGEN plant DNeasy method uses a spin column format for recovery of DNA and is effective for obtaining high molecular weight DNA from leaf, flower (bud), and seed samples of marijuana. The average DNA yield was 125-500 ng per 100 milligrams of fresh plant tissue. The recovered DNA was of polymerase chain reaction (PCR) quality as measured by the ability to generate reproducible amplified fragment length polymorphism (AFLP) profiles. AFLP is a technique used to create a DNA profile for plant varieties and is being applied to marijuana samples by the authors to link growers and distributors of clonal material. The QIAGEN plant DNeasy method was simple, efficient, and reproducible for processing small quantities of marijuana into DNA. PMID:12664992

  19. A rapid and simple method for DNA extraction from yeasts and fungi isolated from Agave fourcroydes.

    PubMed

    Tapia-Tussell, Raul; Lappe, Patricia; Ulloa, Miguel; Quijano-Ramayo, Andrés; Cáceres-Farfán, Mirbella; Larqué-Saavedra, Alfonso; Perez-Brito, Daisy

    2006-05-01

    A simple and easy protocol for extracting high-quality DNA from different yeast and filamentous fungal species is described. This method involves two important steps: first, the disruption of cell walls by mechanical means and freezing; and second, the extraction, isolation, and precipitation of genomic DNA. The absorbance ratios (A(260)/A(280)) obtained ranged from 1.6 to 2.0. The main objective of this procedure is to extract pure DNA from yeast and filamentous fungi, including those with high contents of proteins, polysaccharides, and other complex compounds in their cell walls. The yield and quality of the DNAs obtained were suitable for micro/minisatellite primer-polymerase chain reaction (MSP-PCR) fingerprinting as well as for the sequence of the D1/D2 domain of the 26S rDNA. PMID:16691008

  20. 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 concentration efficiencies among substrates is proposed. PMID:23040243

  1. Optimization of DNA Extraction for RAPD and ISSR Analysis of Arbutus unedo L. Leaves

    PubMed Central

    Sá, Olga; Pereira, José Alberto; Baptista, Paula

    2011-01-01

    Genetic analysis of plants relies on high yields of pure DNA. For the strawberry tree (Arbutus unedo) this represents a great challenge since leaves can accumulate large amounts of polysaccharides, polyphenols and secondary metabolites, which co-purify with DNA. For this specie, standard protocols do not produce efficient yields of high-quality amplifiable DNA. Here, we present for the first time an improved leaf-tissue protocol, based on the standard cetyl trimethyl ammonium bromide protocol, which yields large amounts of high-quality amplifiable DNA. Key steps in the optimized protocol are the addition of antioxidant compounds—namely polyvinyl pyrrolidone (PVP), 1,4-dithiothreitol (DTT) and 2-mercaptoethanol, in the extraction buffer; the increasing of CTAB (3%, w/v) and sodium chloride (2M) concentration; and an extraction with organic solvents (phenol and chloroform) with the incubation of samples on ice. Increasing the temperature for cell lyses to 70 °C also improved both DNA quality and yield. The yield of DNA extracted was 200.0 ± 78.0 μg/μL and the purity, evaluated by the ratio A260/A280, was 1.80 ± 0.021, indicative of minimal levels of contaminating metabolites. The quality of the DNA isolated was confirmed by random amplification polymorphism DNA and by inter-simple sequence repeat amplification, proving that the DNA can be amplified via PCR. PMID:21747730

  2. 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

  3. 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.

  4. 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

  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. 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, metagenomic, transcriptomic and metabolomic projects using samples collected in situ. PMID:22672795

  7. Extraction of DNA by magnetic ionic liquids: tunable solvents for rapid and selective DNA analysis.

    PubMed

    Clark, Kevin D; Nacham, Omprakash; Yu, Honglian; Li, Tianhao; Yamsek, Melissa M; Ronning, Donald R; Anderson, Jared L

    2015-02-01

    DNA extraction represents a significant bottleneck in nucleic acid analysis. In this study, hydrophobic magnetic ionic liquids (MILs) were synthesized and employed as solvents for the rapid and efficient extraction of DNA from aqueous solution. The DNA-enriched microdroplets were manipulated by application of a magnetic field. The three MILs examined in this study exhibited unique DNA extraction capabilities when applied toward a variety of DNA samples and matrices. High extraction efficiencies were obtained for smaller single-stranded and double-stranded DNA using the benzyltrioctylammonium bromotrichloroferrate(III) ([(C8)3BnN(+)][FeCl3Br(-)]) MIL, while the dicationic 1,12-di(3-hexadecylbenzimidazolium)dodecane bis[(trifluoromethyl)sulfonyl]imide bromotrichloroferrate(III) ([(C16BnIM)2C12(2+)][NTf2(-), FeCl3Br(-)]) MIL produced higher extraction efficiencies for larger DNA molecules. The MIL-based method was also employed for the extraction of DNA from a complex matrix containing albumin, revealing a competitive extraction behavior for the trihexyl(tetradecyl)phosphonium tetrachloroferrate(III) ([P6,6,6,14(+)][FeCl4(-)]) MIL in contrast to the [(C8)3BnN(+)][FeCl3Br(-)] MIL, which resulted in significantly less coextraction of albumin. The MIL-DNA method was employed for the extraction of plasmid DNA from bacterial cell lysate. DNA of sufficient quality and quantity for polymerase chain reaction (PCR) amplification was recovered from the MIL extraction phase, demonstrating the feasibility of MIL-based DNA sample preparation prior to downstream analysis. PMID:25582771

  8. Evaluation of Methods for the Extraction and Purification of DNA from the Human Microbiome

    PubMed Central

    Yuan, Sanqing; Cohen, Dora B.; Ravel, Jacques; Abdo, Zaid; Forney, Larry J.

    2012-01-01

    Background DNA extraction is an essential step in all cultivation-independent approaches to characterize microbial diversity, including that associated with the human body. A fundamental challenge in using these approaches has been to isolate DNA that is representative of the microbial community sampled. Methodology/Principal Findings In this study, we statistically evaluated six commonly used DNA extraction procedures using eleven human-associated bacterial species and a mock community that contained equal numbers of those eleven species. These methods were compared on the basis of DNA yield, DNA shearing, reproducibility, and most importantly representation of microbial diversity. The analysis of 16S rRNA gene sequences from a mock community showed that the observed species abundances were significantly different from the expected species abundances for all six DNA extraction methods used. Conclusions/Significance Protocols that included bead beating and/or mutanolysin produced significantly better bacterial community structure representation than methods without both of them. The reproducibility of all six methods was similar, and results from different experimenters and different times were in good agreement. Based on the evaluations done it appears that DNA extraction procedures for bacterial community analysis of human associated samples should include bead beating and/or mutanolysin to effectively lyse cells. PMID:22457796

  9. 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

  10. 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 ...

  11. 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

  12. DNA damage mediated transcription arrest: Step back to go forward.

    PubMed

    Mullenders, Leon

    2015-12-01

    The disturbance of DNA helix conformation by bulky DNA damage poses hindrance to transcription elongating due to stalling of RNA polymerase at transcription blocking lesions. Stalling of RNA polymerase provokes the formation of R-loops, i.e. the formation of a DNA-RNA hybrid and a displaced single stranded DNA strand as well as displacement of spliceosomes. R-loops are processed into DNA single and double strand breaks by NER factors depending on TC-NER factors leading to genome instability. Moreover, stalling of RNA polymerase induces a strong signal for cell cycle arrest and apoptosis. These toxic and mutagenic effects are counteracted by a rapid recruitment of DNA repair proteins to perform transcription coupled nucleotide excision repair (TC-NER) to remove the blocking DNA lesions and to restore transcription. Recent studies have highlighted the role of backtracking of RNA polymerase to facilitate TC-NER and identified novel factors that play key roles in TC-NER and in restoration of transcription. On the molecular level these factors facilitate stability of the repair complex by promotion and regulation of various post-translational modifications of NER factors and chromatin substrate. In addition, the continuous flow of new factors that emerge from screening assays hints to several regulatory levels to safeguard the integrity of transcription elongation after disturbance by DNA damage that have yet to be explored. PMID:26422136

  13. Extracting biological knowledge from DNA sequences

    SciTech Connect

    De La Vega, F.M.; Thieffry, D.; Collado-Vides, J.

    1996-12-31

    This session describes the elucidation of information from dna sequences and what challenges computational biologists face in their task of summarizing and deciphering the human genome. Techniques discussed include methods from statistics, information theory, artificial intelligence and linguistics. 1 ref.

  14. An assessment of the efficiency of fungal DNA extraction methods for maximizing the detection of medically important fungi using PCR.

    PubMed

    Karakousis, A; Tan, L; Ellis, D; Alexiou, H; Wormald, P J

    2006-04-01

    To date, no single reported DNA extraction method is suitable for the efficient extraction of DNA from all fungal species. The efficiency of extraction is of particular importance in PCR-based medical diagnostic applications where the quantity of fungus in a tissue biopsy may be limited. We subjected 16 medically relevant fungi to physical, chemical and enzymatic cell wall disruption methods which constitutes the first step in extracting DNA. Examination by light microscopy showed that grinding with mortar and pestle was the most efficient means of disrupting the rigid fungal cell walls of hyphae and conidia. We then trialled several published DNA isolation protocols to ascertain the most efficient method of extraction. Optimal extraction was achieved by incorporating a lyticase and proteinase K enzymatic digestion step and adapting a DNA extraction procedure from a commercial kit (MO BIO) to generate high yields of high quality DNA from all 16 species. DNA quality was confirmed by the successful PCR amplification of the conserved region of the fungal 18S small-subunit rRNA multicopy gene. PMID:16099520

  15. Extraction of High Quality DNA from Seized Moroccan Cannabis Resin (Hashish)

    PubMed Central

    El Alaoui, Moulay Abdelaziz; Melloul, Marouane; Alaoui Amine, Sanaâ; Stambouli, Hamid; El Bouri, Aziz; Soulaymani, Abdelmajid; El Fahime, Elmostafa

    2013-01-01

    The extraction and purification of nucleic acids is the first step in most molecular biology analysis techniques. The objective of this work is to obtain highly purified nucleic acids derived from Cannabis sativa resin seizure in order to conduct a DNA typing method for the individualization of cannabis resin samples. To obtain highly purified nucleic acids from cannabis resin (Hashish) free from contaminants that cause inhibition of PCR reaction, we have tested two protocols: the CTAB protocol of Wagner and a CTAB protocol described by Somma (2004) adapted for difficult matrix. We obtained high quality genomic DNA from 8 cannabis resin seizures using the adapted protocol. DNA extracted by the Wagner CTAB protocol failed to give polymerase chain reaction (PCR) amplification of tetrahydrocannabinolic acid (THCA) synthase coding gene. However, the extracted DNA by the second protocol permits amplification of THCA synthase coding gene using different sets of primers as assessed by PCR. We describe here for the first time the possibility of DNA extraction from (Hashish) resin derived from Cannabis sativa. This allows the use of DNA molecular tests under special forensic circumstances. PMID:24124454

  16. Purification of crime scene DNA extracts using centrifugal filter devices

    PubMed Central

    2013-01-01

    Background The success of forensic DNA analysis is limited by the size, quality and purity of biological evidence found at crime scenes. Sample impurities can inhibit PCR, resulting in partial or negative DNA profiles. Various DNA purification methods are applied to remove impurities, for example, employing centrifugal filter devices. However, irrespective of method, DNA purification leads to DNA loss. Here we evaluate the filter devices Amicon Ultra 30 K and Microsep 30 K with respect to recovery rate and general performance for various types of PCR-inhibitory crime scene samples. Methods Recovery rates for DNA purification using Amicon Ultra 30 K and Microsep 30 K were gathered using quantitative PCR. Mock crime scene DNA extracts were analyzed using quantitative PCR and short tandem repeat (STR) profiling to test the general performance and inhibitor-removal properties of the two filter devices. Additionally, the outcome of long-term routine casework DNA analysis applying each of the devices was evaluated. Results Applying Microsep 30 K, 14 to 32% of the input DNA was recovered, whereas Amicon Ultra 30 K retained 62 to 70% of the DNA. The improved purity following filter purification counteracted some of this DNA loss, leading to slightly increased electropherogram peak heights for blood on denim (Amicon Ultra 30 K and Microsep 30 K) and saliva on envelope (Amicon Ultra 30 K). Comparing Amicon Ultra 30 K and Microsep 30 K for purification of DNA extracts from mock crime scene samples, the former generated significantly higher peak heights for rape case samples (P-values <0.01) and for hairs (P-values <0.036). In long-term routine use of the two filter devices, DNA extracts purified with Amicon Ultra 30 K were considerably less PCR-inhibitory in Quantifiler Human qPCR analysis compared to Microsep 30 K. Conclusions Amicon Ultra 30 K performed better than Microsep 30 K due to higher DNA recovery and more efficient removal of PCR-inhibitory substances. The different performances of the filter devices are likely caused by the quality of the filters and plastic wares, for example, their DNA binding properties. DNA purification using centrifugal filter devices can be necessary for successful DNA profiling of impure crime scene samples and for consistency between different PCR-based analysis systems, such as quantification and STR analysis. In order to maximize the possibility to obtain complete STR DNA profiles and to create an efficient workflow, the level of DNA purification applied should be correlated to the inhibitor-tolerance of the STR analysis system used. PMID:23618387

  17. Techniques of dental DNA extraction: Some operative experiences.

    PubMed

    Pinchi, Vilma; Torricelli, Francesca; Nutini, A Lucia; Conti, Marco; Iozzi, Sara; Norelli, Gian-Aristide

    2011-01-30

    This study presents and discusses the possibility of multiple dental DNA extraction at different times using an endodontic technique. We chose a mixed sample of twenty teeth (decayed, filled, different kinds of teeth, etc.) and performed two different accesses to the pulp cavity to collect samples of dental tissue useful for DNA extraction. The Identifiler(®) and Minifiler(®) kits were used respectively for the first and second genetic analysis. The research's most relevant findings are the possibility of successfully repeating dental DNA extractions, and that a dental element can be considered as a DNA source that can be reused even after prolonged time elapses. The study's results document a simple and efficient methodology for dental identification based on the choice of dental elements, endodontic techniques and the use of the Minifiler(®) kit for genetic analysis. PMID:20558019

  18. DNA extraction protocol for rapid PCR detection of pathogenic bacteria.

    PubMed

    Brewster, Jeffrey D; Paoli, George C

    2013-11-01

    Twelve reagents were evaluated to develop a direct DNA extraction method suitable for PCR detection of foodborne bacterial pathogens. Many reagents exhibited strong PCR inhibition, requiring significant dilution of the extract with a corresponding reduction in sensitivity. Most reagents also exhibited much lower recovery of DNA from the gram-positive test organism (Listeria monocytogenes) than from the gram-negative organism (Escherichia coli O157:H7), preventing unbiased detection and quantitation of both organisms. The 5× HotSHOT+Tween reagent exhibited minimal inhibition and high extraction efficiency for both test organisms, providing a 15-min single-tube DNA-extraction protocol suitable for highly sensitive quantitative PCR assays. PMID:23872000

  19. Yeast-based recombineering of DNA fragments into plant transformation vectors by one-step transformation.

    PubMed

    Nagano, Yukio; Takao, Syoko; Kudo, Takahiro; Iizasa, Ei'ichi; Anai, Toyoaki

    2007-12-01

    T-DNA binary vectors are often used in plant transformation experiments. Because they are usually very large and have few restriction sites suitable for DNA ligation reactions, cloning DNA fragments into these vectors is difficult. We provide herein an alternative to cloning DNA fragments into very large vectors. Our yeast-based recombineering method enables DNA fragments to be cloned into certain types of T-DNA binary vectors by one-step transformation without the requirement of specific recombination sites or precisely positioned restriction ends, thus making the cloning process more flexible. Moreover, this method is inexpensive and is applicable to multifragment cloning. PMID:17680244

  20. Effect of DNA Extraction Methods and Sampling Techniques on the Apparent Structure of Cow and Sheep Rumen Microbial Communities

    PubMed Central

    Henderson, Gemma; Cox, Faith; Kittelmann, Sandra; Miri, Vahideh Heidarian; Zethof, Michael; Noel, Samantha J.; Waghorn, Garry C.; Janssen, Peter H.

    2013-01-01

    Molecular microbial ecology techniques are widely used to study the composition of the rumen microbiota and to increase understanding of the roles they play. Therefore, sampling and DNA extraction methods that result in adequate yields of microbial DNA that also accurately represents the microbial community are crucial. Fifteen different methods were used to extract DNA from cow and sheep rumen samples. The DNA yield and quality, and its suitability for downstream PCR amplifications varied considerably, depending on the DNA extraction method used. DNA extracts from nine extraction methods that passed these first quality criteria were evaluated further by quantitative PCR enumeration of microbial marker loci. Absolute microbial numbers, determined on the same rumen samples, differed by more than 100-fold, depending on the DNA extraction method used. The apparent compositions of the archaeal, bacterial, ciliate protozoal, and fungal communities in identical rumen samples were assessed using 454 Titanium pyrosequencing. Significant differences in microbial community composition were observed between extraction methods, for example in the relative abundances of members of the phyla Bacteroidetes and Firmicutes. Microbial communities in parallel samples collected from cows by oral stomach-tubing or through a rumen fistula, and in liquid and solid rumen digesta fractions, were compared using one of the DNA extraction methods. Community representations were generally similar, regardless of the rumen sampling technique used, but significant differences in the abundances of some microbial taxa such as the Clostridiales and the Methanobrevibacter ruminantium clade were observed. The apparent microbial community composition differed between rumen sample fractions, and Prevotellaceae were most abundant in the liquid fraction. DNA extraction methods that involved phenol-chloroform extraction and mechanical lysis steps tended to be more comparable. However, comparison of data from studies in which different sampling techniques, different rumen sample fractions or different DNA extraction methods were used should be avoided. PMID:24040342

  1. Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species.

    PubMed

    Healey, Adam; Furtado, Agnelo; Cooper, Tal; Henry, Robert J

    2014-01-01

    Next-generation sequencing technologies rely on high quality DNA that is suitable for library preparation followed by sequencing. Some plant species store large amounts of phenolics and polysaccharides within their leaf tissue making genomic DNA extraction difficult. While many DNA extraction methods exist that contend with the presence of phenolics and polysaccharides, these methods rely on long incubations, multiple precipitations or commercially available kits to produce high molecular weight and contaminant-free DNA. In this protocol, we describe simple modifications to the established CTAB- based extraction method that allows for reliable isolation of high molecular weight genomic DNA from difficult to isolate plant species Corymbia (a eucalypt) and Coffea (coffee). The simplified protocol does not require multiple clean up steps or commercial based kits, and the isolated DNA passed stringent quality control standards for whole genome sequencing on Illumina HiSeq and TruSeq sequencing platforms. PMID:25053969

  2. Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

    PubMed Central

    Patel, Yogin; Gowda, Vrushab; Yan, Shan

    2012-01-01

    On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop. Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle. The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, γ-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint 4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling 9-12. Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure. PMID:23149695

  3. 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

  4. Establishing a novel automated magnetic bead-based method for the extraction of DNA from a variety of forensic samples.

    PubMed

    Witt, Sebastian; Neumann, Jan; Zierdt, Holger; Gébel, Gabriella; Röscheisen, Christiane

    2012-09-01

    Automated systems have been increasingly utilized for DNA extraction by many forensic laboratories to handle growing numbers of forensic casework samples while minimizing the risk of human errors and assuring high reproducibility. The step towards automation however is not easy: The automated extraction method has to be very versatile to reliably prepare high yields of pure genomic DNA from a broad variety of sample types on different carrier materials. To prevent possible cross-contamination of samples or the loss of DNA, the components of the kit have to be designed in a way that allows for the automated handling of the samples with no manual intervention necessary. DNA extraction using paramagnetic particles coated with a DNA-binding surface is predestined for an automated approach. For this study, we tested different DNA extraction kits using DNA-binding paramagnetic particles with regard to DNA yield and handling by a Freedom EVO(®)150 extraction robot (Tecan) equipped with a Te-MagS magnetic separator. Among others, the extraction kits tested were the ChargeSwitch(®)Forensic DNA Purification Kit (Invitrogen), the PrepFiler™Automated Forensic DNA Extraction Kit (Applied Biosystems) and NucleoMag™96 Trace (Macherey-Nagel). After an extensive test phase, we established a novel magnetic bead extraction method based upon the NucleoMag™ extraction kit (Macherey-Nagel). The new method is readily automatable and produces high yields of DNA from different sample types (blood, saliva, sperm, contact stains) on various substrates (filter paper, swabs, cigarette butts) with no evidence of a loss of magnetic beads or sample cross-contamination. PMID:22310206

  5. 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.

  6. Automatable full demineralization DNA extraction procedure from degraded skeletal remains.

    PubMed

    Amory, Sylvain; Huel, René; Bilić, Ana; Loreille, Odile; Parsons, Thomas J

    2012-05-01

    During the 7 year period from 2002 to 2009 a high volume, silica-binding DNA extraction protocol for bone, based on modified QIAGEN's Blood Maxi Kit protocol was highly successful permitting the DNA matching of >14,500 missing persons from former Yugoslavia. This method, however, requires large amount of bone material and large volumes of reagents. The logical evolution was to develop a more efficient extraction protocol for bone samples that uses significantly less starting material while increasing the success in obtaining DNA results from smaller, more challenging samples. In this study we compared the performance of ICMP's original protocol against an automatable full demineralization approach. In order to provide reliable results and to simulate a wide variety of cases, we analyzed 40 bone samples in a comparative study based on DNA concentrations and quality of resulting STR profiles. The new protocol results in the dissolution of the entire bone powder sample, thus eliminating the possibility that DNA is left behind, locked in remaining solid bone matrix. For the majority of samples tested, the DNA concentrations obtained from half a gram of fully digested bone material were equivalent to or greater than the ones obtained from 2g of partially demineralized bone powder. Furthermore, the full demineralization process significantly increases the proportion of full profiles reflecting the correlation with better DNA quality. This method has been adapted for the QIAcube robotic platform. The performance of this automated full demineralization protocol is similar to the manual version and increases overall lab throughput. It also simplifies the process by eliminating quality control procedures that are advisable in manual procedures, and overall reduces the chance of human error. Finally we described a simple and efficient post-extraction clean-up method that can be applied to DNA extracts obtained from different protocols. This protocol has also been adjusted for the QIAcube platform. PMID:21885362

  7. A semi-automated protocol for Archaea DNA extraction from stools

    PubMed Central

    2013-01-01

    Background The PCR-based detection of archaea DNA in human specimens relies on efficient DNA extraction. We previously designed one such protocol involving only manual steps. In an effort to reduce the workload involved, we compared this manual protocol to semi-automated and automated protocols for archaea DNA extraction from human specimens. Findings We tested 110 human stool specimens using each protocol. An automated protocol using the EZ1 Advanced XL extractor with the V 1.066069118 Qiagen DNA bacteria card and the EZ1® DNA Tissue Kit (Qiagen, Courtaboeuf, France) yielded 35/110 (32%) positives for the real-time PCR detection of the Methanobrevibacter smithii 16S rRNA gene, with average Ct values of 36.1. A semi-automated protocol combining glass-powder crushing, overnight proteinase K digestion and lysis in the buffer from the EZ1 kit yielded 90/110 (82%) positive specimens (P = 0.001) with an average Ct value of 27.4 (P = 0.001). The manual protocol yielded 100/110 (91%) positive specimens (P = 0.001) with an average Ct value of 30.33 (P = 0.001). However, neither the number of positive specimens nor the Ct values were significantly different between the manual protocol and the semi-automated protocol (P > 0.1 and P > 0.1). Conclusion Proteinase K digestion and glass powder crushing dramatically increase the extraction yield of archaea DNA from human stools. The semi-automated protocol described here was more rapid than the manual protocol and yielded significantly more archaeal DNA. It could be applied for extracting total stool DNA for further PCR amplification. PMID:23651536

  8. 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.

  9. The NucleoSpin® DNA Clean-up XS kit for the concentration and purification of genomic DNA extracts: an alternative to microdialysis filtration.

    PubMed

    Hudlow, William R; Krieger, Robert; Meusel, Markus; Sehhat, Joshua C; Timken, Mark D; Buoncristiani, Martin R

    2011-06-01

    Traditionally, DNA extracts from biological evidence items have been concentrated and rinsed using microdialysis filtration units, including the Centricon(®) and Microcon(®) centrifugal filter devices. As an alternative to microdialysis filtration, we present an optimized method for using NucleoSpin(®) XS silica columns to concentrate and clean-up aqueous extracts from the organic extraction of DNA from biological samples. The method can be used with standard organic extraction and dithiothreitol (DTT)-based differential extraction methods with no modifications to these methods prior to the concentration and clean-up step. Extracts from laboratory-prepared bloodstains, saliva and semen stains have been successfully amplified with both qPCR and STR assays. Finally, the total time to process a set of samples with the NucleoSpin(®) XS column is approximately 30 min vs. approximately 1.5h with the Centricon(®) YM-100 filter device. PMID:20457101

  10. 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

  11. Evaluation of the ISO Standard 11063 DNA Extraction Procedure for Assessing Soil Microbial Abundance and Community Structure

    PubMed Central

    Griffiths, Robert; Dequiedt, Samuel; Lelievre, Mlanie; Regnier, Tiffanie; Nowak, Virginie; Bailey, Mark; Lemanceau, Philippe; Bispo, Antonio; Chabbi, Abad; Maron, Pierre-Alain; Mougel, Christophe; Ranjard, Lionel

    2012-01-01

    Soil DNA extraction has become a critical step in describing microbial biodiversity. Historically, ascertaining overarching microbial ecological theories has been hindered as independent studies have used numerous custom and commercial DNA extraction procedures. For that reason, a standardized soil DNA extraction method (ISO-11063) was previously published. However, although this ISO method is suited for molecular tools such as quantitative PCR and community fingerprinting techniques, it has only been optimized for examining soil bacteria. Therefore, the aim of this study was to assess an appropriate soil DNA extraction procedure for examining bacterial, archaeal and fungal diversity in soils of contrasting land-use and physico-chemical properties. Three different procedures were tested: the ISO-11063 standard; a custom procedure (GnS-GII); and a modified ISO procedure (ISOm) which includes a different mechanical lysis step (a FastPrep -24 lysis step instead of the recommended bead-beating). The efficacy of each method was first assessed by estimating microbial biomass through total DNA quantification. Then, the abundances and community structure of bacteria, archaea and fungi were determined using real-time PCR and terminal restriction fragment length polymorphism approaches. Results showed that DNA yield was improved with the GnS-GII and ISOm procedures, and fungal community patterns were found to be strongly dependent on the extraction method. The main methodological factor responsible for differences between extraction procedure efficiencies was found to be the soil homogenization step. For integrative studies which aim to examine bacteria, archaea and fungi simultaneously, the ISOm procedure results in higher DNA recovery and better represents microbial communities. PMID:22984486

  12. HHS guidance on synthetic DNA is the right step.

    PubMed

    Gronvall, Gigi Kwik

    2010-12-01

    Synthetic biology has advanced to the point where some pathogens can be manufactured from scratch. This technical leap has beneficent implications for medical research and vaccine design, but it also raises concerns that the technology could be used to produce a deadly pathogen for nefarious use. Addressing these concerns, the Department of Health and Human Services (HHS) released their Screening Framework Guidance for Providers of Synthetic Double-Stranded DNA on October 13, 2010. They took the right approach: The oversight framework for gene synthesis companies included in this guidance is adaptable to new technical developments and changing risks, it can be implemented immediately, it can be readily adopted by other countries, and it will cost little. Though there have been some calls to increase the regulatory controls on synthetic biology, these should be resisted. For now, at least, the oversight is appropriate to the risks. PMID:21142764

  13. 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)

  14. 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.

  15. An expedient and versatile protocol for extracting high-quality DNA from plant leaves.

    PubMed

    Vallejos, C Eduardo

    2007-01-01

    INTRODUCTIONProtocols for extracting plant genomic DNA have to contend with two major challenges: preventing the oxidation of phenolic substances that can react with nucleic acids and proteins, and eliminating polysaccharides that interfere with downstream enzymatic manipulations of DNA. A protocol has been developed in which the above-mentioned substances are removed from the sample before the DNA is removed from the chromatin structure. This protocol requires only two tubes during extraction and a single precipitation step to yield high-quality DNA. DNA extracted with this method has virtually no protein or phenolic contaminants (A(260)/A(280)>1.8), is RNA-free, contains high-molecular-weight fragments according to CHEF electrophoresis, and is fully digestible by restriction enzymes. This protocol has been successfully tested with leaf samples from common bean and corn; with leaves known to be rich in phenolics (tomato and bell pepper), pectins (sweet orange, grapefruit, and peach), or both substances (Muscadine grape); and with leaves that contain latex in the sap (papaya). PMID:21357104

  16. 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

  17. Optimization and validation of DNA extraction and real-time PCR assay for the quantitative measurement of residual host cell DNA in biopharmaceutical products.

    PubMed

    Hu, B; Sellers, J; Kupec, J; Ngo, W; Fenton, S; Yang, T-Y; Grebanier, A

    2014-01-01

    Host cell DNA contamination occurs during the production of biopharmaceuticals and must be controlled and monitored for the purity and safety of the drug products. A sodium iodide-based DNA extraction and a subsequent real time PCR assay were developed and validated for the quantitative measurement of residual host cell DNA impurity in monoclonal antibody therapeutic products. A sodium iodide-based commercial kit was optimized for the removal of interfering matrices. Several incubation steps from the kit protocol were combined and a neutralization buffer was introduced to protein digestion step to eliminate any precipitation from the detergent. The elimination of the two washing steps significantly reduced assay variability from loss of DNA pellets. The optimized DNA extraction procedure can recover DNA close to 100% for DNA concentrations from 10 to 100,000pg/mL. Of the published sequences of repetitive interspersed nuclear elements, we identified a nucleotide mismatch from the published CHO probe. Correction of this nucleotide increased DNA amplification by a thousand fold. The optimized assay was further validated for the quantitation of residual CHO DNA according to ICH guidelines with preset assay acceptance criteria. The method met all assay acceptance criteria and was found linear, accurate and precise for the quantitation of residual CHO in the linear range of 10-100,000pg DNA/mL. LOQ was measured at 10pg DNA/mL and LOD at 1pg DNA/mL. No matrix interference to our validated assay was detected from bioreactor harvest, Protein A eluate or eluate from ion exchange columns. PMID:24042121

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

    PubMed

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

    1994-05-01

    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) PMID:8017936

  19. Evidence on DNA slippage step-length distribution

    NASA Astrophysics Data System (ADS)

    Borštnik, Branko; Pumpernik, Danilo

    2005-03-01

    A simple model based on a master equation is constructed in order to reveal the details of the mutational events modifying simple sequence repeats in the human genome, A database of simple repeats together with their flanking sequences comprising approximately 105 entries from all 24 human chromosomes was constructed. By aligning the pairs of fragments of sequences containing the repeat elements, the matrices that count the number of slippage events were evaluated. The counts were then used as a target to be reproduced by our theoretical model, in which the elongation and shortening of the repeats proceed through a mechanism in which the step lengths exhibit a decaying distribution in the form of an inverse power law rather than through one nucleotide extension or deletion, which was the most frequent supposition in previous studies.

  20. 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

  1. Convenient determination of DNA extraction efficiency using an external DNA recovery standard and quantitative-competitive PCR.

    PubMed

    Mumy, Karen L; Findlay, Robert H

    2004-05-01

    Molecular biology techniques have advanced the field of microbial ecology through the analysis of nucleic acids. Most techniques that use DNA or RNA require their extraction from environmental matrices, which can be tedious and inefficient. While a number of extraction methods, both laboratory-based and commercially available, have been developed, none of these include a convenient method to determine extraction efficiency. We have developed an external DNA recovery standard, Lambda DNA (target DNA) contained within pBR322, allowing routine determinations of DNA recovery efficiency. Target DNA was added to sediments as whole cells, total DNA extracted using commercial DNA extraction/purification kits and the amount of target DNA recovered quantified by quantitative-competitive PCR (QC-PCR). Three commercially available kits (UltraClean Soil DNA, FastDNA SPIN and Soil Master DNA Extraction) were evaluated for recovery efficiency. Recoveries for the three kits ranged from undetectable to 43.3% with average recoveries of 14.9+/-16.0%, 28.3+/-10.5% and 2.4+/-0.1% (UltraClean, FastDNA and Soil Master, respectively). Quantification of target DNA proved robust in sediments heavily polluted with polycyclic aromatic hydrocarbons and the external recovery standard could be detected following extraction and amplification from as few as 1 x 10(3) cells added to 0.5 g sediment (wet weight). The external DNA recovery standard was also added directly to the sediment as purified plasmid DNA prior to extraction. It was recovered with similar efficiency as when added as whole cells, suggesting its usefulness in estimating DNA recovery in ribosomal DNA studies. These results show that, while the commercial kits offer expedited sample processing, the extraction efficiencies vary on a sample-by-sample basis and were <100%. Therefore, quantitative DNA studies require an estimation of DNA recovery. PMID:15063066

  2. Comparisons of Three Automated Systems for Genomic DNA Extraction in a Clinical Diagnostic Laboratory

    PubMed Central

    Lee, Jong-Han; Park, Yongjung; Choi, Jong Rak; Lee, Eun Kyung

    2010-01-01

    Purpose The extraction of nucleic acid is initially a limiting step for successful molecular-based diagnostic workup. This study aims to compare the effectiveness of three automated DNA extraction systems for clinical laboratory use. Materials and Methods Venous blood samples from 22 healthy volunteers were analyzed using QIAamp Blood Mini Kit (Qiagen), MagNA Pure LC Nucleic Acid Isolation Kit I (Roche), and Magtration-Magnazorb DNA common kit-200N (PSS). The concentration of extracted DNAs was measured by NanoDrop ND-1000 (PeqLab). Also, extracted DNAs were confirmed by applying in direct agarose gel electrophoresis and were amplified by polymerase chain reaction (PCR) for human beta-globin gene. Results The corrected concentrations of extracted DNAs were 25.42 8.82 ng/L (13.49-52.85 ng/L) by QIAamp Blood Mini Kit (Qiagen), and 22.65 14.49 ng/L (19.18-93.39 ng/L) by MagNA Pure LC Nucleic Acid Isolation Kit I, and 22.35 6.47 ng/L (12.57-35.08 ng/L) by Magtration-Magnazorb DNA common kit-200N (PSS). No statistically significant difference was noticed among the three commercial kits (p > 0.05). Only the mean value of DNA purity through PSS was slightly lower than others. All the extracted DNAs were successfully identified in direct agarose gel electrophoresis. And all the product of beta-globin gene PCR showed a reproducible pattern of bands. Conclusion The effectiveness of the three automated extraction systems is of an equivalent level and good enough to produce reasonable results. Each laboratory could select the automated system according to its clinical and laboratory conditions. PMID:20046522

  3. Implication of DNA polymerase lambda in alignment-based gap filling for nonhomologous DNA end joining in human nuclear extracts.

    PubMed

    Lee, Jae Wan; Blanco, Luis; Zhou, Tong; Garcia-Diaz, Miguel; Bebenek, Katarzyna; Kunkel, Thomas A; Wang, Zhigang; Povirk, Lawrence F

    2004-01-01

    Accurate repair of free radical-mediated DNA double-strand breaks by the nonhomologous end joining pathway requires replacement of fragmented nucleotides in the aligned ends by a gap-filling DNA polymerase. Nuclear extracts of human HeLa cells, supplemented with recombinant XRCC4-DNA ligase IV complex (XRCC4/ligase IV), were capable of accurately rejoining model double-strand break substrates with a 1- or 2-base gap, and the gap-filling step was dependent on XRCC4/ligase IV. To determine what polymerase was responsible for gap filling, end joining was examined in the presence of polyclonal antibodies against each of two prime candidate enzymes, DNA polymerases mu and lambda, both of which were present in the extracts. For a DNA substrate with partially complementary 3' overhangs and a 2-base gap, antibodies to polymerase lambda completely eliminated both gap filling and accurate end joining, whereas antibodies to polymerase mu had little effect. Immunodepletion of polymerase lambda, but not polymerase mu, likewise blocked both gap filling and end joining, and both functions could be restored by addition of recombinant polymerase lambda. Recombinant polymerase mu, and a truncated polymerase lambda lacking the Brca1 C-terminal domain, were at least 10-fold less active in restoring gap filling to the immunodepleted extracts, and polymerase beta was completely inactive. The results suggest that polymerase lambda is the primary gap-filling polymerase for accurate nonhomologous end joining, and that the Brca1 C-terminal domain is required for this activity. PMID:14561766

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

    NASA Astrophysics Data System (ADS)

    Morales, Mercedes C.

    In this work, microfluidic platforms have been designed and evaluated to demonstrate microscale DNA purification via organic (phenol) extraction as well as analyte trapping and concentration using a transverse electrokinetic force balance. First, in order to evaluate DNA purification via phenol extraction in a microdevice, an aqueous phase containing protein and DNA and an immiscible receiving organic phase were utilized to evaluate microfluidic DNA extraction under both stratified and droplet-based flow conditions using a serpentine microfluidic device. The droplet based flow resulted in a significant improvement of protein partitioning from the aqueous phase due to the flow recirculation inside each droplet improving material convective transport into the organic phase. The plasmid recovery from bacterial lysates using droplet-based flow was high (>92%) and comparable to the recovery achieved using commercial DNA purification kits and standard macroscale phenol extraction. Second, a converging Y-inlet microfluidic channel with integrated coplanar electrodes was used to investigate transverse DNA and protein migration under uniform direct current (DC) electric fields. Negatively charged samples diluted in low and high ionic strength buffers were co-infused with a receiving buffer of the same ionic strength into a main channel where transverse electric fields were applied. Experimental results demonstrated that charged analytes could traverse the channel width and accumulate at the positive bias electrode in a low electroosmotic mobility and high electrophoretic mobility condition (high ionic strength buffer) or migrated towards an equilibrium position within the channel when both electroosmotic mobility and electrophoretic mobility are high (low ionic strength buffer). The different behaviors are the result of a balance between the electrophoretic force and a drag force induced by a recirculating electroosmotic flow generated across the channel width due to the 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.

  5. 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

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

    NASA Astrophysics Data System (ADS)

    Fotouhi, Gareth

    DNA sample preparation is essential for genetic analysis. However, rapid and easy-to-use methods are a major challenge to obtaining genetic information. Furthermore, DNA sample preparation technology must follow the growing need for point-of-care (POC) diagnostics. The current use of centrifuges, large robots, and laboratory-intensive protocols has to be minimized to meet the global challenge of limited access healthcare by bringing the lab to patients through POC devices. To address these challenges, a novel extraction method of genomic DNA from human samples is presented by using heat-cured polyethyleneimine-coated microtips generating a high electric field. The microtip extraction method is based on recent work using an electric field and capillary action integrated into an automated device. The main challenges to the method are: (1) to obtain a stable microtip surface for the controlled capture and release of DNA and (2) to improve the recovery of DNA from samples with a high concentration of inhibitors, such as human samples. The present study addresses these challenges by investigating the heat curing of polyethyleneimine (PEI) coated on the surface of the microtip. Heat-cured PEI-coated microtips are shown to control the capture and release of DNA. Protocols are developed for the extraction and purification of DNA from human samples. Heat-cured PEI-coated microtip methods of DNA sample preparation are used to extract genomic DNA from human samples. It is discovered through experiment that heat curing of a PEI layer on a gold-coated surface below 150°C could inhibit the signal of polymerase chain reaction (PCR). Below 150°C, the PEI layer is not completely cured and dissolved off the gold-coated surface. Dissolved PEI binds with DNA to inhibit PCR. Heat curing of a PEI layer above 150°C on a gold-coated surface prevents inhibition to PCR and gel electrophoresis. In comparison to gold-coated microtips, the 225°C-cured PEI-coated microtips improve the 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.

  7. DNA Extraction from Protozoan Oocysts/Cysts in Feces for Diagnostic PCR

    PubMed Central

    2014-01-01

    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

  8. Nondestructive DNA extraction method for mitochondrial DNA analyses of museum specimens.

    PubMed

    Rohland, Nadin; Siedel, Heike; Hofreiter, Michael

    2004-05-01

    Museum specimens have provided the material for a large proportion of ancient DNA studies conducted during the last 20 years. However, a major drawback of the genetic analyses is that the specimens investigated are usually damaged, as parts of skin, bone, or a tooth have to be removed for DNA extraction. To get around these limitations, we have developed a nondestructive extraction method for bone, tooth, and skin samples. We found that it is possible to amplify mitochondrial DNA (mtDNA) sequences up to at least 414 bp long from samples up to 164 years old. Using this method, almost 90% (35 of 40) of the investigated samples yielded amplifiable mtDNA. Moreover, we found that repeated extractions of the same samples allowed amplifications of the expected length for all samples at least three times and for some samples up to at least five times. Thus this method opens up the possibility to repeatedly use museum collections for mtDNA analyses without damaging the specimens and thus without reducing the value of irreplaceable collections for morphological analyses. PMID:15152601

  9. 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

  10. Comparison of eight methods of genomic DNA extraction from babassu.

    PubMed

    Viana, J P G; Borges, A N C; Lopes, A C A; Gomes, R L F; Britto, F B; Lima, P S C; Valente, S E S

    2015-01-01

    Babassu (Orbignya phalerata Martius) is one of the most important palms in Brazil because of the largest morphological variation, wide geographic distribution, and high socio-economic importance. The diversity present in babassu germplasm should be protected against loss to ensure their use with high productivity. Study of the available variability in populations of babassu is necessary to develop conservation strategies. The study of genetic variability can be conducted using molecular markers and many of these studies require significant quantity of high-quality DNA. The present study aimed to effect comparison among eight DNA extraction methods in case of O. phalerata. The quality and concentration of nucleic acids were analyzed by spectrophotometry and integrity of DNA was ascertained by agarose gel electrophoresis. The spectrophotometry revealed that some methods resulted in high levels of concentration of nucleic acids, in which values of the ratio A260/280 and A260/230 were outside the range of purity. The agarose gel electrophoresis established the concentration and integrity of DNA. The methods of Murray and Thompson (1980) and Ferreira and Grattapaglia (1998) did not result in satisfactory quantities of DNA. Conversely, the method proposed by Khanuja et al. (1999) resulted in DNA of adequate quality and quantity that could be satisfactorily used for amplification reactions performed with two ISSR primers. PMID:26782447

  11. 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.

  12. 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

  13. Isolation of plant DNA for PCR and genotyping using organic extraction and CTAB.

    PubMed

    Springer, Nathan M

    2010-11-01

    A general difficulty in isolation of DNA from plant cells is the presence of a cell wall. It is necessary to degrade plant cell walls, either physically or enzymatically, in order to effectively isolate plant DNA. Additionally, some tissues (such as endosperm) or some species contain high levels of starches or phenolic compounds that can complicate DNA isolation. A number of plant DNA isolation protocols are designed to overcome species-specific difficulties. This is a relatively simple protocol that uses an extraction buffer containing cetyltrimethylammonium bromide (CTAB); it can be used for many plant species. It provides a substantial amount of high-quality DNA that is suitable for polymerase chain reaction (PCR) procedures and is stable for long periods of time. The cost per sample is very low. In addition, this protocol is relatively robust and can be performed by individuals who have had relatively little training. A typical undergraduate student can perform ~200-300 isolations in a day using this protocol. The disadvantages are that it requires a freeze-dryer and a mill or paint-shaker-like device and that it utilizes an organic extraction step, requiring the use of a fume hood. PMID:21041388

  14. 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

  15. 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

  16. 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

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

    PubMed Central

    Fujikura, Kohei

    2016-01-01

    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.

  18. 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.

  19. 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

  20. 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

  1. 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

  2. Using Concrete & Representational Experiences to Understand the Structure of DNA: A Four-Step Instructional Framework

    ERIC Educational Resources Information Center

    Harrell, Pamela Esprivalo; Richards, Debbie; Collins, James; Taylor, Sarah

    2005-01-01

    A description of learning experience that uses a four-step instrumentational framework involving concrete and representational experiences to promote conceptual understanding of abstract biological concepts by a series of closely-related activities is presented. The students are introduced to the structure and implications of DNA using four

  3. A Single-Step Method for Rapid Extraction of Total Lipids from Green Microalgae

    PubMed Central

    Axelsson, Martin; Gentili, Francesco

    2014-01-01

    Microalgae produce a wide range of lipid compounds of potential commercial interest. Total lipid extraction performed by conventional extraction methods, relying on the chloroform-methanol solvent system are too laborious and time consuming for screening large numbers of samples. In this study, three previous extraction methods devised by Folch et al. (1957), Bligh and Dyer (1959) and Selstam and Öquist (1985) were compared and a faster single-step procedure was developed for extraction of total lipids from green microalgae. In the single-step procedure, 8 ml of a 2∶1 chloroform-methanol (v/v) mixture was added to fresh or frozen microalgal paste or pulverized dry algal biomass contained in a glass centrifuge tube. The biomass was manually suspended by vigorously shaking the tube for a few seconds and 2 ml of a 0.73% NaCl water solution was added. Phase separation was facilitated by 2 min of centrifugation at 350 g and the lower phase was recovered for analysis. An uncharacterized microalgal polyculture and the green microalgae Scenedesmus dimorphus, Selenastrum minutum, and Chlorella protothecoides were subjected to the different extraction methods and various techniques of biomass homogenization. The less labour intensive single-step procedure presented here allowed simultaneous recovery of total lipid extracts from multiple samples of green microalgae with quantitative yields and fatty acid profiles comparable to those of the previous methods. While the single-step procedure is highly correlated in lipid extractability (r2 = 0.985) to the previous method of Folch et al. (1957), it allowed at least five times higher sample throughput. PMID:24586930

  4. The mechanism of the translocation step in DNA replication by DNA polymerase I: a computer simulation analysis.

    PubMed

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

    2010-01-13

    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. PMID:20152155

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

    PubMed Central

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

    2012-01-01

    Summary 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. PMID:20152155

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

    PubMed

    Morin, Jos A; Cao, Francisco J; Lzaro, Jos M; Arias-Gonzalez, J Ricardo; Valpuesta, Jos M; Carrascosa, Jos L; Salas, Margarita; Ibarra, Borja

    2015-04-20

    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

  7. 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.

  8. Microfluidic chip for stacking, separation and extraction of multiple DNA fragments.

    PubMed

    Wu, Ruige; Seah, Y P; Wang, Zhiping

    2016-03-11

    A disposable integrated microfluidic device was developed for rapid sample stacking, separation and extraction of multiple DNA fragments from a relatively large amount of sample. Isotachophoresis hyphenated gel electrophoresis (ITP-GE) was used to pre-concentrate and separate DNA fragments, followed by extraction of pure DNA fragments with electroelution on-chip. DNA fragments of 200bp, 500bp and 1kbp were successfully separated and collected in the extraction chamber within 25min. The extraction efficiency obtained from the chip was 49.9%, 52.1% and 53.7% for 200bp, 500bp and 1kbp DNA fragments, respectively. The extracted DNA fragments exhibited compatibility with downstream enzymatic reactions, for example PCR. The chip was also used to extract DNA fragments with specific size range from sheared genomic DNA and demonstrated similar performance to that using traditional gel cutting method. The whole assay can finish in 32min, 6 times faster than traditional method. PMID:26879456

  9. Development of a rapid, 96-well alkaline based differential DNA extraction method for sexual assault evidence.

    PubMed

    Hudlow, William R; Buoncristiani, Martin R

    2012-01-01

    We present a rapid alkaline lysis procedure for the extraction of DNA from sexual assault evidence that generates purified sperm fraction extracts that yield STR typing results similar to those obtained from the traditional organic/dithiothreitol differential extraction. Specifically, a sodium hydroxide based differential extraction method has been developed in a single-tube format and further optimized in a 96-well format. The method yields purified extracts from a small sample set (≈ 2-6 swabs) in approximately 2h and from a larger sample set (up to 96 swabs) in approximately 4h. While conventional differential extraction methods require vigorous sample manipulation to remove the spermatozoa from the substrate, the method described here exploits the propensity of sperm to adhere to a substrate and does not require any manipulation of the substrate after it is sampled. For swabs, sample handling is minimized by employing a process where the tip of the swab, including the shaft, is transferred to the appropriate vessel eliminating the need for potentially hazardous scalpels to separate the swab material from the shaft. The absence of multiple handling steps allows the process to be semi-automated, however the procedure as described here does not require use of a robotic system. This method may provide forensic laboratories a cost-effective tool for the eradication of backlogs of sexual assault evidence, and more timely service to their client agencies. In addition, we have demonstrated that a modification of the procedure can be used to retrieve residual sperm-cell DNA from previously extracted swabs. PMID:21288791

  10. 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

  11. 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

  12. Rapid microfluidic solid-phase extraction system for hyper-methylated DNA enrichment and epigenetic analysis

    PubMed Central

    De, Arpita; Sparreboom, Wouter; van den Berg, Albert; Carlen, Edwin T.

    2014-01-01

    Genetic sequence and hyper-methylation profile information from the promoter regions of tumor suppressor genes are important for cancer disease investigation. Since hyper-methylated DNA (hm-DNA) is typically present in ultra-low concentrations in biological samples, such as stool, urine, and saliva, sample enrichment and amplification is typically required before detection. We present a rapid microfluidic solid phase extraction (μSPE) system for the capture and elution of low concentrations of hm-DNA (≤1 ng ml−1), based on a protein-DNA capture surface, into small volumes using a passive microfluidic lab-on-a-chip platform. All assay steps have been qualitatively characterized using a real-time surface plasmon resonance (SPR) biosensor, and quantitatively characterized using fluorescence spectroscopy. The hm-DNA capture/elution process requires less than 5 min with an efficiency of 71% using a 25 μl elution volume and 92% efficiency using a 100 μl elution volume. PMID:25538809

  13. 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

  14. 'Direct PCR' optimization yields a rapid, cost-effective, nondestructive and efficient method for obtaining DNA barcodes without DNA extraction.

    PubMed

    Wong, Wing Hing; Tay, Ywee Chieh; Puniamoorthy, Jayanthi; Balke, Michael; Cranston, Peter S; Meier, Rudolf

    2014-11-01

    Macroinvertebrates that are collected in large numbers pose major problems in basic and applied biodiversity research: identification to species via morphology is often difficult, slow and/or expensive. DNA barcodes are an attractive alternative or complementary source of information. Unfortunately, obtaining DNA barcodes from specimens requires many steps and thus time and money. Here, we promote a short cut to DNA barcoding, that is, a nondestructive PCR method that skips DNA extraction ('direct PCR') and that can be used for a broad range of invertebrate taxa. We demonstrate how direct PCR can be optimized for the larvae and adults of nonbiting midges (Diptera: Chironomidae), a typical invertebrate group that is abundant, contains important bioindicator species, but is difficult to identify based on morphological features. After optimization, direct PCR yields high PCR success rates (>90%), preserves delicate morphological features (e.g. details of genitalia, and larval head capsules) while allowing for the recovery of genomic DNA. We also document that direct PCR can be successfully optimized for a wide range of other invertebrate taxa that need routine barcoding (flies: Culicidae, Drosophilidae, Dolichopodidae, Sepsidae; sea stars: Oreasteridae). Key for obtaining high PCR success rates is optimizing (i) tissue quantity, (ii) body part, (iii) primer pair and (iv) type of Taq polymerase. Unfortunately, not all invertebrates appear suitable because direct PCR has low success rates for other taxa that were tested (e.g. Coleoptera: Dytiscidae, Copepoda, Hymenoptera: Formicidae and Odonata). It appears that the technique is less successful for heavily sclerotized insects and/or those with many exocrine glands. PMID:24816169

  15. Evaluation of different methods for extracting extracellular DNA from the biofilm matrix.

    PubMed

    Wu, Jianfeng; Xi, Chuanwu

    2009-08-01

    The occurrence of high concentrations of extracellular DNA (eDNA) in the extracellular matrices of biofilms plays an important role in biofilm formation and development and possibly in horizontal gene transfer through natural transformation. Studies have been conducted to characterize the nature of eDNA and its potential function in biofilm development, but it is difficult to extract eDNA from the extracellular matrices of biofilms without any contamination from genomic DNA released by cell lysis during the extraction process. In this report, we compared several different extraction methods in order to obtain highly pure eDNA from different biofilm samples. After different extraction methods were explored, it was concluded that using chemical treatment or enzymatic treatment of biofilm samples may obtain larger amounts of eDNA than using the simple filtration method. There was no detectable cell lysis when the enzymatic treatment methods were used, but substantial cell lysis was observed when the chemical treatment methods were used. These data suggest that eDNA may bind to other extracellular polymers in the biofilm matrix and that enzymatic treatment methods are effective and favorable for extracting eDNA from biofilm samples. Moreover, randomly amplified polymorphic DNA analysis of eDNA in Acinetobacter sp. biofilms and Acinetobacter sp. genomic DNA and DNA sequencing analysis revealed that eDNA originated from genomic DNA but was not structurally identical to the genomic DNA. PMID:19561191

  16. Evaluation of Different Methods for Extracting Extracellular DNA from the Biofilm Matrix▿

    PubMed Central

    Wu, Jianfeng; Xi, Chuanwu

    2009-01-01

    The occurrence of high concentrations of extracellular DNA (eDNA) in the extracellular matrices of biofilms plays an important role in biofilm formation and development and possibly in horizontal gene transfer through natural transformation. Studies have been conducted to characterize the nature of eDNA and its potential function in biofilm development, but it is difficult to extract eDNA from the extracellular matrices of biofilms without any contamination from genomic DNA released by cell lysis during the extraction process. In this report, we compared several different extraction methods in order to obtain highly pure eDNA from different biofilm samples. After different extraction methods were explored, it was concluded that using chemical treatment or enzymatic treatment of biofilm samples may obtain larger amounts of eDNA than using the simple filtration method. There was no detectable cell lysis when the enzymatic treatment methods were used, but substantial cell lysis was observed when the chemical treatment methods were used. These data suggest that eDNA may bind to other extracellular polymers in the biofilm matrix and that enzymatic treatment methods are effective and favorable for extracting eDNA from biofilm samples. Moreover, randomly amplified polymorphic DNA analysis of eDNA in Acinetobacter sp. biofilms and Acinetobacter sp. genomic DNA and DNA sequencing analysis revealed that eDNA originated from genomic DNA but was not structurally identical to the genomic DNA. PMID:19561191

  17. Optimization of a One-Step Heat-Inducible In Vivo Mini DNA Vector Production System

    PubMed Central

    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, achieving an overall LCC DNA minivector production efficiency of ∼90%.We optimized a robust technology conferring rapid, scalable, one-step in vivo production of LCC DNA minivectors with potential application to gene transfer-mediated therapeutics. PMID:24586704

  18. 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, achieving an overall LCC DNA minivector production efficiency of ∼ 90%.We optimized a robust technology conferring rapid, scalable, one-step in vivo production of LCC DNA minivectors with potential application to gene transfer-mediated therapeutics. PMID:24586704

  19. Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: effective recovery of bacterial and archaeal DNA using mechanical cell lysis.

    PubMed

    Salonen, Anne; Nikkilä, Janne; Jalanka-Tuovinen, Jonna; Immonen, Outi; Rajilić-Stojanović, Mirjana; Kekkonen, Riina A; Palva, Airi; de Vos, Willem M

    2010-05-01

    Several different protocols are used for fecal DNA extraction, which is an integral step in all phylogenetic and metagenomic approaches to characterize the highly diverse intestinal ecosystem. We compared four widely used methods, and found their DNA yields to vary up to 35-fold. Bacterial, archaeal and human DNA was quantified by real-time PCR, and a compositional analysis of different extracts was carried out using the Human Intestinal Tract Chip, a 16S rRNA gene-based phylogenetic microarray. The overall microbiota composition was highly similar between the methods in contrast to the profound differences between the subjects (Pearson correlations >0.899 and 0.735, respectively). A detailed comparative analysis of mechanical and enzymatic methods showed that despite their overall similarity, the mechanical cell disruption by repeated bead beating showed the highest bacterial diversity and resulted in significantly improved DNA extraction efficiency of archaea and some bacteria, including Clostridium cluster IV. By applying the mechanical disruption method a high prevalence (67%) of methanogenic archaea was detected in healthy subjects (n=24), exceeding the typical values reported previously. The assessment of performance differences between different methodologies serves as a concrete step towards the comparison and reliable meta-analysis of the results obtained in different laboratories. PMID:20171997

  20. Microbial DNA extraction from intestinal biopsies is improved by avoiding mechanical cell disruption

    PubMed Central

    Carbonero, Franck; Nava, Gerardo M.; Benefiel, Ann C.; Greenberg, Eugene; Gaskins, H. Rex

    2011-01-01

    Currently, standard protocols for microbial DNA extraction from intestinal tissues do not exist. We assessed the efficiency of a commercial kit with and without mechanical disruption. Better quality DNA was obtained without mechanical disruption. Thus, it appears that bead-beating is not required for efficient microbial DNA extraction from intestinal biopsies. PMID:21820015

  1. Highly Effective DNA Extraction Method from Fresh, Frozen, Dried and Clotted Blood Samples

    PubMed Central

    Samadi Shams, Sara; Zununi Vahed, Sepideh; Soltanzad, Farzaneh; Kafil, Vala; Barzegari, Abolfazl; Atashpaz, Sina; Barar, Jaleh

    2011-01-01

    Introduction Today, with the tremendous potential of genomics and other recent advances in science, the role of science to improve reliable DNA extraction methods is more relevant than ever before. The ideal process for genomic DNA extraction demands high quantities of pure, integral and intact genomic DNA (gDNA) from the sample with minimal co-extraction of inhibitors of downstream processes. Here, we report the development of a very rapid, less-hazardous, and high throughput protocol for extracting of high quality DNA from blood samples. Methods Dried, clotted and ethylene diamine tetra-acetic acid (EDTA) treated fresh and frozen blood samples were extracted using this method in which the quality and integrity of the extracted DNA were corroborated by agarose gel electrophoresis, PCR reaction and DNA digestion using restricted enzyme. The UV spectrophotometric and gel electrophoresis analysis resulted in high A260/A280 ratio (>1.8) with high intactness of DNA. Results PCR and DNA digestion experiments indicated that the final solutions of extracted DNA contained no inhibitory substances, which confirms that the isolated DNA is of good quality. Conclusion The high quality and quantity of current method, no enzymatic processing and accordingly its low cost, make it appropriate for DNA extraction not only from human but also from animal blood samples in any molecular biology labs. PMID:23678425

  2. 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

  3. Methods for rapid and effective PCR-based detection of 'Candidatus Liberibacter solanacearum' from the insect vector Bactericera cockerelli: streamlining the DNA extraction/purification process.

    PubMed

    Lévy, Julien; Hancock, Joseph; Ravindran, Aravind; Gross, Dennis; Tamborindeguy, Cecilia; Pierson, Elizabeth

    2013-06-01

    This study provides a protocol for rapid DNA isolation from psyllid vectors (Bactericera cockerelli and Diaphorina citri) that can be used directly with DNA-based methods for the detection of 'Candidatus (Ca.) Liberibacter solanacearum,' the bacterial causal agent of potato zebra chip disease and eventually for 'Ca. Liberibacter asiaticus' the causal agent of huanglongbing disease in citrus. The fast DNA extraction protocol was designed to work with conventional polymerase chain reaction (cPCR) DNA amplification as well as Loop mediated PCR DNA amplification. Direct cPCR of the psyllid 28S rDNA gene from samples prepared using the fast DNA extraction method was as reliable as from samples prepared using standard DNA purification (> 97% from live insects) as tested in B. cockerelli. However, samples prepared using the fast DNA extraction method had to be diluted 1:100 in sterile water for reliable amplification, presumably to dilute PCR inhibitors in the crude extract. Similarly, both cPCR and loop mediated PCR DNA amplification detected 'Ca. Liberibacter' in psyllids infected with either the zebra chip or huanglongbing pathogen equally well from diluted samples prepared using the fast DNA extraction method or from samples prepared using a DNA purification step. In addition to being reliable, the time required to complete the fast DNA extraction for 10 samples was on average approximately 5 min and required no special reagents or laboratory equipment. Thus, the fast DNA extraction method shows strong promise as a rapid, reliable, and expedient method when coupled with PCR-based analyses for detection of 'Ca. Liberibacter' pathogens in psyllids. PMID:23865212

  4. 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 electrolyte ions. Conversely, the trailing electrolyte ions have a slow electrophoretic mobility, so they lag behind the sample, thus trapping the species of interest between the LE and TE streams. In a typical isotachophoresis configuration, the electric field is applied in a direction parallel to the direction of flow. The species then form bands that stretch across the width of the channel. A major limitation of that approach is that only a finite amount of sample can be processed at once, and the sample must be processed in batches. For our purposes, a form of free-flow isotachophoresis is more convenient, where the DNA forms a band parallel to the edges of the channel. To achieve this, in our chip, the electric field is applied transversely. This creates a force perpendicular to the direction of flow, which causes the different ions to migrate across the flow direction. Because the mobility of the DNA is between the mobility of the leading and the trailing electrolyte, the DNA is focused in a tight band near the center of the channel. The stream of DNA can then be directed to a different output to produce a highly concentrated outlet stream without batch processing. One hurdle that must be overcome for successful ITP is isolating the electrochemical reactions that result from the application of high voltage for the actual process of isotachophoresis. The electrochemical reactions that occur around metal electrodes produce bubbles and pH changes that are detrimental to successful ITP. The design of the chips we use incorporates polyacrylamide gels to serve as electrodes along the central channel. For our design, the metal electrodes are located away from the chip, and high conductivity buffer streams carry the potential to the chip, functioning as a 'liquid electrode.' The stream then runs alongside a gel barrier. The gel electrode permits ion transfer while simultaneously isolating the separation chamber from any contaminants in the outer, 'liquid electrode' streams. The difference in potential from one side of the chip to the other creates an electric field. This field traverses the inner, separation channel, containing the leading electrolyte, the trailing electrolyte, and the sample of interest (DNA). To increase the ease of use of the chips, a newer chip design has been fabricated. This design has wire electrodes integrated on the chip, rather than elsewhere. To keep the pH changes and bubbling isolated from the separation channel, the chip contains deeper wells near the electrodes so that the flowing buffer can wash away any gases that form around the electrode. This design is significantly more compact because it eliminates the cumbersome electrode boxes. Eliminating the electrode boxes also decreases the required voltage, making the experiments safer. This happens because when the 'liquid electrode' streams travel through small diameter tubing, they lose much of their voltage due to the electrical resistance of the fluid in the tubing.

  5. One-step purification of metallothionein extracted from two different sources.

    PubMed

    Honda, Rubens T; Araújo, Roziete Mendes; Horta, Bruno Brasil; Val, Adalberto L; Demasi, Marilene

    2005-06-25

    We describe a one-step purification of hepatic metallothionein from the Amazon fish Colossoma macropomum injected with cadmium and from the copper-loaded metallothionein from the yeast Saccharomyces cerevisiae, performed by affinity chromatography through metal-chelating columns. Yeast metallothionein was purified from Cu2+-loaded resin and eluted by a continuous EDTA gradient whereas hepatic metallothionein extracted from fishes was purified by Ni2+-loaded resin and eluted by a continuous imidazol gradient. Purified metallothioneins were evaluated by SDS-PAGE and characterized by UV spectra of the apo- and Cd2+-loaded protein. This method allowed high purity and yield as well as rapid one-step extraction of both metal-loaded and apoprotein. PMID:15899374

  6. 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

  7. 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 workflow requiring no additional equipment. PMID:25067909

  8. Step-wise DNA relaxation and decatenation by NaeI-43K.

    PubMed Central

    Jo, K; Topal, M D

    1998-01-01

    Nae I protein was originally isolated for its restriction endonuclease properties. Nae I was later discovered to either relax or cleave supercoiled DNA, depending upon whether Nae I position 43 contains a lysine (43K) or leucine (43L) respectively. Nae I-43K DNA relaxation activity appears to be the product of coupling separate endonuclease and ligase domains within the same polypeptide. Whereas Nae I relaxes supercoiled DNA like a topoisomerase, even forming a transient covalent intermediate with the substrate DNA, Nae I shows no obvious sequence similarity to the topoisomerases. To further characterize the topoisomerase activity of Nae I, we report here that Nae I-43K changes the linking number of a single negatively supercoiled topoisomer of pBR322 by units of one and therefore is a type I topoisomerase. Positively supercoiled pBR322 was resistant to Nae I-43K. At low salt concentration Nae I-43K was processive; non-saturating amounts of enzyme relaxed a fraction of the DNA. At high salt concentration the same non-saturating amounts of Nae I-43K partially relaxed all the DNA in a step-wise fashion to give a Gaussian distribution of topoisomers, demonstrating a switch from a processive to a distributive mode of action. Nae I-43K decatenated kinetoplast DNA containing nicked circles, implying that Nae I-43K can cleave opposite a nick. The products of the reaction are decatenated nicked circles under both processive and distributive conditions. The behavior of Nae I-43K is consistent with that of a prokaryotic type I topoisomerase. PMID:9580689

  9. Early Steps in the DNA Base Excision/Single-Strand Interruption Repair Pathway in Mammalian Cells

    PubMed Central

    Hegde, Muralidhar L.; Hazra, Tapas K.; Mitra, Sankar

    2009-01-01

    Base excision repair (BER) is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged (oxidized or alkylated) or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species (ROS). BER involves 4–5 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease (APE) to generate 3′ OH terminus at the damage site, followed by repair synthesis with a DNA polymerase and nick sealing by a DNA ligase. This pathway is also responsible for repairing DNA single-strand breaks with blocked termini directly generated by ROS. Nearly all glycosylases, far fewer than their substrate lesions particularly for oxidized bases, have broad and overlapping substrate range, and could serve as back-up enzymes in vivo. In contrast, mammalian cells encode only one APE, APE1, unlike two APEs in lower organisms. In spite of overall similarity, BER with distinct subpathways in the mammals is more complex than in E. coli. The glycosylases form complexes with downstream proteins to carry out efficient repair via distinct subpathways one of which, responsible for repair of strand breaks with 3′ phosphate termini generated by the NEIL family glycosylases or by ROS, requires the phosphatase activity of polynucleotide kinase instead of APE1. Different complexes may utilize distinct DNA polymerases and ligases. Mammalian glycosylases have nonconserved extensions at one of the termini, dispensable for enzymatic activity but needed for interaction with other BER and non BER proteins for complex formation and organelle targeting. The mammalian enzymes are sometimes covalently modified that may affect activity and complex formation. The focus of this review are the early steps in mammalian BER for oxidized damage. PMID:18166975

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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.

  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. Sequential steps in DNA replication are inhibited to ensure reduction of ploidy in meiosis.

    PubMed

    Hua, Hui; Namdar, Mandana; Ganier, Olivier; Gregan, Juraj; Méchali, Marcel; Kearsey, Stephen E

    2013-03-01

    Meiosis involves two successive rounds of chromosome segregation without an intervening S phase. Exit from meiosis I is distinct from mitotic exit, in that replication origins are not licensed by Mcm2-7 chromatin binding, but spindle disassembly occurs during a transient interphase-like state before meiosis II. The absence of licensing is assumed to explain the block to DNA replication, but this has not been formally tested. Here we attempt to subvert this block by expressing the licensing control factors Cdc18 and Cdt1 during the interval between meiotic nuclear divisions. Surprisingly, this leads only to a partial round of DNA replication, even when these factors are overexpressed and effect clear Mcm2-7 chromatin binding. Combining Cdc18 and Cdt1 expression with modulation of cyclin-dependent kinase activity, activation of Dbf4-dependent kinase, or deletion of the Spd1 inhibitor of ribonucleotide reductase has little additional effect on the extent of DNA replication. Single-molecule analysis indicates this partial round of replication results from inefficient progression of replication forks, and thus both initiation and elongation replication steps may be inhibited in late meiosis. In addition, DNA replication or damage during the meiosis I-II interval fails to arrest meiotic progress, suggesting absence of checkpoint regulation of meiosis II entry. PMID:23303250

  18. Sequential steps in DNA replication are inhibited to ensure reduction of ploidy in meiosis

    PubMed Central

    Hua, Hui; Namdar, Mandana; Ganier, Olivier; Gregan, Juraj; Méchali, Marcel; Kearsey, Stephen E.

    2013-01-01

    Meiosis involves two successive rounds of chromosome segregation without an intervening S phase. Exit from meiosis I is distinct from mitotic exit, in that replication origins are not licensed by Mcm2-7 chromatin binding, but spindle disassembly occurs during a transient interphase-like state before meiosis II. The absence of licensing is assumed to explain the block to DNA replication, but this has not been formally tested. Here we attempt to subvert this block by expressing the licensing control factors Cdc18 and Cdt1 during the interval between meiotic nuclear divisions. Surprisingly, this leads only to a partial round of DNA replication, even when these factors are overexpressed and effect clear Mcm2-7 chromatin binding. Combining Cdc18 and Cdt1 expression with modulation of cyclin-dependent kinase activity, activation of Dbf4-dependent kinase, or deletion of the Spd1 inhibitor of ribonucleotide reductase has little additional effect on the extent of DNA replication. Single-molecule analysis indicates this partial round of replication results from inefficient progression of replication forks, and thus both initiation and elongation replication steps may be inhibited in late meiosis. In addition, DNA replication or damage during the meiosis I–II interval fails to arrest meiotic progress, suggesting absence of checkpoint regulation of meiosis II entry. PMID:23303250

  19. Steps and Bumps: Precision Extraction of Discrete States of Molecular Machines

    PubMed Central

    Little, Max A.; Steel, Bradley C.; Bai, Fan; Sowa, Yoshiyuki; Bilyard, Thomas; Mueller, David M.; Berry, Richard M.; Jones, Nick S.

    2011-01-01

    We report statistical time-series analysis tools providing improvements in the rapid, precision extraction of discrete state dynamics from time traces of experimental observations of molecular machines. By building physical knowledge and statistical innovations into analysis tools, we provide techniques for estimating discrete state transitions buried in highly correlated molecular noise. We demonstrate the effectiveness of our approach on simulated and real examples of steplike rotation of the bacterial flagellar motor and the F1-ATPase enzyme. We show that our method can clearly identify molecular steps, periodicities and cascaded processes that are too weak for existing algorithms to detect, and can do so much faster than existing algorithms. Our techniques represent a step in the direction toward automated analysis of high-sample-rate, molecular-machine dynamics. Modular, open-source software that implements these techniques is provided. PMID:21767501

  20. 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...

  1. 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 ...

  2. Method for extraction of high-quantity and -quality cell-free DNA from amniotic fluid.

    PubMed

    Lapaire, Olav; Johnson, Kirby L; Bianchi, Diana W

    2008-01-01

    Circulating cell-free fetal deoxyribonucleic acids (cffDNAs) are promising biomarkers with various potential clinical applications. Second and third trimester amniotic fluid (AF) is a rich source of cffDNAs. Further improvements to the original protocol for the extraction of cffDNAs from AF supernatant resulted in statistically significant higher yields of high-quality cffDNAs, allowing for a substantial majority of samples to be analyzed with subsequent molecular methods (e.g., comparative genomic hybridization microarrays) to further assess for genetic abnormalities. Several advantages have been realized with the optimized protocol. In addition to an improved yield from a greater proportion of samples compared with the original protocol, the current method, using large silico-membranes, allows for the extraction of cffDNAs from up to 10 samples in <3 h. The replacement of the original lysis buffer eliminates the need for a heating bath during the lysis step, and fewer overall steps are involved in the protocol (e.g., to reduce potential contamination). The improvements in the yield with the current protocol make it possible to augment current standard of care through the analysis of this previously unappreciated source of genetic material. Furthermore, the improvements allow for exploration of widely unknown genetic, pathophysiological, and kinetic issues of cell-free fetal DNA in AF. PMID:18425491

  3. 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...

  4. 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...

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. Embryonic extracts derived from the nematode Caenorhabditis elegans remove uracil from DNA by the sequential action of uracil-DNA glycosylase and AP (apurinic/apyrimidinic) endonuclease.

    PubMed Central

    Shatilla, Andrea; Ramotar, Dindial

    2002-01-01

    DNA bases continuously undergo modifications in response to endogenous reactions such as oxidation, alkylation or deamination. The modified bases are primarily removed by DNA glycosylases, which cleave the N-glycosylic bond linking the base to the sugar, to generate an apurinic/apyrimidinic (AP) site, and this latter lesion is highly mutagenic. Previously, no study has demonstrated the processing of these lesions in the nematode Caenorhabditis elegans. Herein, we report the existence of uracil-DNA glycosylase and AP endonuclease activities in extracts derived from embryos of C. elegans. These enzyme activities were monitored using a defined 5'-end (32)P-labelled 42-bp synthetic oligonucleotide substrate bearing a single uracil residue opposite guanine at position 21. The embryonic extract rapidly cleaved the substrate in a time-dependent manner to produce a 20-mer product. The extract did not excise adenine or thymine opposite guanine, although uracil opposite either adenine or thymine was processed. Addition of the highly specific inhibitor of uracil-DNA glycosylase produced by Bacillus subtilis to the extract prevented the formation of the 20-mer product, indicating that removal of uracil is catalysed by uracil-DNA glycosylase. The data suggest that the 20-mer product was generated by a sequential reaction, i.e., removal of the uracil base followed by 5'-cleavage of the AP site. Further analysis revealed that product formation was dependent upon the presence of Mg(2+), suggesting that cleavage of the AP site, following uracil excision, is carried out by a Mg(2+)-dependent AP endonuclease. It would appear that these activities correspond to the first two steps of a putative base-excision-repair pathway in C. elegans. PMID:11966472

  13. 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

  14. Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast.

    PubMed

    Tebbe, C C; Vahjen, W

    1993-08-01

    A two-step protocol for the extraction and purification of total DNA from soil samples was developed. Crude DNA extracts (100 microliters from 5 g of soil) were contaminated with humic acids at concentrations of 0.7 to 3.3 micrograms/microliters, depending on the type of soil extracted. The coextracted humic acid fraction of a clay silt was similar to a commercially available standard humic acid mixture, as determined by electrophoretic mobility in agarose gels, UV fluorescence, and inhibition assays with DNA-transforming enzymes. Restriction endonucleases were inhibited at humic acid concentrations of 0.5 to 17.2 micrograms/ml for the commercial product and 0.8 to 51.7 micrograms/ml for the coextracted humic acids. DNase I was less susceptible (MIC of standard humic acids, 912 micrograms/ml), and RNase could not be inhibited at all (MIC, > 7.6 mg/ml). High inhibitory susceptibilities for humic acids were observed with Taq polymerase. For three Taq polymerases from different commercial sources, MICs were 0.08 to 0.64 micrograms of the standard humic acids per ml and 0.24 to 0.48 micrograms of the coextracted humic acids per ml. The addition of T4 gene 32 protein increased the MIC for one Taq polymerase to 5.12 micrograms/ml. Humic acids decreased nonradioactive detection in DNA-DNA slot blot hybridizations at amounts of 0.1 micrograms and inhibited transformation of competent Escherichia coli HB101 with a broad-host-range plasmid, pUN1, at concentrations of 100 micrograms/ml. Purification of crude DNA with ion-exchange chromatography resulted in removal of 97% of the initially coextracted humic acids.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7690221

  15. 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

  16. Extraction of high-quality DNA from ethanol-preserved tropical plant tissues

    PubMed Central

    2014-01-01

    Background Proper conservation of plant samples, especially during remote field collection, is essential to assure quality of extracted DNA. Tropical plant species contain considerable amounts of secondary compounds, such as polysaccharides, phenols, and latex, which affect DNA quality during extraction. The suitability of ethanol (96% v/v) as a preservative solution prior to DNA extraction was evaluated using leaves of Jatropha curcas and other tropical species. Results Total DNA extracted from leaf samples stored in liquid nitrogen or ethanol from J. curcas and other tropical species (Theobroma cacao, Coffea arabica, Ricinus communis, Saccharum spp., and Solanum lycopersicon) was similar in quality, with high-molecular-weight DNA visualized by gel electrophoresis. DNA quality was confirmed by digestion with EcoRI or HindIII and by amplification of the ribosomal gene internal transcribed spacer region. Leaf tissue of J. curcas was analyzed by light and transmission electron microscopy before and after exposure to ethanol. Our results indicate that leaf samples can be successfully preserved in ethanol for long periods (30 days) as a viable method for fixation and conservation of DNA from leaves. The success of this technique is likely due to reduction or inactivation of secondary metabolites that could contaminate or degrade genomic DNA. Conclusions Tissue conservation in 96% ethanol represents an attractive low-cost alternative to commonly used methods for preservation of samples for DNA extraction. This technique yields DNA of equivalent quality to that obtained from fresh or frozen tissue. PMID:24761774

  17. A comparative evaluation of four DNA extraction protocols from whole blood sample.

    PubMed

    Ghaheri, M; Kahrizi, D; Yari, K; Babaie, A; Suthar, R S; Kazemi, E

    2016-01-01

    All organisms have Deoxyribonucleic acid (DNA) within their cells. DNA is a complex molecule that contains all of the information necessary to build and maintain an organism. DNA extraction is one of the most basic and essential techniques in the study of DNA that allow huge advances in molecular biology, biotechnology and bioinformatics laboratories. Whole blood samples are one of the main sources used to obtain DNA and there are many different protocols available in this issue. In current research, compared four DNA extraction protocols from blood samples; include modified phenol-chloroform protocol, two salting-out and enzyme free method and from commercial kit. The extracted DNAs by these protocols were analyzed according to their time demands, quality and quantity, toxicity and functionality in PCR method. Also the quality and quantity of the extracted DNA were surveyed by gel electrophoresis and Nanodrop spectrophotometry methods. It was observed that there are not significantly differences between these methods about DNA Purity (A260/A280), but the DNA yield (ng DNA/μl) of phenol/chloroform method was higher than other methods. In addition, phenol/chloroform was the most toxic method and it takes more time than other methods. Roche diagnostics GmbH kit was the most expensive among the four methods but the least extraction time was required and it was the safest method. PMID:27064884

  18. 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 ...

  19. [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

    Harl, Alexandre; Lion, Mava; 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 ISO15189 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 ISO15189 and the accreditation technical guide in Human Health SH-GTA-04. PMID:24113450

  20. Modification of a commercial DNA extraction kit for safe and rapid recovery of DNA and RNA simultaneously from soil, without the use of harmful solvents.

    PubMed

    Tournier, E; Amenc, L; Pablo, A L; Legname, E; Blanchart, E; Plassard, C; Robin, A; Bernard, L

    2015-01-01

    An optimized method, based on the coupling of two commercial kits, is described for the extraction of soil nucleic acids, with simultaneous extraction and purification of DNA and RNA following a cascade scheme and avoiding the use of harmful solvents. The protocol canmonitor the variations in the recovery yield of DNA and RNA from soils of various types.The quantitative version of the protocol was obtained by testing the starting soil quantity, the grinding parameters and the final elution volumes, in order to avoid saturation of both kits. •A first soil-crushing step in liquid nitrogen could be added for the assessment of fungal parameters.•The protocol was efficienton different tropical soils, including Andosol, while their high contents of clays, including poorly crystalline clays, and Fe and Al oxides usually make the nucleic acid extraction more difficult.•The RNA recovery yield from the previous tropical soils appeared to correlate better to soil respiration than DNA, which is positively influenced by soil clay content. PMID:26150987

  1. Comparison of several methods for the extraction of DNA from potatoes and potato-derived products.

    PubMed

    Smith, Donna S; Maxwell, Philip W; De Boer, Solke H

    2005-12-28

    Eight methods were compared for the extraction of DNA from raw potato tubers, and nine methods were evaluated for the extraction of DNA from dehydrated potato slices, potato flakes, potato flour, potato starch, and two ready-to-eat potato snack foods. Extracts were assessed for yield using a fluorescence-based DNA quantification assay. Real-time amplification of an endogenous gene, sucrose synthase (sus), was used to assess extract and template quality. A CTAB-based method extracted the highest DNA yields from the tuber material. An in-house method, which utilized the Kingfisher magnetic particle processor, yielded the highest template quality from the tubers. For most of the tuber samples, the Kingfisher and CTAB methods recovered the highest levels of amplifiable sus. DNA yields for potato-derived foods generally decreased with the extent that the product had been processed. The methods that utilized the magnetic particle processor delivered the highest template quality from one of the snack products that was particularly high in fat. For most of the remaining processed products, the levels of amplifiable target DNA recovered were roughly correlated with total DNA recovery, indicating that overall yield had greater influence over sus amplification than template quality. The Wizard method was generally the best method for the extraction of DNA from most of the potato-derived foods. PMID:16366665

  2. 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

  3. EVALUATION OF FOUR DIFFERENT DNA EXTRACTION METHODS IN COAGULASE-NEGATIVE STAPHYLOCOCCI CLINICAL ISOLATES

    PubMed Central

    de Oliveira, Caio Fernando; Paim, Thiago Galvão da Silva; Reiter, Keli Cristine; Rieger, Alexandre; D'azevedo, Pedro Alves

    2014-01-01

    Currently there are several methods to extract bacterial DNA based on different principles. However, the amount and the quality of the DNA obtained by each one of those methods is highly variable and microorganism dependent, as illustrated by coagulase-negative staphylococci (CoNS) which have a thick cell wall that is difficult to lyse. This study was designed to compare the quality and the amount of CoNS DNA, extracted by four different techniques: two in-house protocols and two commercial kits. DNA amount and quality determination was performed through spectrophotometry. The extracted DNA was also analyzed using agarose gel electrophoresis and by PCR. 267 isolates of CoNS were used in this study. The column method and thermal lyses showed better results with regard to DNA quality (mean ratio of A260/280 = 1.95) and average concentration of DNA (), respectively. All four methods tested provided appropriate DNA for PCR amplification, but with different yields. DNA quality is important since it allows the application of a large number of molecular biology techniques, and also it's storage for a longer period of time. In this sense the extraction method based on an extraction column presented the best results for CoNS. PMID:24553605

  4. New rapid DNA extraction method with Chelex from Venturia inaequalis spores.

    PubMed

    Turan, Ceren; Nanni, Irene Maja; Brunelli, Agostino; Collina, Marina

    2015-08-01

    The objective of this study was to develop a rapid method to isolate DNA from Venturia inaequalis spores for use in diagnostic DNA mutation analysis. Chelex-100 resin was evaluated and compared with a well established DNA exctraction method, utilizing CTAB in order to have a robust comparison. In this research we demonstrated that Chelex-100 efficiently makes extraction of the DNA from V. inaequalis spores available for direct use in molecular analyses. Also, the quantity and quality of extracted DNA were shown to be adequate for PCR analysis. Comparatively, the quality of DNA samples isolated using Chelex method was better than those extracted using CTAB. In conclusion, the Chelex method is recommended for PCR experiments considering its simplicity and cost-effectiveness. PMID:26079986

  5. Complete sequence analysis of 18S rDNA based on genomic DNA extraction from individual Demodex mites (Acari: Demodicidae).

    PubMed

    Zhao, Ya-E; Xu, Ji-Ru; Hu, Li; Wu, Li-Ping; Wang, Zheng-Hang

    2012-05-01

    The study for the first time attempted to accomplish 18S ribosomal DNA (rDNA) complete sequence amplification and analysis for three Demodex species (Demodex folliculorum, Demodex brevis and Demodex canis) based on gDNA extraction from individual mites. The mites were treated by DNA Release Additive and Hot Start II DNA Polymerase so as to promote mite disruption and increase PCR specificity. Determination of D. folliculorum gDNA showed that the gDNA yield reached the highest at 1 mite, tending to descend with the increase of mite number. The individual mite gDNA was successfully used for 18S rDNA fragment (about 900 bp) amplification examination. The alignments of 18S rDNA complete sequences of individual mite samples and those of pooled mite samples ( ≥ 1000mites/sample) showed over 97% identities for each species, indicating that the gDNA extracted from a single individual mite was as satisfactory as that from pooled mites for PCR amplification. Further pairwise sequence analyses showed that average divergence, genetic distance, transition/transversion or phylogenetic tree could not effectively identify the three Demodex species, largely due to the differentiation in the D. canis isolates. It can be concluded that the individual Demodex mite gDNA can satisfy the molecular study of Demodex. 18S rDNA complete sequence is suitable for interfamily identification in Cheyletoidea, but whether it is suitable for intrafamily identification cannot be confirmed until the ascertainment of the types of Demodex mites parasitizing in dogs. PMID:22414329

  6. 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

  7. Semi-quantitative detection of cytomegalovirus DNA from native serum and plasma by nested PCR: influence of DNA extraction procedures.

    PubMed

    Hamprecht, K; Mikeler, E; Jahn, G

    1997-12-01

    The diagnostic implications of different procedures of DNA extraction were examined for the detection of HCMV DNA from sera and plasma of immunosuppressed patients. The detection limit of HCMV plasmid DNA from cell free seronegative plasma and serum by limiting dilution nested PCR decreased in the following sequence: phenol/chloroform > NaI-single tube method > proteinase K digestion equal to amplification of native sera and plasma. Nested PCR from native sera and plasma performed well and surpassed the proteinase K method in sensitivity for detection of serum DNAemia. Semi-quantitative determination of HCMV DNA titer present in native sera of immunosuppressed patients did not seem to be correlated to HCMV disease. When compared to the persistence of leukoDNAemia, the viral DNA titer in native plasma could only be observed in the acute phase of HCMV infection, an important phenomenon for diagnostic purposes. Correlation of serum DNAemia to virus culture revealed low positive and high negative predictive values. Predictive values of nested PCR from native sera for HCMV infection were not lower than those following organic DNA extraction. Despite its low correlation to viremia and virus isolation from any site, nested PCR from organic DNA extracts of serum or plasma is the most sensitive diagnostic tool of an ongoing HCMV infection. Additionally, semi-quantitative end point dilution nested PCR from native serum or plasma promises to be a rapid and easy tool for the monitoring of antiviral therapy. PMID:9504758

  8. 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

  9. DNA extraction of ancient animal hard tissue samples via adsorption to silica particles.

    PubMed

    Rohland, Nadin

    2012-01-01

    A large number of subfossil and more recent skeletal remains, many of which are stored in museums and private collections, are potentially accessible for DNA sequence analysis. In order to extract the small amount of DNA preserved in these specimens, an efficient DNA release and purification method is required. In this chapter, I describe an efficient and straightforward purification and concentration method that uses DNA adsorption to a solid surface of silica particles. Comparative analysis of extraction methods has shown that this method works reliably for ancient as well as younger, museum-preserved specimens. PMID:22237517

  10. 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

  11. Optimization of HPV DNA detection in urine by improving collection, storage, and extraction.

    PubMed

    Vorsters, A; Van den Bergh, J; Micalessi, I; Biesmans, S; Bogers, J; Hens, A; De Coster, I; Ieven, M; Van Damme, P

    2014-11-01

    The benefits of using urine for the detection of human papillomavirus (HPV) DNA have been evaluated in disease surveillance, epidemiological studies, and screening for cervical cancers in specific subgroups. HPV DNA testing in urine is being considered for important purposes, notably the monitoring of HPV vaccination in adolescent girls and young women who do not wish to have a vaginal examination. The need to optimize and standardize sampling, storage, and processing has been reported.In this paper, we examined the impact of a DNA-conservation buffer, the extraction method, and urine sampling on the detection of HPV DNA and human DNA in urine provided by 44 women with a cytologically normal but HPV DNA-positive cervical sample. Ten women provided first-void and midstream urine samples. DNA analysis was performed using real-time PCR to allow quantification of HPV and human DNA.The results showed that an optimized method for HPV DNA detection in urine should (a) prevent DNA degradation during extraction and storage, (b) recover cell-free HPV DNA in addition to cell-associated DNA, (c) process a sufficient volume of urine, and (d) use a first-void sample.In addition, we found that detectable human DNA in urine may not be a good internal control for sample validity. HPV prevalence data that are based on urine samples collected, stored, and/or processed under suboptimal conditions may underestimate infection rates. PMID:24916950

  12. 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

  13. DNA extraction protocol for biological ingredient analysis of Liuwei Dihuang Wan.

    PubMed

    Cheng, Xinwei; Chen, Xiaohua; Su, Xiaoquan; Zhao, Huanxin; Han, Maozhen; Bo, Cunpei; Xu, Jian; Bai, Hong; Ning, Kang

    2014-06-01

    Traditional Chinese medicine (TCM) preparations are widely used for healthcare and clinical practice. So far, the methods commonly used for quality evaluation of TCM preparations mainly focused on chemical ingredients. The biological ingredient analysis of TCM preparations is also important because TCM preparations usually contain both plant and animal ingredients, which often include some mis-identified herbal materials, adulterants or even some biological contaminants. For biological ingredient analysis, the efficiency of DNA extraction is an important factor which might affect the accuracy and reliability of identification. The component complexity in TCM preparations is high, and DNA might be destroyed or degraded in different degrees after a series of processing procedures. Therefore, it is necessary to establish an effective protocol for DNA extraction from TCM preparations. In this study, we chose a classical TCM preparation, Liuwei Dihuang Wan (LDW), as an example to develop a TCM-specific DNA extraction method. An optimized cetyl trimethyl ammonium bromide (CTAB) method (TCM-CTAB) and three commonly-used extraction kits were tested for extraction of DNA from LDW samples. Experimental results indicated that DNA with the highest purity and concentration was obtained by using TCM-CTAB. To further evaluate the different extraction methods, amplification of the second internal transcribed spacer (ITS2) and the chloroplast genome trnL intron was carried out. The results have shown that PCR amplification was successful only with template of DNA extracted by using TCM-CTAB. Moreover, we performed high-throughput 454 sequencing using DNA extracted by TCM-CTAB. Data analysis showed that 3-4 out of 6 prescribed species were detected from LDW samples, while up to 5 contaminating species were detected, suggesting TCM-CTAB method could facilitate follow-up DNA-based examination of TCM preparations. PMID:24838067

  14. 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

  15. Evaluation of three methods for effective extraction of DNA from human hair.

    PubMed

    Suenaga, Emi; Nakamura, Hiroshi

    2005-06-01

    In this paper we evaluate three different methods for extracting DNA from human hair i.e. the Chelex method, the QIAamp DNA Mini Kit method and the ISOHAIR method. Analysis of DNA prepared from dyed hairs with the ISOHAIR method suggested that the DNA extracts contained PCR inhibitors. On the other hand, few inhibition was observed when DNA from dyed hairs were extracted using the Chelex method and the QIAamp DNA Mini Kit method. In conclusion, the Chelex method is recommended for PCR experiments in view of its simplicity and cost-effectiveness. To assess the reliability of the Chelex method for the extraction of genomic DNA from both natural and dyed hair samples, minisatellite variant repeat (MVR)-polymerase chain reaction (PCR) patterns of Chelex-extracted DNA were compared using hairs (three natural black hairs and three dyed hairs) with buccal swabs from six individuals. Complete agreement was observed between hair and swab samples in each individual, proving the utility of the Chelex method. PMID:15866502

  16. 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

  17. Comparison of methods for total community DNA extraction and purification from compost.

    PubMed

    Yang, Zh H; Xiao, Y; Zeng, G M; Xu, Zh Y; Liu, Y Sh

    2007-03-01

    The differences on DNA yield and purity of three different DNA extraction protocols were compared with regard to the use for PCR and other molecular analyses. Total DNA was extracted from compost by the three protocols, and then was purified by spin-bind cartridges after being precipitated by PEG8000. The detection performed on a nucleic acid and protein analyzer showed that all three methods produced high DNA yields. The agarose gel electrophoresis showed that the fragments of crude and purified DNA had a length of about 23 kb. A eubacterial 16S rRNA gene-targeted primer pair was used for PCR amplification, and full length 16S rDNAs were amplified from all the purified DNA samples. After being digested by restriction endonucleases, the restriction map of amplified rDNA showed identical genetic diversity. The products of PCR using primer pair GC341F and 907R were also used for denaturing gradient gel electrophoresis analysis. The results indicated that high-quality DNA was extracted from compost by the three protocols, and each of the protocols is adapted to extract microbial genome DNA from compost expediently and cheaply. PMID:17115207

  18. Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens

    PubMed Central

    2010-01-01

    Background The influence of diet on intestinal microflora has been investigated mainly using conventional microbiological approaches. Although these studies have advanced knowledge on human intestinal microflora, it is imperative that new methods are applied to facilitate scientific progress. Culture-independent molecular fingerprinting method of Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis (PCR-DGGE) has been used to study microbial communities in a variety of environmental samples. However, these protocols must be optimized prior to their application in order to enhance the quality and accuracy of downstream analyses. In this study, the relative efficacy of four commercial DNA extraction kits (Mobio Ultra Clean Fecal DNA Isolation Kit, M; QIAamp DNA Stool Mini Kit, Q; FastDNA SPIN Kit, FSp; FastDNA SPIN Kit for Soil, FSo) were evaluated. Further, PCR-DGGE technique was also assessed for its feasibility in detecting differences in human intestinal bacterial fingerprint profiles. Method Total DNA was extracted from varying weights of human fecal specimens using four different kits, followed by PCR amplification of bacterial 16S rRNA genes, and DGGE separation of the amplicons. Results Regardless of kit, maximum DNA yield was obtained using 10 to 50 mg (wet wt) of fecal specimens and similar DGGE profiles were obtained. However, kits FSp and FSo extracted significantly larger amounts of DNA per g dry fecal specimens and produced more bands on their DGGE profiles than kits M and Q due to their use of bead-containing lysing matrix and vigorous shaking step. DGGE of 16S rRNA gene PCR products was suitable for capturing the profiles of human intestinal microbial community and enabled rapid comparative assessment of inter- and intra-subject differences. Conclusion We conclude that extraction kits that incorporated bead-containing lysing matrix and vigorous shaking produced high quality DNA from human fecal specimens (10 to 50 mg, wet wt) that can be resolved as bacterial community fingerprints using PCR-DGGE technique. Subsequently, PCR-DGGE technique can be applied for studying variations in human intestinal microbial communities. PMID:20492702

  19. A rapid column-based ancient DNA extraction method for increased sample throughput.

    PubMed

    Rohland, Nadin; Siedel, Heike; Hofreiter, Michael

    2010-07-01

    Genetic analyses using museum specimens and ancient DNA from fossil samples are becoming increasingly important in phylogenetic and especially population genetic studies. Recent progress in ancient DNA sequencing technologies has substantially increased DNA sequence yields and, in combination with barcoding methods, has enabled large-scale studies using any type of DNA. Moreover, more and more studies now use nuclear DNA sequences in addition to mitochondrial ones. Unfortunately, nuclear DNA is, due to its much lower copy number in living cells compared to mitochondrial DNA, much more difficult to obtain from low-quality samples. Therefore, a DNA extraction method that optimizes DNA yields from low-quality samples and at the same time allows processing many samples within a short time frame is immediately required. In fact, the major bottleneck in the analysis process using samples containing low amounts of degraded DNA now lies in the extraction of samples, as column-based methods using commercial kits are fast but have proven to give very low yields, while more efficient methods are generally very time-consuming. Here, we present a method that combines the high DNA yield of batch-based silica extraction with the time-efficiency of column-based methods. Our results on Pleistocene cave bear samples show that DNA yields are quantitatively comparable, and in fact even slightly better than with silica batch extraction, while at the same time the number of samples that can conveniently be processed in parallel increases and both bench time and costs decrease using this method. Thus, this method is suited for harvesting the power of high-throughput sequencing using the DNA preserved in the millions of paleontological and museums specimens. PMID:21565072

  20. 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

  1. 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

  2. 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...

  3. A simple method for extracting DNA from Cryptosporidium oocysts using the anionic surfactant LSS.

    PubMed

    Sekikawa, Takahiro; Kawasaki, Yu; Katayama, Yasuto; Iwahori, Keisuke

    2011-12-15

    Detection of low amounts of Cryptosporidium oocysts in raw water sources is considered an important component in the management, prevention and control of Cryptosporidium in drinking water supplies as Cryptosporidium causes massive waterborne outbreaks worldwide. As Cryptosporidium has a robust oocyst that is extremely resistant to chlorine and other drinking water disinfectants, both the freeze-thaw method and DNA extraction kits have been commonly used for extracting and purifying DNA from the oocyst. However, the DNA extraction procedures are time consuming and costly. Therefore, a simple and low-cost method to extract and purify DNA from the robust oocyst has been required. In this study, we discussed a simple method for detecting Cryptosporidium DNA with the anionic surfactant, n-lauroylsarcosine sodium salt (LSS) using the loop-mediated isothermal amplification (LAMP) to eliminate the need for the freeze-thaw method and the DNA extraction kits. As a result, Bst DNA polymerase was inhibited by 0.1% LSS but not 0.01% LSS and 5% Triton X-100 or Tween 20. Although DNA was extracted from the oocysts by incubating with 0.1% LSS at 90°C for 15 min, Bst DNA polymerase was inhibited by 0.1% LSS. The inhibition by 0.1% LSS was suppressed by adding 5% of the nonionic surfactants, Triton X-100 or Tween 20. The concentration of LSS in a LAMP tube was 0.01% while that in an incubation tube was 0.1%, because LSS in an incubation tube was diluted by a factor of 10 at the DNA amplification process. Therefore, we found that ten oocysts of Cryptosporidium parvum could be detected by incubation with 0.1% LSS, without removing LSS or adding the nonionic surfactants in the LAMP method. PMID:21924387

  4. A simple method of genomic DNA extraction from human samples for PCR-RFLP analysis.

    PubMed

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

    2013-12-01

    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

  5. Fabrication of two-weir structure-based packed columns for on-chip solid-phase extraction of DNA.

    PubMed

    Zhong, Runtao; Liu, Dayu; Yu, Linfen; Ye, Nannan; Dai, Zhongpeng; Qin, Jianhua; Lin, Bingcheng

    2007-08-01

    Microchip-based packed column SPE of DNA was performed using the microfabricated two-weir structure within a microchannel. We developed two methods to fabricate the two-weir structured glass chips: a "two-side etching/alignment" method and a simplified "one-side/two-step etching" method. The former method required a straightforward alignment step, while the latter approach comprised a simplified wet etching process using paraffin wax as the temporary protective layer. Both methods were convenient and rapid as compared to the previous approaches. Through a reversibly sealed bead-introduction channel, beads can be fed into or out of the chip columns, thus enabling refreshment of the packing materials. Using the proposed chip columns, highly efficient lambda-DNA extractions (average recovery >80%) were performed with good chip-to-chip reproducibility (RSD <10%). The on-chip SPE procedure was completed within 15 min at the flow rate of 3 microL/min and the bulk of the loaded DNA was eluted within a small volume of approximately 8 microL. Application of the microchip-based packed columns was demonstrated by purifying PCR-amplifiable genomic DNA from human hepatocellular carcinoma (HepG2) cells and human whole blood samples. PMID:17640088

  6. 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

  7. Comparison of protocols for DNA extraction from long-term preserved formalin fixed tissues.

    PubMed

    Paireder, Stefan; Werner, Bettina; Bailer, Josef; Werther, Wolfgang; Schmid, Erich; Patzak, Beatrix; Cichna-Markl, Margit

    2013-08-15

    The current study compared the applicability of protocols to extract DNA from formalin fixed heart tissues that have been preserved for more than 50 years. Ten methods were tested: a cetyltrimethylammonium bromide (CTAB) standard protocol, seven variants of this standard protocol, and two commercial kits. In the case of younger specimens (fixed in 1951, 1934, or 1914), extracts with DNA concentrations ≥ 10.0 ng/μl were obtained with the standard CTAB protocol, two variants of the standard protocol including prolonged tissue digestion (72 h instead of 1-2h), and a commercial kit particularly recommended for DNA extraction from formalin fixed paraffin embedded tissues (FFPE Kit). With the FFPE Kit, DNA could also be extracted from older tissues (fixed in 1893, 1850/1851, or before 1820). In general, the purity of the DNA extracts, assessed from the ratio of the absorbance at 260 and 280 nm, was not very high. In spite of their rather low purity, the DNA extracts could, however, be used to amplify a 122-bp sequence and, in most cases, also a 171-bp sequence of the gene coding for human albumin by the polymerase chain reaction (PCR). PMID:23603300

  8. 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

  9. Comparison of six extraction techniques for isolation of DNA from filamentous fungi.

    PubMed

    van Burik, J A; Schreckhise, R W; White, T C; Bowden, R A; Myerson, D

    1998-10-01

    Filamentous fungi have a sturdy cell wall which is resistant to the usual DNA extraction procedures. We determined the DNA extraction procedure with the greatest yield of high quality fungal DNA and the least predilection for cross-contamination of equipment between specimens. Each of six extraction methods was performed using Aspergillus fumigatus hyphae. The six methods were: (1) glass bead pulverization with vortexing; (2) grinding with mortar and pestle followed by glass bead pulverization; (3) glass bead pulverization using 1% hydroxyacetyl trimethyl ammonium bromide (CTAB) buffer in a water bath sonicator; (4) water bath sonication in CTAB buffer; (5) grinding followed by incubation with CTAB; and (6) lyticase enzymatic cell lysis. Genomic DNA yields were measured by spectrophotometry and by visual reading of 2% agarose gels, with shearing assessed by the migration of the DNA on the gel. Genomic fungal DNA yields were highest for Method 1, followed by Methods 5 approximately = to 2 >3 approximately = to 4 approximately = to 6. Methods 2 and 5, both of which involved grinding with mortar and pestle, led to shearing of the genomic DNA in one of two trials each. We conclude that the use of glass beads with extended vortexing is optimal for extraction of microgramme amounts of DNA from filamentous fungal cultures. PMID:10075499

  10. 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

  11. Enzymatic activities involved in the DNA resynthesis step of nucleotide excision repair are firmly attached to chromatin.

    PubMed

    Bouayadi, K; van der Leer-van Hoffen, A; Balajee, A S; Natarajan, A T; van Zeeland, A A; Mullenders, L H

    1997-03-01

    In this study the role of nuclear architecture in nucleotide excision repair (NER) was investigated by gentle dismantling of the cell and probing the capability of chromatin to carry out repair in vitro. The rationale behind this approach is that compartmentalization of NER at nuclear structures would make the enzymatic activities refractory to extraction by buffers that solubilize cellular membranes. In order to obtain intact chromatin primary human fibroblasts were encapsulated in agarose microbeads and lysed in isotonic buffers containing the non-ionic detergent Triton X-100. Under these conditions the majority of cellular proteins diffuse out of the beads, but the remaining chromatin is able to replicate and to transcribe DNA in the presence of triphosphates and Mg2+. UV irradiation of confluent repair-proficient human fibroblasts prior to lysis stimulated the incorporation of deoxynucleotide triphosphates in Triton X-100-isolated chromatin, even under stringent lysis conditions. In addition, experiments with UV-sensitive xeroderma pigmentosum (complementation groups A and C) and Cockayne's syndrome fibroblasts (complementation group A) revealed that this repair synthesis was due to global genome repair activity. Transcription-coupled repair was only detectable in cells permeabilized by streptolysin O (SLO). Repair synthesis in Triton X-100-isolated chromatin amounted to 15% of the total repair synthesis as measured in SLO-permeabilized cells. To allow the detection of these activities in vitro, presynthesis complexes have to be formed in intact cells, indicating that chromatin from Triton X-100-lysed cells is unable to initiate NER in vitro. Our data indicate that the components involved in the resynthesis step of NER are tightly associated with chromatin. A substantial fraction of total proliferating cell nuclear antigen (PCNA), which is required for the resynthesis step in NER, has been reported to become Triton X-100 non-extractable and tightly associated with nuclear structures after UV irradiation of cells. We propose that Triton X-100-resistant repair synthesis might be mediated by this chromatin-bound fraction of total PCNA. PMID:9023118

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. Initiation of strand incision at G:T and O6-methylguanine:T base mismatches in DNA by human cell extracts

    PubMed Central

    Lari, Sibghat-Ullah; Day, Rufus S.; Dobler, Kelly; Paterson, Malcolm C.

    2001-01-01

    Extracts of the human glioma cell line A1235 (lacking O6-methylguanine-DNA methyltransferase) are known to restore a G:T mismatch to a normal G:C pair in a G:T-containing model (45 bp) DNA substrate. Herein we demonstrate that substitution of G:T with O6-methylguanine:T (m6G:T) results in extract-induced intra-strand incision in the DNA at an efficiency comparable to that of complete repair of the G:T-containing substrate, although the m6G:T mispair serves as a poor substrate for later repair steps (e.g. gap filling, as judged by defective DNA repair synthesis). The A1235 extract, when supplemented with ATP and the four normal dNTPs, incises 5′ to the mismatched T, as inferred by the generation of a single-stranded 20mer fragment. Unlike its parental (A1235) counterpart, an extract of the alkylation-tolerant derivative cell line A1235-MR4 produces no 20mer fragment, even when thymine-DNA glycosylase (TDG) is added to the reaction mixture. In contrast, the A1235 extract, when augmented with TDG, catalyzes enhanced incision at m6G:T in the 45 bp DNA, yielding 5–10-fold greater 20mer than that of either extract or TDG alone. Interestingly, the absence of m6G:T incision activity in the A1235-MR4 extract is similar to that seen for extracts of several known mismatch repair-deficient cell lines of colon tumor origin. Together these results suggest that derivative A1235-MR4 cells are defective in m6G:T incision activity and that the efficiency of this activity in the parental (A1235) cells may depend on the presence of several ill-defined mismatch repair recognition proteins along with TDG and ATP. PMID:11376160

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

    PubMed

    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

  20. 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

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

    PubMed

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

    2014-02-01

    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

  2. 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

  3. 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

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

    PubMed

    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

  5. 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

  6. 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

  7. Extraction of DNA from amniotic fluid cells for the early prenatal diagnosis of genetic disease.

    PubMed

    Rebello, M T; Hackett, G; Smith, J; Loeffler, F E; Robson, S; MacLachlan, N; Beard, R W; Rodeck, C H; Williamson, R; Coleman, D V

    1991-01-01

    Ten-ml samples of amniotic fluid were taken from pregnancies being terminated at 8-14 weeks' gestation. DNA was extracted from the amniotic cells by sequential centrifugation and analysed using the polymerase chain reaction (PCR). Fifteen samples were analysed for evidence of maternal contamination using Mfd5 oligonucleotide primers for repeat polymorphisms. Ten amniotic fluid samples were tested for the Delta-F508 deletion characteristic of cystic fibrosis to demonstrate a diagnostic application for the technique. In each case, DNA extracted from fetal tissue from the same pregnancy was included in the controls. In 14 of the 15 cases tested with the Mfd5 primers, both the amniotic fluid DNA and the fetal DNA showed no evidence of contaminating DNA. In one case, neither the amniotic fluid cells nor the fetal cells yielded results. In nine of the ten cases tested with the Delta-F508 primers, the amniotic fluid cell DNA provided accurate information about the genetic status of the fetus; in the tenth, the fetal DNA failed to amplify. The results indicate that adequate DNA can be extracted from amniotic fluid from 8 weeks' gestation onward and these samples are suitable for prenatal diagnosis using PCR. PMID:2027853

  8. Automated DNA extraction from genetically modified maize using aminosilane-modified bacterial magnetic particles.

    PubMed

    Ota, Hiroyuki; Lim, Tae-Kyu; Tanaka, Tsuyoshi; Yoshino, Tomoko; Harada, Manabu; Matsunaga, Tadashi

    2006-09-18

    A novel, automated system, PNE-1080, equipped with eight automated pestle units and a spectrophotometer was developed for genomic DNA extraction from maize using aminosilane-modified bacterial magnetic particles (BMPs). The use of aminosilane-modified BMPs allowed highly accurate DNA recovery. The (A(260)-A(320)):(A(280)-A(320)) ratio of the extracted DNA was 1.9+/-0.1. The DNA quality was sufficiently pure for PCR analysis. The PNE-1080 offered rapid assay completion (30 min) with high accuracy. Furthermore, the results of real-time PCR confirmed that our proposed method permitted the accurate determination of genetically modified DNA composition and correlated well with results obtained by conventional cetyltrimethylammonium bromide (CTAB)-based methods. PMID:16621089

  9. Dichlorvos exposure impedes extraction and amplification of DNA from insects in museum collections

    PubMed Central

    2010-01-01

    Background The insecticides dichlorvos, paradichlorobenzene and naphthalene have been commonly used to eradicate pest insects from natural history collections. However, it is not known how these chemicals affect the DNA of the specimens in the collections. We thus tested the effect of dichlorvos, paradichlorobenzene and naphthalene on DNA of insects (Musca domestica) by extracting and amplifying DNA from specimens exposed to insecticides in two different concentrations over increasing time intervals. Results The results clearly show that dichlorvos impedes both extraction and amplification of mitochondrial and nuclear DNA after relatively short time, whereas paradichlorobenzene and naphthalene do not. Conclusion Collections treated with paradichlorobenzene and naphthalene, are better preserved concerning DNA, than those treated with dichlorvos. Non toxic pest control methods should, however, be preferred due to physical damage of specimens and putative health risks by chemicals. PMID:20148102

  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. A comparison between direct PCR and extraction to generate DNA profiles from samples retrieved from various substrates.

    PubMed

    Swaran, Yuvaneswari Chandramoulee; Welch, Lindsey

    2012-05-01

    Direct PCR generates DNA profiles from samples without using the extraction process. During sample extraction, DNA may be lost due to the methods used, which can affect the quality of the DNA profile obtained. This is not the case with direct PCR, where the sample is transferred directly into the PCR tube. Here, we report on the ability of direct PCR to generate DNA profiles from low amounts of control DNA retrieved from various surfaces using PowerPlex 16 HS. A comparison is made with samples undergoing a preliminary extraction stage using QiaAmp DNA Micro kits. Samples subjected to direct PCR generated DNA profiles with higher peak heights and lower allele dropout on all the different substrates tested when compared to the samples subjected to extraction. The amount of DNA retrieved from each substrate also varied even though the same amount of starting material was deposited, proving that the type of substrate can affect the retrieval of DNA. PMID:21925992

  12. Inhibitory effect of monogalactosyldiacylglycerol, extracted from spinach using supercritical CO2, on mammalian DNA polymerase activity.

    PubMed

    Iijima, Hiroshi; Musumi, Keiichi; Hada, Takahiko; Maeda, Naoki; Yonezawa, Yuko; Yoshida, Hiromi; Mizushina, Yoshiyuki

    2006-03-01

    We investigated the effective extraction of monogalactosyldiacylglycerol (MGDG) from dried spinach (Spinacia oleracea) using supercritical fluid carbon dioxide (SC-CO(2)) with a modifier/entrainer. The yield of MGDG in the SC-CO(2) extract was not influenced by increasing temperature at a constant pressure, although the total extract yield was decreased. The total extract yield and MGDG yield in the extract from commercially purchased spinach (unknown subspecies), were greatly influenced by lower pressure. In a modifier (i.e., ethanol) concentration range of 2.5-20%, both the extract and MGDG yield increased as the ethanol concentration rose. The highest total extract yield (69.5 mg/g of spinach) and a good MGDG yield (16.3 mg/g of spinach) were obtained at 80 degrees C, 25 MPa, and 20% ethanol. The highest MGDG concentration (76.0% in the extract) was obtained at 80 degrees C, 25 MPa, and 2.5% ethanol, although the total extract yield under these conditions was low (5.2 mg/g of spinach). The optimal conditions for the extraction of MGDG were 80 degrees C, 20 MPa, and 10% ethanol. Of the 11 subspecies of spinach tested under these conditions, "Ujyou" had the highest concentration of MGDG. The total extract yield and MGDG concentration of Ujyou were 20.4 mg of the extract/g of spinach and 70.5%, respectively. The concentration of MGDG was higher in the SC-CO(2) extract than in the extract obtained using solvents such as methanol and n-hexane. The extract of Ujyou, which was the optimal subspecies for the extraction of MGDG, inhibited the activity of calf DNA polymerase alpha with IC(50) values of 145 microg/mL but was not effective against DNA polymerase beta. PMID:16506811

  13. 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 are at least twice as many ionic protein-DNA contacts in the fully wrapped complex than in the weakly bent intermediate. The following picture emerges from this analysis: in the bimolecular step, the observed [KCl]-dependence is consistent with the number of DNA counterions expected to be released when IHF binds nonspecifically to DNA whereas in the unimolecular reorganization step, the weak [KCl]-dependence suggests that two effects cancel one another. On one hand, formation of additional protein-DNA contacts in the fully wrapped complex releases bound counterions into bulk solution, which is entropically favored by decreasing [salt]. On the other hand, formation of the fully wrapped complex also releases tightly bound water molecules, which is osmotically favored by increasing [salt]. More generally, our global analysis strategy is applicable to other protein-DNA complexes, and opens up the possibility of measuring DNA bending rates in complexes where the unimolecular and bimolecular steps are not easily separable.

  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. Lysis efficiency of standard DNA extraction methods for Halococcus spp. in an organic rich environment.

    PubMed

    Leuko, S; Goh, F; Ibáñez-Peral, R; Burns, B P; Walter, M R; Neilan, B A

    2008-03-01

    The extraction of nucleic acids from a given environment marks a crucial and essential starting point in any molecular investigation. Members of Halococcus spp. are known for their rigid cell walls, and are thus difficult to lyse and could potentially be overlooked in an environment. Furthermore, the lack of a suitable lysis method hinders subsequent molecular analysis. The effects of six different DNA extraction methods were tested on Halococcus hamelinensis, Halococcus saccharolyticus and Halobacterium salinarum NRC-1 as well as on an organic rich, highly carbonated sediment from stromatolites spiked with Halococcus hamelinensis. The methods tested were based on physical disruption (boiling and freeze/thawing), chemical lysis (Triton X-100, potassium ethyl xanthogenate (XS) buffer and CTAB) and on enzymatic lysis (lysozyme). Results showed that boiling and freeze/thawing had little effect on the lysis of both Halococcus strains. Methods based on chemical lysis (Triton X-100, XS-buffer, and CTAB) showed the best results, however, Triton X-100 treatment failed to produce visible DNA fragments. Using a combination of bead beating, chemical lysis with lysozyme, and thermal shock, lysis of cells was achieved however DNA was badly sheared. Lysis of cells and DNA extraction of samples from spiked sediment proved to be difficult, with the XS-buffer method indicating the best results. This study provides an evaluation of six commonly used methods of cell lysis and DNA extraction of Halococcus spp., and the suitability of the resulting DNA for molecular analysis. PMID:18087671

  16. 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

  17. 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

  18. Imported Malaria in United Arab Emirates: Evaluation of a New DNA Extraction Technique Using Nested PCR

    PubMed Central

    Khalil, Marwa M.; Abdouh, Ahmed S.; Doleh, Wafaa F.; Al Muthanna, Abdul Aziz M.

    2009-01-01

    Local malaria transmission in the United Arab Emirates (UAE) came to an end in 1997. Nevertheless, UAE has been subjected to substantial importation of malaria cases from abroad, concerning both UAE nationals and immigrants from malarious countries with a total number of 2,119 cases in 2007. To evaluate a new DNA extraction technique using nested PCR, blood samples were collected from 132 individuals who presented to Infectious Diseases Department in Rashid Hospital, Dubai, and Central Department of Malaria Control with fever and persistent headache. Giemsa-stained blood films and ELISA test for malaria antibodies were carried out for detection of Plasmodium infection. Plasmodium infections were identified with the genus-specific primer set and species differentiation using nested PCR. A rapid procedure for diagnosis of malaria infections directly from dried blood spots using for the first time DNA extract from FTA Elute cards was evaluated in contrast to extraction techniques using FTA classic cards and rapid boiling technique. Our new simple technique for DNA extraction using FTA Elute cards was very sensitive giving a sensitivity of 100% compared to 94% using FTA classic cards and 62% in the rapid boiling technique. No complex preparation of blood samples was required prior to the amplification. The production cost of DNA isolation in our PCR assay was much less in comparable to that of other DNA extraction protocols. The nested PCR detected plasmodial infection and could differentiate P. falciparum from P. vivax, and also detected the mixed infection. PMID:19724695

  19. 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

  20. 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

  1. Development and validation of one-step ultrasound-assisted extraction for simultaneous determination of multiclass fungicides in soils.

    PubMed

    Merdassa, Yared; Liu, Jing-Fu; Megersa, Negussie

    2015-01-01

    A rapid, efficient, and simple one-step ultrasound-assisted extraction (UAE) method was developed for the analysis of seven fungicides (cymoxanil, metalaxyl, mandipropamid, folpet, chlorothalonil, kresoxim-methyl, and famoxadone) in horticultural soils. Analytes in the samples were determined by HPLC with variable wavelength detection. Key parameters that influence the UAE procedure were optimized, such as the nature and volume of extraction solvent, number of sonication steps, and sonication time. The highest extraction efficiencies in the range of 61.1-87.8% were obtained by using only 7.5 mL of ethyl acetate-hexane (1+1, v/v) and sonicating for 10 min. At 0.5 and 2.0 μg/g fortification levels, satisfactory recoveries (>60%) with RSD<13% were obtained for each analyte, except for folpet (>52%). The method was linear over the range of 0.005 to 10 μg/g and the correlation coefficients (r2) obtained ranged from 0.9955 to 0.9992. The LODs (S/N=3) varied from 0.0015 to 0.006 μg/g. The proposed UAE procedure was compared to classical extractions (shake-flask and Soxhlet extraction) and showed satisfactory extraction efficiencies using shorter time and smaller amounts of organic solvents, thereby minimizing the costs of the analysis and the disposal of waste solvent. PMID:25857897

  2. 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

  3. 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.

  4. Protection from radiation-induced mitochondrial and genomic DNA damage by an extract of Hippophae rhamnoides.

    PubMed

    Shukla, Sandeep Kumar; Chaudhary, Pankaj; Kumar, Indracanti Prem; Samanta, Namita; Afrin, Farhat; Gupta, Manju Lata; Sharma, Upendra Kumar; Sinha, Arun Kumar; Sharma, Yogendra Kumar; Sharma, Rakesh Kumar

    2006-12-01

    Hippophae rhamnoides or seabuckthorn is used extensively in Indian and Tibetan traditional medicine for the treatment of circulatory disorders, ischemic heart disease, hepatic injury, and neoplasia. In the present study, we have evaluated the radioprotective potential of REC-1001, a fraction isolated from the berries of H. rhamnoides. Chemical analysis of the extract indicated that REC-1001 was approximately 68% by weight polyphenols, and contained kaempferol, isorhamnetin, and quercetin. The effect of REC-1001 on modulating radiation-induced DNA damage was determined in murine thymocytes by measuring nonspecific nuclear DNA damage at the whole genome level using the alkaline halo assay and by measuring sequence/gene-specific DNA damage both in nuclear DNA (beta-globin gene) and in mitochondrial DNA using a quantitative polymerase chain reaction. Treatment with 10 Gy resulted in a significant amount of DNA damage in the halo assay and reductions in the amplification of both the beta-globin gene and mitochondrial DNA. REC-1001 dose-dependently reduced the amount of damage detected in each assay, with the maximum protective effects observed at the highest REC-1001 dose evaluated (250 micro g/ml). Studies measuring the nicking of naked plasmid DNA further established the radioprotective effect of REC-1001. To elucidate possible mechanisms of action, the antioxidant properties and the free-radical scavenging activities of REC-1001 were evaluated. REC-1001 dose-dependently scavenged radiation-induced hydroxyl radicals, chemically-generated superoxide anions, stabilized DPPH radicals, and reduced Fe(3+) to Fe(2+). The results of the study indicate that the REC-1001 extract of H. rhamnoides protects mitochondrial and genomic DNA from radiation-induced damage. The polyphenols/flavonoids present in the extract might be responsible for the free radical scavenging and DNA protection afforded by REC-1001. PMID:16948057

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

    PubMed

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

    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. PMID:25257543

  6. 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.

  7. 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

  8. 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

  9. DNA extract characterization process for microbial detection methods development and validation

    PubMed Central

    2012-01-01

    Background Quantitative polymerase chain reaction (qPCR) assays used in pathogen detection require rigorous methods development including characterizing DNA extraction products. A DNA extract characterization process is demonstrated using DNA extracted from five different cells types (two Gram-negatives: Escherichia coli, and Burkholderia thailandensis, spores and vegetative cells from the Gram-positive Bacillus cereus, and yeast Saccharomyces cerevisiae) with six different methods. Results DNA extract quantity (concentration and extraction efficiency) and quality (purity and intactness) varied by cell type and extraction method enabling the demonstration of different DNA characterization methods. DNA purity was measured using UV spectroscopy, where the A260/A280 and A260/A230 ratios are indicators of different contaminants. Reproducibility of UV spectroscopy measurements decreased for DNA concentrations less than 17.5 ng/μL. Forty-seven extracts had concentrations greater than 17.5 ng/μL, 25 had A260/A280 above 2.0, and 28 had A260/A230 ratios below 1.8 indicating RNA and polysaccharide contamination respectively. Based on a qPCR inhibition assay the contaminants did not inhibit PCR. Extract intactness was evaluated using microfluidic gel electrophoresis. Thirty-five samples had concentrations above the limit of quantification (LOQ, roughly 11 ng/ μL), 93.5% of the DNA was larger than 1kb and 1% was smaller than 300 bp. Extract concentrations ranged from 1502.2 ng/μL to below the LOQ when UV spectroscopy, fluorometry, and qPCR were used. LOQ for UV spectroscopic and fluorometric measurements were 3.5 ng/μL and 0.25 ng/μL respectively. The qPCR LOQ varied by cell type (5.72 × 10-3 ng/μL for E. coli, 2.66 × 10-3 ng/μL, for B. cereus, 3.78 × 10-3 ng/μL for B. thailandensis, and 7.67 × 10-4 ng/μL for S. cerevisiae). A number of samples were below the UV spectroscopy (n = 27), flurometry (n = 15), and qPCR (n = 3) LOQ. Conclusion The presented DNA extract characterization process provides measures of DNA quantity and quality applicable to microbial detection methods development and validation studies. Evaluating DNA quality and quantity results in a better understanding of process LOD and contributing factors to suboptimal assay performance. The samples used demonstrated the use of different DNA characterization methods presented but did not encompass the full range of DNA extract characteristics. PMID:23206278

  10. Mite species identification in the production of allergenic extracts for clinical use and in environmental samples by ribosomal DNA amplification.

    PubMed

    Beroiz, B; Couso-Ferrer, F; Ortego, F; Chamorro, M J; Arteaga, C; Lombardero, M; Castañera, P; Hernández-Crespo, P

    2014-09-01

    The identification of allergy-causing mites is conventionally based on morphological characters. However, molecular taxonomy using ribosomal DNA (rDNA) may be particularly useful in the analysis of mite cultures and purified mite fractions in the production of allergenic extracts. Full-length internal transcribed spacers (ITS1 and ITS2) were obtained from Dermatophagoides farinae, Dermatophagoides pteronyssinus, Dermatophagoides microceras and Euroglyphus maynei (Astigmata: Pyroglyphidae), Glycyphagus domesticus and Lepidoglyphus destructor (Astigmata: Glycyphagidae), Tyrophagus fanetzhangorum, Tyrophagus putrescentiae, Tyrophagus longior, Tyrophagus neiswanderi, Acarus farris and Acarus siro (Astigmata: Acaridae), and Blomia tropicalis (Astigmata: Echymopodidae), using mite-specific primers. Polymerase chain reaction (PCR) products were digested with HpaII and RsaI restriction enzymes in order to produce species-specific PCR restricted fragment length polymorphism (RFLP) profiles. A semi-nested re-amplification step was introduced before the RFLP in order to apply the method to environmental samples. Results demonstrate that rDNA sequences can be used for the unambiguous identification of mite species. The PCR-RFLP system allows the identification of species in purified mite fractions when the availability of intact adult mite bodies for morphological identification is limited. This reliable and straightforward PCR-RFLP system and the rDNA sequences obtained can be of use in the identification of allergy-causing mite species. PMID:24617319

  11. 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

  12. One-Step Formation of "Chain-Armor"-Stabilized DNA Nanostructures.

    PubMed

    Cassinelli, Valentina; Oberleitner, Birgit; Sobotta, Jessica; Nickels, Philipp; Grossi, Guido; Kempter, Susanne; Frischmuth, Thomas; Liedl, Tim; Manetto, Antonio

    2015-06-26

    DNA-based self-assembled nanostructures are widely used to position organic and inorganic objects with nanoscale precision. A particular promising application of DNA structures is their usage as programmable carrier systems for targeted drug delivery. To provide DNA-based templates that are robust against degradation at elevated temperatures, low ion concentrations, adverse pH conditions, and DNases, we built 6-helix DNA tile tubes consisting of 24 oligonucleotides carrying alkyne groups on their 3'-ends and azides on their 5'-ends. By a mild click reaction, the two ends of selected oligonucleotides were covalently connected to form rings and interlocked DNA single strands, so-called DNA catenanes. Strikingly, the structures stayed topologically intact in pure water and even after precipitation from EtOH. The structures even withstood a temperature of 95 °C when all of the 24 strands were chemically interlocked. PMID:25980669

  13. 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

  14. 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

  15. A simple and rapid method for extracting bacterial DNA from intestinal microflora for ERIC-PCR detection

    PubMed Central

    Yang, Jin-Long; Wang, Ming-Shu; Cheng, An-Chun; Pan, Kang-Cheng; Li, Chuan-Feng; Deng, Shu-Xuan

    2008-01-01

    AIM: To develop a simple and convenient method for extracting genomic DNA from intestinal microflora for enterobacterial repetitive intergenic consensus (ERIC)-PCR detection. METHODS: Five methods of extracting bacterial DNA, including Tris-EDTA buffer, chelex-100, ultrapure water, 2% sodium dodecyl sulfate and 10% Triton-100 with and without sonication, were compared with the commercial fecal DNA extraction kit method, which is considered as the gold standard for DNA extraction. The comparison was based on the yield and purity of DNA and the indexes of the structure and property of micro-organisms that were reflected by ERIC-PCR. RESULTS: The yield and purity of DNA obtained by the chelex method was similar to that obtained with the fecal DNA kit. The ERIC-PCR results obtained for the DNA extracted by the chelex method and those obtained for DNA extracted with the fecal DNA kit were basically the same. CONCLUSION: The chelex method is recommended for ERIC-PCR experiments in view of its simplicity and cost-effectiveness; and it is suitable for extracting total DNA from intestinal micro-organisms, particularly for handling a large number of samples. PMID:18473413

  16. Comparison of six simple methods for extracting ribosomal and mitochondrial DNA from Toxocara and Toxascaris nematodes.

    PubMed

    Mikaeili, F; Kia, E B; Sharbatkhori, M; Sharifdini, M; Jalalizand, N; Heidari, Z; Zarei, Z; Stensvold, C R; Mirhendi, H

    2013-06-01

    Six simple methods for extraction of ribosomal and mitochondrial DNA from Toxocara canis, Toxocara cati and Toxascaris leonina were compared by evaluating the presence, appearance and intensity of PCR products visualized on agarose gels and amplified from DNA extracted by each of the methods. For each species, two isolates were obtained from the intestines of their respective hosts: T. canis and T. leonina from dogs, and T. cati from cats. For all isolates, total DNA was extracted using six different methods, including grinding, boiling, crushing, beating, freeze-thawing and the use of a commercial kit. To evaluate the efficacy of each method, the internal transcribed spacer (ITS) region and the cytochrome c oxidase subunit 1 (cox1) gene were chosen as representative markers for ribosomal and mitochondrial DNA, respectively. Among the six DNA extraction methods, the beating method was the most cost effective for all three species, followed by the commercial kit. Both methods produced high intensity bands on agarose gels and were characterized by no or minimal smear formation, depending on gene target; however, beating was less expensive. We therefore recommend the beating method for studies where costs need to be kept at low levels. PMID:23499880

  17. Inhibition of oxidative DNA damage in vitro by extracts of brussels sprouts.

    PubMed

    Zhu, C; Poulsen, H E; Loft, S

    2000-08-01

    Cruciferous vegetables have cancer preventive effects which may be due to reduction of oxidative DNA damage. We investigated the effect of an aqueous extract of cooked Brussels sprouts on formation of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) in calf thymus DNA in vitro. Damage was induced by a Fenton reaction, UVC (254 nm), UVA (365 nm), sunlamp light, and methylene blue with visible light. The extract inhibited 8-oxodG formation in all systems except visible light with methylene blue. The IC50 values were 6-20 microg/ml corresponding to the extract of 5-20 g of Brussels sprouts distributed in a volume of 50 L. The protective effect in the Fenton reaction was unaffected by addition of EDTA. After HPLC separation fractions were identified with similar DNA protective effects. Sinigrin, a glucosinolate abundant in Brussels sprouts, co-eluted with the most effective fraction and had DNA protective effects. In comparison with other antioxidants the patterns of effect of the extract in the five damage systems were more similar to that of sodium azide than to those of dimethylsulfoxide and vitamin C. Constituents of Brussels sprouts can protect DNA by direct scavenging, e.g. hydroxyl radical and other oxidants, without prooxidant effects at concentrations potentially achievable by modest intake of the vegetable. PMID:10885626

  18. Multicenter comparative evaluation of five commercial methods for toxoplasma DNA extraction from amniotic fluid.

    PubMed

    Yera, H; Filisetti, D; Bastien, P; Ancelle, T; Thulliez, P; Delhaes, L

    2009-12-01

    Over the past few years, a number of new nucleic acid extraction methods and extraction platforms using chemistry combined with magnetic or silica particles have been developed, in combination with instruments to facilitate the extraction procedure. The objective of the present study was to investigate the suitability of these automated methods for the isolation of Toxoplasma gondii DNA from amniotic fluid (AF). Therefore, three automated procedures were compared to two commercialized manual extraction methods. The MagNA Pure Compact (Roche), BioRobot EZ1 (Qiagen), and easyMAG (bioMérieux) automated procedures were compared to two manual DNA extraction kits, the QIAamp DNA minikit (Qiagen) and the High Pure PCR template preparation kit (Roche). Evaluation was carried out with two specific Toxoplasma PCRs (targeting the 529-bp repeat element), inhibitor search PCRs, and human beta-globin PCRs. The samples each consisted of 4 ml of AF with or without a calibrated Toxoplasma gondii RH strain suspension (0, 1, 2.5, 5, and 25 tachyzoites/ml). All PCR assays were laboratory-developed real-time PCR assays, using either TaqMan or fluorescent resonance energy transfer probes. A total of 1,178 PCRs were performed, including 978 Toxoplasma PCRs. The automated and manual methods were similar in sensitivity for DNA extraction from T. gondii at the highest concentration (25 Toxoplasma gondii cells/ml). However, our results showed that the DNA extraction procedures led to variable efficacy in isolating low concentrations of tachyzoites in AF samples (<5 Toxoplasma gondii cells/ml), a difference that might have repercussions since low parasite concentrations in AF exist and can lead to congenital toxoplasmosis. PMID:19846633

  19. First-passage problems in DNA replication: effects of template tension on stepping and exonuclease activities of a DNA polymerase motor

    NASA Astrophysics Data System (ADS)

    Sharma, Ajeet K.; Chowdhury, Debashish

    2013-09-01

    A DNA polymerase (DNAP) replicates a template DNA strand. It also exploits the template as the track for its own motor-like mechanical movement. In the polymerase mode it elongates the nascent DNA by one nucleotide in each step. However, whenever it commits an error by misincorporating an incorrect nucleotide, it can switch to an exonuclease mode. In the latter mode it excises the wrong nucleotide before switching back to its polymerase mode. We develop a stochastic kinetic model of DNA replication that mimics an in vitro experiment where single-stranded DNA, subjected to a mechanical tension F, is converted to double-stranded DNA by a single DNAP. The F-dependence of the average rate of replication, which depends on the rates of both polymerase and exonuclease activities of the DNAP, is in good qualitative agreement with the corresponding experimental results. We introduce nine novel distinct conditional dwell times of a DNAP. Using the method of first-passage times, we also derive the exact analytical expressions for the probability distributions of these conditional dwell times. The predicted F-dependences of these distributions are, in principle, accessible to single-molecule experiments.

  20. DNA extraction using modified bacterial magnetic particles in the presence of amino silane compound.

    PubMed

    Yoza, Brandon; Matsumoto, Mitsufumi; Matsunaga, Tadashi

    2002-04-11

    Magnetic particles produced by magnetic bacteria have been used to carry out magnetic separation of DNA. Separation was achieved using magnetite coated with 3-aminopropyltriethoxysilane, N-(trimethoxy-silylpropyl) isothiouronium chloride or 3-[2-(2-aminoethyl)-ethylamino]-propyltrimethoxysilane (AEEA). The DNA binding efficiency increased with the number of amino groups present on the silane compounds and was 14 fold higher than with untreated magnetite. Addition of AEEA to aqueous solutions containing coated magnetite increased efficiency due to co-condensation of DNA. From 10(8) Escherichia coli cells, 7.1 microg of DNA was recovered using 100 microg of magnetite. E. coli DNA extracted with modified bacterial magnetite was suitable for restriction enzyme digestion and polymerase chain reaction (PCR). Ten replicate samples of E. coli cells were extracted using an automated magnetic robot and 9.5 microg of DNA was extracted using 100 microg of modified bacterial magnetite and possessed a 1.94 absorbance ratio (260:280 nm). PMID:11861080

  1. 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

  2. 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

  3. 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.

  4. Separation of Y-chromosomal haplotypes from male DNA mixtures via multiplex haplotype-specific extraction.

    PubMed

    Rothe, Jessica; Nagy, Marion

    2015-11-01

    In forensic analysis, the interpretation of DNA mixtures is the subject of ongoing debate and requires expertise knowledge. Haplotype-specific extraction (HSE) is an alternative method that enables the separation of large chromosome fragments or haplotypes by using magnetic beads in conjunction with allele-specific probes. HSE thus allows physical separation of the components of a DNA mixture. Here, we present the first multiplex HSE separation of a Y-chromosomal haplotype consisting of six Yfiler short tandem repeat markers from a mixture of male DNA. PMID:26275613

  5. 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

  6. 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.

  7. Comparative Evaluation of DNA Extraction Methods from Feces of Multiple Host Species for Downstream Next-Generation Sequencing.

    PubMed

    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

  8. 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

  9. Rapid and simple DNA extraction protocol from goat rumen digesta for metagenomic analysis.

    PubMed

    Bashir, Yasir; Rather, Irfan A; Konwar, B K

    2015-11-01

    In contrast to the traditional culturing techniques and microscopy that have led to the identification and characterization of only about 15-20 % of the rumen microbes till date, nucleic acid-based molecular approaches are rapid, reproducible, and allow both the qualitative and quantitative assessment of microbial diversity. The aim of this study was to develop a simple, rapid and effective extraction protocol for the recovery of high-molecular-weight and cloneable metagenomic DNA (mDNA) from goat rumen contents. An efficient method was devised to isolate high-molecular-weight mDNA (&>23kb) that was pure and cloneable after isolation in a relatively short period (3.5 h). This is the first report wherein purification of isolated mDNA could be passed. The purity and cloneability of mDNA was found to be possible with the successful restriction digestion, 16S rDNA PCR amplification of the isolated mDNA and mDNA library construction.The screening of 1600 clones from the metagenomic library revealed one clone with adistinct hydrolytic activity on carboxymethyl cellulose (CMC) agar suggesting its endoglucanase activity. Agarose gel electrophoresis showed aDNA insert of ~1.5kb size on digestion with BamH1. The metagenomic clones offer a prodigious non-conventional means to explore the genetically untapped resources from nature. PMID:26687757

  10. A viral packaging motor varies its DNA rotation and step size to preserve subunit coordination as the capsid fills.

    PubMed

    Liu, Shixin; Chistol, Gheorghe; Hetherington, Craig L; Tafoya, Sara; Aathavan, K; Schnitzbauer, Joerg; Grimes, Shelley; Jardine, Paul J; Bustamante, Carlos

    2014-04-24

    Multimeric, ring-shaped molecular motors rely on the coordinated action of their subunits to perform crucial biological functions. During these tasks, motors often change their operation in response to regulatory signals. Here, we investigate a viral packaging machine as it fills the capsid with DNA and encounters increasing internal pressure. We find that the motor rotates the DNA during packaging and that the rotation per base pair increases with filling. This change accompanies a reduction in the motor's step size. We propose that these adjustments preserve motor coordination by allowing one subunit to make periodic, specific, and regulatory contacts with the DNA. At high filling, we also observe the downregulation of the ATP-binding rate and the emergence of long-lived pauses, suggesting a throttling-down mechanism employed by the motor near the completion of packaging. This study illustrates how a biological motor adjusts its operation in response to changing conditions, while remaining highly coordinated. PMID:24766813

  11. Low Cost Extraction and Isothermal Amplification of DNA for Infectious Diarrhea Diagnosis

    PubMed Central

    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

  12. Step-gate polysilicon nanowires field effect transistor compatible with CMOS technology for label-free DNA biosensor.

    PubMed

    Wenga, G; Jacques, E; Salaün, A-C; Rogel, R; Pichon, L; Geneste, F

    2013-02-15

    Currently, detection of DNA hybridization using fluorescence-based detection technique requires expensive optical systems and complex bioinformatics tools. Hence, the development of new low cost devices that enable direct and highly sensitive detection stimulates a lot of research efforts. Particularly, devices based on silicon nanowires are emerging as ultrasensitive electrical sensors for the direct detection of biological species thanks to their high surface to volume ratio. In this study, we propose innovative devices using step-gate polycrystalline silicon nanowire FET (poly-Si NW FETs), achieved with simple and low cost fabrication process, and used as ultrasensitive electronic sensor for DNA hybridization. The poly-SiNWs are synthesized using the sidewall spacer formation technique. The detailed fabrication procedure for a step-gate NWFET sensor is described in this paper. No-complementary and complementary DNA sequences were clearly discriminated and detection limit to 1 fM range is observed. This first result using this nano-device is promising for the development of low cost and ultrasensitive polysilicon nanowires based DNA sensors compatible with the CMOS technology. PMID:22841443

  13. Second-step transfer of bacteriophage T5 DNA: purification and characterization of the T5 gene A2 protein.

    PubMed Central

    Snyder, C E; Benzinger, R H

    1981-01-01

    Second-step transfer of bacteriophage T5 DNA requires the function of the T5 pre-early proteins A1 and A2. We have isolated and characterized the gene A2 protein as part of an effort to determine the mechanism of second-step transfer. The A2 protein was purified by DNA-cellulose column chromatography followed by gel filtration and ion-exchange column chromatography. The A2 protein's identity was confirmed by two-dimensional gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thin-layer gel filtration in 6 M guanidine hydrochloride demonstrated a molecular weight of 15,000 for the A2 polypeptide. Migration of the A2 protein through gel filtration columns under nondenaturing conditions, in combination with sedimentation behavior, indicated dimerization of the A2 polypeptide. The existence of the A2 dimer was confirmed by protein cross-linking with dimethyl suberimidate and analysis of the cross-linked proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition, degree of polymerization, DNA-binding ability, and physical characteristics of the T5 gene A2 protein are consistent with a function of the A2 protein in DNA transfer. Images PMID:7288924

  14. Dynamic solid phase DNA extraction and PCR amplification in polyester-toner based microchip.

    PubMed

    Duarte, Gabriela R M; Price, Carol W; Augustine, Brian H; Carrilho, Emanuel; Landers, James P

    2011-07-01

    A variety of substrates have been used for fabrication of microchips for DNA extraction, PCR amplification, and DNA fragment separation, including the more conventional glass and silicon as well as alternative polymer-based materials. Polyester represents one such polymer, and the laser-printing of toner onto polyester films has been shown to be effective for generating polyester-toner (PeT) microfluidic devices with channel depths on the order of tens of micrometers. Here, we describe a novel and simple process that allows for the production of multilayer, high aspect-ratio PeT microdevices with substantially larger channel depths. This innovative process utilizes a CO(2) laser to create the microchannel in polyester sheets containing a uniform layer of printed toner, and multilayer devices can easily be constructed by sandwiching the channel layer between uncoated cover sheets of polyester containing precut access holes. The process allows the fabrication of deep channels, with ~270 μm, and we demonstrate the effectiveness of multilayer PeT microchips for dynamic solid phase extraction (dSPE) and PCR amplification. With the former, we found that (i) more than 65% of DNA from 0.6 μL of blood was recovered, (ii) the resultant DNA was concentrated to greater than 3 ng/μL (which was better than other chip-based extraction methods), and (iii) the DNA recovered was compatible with downstream microchip-based PCR amplification. Illustrative of the compatibility of PeT microchips with the PCR process, the successful amplification of a 520 bp fragment of λ-phage DNA in a conventional thermocycler is shown. The ability to handle the diverse chemistries associated with DNA purification and extraction is a testimony to the potential utility of PeT microchips beyond separations and presents a promising new disposable platform for genetic analysis that is low cost and easy to fabricate. PMID:21557576

  15. MtDNA GeneExtractor: a computer tool for mtDNA gene/region information extraction.

    PubMed

    Freitas, Fernando; Oliveira, Sandra; Rocha, Ricardo; Pereira, Luísa

    2009-02-01

    The analysis of considerable numbers of DNA sequences is largely dependent on the development of simple software tools for automatically process the genetic data deposited on public databases. However, there are some difficulties in the automation process due to diverse synonyms being used as qualifiers for genes and some inconsistencies in gene locations between related Primate species, this fact happening even in the carefully curated database RefSeq. Here, we present mtDNA GeneEXtractor, a Windows based computer tool developed for the extraction of information for particular gene/regions from mammal mitochondrial DNA sequences deposited under GenBank format. The tool was quite efficient in retrieving organized information for comparative mtDNA gene/region diversity analyses when tested for the evaluation of transition/transversion ratios in humans and between Primates. Taking phylogenetic information into account to avoid redundancy due to ancestry-sharing, the transition/transversion ratios in the 13 protein-coding genes had a mean value of 12.46 for Primates (from 6.46 in ND2 to 17.04 in COX1) and higher (34.74) but more heterogeneous (ranging from 17.30 in ND5 to 74.39 in ND4) in a worldwide human database. The similar patterns of transition/transversion ratios in all positions and in only four fold degenerate positions show no evidence for selection in the 13 mtDNA protein-coding genes. PMID:19070686

  16. GENESUS: a two-step sequence design program for DNA nanostructure self-assembly.

    PubMed

    Tsutsumi, Takanobu; Asakawa, Takeshi; Kanegami, Akemi; Okada, Takao; Tahira, Tomoko; Hayashi, Kenshi

    2014-01-01

    DNA has been recognized as an ideal material for bottom-up construction of nanometer scale structures by self-assembly. The generation of sequences optimized for unique self-assembly (GENESUS) program reported here is a straightforward method for generating sets of strand sequences optimized for self-assembly of arbitrarily designed DNA nanostructures by a generate-candidates-and-choose-the-best strategy. A scalable procedure to prepare single-stranded DNA having arbitrary sequences is also presented. Strands for the assembly of various structures were designed and successfully constructed, validating both the program and the procedure. PMID:24724843

  17. 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

  18. 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

  19. Effects of Sanionia uncinata extracts in protecting against and inducing DNA cleavage by reactive oxygen species.

    PubMed

    Fernandes, Andréia da Silva; Mazzei, José Luiz; de Alencar, Alexandre Santos; Evangelista, Heitor; Felzenszwalb, Israel

    2011-01-01

    When mosses are exposed to increased quantities of ultraviolet (UV) radiation, they produce more secondary metabolites. Antarctica moss Sanionia uncinata (Hedw.) Loeske has presented high carotenoid contents in response to an increase in UVB radiation. This moss has been recommended as a potential source of antioxidants. In the present work, the protective and enhancing effects of aqueous (AE) and hydroalcoholic (HE) extracts of S. uncinata on the cleavage of supercoiled DNA were evaluated through topological modifications, quantified by densitometry after agarose gel electrophoresis. Total phenolic contents reached 5.89 mg/g. Our data demonstrated that the extract does not induce DNA cleavage. Furthermore, both extracts showed antioxidant activity that protected the DNA against cleavage induced by (i) O(2)(•-), 89% (AE) and 94% (HE) (P<0.05), and (ii) (.)OH, 17% (AE) and 18% (HE). However, the extracts intensified cleavage induced by Fenton-like reactions: (i) Cu(2+)/H(2)O(2), 94% (AE) and 100% (HE) (P<0.05), and (ii) SnCl(2), 62% (AE) and 56% (HE). DNA damages seem to follow different ways: (i) in the presence of Fenton-like reactions could be via reactive oxygen species generation and (ii) with HE/Cu(2+) could have also been triggered by other mechanisms. PMID:22005340

  20. 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...

  1. Comparison of commercial systems for extraction of nucleic acids from DNA/RNA respiratory pathogens.

    PubMed

    Yang, Genyan; Erdman, Dean E; Kodani, Maja; Kools, John; Bowen, Michael D; Fields, Barry S

    2011-01-01

    This study compared six automated nucleic acid extraction systems and one manual kit for their ability to recover nucleic acids from human nasal wash specimens spiked with five respiratory pathogens, representing Gram-positive bacteria (Streptococcus pyogenes), Gram-negative bacteria (Legionella pneumophila), DNA viruses (adenovirus), segmented RNA viruses (human influenza virus A), and non-segmented RNA viruses (respiratory syncytial virus). The robots and kit evaluated represent major commercially available methods that are capable of simultaneous extraction of DNA and RNA from respiratory specimens, and included platforms based on magnetic-bead technology (KingFisher mL, Biorobot EZ1, easyMAG, KingFisher Flex, and MagNA Pure Compact) or glass fiber filter technology (Biorobot MDX and the manual kit Allprep). All methods yielded extracts free of cross-contamination and RT-PCR inhibition. All automated systems recovered L. pneumophila and adenovirus DNA equivalently. However, the MagNA Pure protocol demonstrated more than 4-fold higher DNA recovery from the S. pyogenes than other methods. The KingFisher mL and easyMAG protocols provided 1- to 3-log wider linearity and extracted 3- to 4-fold more RNA from the human influenza virus and respiratory syncytial virus. These findings suggest that systems differed in nucleic acid recovery, reproducibility, and linearity in a pathogen specific manner. PMID:21034773

  2. Nucleotide-excision repair of DNA in cell-free extracts of the yeast Saccharomyces cerevisiae.

    PubMed Central

    Wang, Z; Wu, X; Friedberg, E C

    1993-01-01

    A wide spectrum of DNA lesions are repaired by the nucleotide-excision repair (NER) pathway in both eukaryotic and prokaryotic cells. We have developed a cell-free system in Saccharomyces cerevisiae that supports NER. NER was monitored by measuring repair synthesis in DNA treated with cisplatin or with UV radiation. Repair synthesis in vitro was defective in extracts of rad1, rad2, and rad10 mutant cells, all of which have mutations in genes whose products are known to be required for NER in vivo. Additionally, repair synthesis was complemented by mixing different mutant extracts, or by adding purified Rad1 or Rad10 protein to rad1 or rad10 mutant extracts, respectively. The latter observation demonstrates that the Rad1 and Rad10 proteins directly participate in the biochemical pathway of NER. NER supported by nuclear extracts requires ATP and Mg2+ and is stimulated by polyethylene glycol and by small amounts of whole cell extract containing overexpressed Rad2 protein. The nuclear extracts also contain base-excision repair activity that is present at wild-type levels in rad mutant extracts. This cell-free system is expected to facilitate studies on the biochemical pathway of NER in S. cerevisiae. Images Fig. 1 Fig. 2 Fig. 3 Fig. 5 PMID:8506335

  3. 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

  4. 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.

  5. 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

  6. 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 evaporation depending on the details of each applied approach. PMID:25535629

  7. 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

  8. 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...

  9. Rapid DNA extraction for specific detection and quantitation of Mycobacterium tuberculosis DNA in sputum specimens using Taqman assays.

    PubMed

    Gomez, Diana I; Mullin, Caroline S; Mora-Guzmán, Francisco; Crespo-Solis, J Gonzalo; Fisher-Hoch, Susan P; McCormick, Joseph B; Restrepo, Blanca I

    2011-12-01

    Rapid tuberculosis (TB) detection is critical for disease control, and further quantitation of Mycobacterium tuberculosis (Mtb) in sputum is valuable for epidemiological and clinical studies. We evaluated a simple, robust and cost-efficient in-house DNA extraction and downstream Taqman approach for detection and quantitation of Mtb genomes from sputum of newly-diagnosed TB patients and non-TB controls. DNA was extracted using guanidine isothiocyanate and silica-based spin columns in less than 2 h, stored frozen, and Taqman assays were used to detect Mtb with IS6110 and quantify it targeting RD1 and IS1081. The Taqmans had a sensitivity >95% in 108 culture-confirmed TB patients and specificity of 100% in 43 non-TB controls. Genome counts were correlated with the Mycobacterial Growth Indicator Tubes' (MGIT) time-to-detection values (1/TTD × 1000; rho = 0.66; p < 0.001) in 91 TB patients (33 excluded with MGIT contamination). This linear relationship was nearly identical between mycobacteria isolated from sputum and H37Rv Mtb grown in-vitro to its log phase. TB treatment between 3 and 7 days was associated with lower 1/TTD × 1000 values but not with genome counts. Together, our protocol provides rapid, specific, inexpensive and quantitative detection of Mtb DNA in fresh or stored sputa making it a robust tool for prompt TB diagnosis, and with potential use for clinical and epidemiologic studies. PMID:22088321

  10. Validation of a one-step extraction/methylation method for determination of fatty acids and cholesterol in marine tissues.

    PubMed

    Meier, Sonnich; Mjøs, Svein A; Joensen, Horaldur; Grahl-Nielsen, Otto

    2006-02-01

    A simple and fast direct extraction/methylation with methanolic hydrogen chloride was validated for determination of fatty acids (FA) in marine tissues. Three parameters: reaction time, temperature and presence of non-polar solvent, were studied by an experimental 2(3) full factorial design. The method was validated for five different types of samples; cod liver (high lipid content >60%, mainly triacylglycerol), cod muscle (low lipid content, approximately 1%, mainly phospholipids), cod plasma (lipid content, approximately 2%, mainly lipoprotein complex, high water amount), cod testis (lipid content approximately 3%, high levels of cholesterol), and herring muscle (lipid content approximately 7%). The one-step procedure for extraction/methylation of wet tissues was compared with the traditional procedure of extraction of the lipids by the Folch method (chloroform/methanol, 2:1, v/v), followed by methylation. The two methods gave similar FA profiles. The one-step extraction/methylation procedure gave a higher recovery of the total FA than the traditional procedure. Problems with carry-over peaks of cholesterol from previous samples were avoided by application of extra long GC temperature programs. The cholesterol decomposed to some degree under the preceding methanolysis step, giving several peaks in the chromatograms. The decomposition peaks were identified by mass spectrometry as cholestdienes originating from dehydration of cholesterol, a metylether of cholesterol and a cholesteryl chloride. These cholesterol artefacts can be used for quantitative determination of cholesterol in the samples. Standard samples of cholesterol were determined with high accuracy, (R(2)>0.99), and cholesterol in cod plasma was compared with good agreement (R(2)=0.97) to an enzymatic method. PMID:16343517

  11. Comparison of alkaline lysis with electroextraction and optimization of electric pulses to extract plasmid DNA from Escherichia coli.

    PubMed

    Haberl, Saša; Jarc, Marko; Strancar, Aleš; Peterka, Matjaž; Hodžić, Duša; Miklavčič, Damijan

    2013-11-01

    The use of plasmid DNA (pDNA) as a pharmaceutical tool has increased since it represents a safer vector for gene transfer compared to viral vectors. Different pDNA extraction methods have been described; among them is alkaline lysis, currently the most commonly used. Although alkaline lysis represents an established method for isolation of pDNA, some drawbacks are recognized, such as entrapment of pDNA in cell debris, leading to lower pDNA recovery; the time-consuming process; and increase of the volume due to the buffers used, all leading to increased cost of production. We compared the concentration of extracted pDNA when two methods for extracting pDNA from Escherichia coli were used: alkaline lysis and a method based on membrane electroporation, electroextraction. At the same time, we also studied the effect of different pulse protocols on bacterial inactivation. The concentration of pDNA was assayed with anion exchange chromatography. When alkaline lysis was used, two incubations of lysis time (5 and 10 min) were compared in terms of the amount of isolated pDNA. We did not observe any difference in pDNA concentration regardless of incubation time used. In electroextraction, different pulse protocols were used in order to exceed the pDNA concentration obtained by alkaline lysis. We show that electroextraction gives a higher concentration of extracted pDNA than alkaline lysis, suggesting the use of electroporation as a potentially superior method for extracting pDNA from E. coli. In addition, electroextraction represents a quicker alternative to alkaline lysis for extracting pDNA. PMID:23831957

  12. Rapid One-Step Selection Method for Generating Nucleic Acid Aptamers: Development of a DNA Aptamer against α-Bungarotoxin

    PubMed Central

    Lauridsen, Lasse H.; Shamaileh, Hadi A.; Edwards, Stacey L.; Taran, Elena; Veedu, Rakesh N.

    2012-01-01

    Background Nucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen®, an inhibitor of vascular endothelial growth factor (VEGF) for the treatment of age related macular degeneration (AMD). Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection, possibly in one-step, technique is required for developing aptamers in limited time period. Principal Findings Herein, we present a simple one-step selection of DNA aptamers against α-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing followed by PCR enrichment of the selected aptamers. One round of selection successfully identified a DNA aptamer sequence with a binding affinity of 7.58 µM. Conclusion We have demonstrated a one-step method for rapid production of nucleic acid aptamers. Although the reported binding affinity is in the low micromolar range, we believe that this could be further improved by using larger targets, increasing the stringency of selection and also by combining a capillary electrophoresis separation prior to the one-step selection. Furthermore, the method presented here is a user-friendly, cheap and an easy way of deriving an aptamer unlike the time consuming conventional SELEX-based approach. The most important application of this method is that chemically-modified nucleic acid libraries can also be used for aptamer selection as it requires only one enzymatic step. This method could equally be suitable for developing RNA aptamers. PMID:22860007

  13. D Object Coordinates Extraction by Radargrammetry and Multi Step Image Matching

    NASA Astrophysics Data System (ADS)

    Eftekhari, A.; Ghannadi, M. A.; Motagh, M.; Saadat Seresht, M.

    2013-09-01

    Nowadays by high resolution SAR imaging systems as Radarsat-2, TerraSAR-X and COSMO-skyMed, three-dimensional terrain data extraction using SAR images is growing. InSAR and Radargrammetry are two most common approaches for removing 3D object coordinate from SAR images. Research has shown that extraction of terrain elevation data using satellite repeat pass interferometry SAR technique due to atmospheric factors and the lack of coherence between the images in areas with dense vegetation cover is a problematic. So the use of Radargrammetry technique can be effective. Generally height derived method by Radargrammetry consists of two stages: Images matching and space intersection. In this paper we propose a multi-stage algorithm founded on the combination of feature based and area based image matching. Then the RPCs that calculate for each images use for extracting 3D coordinate in matched points. At the end, the coordinates calculating that compare with coordinates extracted from 1 meters DEM. The results show root mean square errors for 360 points are 3.09 meters. We use a pair of spotlight TerraSAR-X images from JAM (IRAN) in this article.

  14. D Object Coordinates Extraction by Radargrammetry and Multi Step Image Matching

    NASA Astrophysics Data System (ADS)

    Eftekhari, A.; Ghannadi, M. A.; Motagh, M.; Saadat Seresht, M.

    2014-11-01

    Nowadays by high resolution SAR imaging systems as Radarsat-2, TerraSAR-X and COSMO-skyMed, three-dimensional terrain data extraction using SAR images is growing. InSAR and Radargrammetry are two most common approaches for removing 3D object coordinate from SAR images. Research has shown that extraction of terrain elevation data using satellite repeat pass interferometry SAR technique due to atmospheric factors and the lack of coherence between the images in areas with dense vegetation cover is a problematic. So the use of Radargrammetry technique can be effective. Generally height derived method by Radargrammetry consists of two stages: Images matching and space intersection. In this paper we propose a multi-stage algorithm founded on the combination of feature based and area based image matching. Then the RPCs that calculate for each images use for extracting 3D coordinate in matched points. At the end, the coordinates calculating that compare with coordinates extracted from 1 meters DEM. The results show root mean square errors for 360 points are 3.09 meters. We use a pair of spotlight TerraSAR-X images from JAM (IRAN) in this article.

  15. DNA mismatch repair in Xenopus egg extracts: repair efficiency and DNA repair synthesis for all single base-pair mismatches.

    PubMed Central

    Varlet, I; Radman, M; Brooks, P

    1990-01-01

    Repair of all 12 single base-pair mismatches by Xenopus egg extracts was measured by a physical assay with a sequence containing four overlapping restriction sites. The heteroduplex substrates, derivatives of M13 phage DNA, differed in sequence at the mismatch position only and permitted measurement of repair to both strands. The efficiency of repair varied about 4-fold between the most and least effectively repaired mismatches. Repair was most active with C/A and T/C mismatches but the efficiency varied depending on the orientation of the mismatch. Mismatch-specific DNA repair synthesis was also observed but the extent of repair was not always predictive of the extent of synthesis, suggesting the presence of different repair systems or different modes of mismatch recognition. Images PMID:2236005

  16. Antioxidant Activity of Lawsonia inermis Extracts Inhibits Chromium(VI)-Induced Cellular and DNA Toxicity

    PubMed Central

    Guha, Gunjan; Rajkumar, V.; Kumar, R. Ashok; Mathew, Lazar

    2011-01-01

    Hexavalent chromium Cr(VI) is a very strong oxidant which consequently causes high cytotoxicity through oxidative stress. Prevention of Cr(VI)-induced cellular damage has been sought in this study in aqueous and methanolic extracts of Lawsonia inermis Linn. (Lythraceae), commonly known as Henna. The extracts showed significant (P < .05) potential in scavenging free radicals (DPPH• and ABTS•+) and Fe3+, and in inhibiting lipid peroxidation. DNA damage caused by exposure of pBR322 to Cr(VI)-UV is markedly inhibited by both extracts in varying degrees. A distinct decline in Cr(VI)-induced cytotoxicity was noticed in MDA-MB-435S (human breast carcinoma) cells with an increase in dosage of both extracts individually. Furthermore, both extracts proved to contain a high content of phenolic compounds which were found to have a strong and significant (P < .05) positive correlation to the radical scavenging potential, lipid peroxidation inhibition capacity and cyto-protective efficiency against Cr(VI)-induced oxidative cellular damage. HPLC analysis identified some of the major phenolic compounds in both extracts, which might be responsible for the antioxidant potential and the properties of DNA and cyto-protection. This study contributes to the search for natural resources that might yield potent therapeutic drugs against Cr(VI)-induced oxidative cell damage. PMID:20008460

  17. 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

  18. 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

  19. Successful use of saliva without DNA extraction for detection of macrolide-resistant Mycoplasma pneumoniae DNA in children using LNA probe-based real-time PCR.

    PubMed

    Komatsu, Haruki; Tsunoda, Tomoyuki; Inui, Ayano; Sogo, Tsuyoshi; Fujisawa, Tomoo; Imura, Motoki; Tateno, Akihiko

    2013-12-01

    It is not known that saliva is useful to diagnose Mycoplasma pneumoniae (M. pneumoniae) infection by PCR. We evaluated prospectively whether crude saliva samples without the DNA extraction process were useful for the detection of M. pneumoniae DNA in a locked nucleic acid (LNA) probe-based real-time PCR assay. Fifty-one clinical specimens (29 sputum, 22 saliva) that were positive by conventional M. pneumoniae-specific PCR were evaluated in this study. We designed an LNA probe-based real-time PCR that could discriminate the mutant strain (A2063G mutation) from the wild-type strain. All the 51 samples treated with DNA extraction were positive using the LNA probe-based real-time PCR. The results of the real-time PCR with DNA extraction were consistent with the sequence analysis. Of the 51 samples without DNA extraction, on the other hand, 41 (80.4%) were positive by real-time PCR. Of 29 sputum samples without DNA extraction, 23 (79.3%) were positive by real-time PCR; of the 22 saliva samples without DNA extraction, 18 (81.8%) were positive by real-time PCR. There was a statistically significant difference in the amplified DNA levels with extraction between the direct real-time PCR-positive samples (mean ± SD, 7.5 ± 1.6 log copies/ml) and PCR-negative samples (4.2 ± 0.8 log copies/ml) (P < 0.001). Saliva was useful for a template for PCR as well as sputum. In addition, crude samples were useful for real-time PCR when the samples had medium or high DNA levels. However, samples with low DNA levels sometimes showed false-negative results in direct real-time PCR. PMID:23771735

  20. Comparison of Eleven Methods for Genomic DNA Extraction Suitable for Large-Scale Whole-Genome Genotyping and Long-Term DNA Banking Using Blood Samples

    PubMed Central

    Psifidi, Androniki; Dovas, Chrysostomos I.; Bramis, Georgios; Lazou, Thomai; Russel, Claire L.; Arsenos, Georgios; Banos, Georgios

    2015-01-01

    Over the recent years, next generation sequencing and microarray technologies have revolutionized scientific research with their applications to high-throughput analysis of biological systems. Isolation of high quantities of pure, intact, double stranded, highly concentrated, not contaminated genomic DNA is prerequisite for successful and reliable large scale genotyping analysis. High quantities of pure DNA are also required for the creation of DNA-banks. In the present study, eleven different DNA extraction procedures, including phenol-chloroform, silica and magnetic beads based extractions, were examined to ascertain their relative effectiveness for extracting DNA from ovine blood samples. The quality and quantity of the differentially extracted DNA was subsequently assessed by spectrophotometric measurements, Qubit measurements, real-time PCR amplifications and gel electrophoresis. Processing time, intensity of labor and cost for each method were also evaluated. Results revealed significant differences among the eleven procedures and only four of the methods yielded satisfactory outputs. These four methods, comprising three modified silica based commercial kits (Modified Blood, Modified Tissue, Modified Dx kits) and an in-house developed magnetic beads based protocol, were most appropriate for extracting high quality and quantity DNA suitable for large-scale microarray genotyping and also for long-term DNA storage as demonstrated by their successful application to 600 individuals. PMID:25635817

  1. Comparison of eleven methods for genomic DNA extraction suitable for large-scale whole-genome genotyping and long-term DNA banking using blood samples.

    PubMed

    Psifidi, Androniki; Dovas, Chrysostomos I; Bramis, Georgios; Lazou, Thomai; Russel, Claire L; Arsenos, Georgios; Banos, Georgios

    2015-01-01

    Over the recent years, next generation sequencing and microarray technologies have revolutionized scientific research with their applications to high-throughput analysis of biological systems. Isolation of high quantities of pure, intact, double stranded, highly concentrated, not contaminated genomic DNA is prerequisite for successful and reliable large scale genotyping analysis. High quantities of pure DNA are also required for the creation of DNA-banks. In the present study, eleven different DNA extraction procedures, including phenol-chloroform, silica and magnetic beads based extractions, were examined to ascertain their relative effectiveness for extracting DNA from ovine blood samples. The quality and quantity of the differentially extracted DNA was subsequently assessed by spectrophotometric measurements, Qubit measurements, real-time PCR amplifications and gel electrophoresis. Processing time, intensity of labor and cost for each method were also evaluated. Results revealed significant differences among the eleven procedures and only four of the methods yielded satisfactory outputs. These four methods, comprising three modified silica based commercial kits (Modified Blood, Modified Tissue, Modified Dx kits) and an in-house developed magnetic beads based protocol, were most appropriate for extracting high quality and quantity DNA suitable for large-scale microarray genotyping and also for long-term DNA storage as demonstrated by their successful application to 600 individuals. PMID:25635817

  2. To beat or not to beat a tick: comparison of DNA extraction methods for ticks (Ixodes scapularis)

    PubMed Central

    Daniels, Thomas J.

    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

  3. 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

  4. 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

  5. Antioxidant Activity and Protection from DNA Damage by Water Extract from Pine (Pinus densiflora) Bark

    PubMed Central

    Jiang, Yunyao; Han, Woong; Shen, Ting; Wang, Myeong-Hyeon

    2012-01-01

    Water extract from Pinus densiflora (WPD) was investigated for its antioxidant activity and its ability to provide protection from DNA damage. A series of antioxidant assays, including a 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging assay, a reducing power assay, a metal-chelating assay, a superoxide radical scavenging assay, and a nitrite scavenging ability, as well as a DNA damage protection assay were performed. Total phenolic content was found to be 211.32 mg Tan/g WPD. The extract scavenged 50% DPPH free radical at a concentration of 21.35 μg/mL. At that same concentration, the reducing power ability of WPD was higher than that of α-tocopherol. The extract chelated 68.9% ferrous ion at the concentration of 4 mg/mL. WPD showed better nitrite scavenging effect at the lower pH. Meanwhile, WPD exhibited a strong capability for DNA damage protection at 1 mg/mL concentration. Taken together, these data suggest water extract from Pinus densiflora could be used as a suitable natural antioxidant. PMID:24471072

  6. A Two-Step Double Filter Method to Extract Open Water Surfaces from Landsat ETM+ Imagery

    NASA Astrophysics Data System (ADS)

    Wang, Haijing; Kinzelbach, Wolfgang

    2010-05-01

    In arid and semi-arid areas, lakes and temporal ponds play a significant role in agriculture and livelihood of local communities as well as in ecology. Monitoring the changes of these open water bodies allows to draw conclusions on water use as well as climatic impacts and can assist in the formulation of a sustainable resource management strategy. The simultaneous monitoring of larger numbers of water bodies with respect to their stage and area is feasible with the aid of remote sensing. Here the monitoring of lake surface areas is discussed. Landsat TM and ETM+ images provide a medium resolution of 30m, and offer an easily available data source to monitor the long term changes of water surfaces in arid and semi-arid regions. In the past great effort was put into developing simple indices to extract water surfaces from satellite images. However, there is a common problem in achieving accurate results with these indices: How to select a threshold value for water pixels without introducing excessive subjective judgment. The threshold value would also have to vary with location, land features and seasons, allowing for inherent uncertainty. A new method was developed using Landsat ETM+ imaginary (30 meter resolution) to extract open water surfaces. This method uses the Normalized Difference of Vegetation Index (NDVI) as the basis for an objective way of selecting threshold values of Modified Normalized Difference of Water Index (MNDWI) and Stress Degree Days (SDD), which were used as a combined filter to extract open water surfaces. We choose two study areas to verify the method. One study area is in Northeast China, where bigger lakes, smaller muddy ponds and wetlands are interspersed with agricultural land and salt crusts. The other one is Kafue Flats in Zambia, where seasonal floods of the Zambezi River create seasonal wetlands in addition to the more permanent water ponds and river channels. For both sites digital globe images of 0.5 meter resolution are available, which were taken within a few days of Landsat passing dates and which will serve here as ground truth information. On their basis the new method was compared to other available methods for extracting water pixels. Compared to the other methods, the new method can extract water surface not only from deep lakes/reservoirs and wetlands but also from small mud ponds in alkali flats and irrigation ponds in the fields. For the big and deep lakes, the extracted boundary of the lakes fits accurately the observed boundary. Five test sites in the study area in Northeast China with only shallow water surfaces were chosen and tested. The extracted water surfaces were compared with each site's digital globe maps, respectively to determine the accuracy of the method. The comparison shows that the method could extract all completely wet pixels (water area covering 100% of the pixel area) in all test sites. For partially wet pixels (50-100% of pixel area), the model can detect 91% of all pixels. No dry pixels were mistaken by the model as water pixels. Keywords: Remote sensing, Landsat ETM+ imaginary, Water Surface, NDVI, MNDWI, and SDD

  7. High-speed shaking of frozen blood clots for extraction of human and malaria parasite DNA

    PubMed Central

    2011-01-01

    Background Frozen blood clots remaining after serum collection is an often disregarded source of host and pathogen DNA due to troublesome handling and suboptimal outcome. Methods High-speed shaking of clot samples in a cell disruptor manufactured for homogenization of tissue and faecal specimens was evaluated for processing frozen blood clots for DNA extraction. The method was compared to two commercial clot protocols based on a chemical kit and centrifugation through a plastic sieve, followed by the same DNA extraction protocol. Blood clots with different levels of parasitaemia (1-1,000 p/μl) were prepared from parasite cultures to assess sensitivity of PCR detection. In addition, clots retrieved from serum samples collected within two epidemiological studies in Kenya (n = 630) were processed by high speed shaking and analysed by PCR for detection of malaria parasites and the human α-thalassaemia gene. Results High speed shaking succeeded in fully dispersing the clots and the method generated the highest DNA yield. The level of PCR detection of P. falciparum parasites and the human thalassaemia gene was the same as samples optimally collected with an anticoagulant. The commercial clot protocol and centrifugation through a sieve failed to fully dissolve the clots and resulted in lower sensitivity of PCR detection. Conclusions High speed shaking was a simple and efficacious method for homogenizing frozen blood clots before DNA purification and resulted in PCR templates of high quality both from humans and malaria parasites. This novel method enables genetic studies from stored blood clots. PMID:21824391

  8. 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

  9. Repair synthesis by human cell extracts in DNA damaged by cis- and trans-diamminedichloroplatinum(II).

    PubMed Central

    Hansson, J; Wood, R D

    1989-01-01

    DNA damage was induced in closed circular plasmid DNA by treatment with cis- or trans-diamminedichloroplatinum(II). These plasmids were used as substrates in reactions to give quantitative measurements of DNA repair synthesis mediated by cell free extracts from human lymphoid cell lines. Adducts induced by both drugs stimulated repair synthesis in a dose dependent manner by an ATP-requiring process. Measurements by an isopycnic gradient sedimentation method gave an upper limit for the average patch sizes in this in vitro system of around 140 nucleotides. It was estimated that up to 3% of the drug adducts induce the synthesis of a repair patch. The repair synthesis is due to repair of a small fraction of frequent drug adducts, rather than extensive repair of a rare subclass of lesions. Nonspecific DNA synthesis in undamaged plasmids, caused by exonucleolytic degradation and resynthesis, was reduced by repeated purification of intact circular forms. An extract made from cells belonging to xeroderma pigmentosum complementation group A was deficient in repair synthesis in response to the presence of cis- or trans-diamminedichloroplatinum(II) adducts in DNA. Images PMID:2554251

  10. 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

  11. Comparison of DNA Extraction Methods for Microbial Community Profiling with an Application to Pediatric Bronchoalveolar Lavage Samples

    PubMed Central

    Willner, Dana; Daly, Joshua; Whiley, David; Grimwood, Keith; Wainwright, Claire E.; Hugenholtz, Philip

    2012-01-01

    Barcoded amplicon sequencing is rapidly becoming a standard method for profiling microbial communities, including the human respiratory microbiome. While this approach has less bias than standard cultivation, several steps can introduce variation including the type of DNA extraction method used. Here we assessed five different extraction methods on pediatric bronchoalveolar lavage (BAL) samples and a mock community comprised of nine bacterial genera to determine method reproducibility and detection limits for these typically low complexity communities. Additionally, using the mock community, we were able to evaluate contamination and select a relative abundance cut-off threshold based on the geometric distribution that optimizes the trade off between detecting bona fide operational taxonomic units and filtering out spurious ones. Using this threshold, the majority of genera in the mock community were predictably detected by all extraction methods including the hard-to-lyse Gram-positive genus Staphylococcus. Differences between extraction methods were significantly greater than between technical replicates for both the mock community and BAL samples emphasizing the importance of using a standardized methodology for microbiome studies. However, regardless of method used, individual patients retained unique diagnostic profiles. Furthermore, despite being stored as raw frozen samples for over five years, community profiles from BAL samples were consistent with historical culturing results. The culture-independent profiling of these samples also identified a number of anaerobic genera that are gaining acceptance as being part of the respiratory microbiome. This study should help guide researchers to formulate sampling, extraction and analysis strategies for respiratory and other human microbiome samples. PMID:22514642

  12. 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

  13. Evaluation of adsorption and desorption steps in the solid-phase extraction of explosives using carbon/silica gel nanocomposites.

    PubMed

    Tomaszewski, Waldemar; Gun'ko, Vladimir M

    2015-07-01

    New series of carbon/silica gel nanocomposites, carbosils, prepared by the carbonization of starch bound to silica gel, and carbosils additionally silylated with octadecyldimethylchlorosilane were synthesized. These materials were applied as adsorbents in the solid-phase extraction of explosive nitrate esters and nitroaromatics from aqueous solutions. The adsorption and desorption steps were evaluated separately. It was found that both the molecular properties of explosives (dipole moments, orbital energies, solvation effects) and textural properties influenced by carbon deposits or octadecyl moieties have a large impact on the recovery rates. It was shown that the composites with moderate content of carbon deposits or with the highest amounts of carbon deposits and additionally silylated can be used as materials tailored for extraction of explosives from the aqueous solutions. PMID:25914305

  14. 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

  15. Simultaneous and selective preconcentration of trace Cu and Ag by one-step displacement cloud point extraction for FAAS determination.

    PubMed

    Gao, Ying; Wu, Peng; Li, Wei; Xuan, Yuelan; Hou, Xiandeng

    2010-04-15

    A simple, rapid and selective one-step displacement cloud point extraction (D-CPE) procedure was developed for the simultaneous preconcentration of trace Cu and Ag followed by flame atomic absorption spectrometric (FAAS) measurement. In this proposed procedure, Ni-DDTC complex instead of DDTC was used to achieve one-step extraction. Since the stability of Cu-DDTC and Ag-DDTC is higher than that of Ni-DDTC, simultaneous preconcentration of Cu and Ag can be achieved in a single step. Up to 15,000 mg L(-1) Co(2+), 10,000 mg L(-1) Zn(2+), 5000 mg L(-1) Cd(2+) and 5000 mg L(-1) Al(3+) did not cause significant interferences for the determination of 50 microg L(-1) Cu by FAAS, while 8000 mg L(-1) Zn(2+), 4000 mg L(-1) Co(2+), 4000 mg L(-1) Cd(2+) and 5000 mg L(-1) Al(3+) had no distinct effect on the determination of 80 microg L(-1) Ag. The limits of detection (LODs) of 0.5 microg L(-1) for Cu and 1.0 microg L(-1) for Ag, respectively, were obtained by FAAS detection. The accuracy of the proposed method is validated by analyzing certified reference materials (GBW 07405, GBW 07311 and GBW 07260) with satisfactory results. Furthermore, the one-step D-CPE was also applied for the preconcentration/separation of Cu and Ag in high-purity salts and artificial seawater, followed by FAAS or inductively coupled plasma-mass spectrometry (ICP-MS) determination. PMID:20188966

  16. 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

  17. 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

  18. 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

  19. 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.

  20. Effects of deletion and site-directed mutations on ligation steps of NAD+-dependent DNA ligase: a biochemical analysis of BRCA1 C-terminal domain.

    PubMed

    Feng, Hong; Parker, Jeremy M; Lu, Jing; Cao, Weiguo

    2004-10-01

    DNA strand joining entails three consecutive steps: enzyme adenylation to form AMP-ligase, substrate adenylation to form AMP-DNA, and nick closure. In this study, we investigate the effects on ligation steps by deletion and site-directed mutagenesis of the BRCA1 C-terminal (BRCT) domain using NAD(+)-dependent DNA ligase from Thermus species AK16D. Deletion of the BRCT domain resulted in substantial loss of ligation activity, but the mutant was still able to form an AMP-ligase intermediate, suggesting that the defects caused by deletion of the entire BRCT domain occur primarily at steps after enzyme adenylation. The lack of AMP-DNA accumulation by the domain deletion mutant as compared to the wild-type ligase indicates that the BRCT domain plays a role in the substrate adenylation step. Gel mobility shift analysis suggests that the BRCT domain and helix-hairpin-helix subdomain play a role in DNA binding. Similar to the BRCT domain deletion mutant, the G617I mutant showed a low ligation activity and lack of accumulation of AMP-DNA intermediate. However, the G617I mutant was only weakly adenylated, suggesting that a point mutation in the BRCT domain could also affect the enzyme adenylation step. The significant reduction of ligation activity by G634I appears to be attributable to a defect at the substrate adenylation step. The greater ligation of mismatched substrates by G638I is accountable by accelerated conversion of the AMP-DNA intermediate to a ligation product at the final nick closure step. The mutational effects of the BRCT domain on ligation steps in relation to protein-DNA and potential protein-protein interactions are discussed. PMID:15449954

  1. Development of an efficient fungal DNA extraction method to be used in random amplified polymorphic DNA-PCR analysis to differentiate cyclopiazonic acid mold producers.

    PubMed

    Sánchez, Beatriz; Rodríguez, Mar; Casado, Eva M; Martín, Alberto; Córdoba, Juan J

    2008-12-01

    A variety of previously established mechanical and chemical treatments to achieve fungal cell lysis combined with a semiautomatic system operated by a vacuum pump were tested to obtain DNA extract to be directly used in randomly amplified polymorphic DNA (RAPD)-PCR to differentiate cyclopiazonic acid-producing and -nonproducing mold strains. A DNA extraction method that includes digestion with proteinase K and lyticase prior to using a mortar and pestle grinding and a semiautomatic vacuum system yielded DNA of high quality in all the fungal strains and species tested, at concentrations ranging from 17 to 89 ng/microl in 150 microl of the final DNA extract. Two microliters of DNA extracted with this method was directly used for RAPD-PCR using primer (GACA)4. Reproducible RAPD fingerprints showing high differences between producer and nonproducer strains were observed. These differences in the RAPD patterns did not differentiate all the strains tested in clusters by cyclopiazonic acid production but may be very useful to distinguish cyclopiazonic acid producer strains from nonproducer strains by a simple RAPD analysis. Thus, the DNA extracts obtained could be used directly without previous purification and quantification for RAPD analysis to differentiate cyclopiazonic acid producer from nonproducer mold strains. This combined analysis could be adaptable to other toxigenic fungal species to enable differentiation of toxigenic and non-toxigenic molds, a procedure of great interest in food safety. PMID:19244904

  2. Development of an Ammonium Sulfate DNA Extraction Method for Obtaining Amplifiable DNA in a Small Number of Cells and Its Application to Clinical Specimens

    PubMed Central

    Oh, Seo Young; Kim, Wook Youn; Hwang, Tae Sook; Han, Hye Seung; Lim, So Dug; Kim, Wan Seop

    2013-01-01

    DNA extraction from microdissected cells has become essential for handling clinical specimens with advances in molecular pathology. Conventional methods have limitations for extracting amplifiable DNA from specimens containing a small number of cells. We developed an ammonium sulfate DNA extraction method (A) and compared it with two other methods (B and C). DNA quality and quantity, β-globin amplification, and detectability of two cancer associated gene mutations were evaluated. Method A showed the best DNA yield, particularly when the cell number was very low. Amplification of the β-globin gene using DNA from the SNU 790 cell line and papillary thyroid carcinoma (PTC) cells extracted with Method A demonstrated the strongest band. BRAFV600E mutation analysis using ethanol-fixed PTC cells from a patient demonstrated both a “T” peak increase and an adjacent “A” peak decrease when 25 and 50 cells were extracted, whereas mutant peaks were too low to be analyzed using the other two methods. EGFR mutation analysis using formalin-fixed paraffin-embedded lung cancer tissues demonstrated a mutant peak with Method A, whereas the mutant peak was undetectable with Methods B or C. Method A yielded the best DNA quantity and quality with outstanding efficiency, particularly when paucicellular specimens were used. PMID:23691506

  3. Fractionation study in bioleached metallurgy wastes using six-step sequential extraction.

    PubMed

    Krasnodebska-Ostrega, Beata; Pałdyna, Joanna; Kowalska, Joanna; Jedynak, Łukasz; Golimowski, Jerzy

    2009-08-15

    The stored metallurgy wastes contain residues from ore processing operations that are characterized by relatively high concentrations of heavy metals. The bioleaching process makes use of bacteria to recover elements from industrial wastes and to decrease potential risk of environmental contamination. Wastes were treated by solutions containing bacteria. In this work, the optimized six-stage sequential extraction procedure was applied for the fractionation of Ni, Cr, Fe, Mn, Cu and Zn in iron-nickel metallurgy wastes deposited in Southern Poland (Szklary). Fractionation and total concentrations of elements in wastes before and after various bioleaching treatments were studied. Analyses of the extracts were performed by ICP-MS and FAAS. To achieve the most effective bioleaching of Zn, Cr, Ni, Cu, Mn, Fe the usage of both autotrophic and heterotrophic bacteria in sequence, combined with flushing of the residue after bioleaching is required. 80-100% of total metal concentrations were mobilized after the proposed treatment. Wastes treated according to this procedure could be deposited without any risk of environmental contamination and additionally the metals could be recovered for industrial purposes. PMID:19171427

  4. Pentacyclic triterpenes in birch bark extract inhibit early step of herpes simplex virus type 1 replication.

    PubMed

    Heidary Navid, M; Laszczyk-Lauer, M N; Reichling, J; Schnitzler, P

    2014-09-25

    Antiviral agents frequently applied for treatment of herpesvirus infections include acyclovir and its derivatives. The antiviral effect of a triterpene extract of birch bark and its major pentacyclic triterpenes, i.e. betulin, lupeol and betulinic acid against acyclovir-sensitive and acyclovir-resistant HSV type 1 strains was examined. The cytotoxic effect of a phytochemically defined birch bark triterpene extract (TE) as well as different pentacyclic triterpenes was analyzed in cell culture, and revealed a moderate cytotoxicity on RC-37 cells. TE, betulin, lupeol and betulinic acid exhibited high levels of antiviral activity against HSV-1 in viral suspension tests with IC50 values ranging between 0.2 and 0.5 ?g/ml. Infectivity of acyclovir-sensitive and clinical isolates of acyclovir-resistant HSV-1 strains was significantly reduced by all tested compounds and a direct concentration- and time-dependent antiherpetic activity could be demonstrated. In order to determine the mode of antiviral action, TE and the compounds were added at different times during the viral infection cycle. Addition of these drugs to uninfected cells prior to infection or to herpesvirus-infected cells during intracellular replication had low effect on virus multiplication. Minor virucidal activity of triterpenes was observed, however both TE and tested compounds exhibited high anti-herpetic activity when viruses were pretreated with these drugs prior to infection. Pentacyclic triterpenes inhibit acyclovir-sensitive and acyclovir-resistant clinical isolates of HSV-1 in the early phase of infection. PMID:25172789

  5. 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

  6. 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

  7. 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...

  8. DNA extraction from arborescent monocots and how to deal with other challenging hosts.

    PubMed

    Harrison, Nigel A; Davis, Robert E; Helmick, Ericka E

    2013-01-01

    Detection of pathogen DNA by polymerase chain reaction (PCR) assays is the most widely used method for diagnosing phytoplasma diseases. Reliable and efficient detection of phytoplasmas, especially in woody perennial plants, is challenging due to the unusually low abundance and sporadic distribution of phytoplasmas within infected host tissues. Detection success depends largely upon the host species and sampling procedures and, to a lesser extent, on the protocol used for DNA extraction. Here we describe a simple, straightforward, nondestructive stem sampling protocol to confirm phytoplasma infection of palms and other arborescent monocots of large stature. The protocol requires minimal processing of excised tissues and yields phytoplasma DNA preparations in suitable quantity for reliable detection by nested PCR assays. PMID:22987413

  9. 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

  10. SLiCE: a novel bacterial cell extract-based DNA cloning method

    PubMed Central

    Zhang, Yongwei; Werling, Uwe; Edelmann, Winfried

    2012-01-01

    We describe a novel cloning method termed SLiCE (Seamless Ligation Cloning Extract) that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (≥15 bp) with or without flanking heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments from Bacteria Artificial Chromosomes (BACs) or other sources. SLiCE is highly cost effective as a number of standard laboratory bacterial strains can serve as sources for SLiCE extract. In addition, the cloning efficiencies and capabilities of these strains can be greatly improved by simple genetic modifications. As an example, we modified the DH10B Escherichia coli strain to express an optimized λ prophage Red recombination system. This strain, termed PPY, facilitates SLiCE with very high efficiencies and demonstrates the versatility of the method. PMID:22241772

  11. 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

  12. Cytotoxic and DNA interaction activities of extracts from medicinal plants used in Argentina.

    PubMed

    Mongelli, E; Pampuro, S; Coussio, J; Salomon, H; Ciccia, G

    2000-07-01

    Eight crude extracts from seven Argentine plants with cancer-related ethnobotanical uses have been subjected to a bioscreening study to detect cytotoxic activity. The plants studied were: Aristolochia triangularis, Baccharis grisebachii, Bolax gummifera, Eupatorium hecatanthum, Erythrina crista-galli, Pterocaulon polystachium and Salpichroa origanifolia. Crown gall tumour inhibition, DNA interaction and cytotoxicity towards KB cells were assayed using the potato disc, the DNA-methyl green (DNA-MG) and the KB cells cytotoxicity bioassays respectively. The results obtained indicate that A. triangularis (ED50=47 microg/ml), B. gummifera (ED50=32 microg/ml) and E. hecatanthum (ED50=35 microg/ml) contained cytotoxic compounds against KB cells. All of the plants studied inhibited the growth of crown gall tumours, showing correlation between the experimental data and the uses reported for these plants. Moreover, the results obtained for the extracts of E. hecatanthum and P. polystachium indicate the presence of compounds that interact with DNA (48 and 22% of absorbance decrease, respectively). The results obtained suggest that cytotoxicity could play an important role in the activities claimed for the plants under study. PMID:10904157

  13. Protocols for metagenomic DNA extraction and Illumina amplicon library preparation for faecal and swab samples.

    PubMed

    Vo, A-T E; Jedlicka, J A

    2014-11-01

    Next-generation sequencing (NGS) technology has extraordinarily enhanced the scope of research in the life sciences. To broaden the application of NGS to systems that were previously difficult to study, we present protocols for processing faecal and swab samples into amplicon libraries amenable to Illumina sequencing. We developed and tested a novel metagenomic DNA extraction approach using solid phase reversible immobilization (SPRI) beads on Western Bluebird (Sialia mexicana) samples stored in RNAlater. Compared with the MO BIO PowerSoil Kit, the current standard for the Human and Earth Microbiome Projects, the SPRI-based method produced comparable 16S rRNA gene PCR amplification from faecal extractions but significantly greater DNA quality, quantity and PCR success for both cloacal and oral swab samples. We furthermore modified published protocols for preparing highly multiplexed Illumina libraries with minimal sample loss and without post-adapter ligation amplification. Our library preparation protocol was successfully validated on three sets of heterogeneous amplicons (16S rRNA gene amplicons from SPRI and PowerSoil extractions as well as control arthropod COI gene amplicons) that were sequenced across three independent, 250-bp, paired-end runs on Illumina's MiSeq platform. Sequence analyses revealed largely equivalent results from the SPRI and PowerSoil extractions. Our comprehensive strategies focus on maximizing efficiency and minimizing costs. In addition to increasing the feasibility of using minimally invasive sampling and NGS capabilities in avian research, our methods are notably not avian-specific and thus applicable to many research programmes that involve DNA extraction and amplicon sequencing. PMID:24774752

  14. 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

  15. 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

  16. 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

  17. 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.

  18. Optimization of DNA extraction from seeds and leaf tissues of Chrysanthemum (Chrysanthemum indicum) for polymerase chain reaction

    PubMed Central

    Hasan, Saba; Prakash, Jyoti; Vashishtha, Abhinav; Sharma, Agnivesh; Srivastava, Kuldeep; Sagar, Faizuddin; Khan, Nausheen; Dwivedi, Keshav; Jain, Payal; Shukla, Saransh; Gupta, Swati Prakash; Mishra, Saumya

    2012-01-01

    Chrysanthemums constitute approximately 30 species of perennial flowering plants, belonging to the family Asteraceae, native to Asia and Northeastern Europe. Chrysanthemum is a natural cosmetic additive extracted from Chinese herb by modern biochemical technology. It has the properties of anti-bacterial, anti-viral, reducing (detoxification) and anti-inflammation. It possesses antioxidant characteristics, which could assist in minimizing free-radical induced damage. Therefore, it is widely used in skin and hair care products. Chemical composition of this herbal remedy includes kikkanols, sesquiterpenes, flavonoids, various essential oils containing camphor, cineole, sabinol, borneole and other elements that interfere with DNA, causing erroneous or no PCR products. In the present study, testing and modification of various standard protocols for isolation of high-quality DNA from leaf tissues and seeds of C. indicum was done. It was observed that the DNA obtained from seeds and leaf tissues with a modified cetyltrimethylammonium bromide buffer protocol was of good quality, with no colored pigments and contaminants. Also, DNA could be extracted from leaf tissues without using liquid nitrogen. Quality of DNA extracted from seeds was much better as compared to that extracted from leaf tissues. The extracted DNA was successfully amplified by PCR using arbitrary RAPD primers. The same protocol will probably be useful for extraction of high-molecular weight DNA from other plant materials containing large amounts of secondary metabolites and essential oils. PMID:22493524

  19. Optimization of Toxoplasma gondii DNA extraction from amniotic fluid using NucliSENS easyMAG and comparison with QIAamp DNA minikit.

    PubMed

    Faucher, B; Miermont, F; Ranque, S; Franck, J; Piarroux, R

    2012-06-01

    Antenatal diagnosis of congenital toxoplasmosis relies on PCR in amniotic fluid. Because parasitic load is often low, DNA extraction must be optimized. Manual methods remain widespread although automated methods appear more effective. This study aimed at optimizing an automated method and at comparing it with a widespread manual method: QIAamp DNA minikit. To optimize NucliSens easyMAG, we evaluated the addition of proteinase K pre-treatment and the increase of the amount of silica particles used for the extraction. The optimized method was then compared to QIAamp DNA minikit on samples containing less than 25 tachyzoites/ml. NucliSens easyMAG DNA yield was improved after proteinase K pre-treatment (p < 0.01), but not with a higher silica particle input. The optimized method yielded more positive PCRs than the manual method, especially for samples containing 5 tachyzoites/ml or less (71% vs 26%, p < 10(-4)). The DNA amount in samples found positive by PCR was higher after optimized automated extraction than after manual extraction (p < 10(-4)). Proteinase K pre-treatment should be added to extract DNA from amniotic fluid using NucliSens easyMAG. Using this optimized automated method rather than manual methods would improve the sensitivity of Toxoplasma PCR and simplify the daily workflow. PMID:21898160

  20. 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…

  1. 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

  2. Evaluation of Sample Stability and Automated DNA Extraction for Fetal Sex Determination Using Cell-Free Fetal DNA in Maternal Plasma

    PubMed Central

    Ordoñez, Elena; Rueda, Laura; Cañadas, M. Paz; Fuster, Carme; Cirigliano, Vincenzo

    2013-01-01

    Objective. The detection of paternally inherited sequences in maternal plasma, such as the SRY gene for fetal sexing or RHD for fetal blood group genotyping, is becoming part of daily routine in diagnostic laboratories. Due to the low percentage of fetal DNA, it is crucial to ensure sample stability and the efficiency of DNA extraction. We evaluated blood stability at 4°C for at least 24 hours and automated DNA extraction, for fetal sex determination in maternal plasma. Methods. A total of 158 blood samples were collected, using EDTA-K tubes, from women in their 1st trimester of pregnancy. Samples were kept at 4°C for at least 24 hours before processing. An automated DNA extraction was evaluated, and its efficiency was compared with a standard manual procedure. The SRY marker was used to quantify cfDNA by real-time PCR. Results. Although lower cfDNA amounts were obtained by automated DNA extraction (mean 107,35 GE/mL versus 259,43 GE/mL), the SRY sequence was successfully detected in all 108 samples from pregnancies with male fetuses. Conclusion. We successfully evaluated the suitability of standard blood tubes for the collection of maternal blood and assessed samples to be suitable for analysis at least 24 hours later. This would allow shipping to a central reference laboratory almost from anywhere in Europe. PMID:24222898

  3. 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

  4. 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...

  5. Validation of the Isotropic Fractionator: Comparison with Unbiased Stereology and DNA Extraction for Quantification of Glial Cells

    PubMed Central

    Bahney, Jami; von Bartheld, Christopher S.

    2014-01-01

    Background The “isotropic fractionator” (IF) is a novel cell counting technique that homogenizes fixed tissue, recovers cell nuclei in solution, and samples and quantifies nuclei by extrapolation. Studies using this technique indicate that the ratio of glia to neurons in the human brain is approximately 1:1 rather than the 10:1 or 50:1 ratio previously assumed. Although some results obtained with the IF have been similar to those obtained by stereology, the IF has never been calibrated or validated. It is conceivable that only a fraction of glial cell nuclei are recovered intact or recognized after the homogenization step. New Method To rule out this simple explanation for the claim of a 1:1 glia-neuron ratio, we compared cell numbers obtained from adjacent, weight-normalized samples of human and macaque monkey white matter using three techniques: the IF, unbiased stereology of histological sections in exhaustively sectioned samples, and cell numbers calculated from DNA extraction. Results and comparison of methods In primate forebrains, the IF yielded 73,000–90,000 nuclei/mg white matter, unbiased stereology yielded 75,000–92,000 nuclei/mg, with coefficients of error ranging from 0.013–0.063, while DNA extraction yielded only 4,000–23,000 nuclei/mg in fixed white matter tissues. Conclusions Since the IF revealed about 100% of the numbers produced by unbiased stereology, there is no significant underestimate of glial cells. This confirms the notion that the human brain overall contains glial cells and neurons with a ratio of about 1:1 far from the originally assumed ratio of 10:1 in favor of glial cells. PMID:24239779

  6. Determination of chloropropanols in foods by one-step extraction and derivatization using pressurized liquid extraction and gas chromatography-mass spectrometry.

    PubMed

    Racamonde, I; González, P; Lorenzo, R A; Carro, A M

    2011-09-28

    3-Chloropropane-1,2-diol (3-MCPD) and 1,3-dichloro-2-propanol (1,3-DCP) were determined for the first time in bakery foods using pressurized liquid extraction (PLE) combined with in situ derivatization and GC-MS analysis. This one-step protocol uses N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) as silylation reagent. Initially, screening experimental design was applied to evaluate the effects of the variables potentially affecting the extraction process, namely extraction time (min) and temperature (°C), number of cycles, dispersant reagent (diatomaceous earth in powder form and as particulate matter with high pore volume Extrelut NT) and percent of flush ethyl acetate volume (%). To reduce the time of analysis and improve the sensitivity, derivatization of the compounds was performed in the cell extraction. Conditions, such as the volume of BSTFA, temperature and time for the in situ derivatization of analytes using PLE, were optimized by a screening design followed to a Doehlert response surface design. The effect of the in-cell dispersants/adsorbents with diatomaceous earth, Florisil and sodium sulfate anhydrous was investigated using a Box-Behnken design. Using the final best conditions, 1 g of sample dispersed with 0.1 g of sodium sulfate anhydrous and 2.5 g diatomaceous earth was extracted with ethyl acetate. 1 g of Florisil, as clean-up adsorbent, and 70 μL of BSTFA were used for 3 min at 70°C. Under the optimum conditions, the calibration curves showed good linearity (R(2)>0.9994) and precision (relative standard deviation, RSD≤2.4%) within the tested ranges. The limits of quantification for 1,3-DCP and 3-MCDP, 1.6 and 1.7 μg kg(-1), respectively, are far below the established limits in the European and American legislations. The accuracy, precision, linearity, and limits of quantification provided make this analytical method suitable for routine control. The method was applied to the analysis of several toasted bread, snacks, cookies and cereal samples, none of which contained chloropropanols at concentrations above the legislation levels. PMID:21875707

  7. Detection of protein-DNA interactions in crude cellular extracts by fluorescence correlation spectroscopy.

    PubMed

    Kobayashi, Tamiyo; Okamoto, Naoaki; Sawasaki, Tatsuya; Endo, Yaeta

    2004-09-01

    Fluorescence correlation spectroscopy (FCS) is a methodology to examine directly the translational diffusion of individual fluorescence-labeled molecules in solutions. Recent studies using FCS have quantified various bimolecular reactions without any need for amplification. To evaluate further the applicability of FCS, we studied the specific binding between proteins and DNA in crude biological samples. Using an automated FCS system that was recently developed in our laboratories and is capable of distinguishing two or more molecular species in a multicomponent analysis, we detected the binding of two representative transcription factors, activator protein-1 (AP-1) and nuclear factor kappa B (NF-kappaB), in nuclear extracts of HeLa cells quantitatively with each sequence-specific DNA. The binding rates of these specific interactions were markedly augmented when cells were treated with tumor necrosis factor alpha which is known to activate both AP-1 and NF-kappaB. We also observed the pyrrolidine-dithiocarbamate-induced reciprocal regulation of these transcription factors. These results indicated that FCS is a useful tool for the analysis of complex interactions of transcription factors with DNA even in crude cellular extracts, suggesting that it is a powerful methodology for the study of a wide variety of molecular events under various experimental conditions. PMID:15301949

  8. Self assembly of functionalised graphene nanostructures by one step reduction of graphene oxide using aqueous extract of Artemisia vulgaris

    NASA Astrophysics Data System (ADS)

    Chettri, Prajwal; Vendamani, V. S.; Tripathi, Ajay; Pathak, Anand P.; Tiwari, Archana

    2016-01-01

    We report self assembly and organisation of graphene sheets to form a macroscopically ordered array of graphene layers using one step reduction of graphene oxide. Aqueous extract of dry leaves of Artemisia vulgaris is used for de-oxygenation of graphene oxide. The reduced graphene oxide samples are investigated for two different reflux times viz. for 6 h and 12 h. It is found that for an efficient reduction of graphene oxide and for the minimal damage to the crystal structure 6 h of reflux is the most favourable. Besides reducing graphene oxide, the phytomolecules also functionalise the graphene layers with electron withdrawing groups by virtue of which three dimensional nanostructures are formed on its surface.

  9. 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...

  10. 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)

  11. 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

  12. Automated and Manual Methods of DNA Extraction for Aspergillus fumigatus and Rhizopus oryzae Analyzed by Quantitative Real-Time PCR▿

    PubMed Central

    Francesconi, Andrea; Kasai, Miki; Harrington, Susan M.; Beveridge, Mara G.; Petraitiene, Ruta; Petraitis, Vidmantas; Schaufele, Robert L.; Walsh, Thomas J.

    2008-01-01

    Quantitative real-time PCR (qPCR) may improve the detection of fungal pathogens. Extraction of DNA from fungal pathogens is fundamental to optimization of qPCR; however, the loss of fungal DNA during the extraction process is a major limitation to molecular diagnostic tools for pathogenic fungi. We therefore studied representative automated and manual extraction methods for Aspergillus fumigatus and Rhizopus oryzae. Both were analyzed by qPCR for their ability to extract DNA from propagules and germinated hyphal elements (GHE). The limit of detection of A. fumigatus and R. oryzae GHE in bronchoalveolar lavage (BAL) fluid with either extraction method was 1 GHE/ml. Both methods efficiently extracted DNA from A. fumigatus, with a limit of detection of 1 × 102 conidia. Extraction of R. oryzae by the manual method resulted in a limit of detection of 1 × 103 sporangiospores. However, extraction with the automated method resulted in a limit of detection of 1 × 101 sporangiospores. The amount of time to process 24 samples by the automated method was 2.5 h prior to transferring for automation, 1.3 h of automation, and 10 min postautomation, resulting in a total time of 4 h. The total time required for the manual method was 5.25 h. The automated and manual methods were similar in sensitivity for DNA extraction from A. fumigatus conidia and GHE. For R. oryzae, the automated method was more sensitive for DNA extraction of sporangiospores, while the manual method was more sensitive for GHE in BAL fluid. PMID:18353931

  13. 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

  14. 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

  15. 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

  16. 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

  17. Rapid, one step staining procedures for analysis of cellular DNA and protein by single and dual laser flow cytometry

    SciTech Connect

    Crissman, H.A.; Steinkamp, J.A.

    1982-01-01

    Detailed, simplified techniques are described for simultaneous staining and analysis of DNA and protein in a number of mammalian cell types. Cell staining involves the addition of appropriate dye mixtures to unfixed or ethanol-fixed cells and subsequent analysis of cell populations in the staining reagents generally within 10 to 20 minutes. The approach is novel in that no centrifugation steps are involved during the staining procedure, thus, eliminating cell clumping and cell loss and making the procedures appropriate for samples containing limited numbers of cells. For single wavelength analysis, staining of DNA and protein in ethanol-fixed cells was accomplished with a dye solution containing propidium iodide, fluorescein isothimocyanate and RNase. After 20 minutes at room temperature cells were analyzed using the 488 nanometer (nm) laser excitation line. For dual laser analysis the following dye combinations were employed without RNase: mithramycin-rhodamine 640, mithramycin-substituted rhodamine isothiocyanate, Hoechst 33342-rhodamine 640 and Hoechst 33342-rhodamine isothiocyanate. Unfixed cells were also stained with the Hoechst 33342-rhodamine 640 dye combination. Mithramycin was excited at 457.9 nm, Hoechst 33342 at 333-363 nm, and rhodamine dyes at 568 nm. Cell types analyzed included Chinese hamster ovary cells, cultured mouse colon 26 cells, mouse embryo forelimb bud cells, and rat cell obtained by lung lavage.

  18. 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

  19. From soil to leaves--aluminum fractionation by single step extraction procedures in polluted and protected areas.

    PubMed

    Frankowski, Marcin; Zioła-Frankowska, Anetta; Siepak, Jerzy

    2013-09-30

    The paper presents the fractionation of aluminum in the samples of soil and plants of different species using a selective single-step extraction method. The study was conducted in the area located near a chemical plant, which for many years served as a post-crystallization leachate disposal site storing chemical waste (sector I), and in the area around the site: in Wielkopolski National Park, Rogalin Landscape Park and toward the infiltration ponds at the "Dębina" groundwater well-field for the city of Poznań (Poland) (sector II). The results of aluminum fractionation in samples of soil, leaves and plants showed heavy pollution with aluminum, especially in the water soluble aluminum fraction - Alsw (maximum concentration of aluminum in soil extract was 234.8 ± 4.8 mg kg(-1), in the leaves of Betula pendula it was 107.4 ± 1.8 mg kg(-1) and in the plants of Artemisia vulgaris (root) and Medicago sativa (leaves) it amounted to 464.7 ± 10.7 mg kg(-1)and 146.8 ± 1.2 mg kg(-1) respectively). In addition, the paper presents the problem of organic aluminum fractionation in biological samples and it shows the relationship between aluminum concentration in soil and the analysed woody and herbaceous species. PMID:23651943

  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. Ancient Microbes from Halite Fluid Inclusions: Optimized Surface Sterilization and DNA Extraction

    PubMed Central

    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

  2. Protective effect of extract of Crataegus pinnatifida pollen on DNA damage response to oxidative stress.

    PubMed

    Cheng, Ni; Wang, Yuan; Gao, Hui; Yuan, Jialing; Feng, Fan; Cao, Wei; Zheng, Jianbin

    2013-09-01

    The protective effect of extract of Crataegus pinnatifida (Rosaceae) pollen (ECPP) on the DNA damage response to oxidative stress was investigated and assessed with an alkaline single-cell gel electrophoresis (SCGE) assay and pBR322 plasmid DNA breaks in site-specific and non-site-specific systems. Total phenolic content, total flavonoid content, individual phenolic compounds, antioxidant activities (1,1-diphenyl-2-picrylhydrazyl (DPPH), radical scavenging activity, FRAP, and chelating activity) were also determined. The results showed that ECPP possessed a strong ability to protect DNA from being damaged by hydroxyl radicals in both the site-specific system and the non-site-specific system. It also exhibited a cytoprotection effect in mouse lymphocytes against H₂O₂-induced DNA damage. These protective effects may be related to its high total phenolic content (17.65±0.97 mg GAE/g), total flavonoid content (8.04±0.97 mg rutin/g), strong free radical scavenging activity and considerable ferrous ion chelating ability (14.48±0.21 mg Na₂EDTA/g). PMID:23871827

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. A microfluidic chip integrating DNA extraction and real-time PCR for the detection of bacteria in saliva

    PubMed Central

    Oblath, Emily A.; Henley, W. Hampton; Alarie, Jean Pierre

    2013-01-01

    A microfluidic chip integrating DNA extraction, amplification, and detection for the identification of bacteria in saliva is described. The chip design integrated a monolithic aluminum oxide membrane (AOM) for DNA extraction with seven parallel reaction wells for real-time polymerase chain reaction (rtPCR) amplification of the extracted DNA. Samples were first heated to lyse target organisms and then added to the chip and filtered through the nanoporous AOM to extract the DNA. PCR reagents were added to each of the wells and the chip was thermocycled. Identification of Streptococcus mutans in a saliva sample is demonstrated along with the detection of 300 fg (100–125 copies) of both methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) genomic DNA (gDNA) spiked into a saliva sample. Multiple target species and strains of bacteria can be simultaneously identified in the same sample by varying the primers and probes used in each of the seven reaction wells. In initial tests, as little as 30 fg (8–12 copies) of MSSA gDNA in buffer has been successfully amplified and detected with this device. PMID:23370016

  11. 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

  12. Rapid and reliable high-throughput methods of DNA extraction for use in barcoding and molecular systematics of mushrooms.

    PubMed

    Dentinger, Bryn T M; Margaritescu, Simona; Moncalvo, Jean-Marc

    2010-07-01

    We present two methods for DNA extraction from fresh and dried mushrooms that are adaptable to high-throughput sequencing initiatives, such as DNA barcoding. Our results show that these protocols yield ∼85% sequencing success from recently collected materials. Tests with both recent (<2 year) and older (>100 years) specimens reveal that older collections have low success rates and may be an inefficient resource for populating a barcode database. However, our method of extracting DNA from herbarium samples using small amount of tissue is reliable and could be used for important historical specimens. The application of these protocols greatly reduces time, and therefore cost, of generating DNA sequences from mushrooms and other fungi vs. traditional extraction methods. The efficiency of these methods illustrates that standardization and streamlining of sample processing should be shifted from the laboratory to the field. PMID:21565067

  13. 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 approach.

  14. 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

  15. Strand specificity of mutagenic bypass replication of DNA containing psoralen monoadducts in a human cell extract.

    PubMed Central

    Thomas, D C; Svoboda, D L; Vos, J M; Kunkel, T A

    1996-01-01

    Psoralens are mutagenic compounds of vegetable origin that are used as photosensitizing agents in the treatment of various skin diseases, blood cell cancer, and autoimmune disorders. To study the mechanism of mutagenicity of psoralens in humans, we examined the efficiency and fidelity of simian virus 40 origin-dependent replication in a human cell extract of M13mp2 DNA randomly treated with the psoralen derivative 4'-hydroxymethyl-4,5',8-trimethyl psoralen plus UVA irradiation. Replication of DNA treated with variable amounts of 4'-hydroxymethyl-4,5',8-trimethyl psoralen and a fixed UVA fluence was inhibited in a concentration-dependent manner. However, covalently closed monomer-length circular replication products were observed. Product analysis by renaturing agarose gel electrophoresis after cross-linking with 250- to 280-nm UV light indicated that approximately 1 of 9 psoralen monoadducts was bypassed during in vitro replication. Introduction of product DNA into Escherichia coli to score replication errors in the lacZalpha reporter gene demonstrated that replication of the damaged DNA was more mutagenic than was replication of undamaged DNA. Sequence analysis of lacZ mutants revealed that damage-dependent replication errors were predominantly T.A-->C.G transitions, transversions at C.G base pairs, and deletions of single A.T base pairs, the last occurring most frequently in homopolymeric runs. A comparison of error specificities with two substrates having the replication origin asymmetrically placed on opposite sides of the mutational target suggests that the lagging-strand replication apparatus is less accurate than the leading-strand replication apparatus for psoralen monoadduct-dependent deletion errors. A model is proposed based on the preferential loopout of the monoadducted base from the strand that templates retrograde discontinuous synthesis. PMID:8628322

  16. 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

  17. 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 microbial DNA extraction method with pretreatment of depletion of host nucleic acid by Benzonase can enable a higher yield of microbial DNA from samples with a higher fraction of host cells to be obtained. The lower airways of patients with IIP without airway infection were inhabited by a great number of bacterium. PMID:27186268

  18. Pathogen Quantitation in Complex Matrices: A Multi-Operator Comparison of DNA Extraction Methods with a Novel Assessment of PCR Inhibition

    PubMed Central

    Pontiroli, Alessandra; Travis, Emma Rachel; Sweeney, Francis Patrick; Porter, David; Gaze, William Hugo; Mason, Sam; Hibberd, Victoria; Holden, Jennifer; Courtenay, Orin; Wellington, Elizabeth Margaret Helen

    2011-01-01

    Background Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB), an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year. Methodology/Principal Findings We report a multi-operator blinded trial for a rigorous comparison of five DNA extraction methods from a variety of soil and faecal samples to assess recovery of M. bovis via real-time PCR detection. The methods included four commercial kits: the QIAamp Stool Mini kit with a pre-treatment step, the FastDNA Spin kit, the UltraClean and PowerSoil soil kits and a published manual method based on phenol:chloroform purification, termed Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and evaluated using a specific real-time PCR assay. A novel inhibition control assay was used alongside spectrophotometric ratios to monitor the level of inhibitory compounds affecting PCR, DNA yield, and purity. There were statistically significant differences in M. bovis detection between methods of extraction and types of environmental samples; no significant differences were observed between operators. Processing times and costs were also evaluated. To improve M. bovis detection further, the two best performing methods, FastDNA Spin kit and Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was adopted, leading to improved sensitivities. Conclusions M. bovis was successfully detected in all environmental samples; DNA extraction using FastDNA Spin kit was the most sensitive method with highest recoveries from all soil types tested. For troublesome faecal samples, we have used and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25105 cells g?1 using Griffiths and 4.25106 cells g?1 using FastDNA Spin kit. PMID:21448453

  19. Distribution of cytotoxic and DNA ADP-ribosylating activity in crude extracts from butterflies among the family Pieridae

    PubMed Central

    Matsumoto, Yasuko; Nakano, Tsuyoshi; Yamamoto, Masafumi; Matsushima-Hibiya, Yuko; Odagiri, Ken-Ichi; Yata, Osamu; Koyama, Kotaro; Sugimura, Takashi; Wakabayashi, Keiji

    2008-01-01

    Cabbage butterflies, Pieris rapae and Pieris brassicae, contain strong cytotoxic proteins, designated as pierisin-1 and -2, against cancer cell lines. These proteins exhibit DNA ADP-ribosylating activity. To determine the distribution of substances with cytotoxicity and DNA ADP-ribosylating activity among other species, crude extracts from 20 species of the family Pieridae were examined for cytotoxicity in HeLa cells and DNA ADP-ribosylating activity. Both activities were detected in extracts from 13 species: subtribes Pierina (Pieris rapae, Pieris canidia, Pieris napi, Pieris melete, Pieris brassicae, Pontia daplidice, and Talbotia naganum), Aporiina (Aporia gigantea, Aporia crataegi, Aporia hippia, and Delias pasithoe), and Appiadina (Appias nero and Appias paulina). All of these extracts contained substances recognized by anti-pierisin-1 antibodies, with a molecular mass of ≈100 kDa established earlier for pierisin-1. Moreover, sequences containing NAD-binding sites, conserved in ADP-ribosyltransferases, were amplified from genomic DNA from 13 species of butterflies with cytotoxicity and DNA ADP-ribosylating activity by PCR. Extracts from seven species, Appias lyncida, Leptosia nina, Anthocharis scolymus, Eurema hecabe, Catopsilia pomona, Catopsilia scylla, and Colias erate, showed neither cytotoxicity nor DNA ADP-ribosylating activity, and did not contain substances recognized by anti-pierisin-1 antibodies. Sequences containing NAD-binding sites were not amplified from genomic DNA from these seven species. Thus, pierisin-like proteins, showing cytotoxicity and DNA ADP-ribosylating activity, are suggested to be present in the extracts from butterflies not only among the subtribe Pierina, but also among the subtribes Aporiina and Appiadina. These findings offer insight to understanding the nature of DNA ADP-ribosylating activity in the butterfly. PMID:18256183

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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

  6. Enhanced fungal DNA-extraction from formalin-fixed, paraffin-embedded tissue specimens by application of thermal energy.

    PubMed

    Rickerts, Volker; Khot, Prasanna D; Ko, Daisy L; Fredricks, David N

    2012-08-01

    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 by PCR in pathology blocks and sequencing it is an alternative approach to determine the cause of IFI. Previous studies identified fungal DNA in only 50% of samples with positive histopathology results, probably due to DNA damage by 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 the incubation temperature from 65°C to 90°C and an additional increase was documented by incubating samples for up to 6 hours at this temperature. The augmented amplification of fungal DNA was associated with improved species identification by the sequencing of the PCR amplicons. This may help illuminate the etiology of IFI and thereby improve patient management by guiding antifungal therapy. PMID:22414380

  7. 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

  8. One-step column chromatographic extraction with gradient elution followed by automatic separation of volatiles, flavonoids and polysaccharides from Citrus grandis.

    PubMed

    Han, Han-Bing; Li, Hui; Hao, Rui-Lin; Chen, Ya-Fei; Ni, He; Li, Hai-Hang

    2014-02-15

    Citrus grandis Tomentosa is widely used in traditional Chinese medicine and health foods. Its functional components include volatiles, flavonoids and polysaccharides which cannot be effectively extracted through traditional methods. A column chromatographic extraction with gradient elution was developed for one-step extraction of all bioactive substances from C. grandis. Dried material was loaded into a column with petroleum ether: ethanol (8:2, PE) and sequentially eluted with 2-fold PE, 3-fold ethanol: water (6:4) and 8-fold water. The elutes was separated into an ether fraction containing volatiles and an ethanol-water fraction containing flavonoids and polysaccharides. The later was separated into flavonoids and polysaccharides by 80% ethanol precipitation of polysaccharides. Through this procedure, volatiles, flavonoids and polysaccharides in C. grandis were simultaneously extracted at 98% extraction rates and simply separated at higher than 95% recovery rates. The method provides a simple and high-efficient extraction and separation of wide range bioactive substances. PMID:24128512

  9. 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.

  10. 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.

  11. Multiplex PCR for the simultaneous identification and detection of Meloidogyne incognita, M. enterolobii, and M. javanica using DNA extracted directly from individual galls.

    PubMed

    Hu, M X; Zhuo, K; Liao, J L

    2011-11-01

    Meloidogyne incognita, M. enterolobii, and M. javanica are the most widespread species of root-knot nematodes in South China, affecting many economically important crops, ornamental plants, and fruit trees. In this study, one pair of Meloidogyne universal primers was designed and three pairs of species-specific primers were employed successfully to rapidly detect and identify M. incognita, M. enterolobii, and M. javanica by multiplex polymerase chain reaction (PCR) using DNA extracted from individual galls. Multiplex PCR from all M. incognita, M. enterolobii, and M. javanica isolates generated two fragments of ≈500 and 1,000, 500 and 200, and 500 and 700 bp, respectively. The 500-bp fragment is the internal positive control fragment of rDNA 28S D2/D3 resulting from the use of the universal primers. Other Meloidogyne spp. included in this study generated only one fragment of ≈500 bp in size. Using this approach, M. incognita, M. enterolobii, and M. javanica were identified and detected using DNA extracted directly from individual galls containing the Meloidogyne spp. at various stages of their life cycle. Moreover, the percentage of positive PCR amplification increased with nematode development and detection was usually easy after the late stage of the second-stage juvenile. The protocol was applied to galls from naturally infested roots and the results were found to be fast, sensitive, robust, and accurate. This present study is the first to provide a definitive diagnostic tool for M. incognita, M. enterolobii, and M. javanica using DNA extracted directly from individual galls using a one-step multiplex PCR technique. PMID:21770774

  12. 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 accuracy and automation. PMID:22947281

  13. Technical reproducibility of single-nucleotide and size-based DNA biomarker assessment using DNA extracted from formalin-fixed, paraffin-embedded tissues.

    PubMed

    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

    2015-05-01

    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

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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

  19. Detection of tomato yellow leaf curl Thailand virus by PCR without DNA extraction.

    PubMed

    Ieamkhang, Supaporn; Riangwong, Lumpueng; Chatchawankanphanich, Orawan

    2005-11-01

    We report the simple and rapid method for detection of tomato yellow leaf curl Thailand virus (TYLCTHV) based on the direct capture of virus particles to the surface of a polymerase chain reaction (PCR) tube. This method allowed PCR without the time-consuming procedures of DNA extraction from infected plant tissue. A small amount of tomato tissue (approximately 10 mg) was ground in extraction buffer to release viruses from plant tissues. The constituents of the plant extract that might inhibit PCR activity were discarded by washing the tube with PBST buffer before adding the PCR mixture to the tube. This method was used for detection of TYLCTHV with plant sap solution diluted up to 1:20,000 and was more sensitive than an enzyme-linked immunosorbent assay (ELISA) method. In addition, this method can be used for detection of TYLCTHV in viruliferous whiteflies. The PCR tubes with captured TYLCTHV could be used for PCR, after storage at 4 degrees C for 4 wk. The method presented here was used for detection of begomoviruses in cucurbit and pepper. In addition, this method was effectively used to detect papaya ringspot virus in papaya and zucchini yellow mosaic virus in cucumber by reverse transcriptase (RT)-PCR. PMID:16230773

  20. 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

  1. Comparative analysis of four methods to extract DNA from paraffin-embedded tissues: effect on downstream molecular applications

    PubMed Central

    2010-01-01

    Background A large portion of tissues stored worldwide for diagnostic purposes is formalin-fixed and paraffin-embedded (FFPE). These FFPE-archived tissues are an extremely valuable source for retrospective (genetic) studies. These include mutation screening in cancer-critical genes as well as pathogen detection. In this study we evaluated the impact of several widely used DNA extraction methods on the quality of molecular diagnostics on FFPE tissues. Findings We compared 4 DNA extraction methods from 4 identically processed FFPE mammary-, prostate-, colon- and lung tissues with regard to PCR inhibition, real time SNP detection and amplifiable fragment size. The extraction methods, with and without proteinase K pre-treatment, tested were: 1) heat-treatment, 2) QIAamp DNA-blood-mini-kit, 3) EasyMAG NucliSens and 4) Gentra Capture-Column-kit. Amplifiable DNA fragment size was assessed by multiplexed 200-400-600 bp PCR and appeared highly influenced by the extraction method used. Proteinase K pre-treatment was a prerequisite for proper purification of DNA from FFPE. Extractions with QIAamp, EasyMAG and heat-treatment were found suitable for amplification of fragments up to 400 bp from all tissues, 600 bp amplification was marginally successful (best was QIAamp). QIAamp and EasyMAG extracts were found suitable for downstream real time SNP detection. Gentra extraction was unsuitable. Hands-on time was lowest for heat-treatment, followed by EasyMAG. Conclusions We conclude that the extraction method plays an important role with regard to performance in downstream molecular applications. PMID:20840759

  2. Determination of O6-butylguanine in DNA by immunoaffinity extraction/gas chromatography-mass spectrometry

    SciTech Connect

    Bonfanti, M.; Magagnotti, C.; Galli, A.; Bagnati, R.; Moret, M.; Gariboldi, P.; Fanelli, R.; Airoldi, L. )

    1990-11-01

    A sensitive, specific, and rapid method for quantitating the minor adduct O6-butylguanine (O6BuG) in hydrolyzed DNA has been developed by combining immunoaffinity chromatography and high resolution gas chromatography-negative ion chemical ionization-mass spectrometry. Polyclonal antibodies raised against O6BuG were coupled to CNBr-activated Sepharose 4B and used for sample clean-up and extraction of the specific O6-alkylguanine. After addition of O6BuG and its deuterium labeled analogue (O6BuG-D7), used as internal standard, hydrolyzed DNA was applied on the immunoaffinity column and washed with water, and the immunoadsorbed butylated guanines were eluted with acetone/water cetome/water (95/5) before gas chromatographic derivatization. O6BuG and O6BuG-D7 were analyzed and quantitated by high resolution gas chromatography-negative ion chemical ionization-mass spectrometry as their pentafluorobenzyl-trimethylsilyl derivatives. Immunoaffinity column capacity and O6BuG recovery from this column were 1.53 nmol O6BuG/column and 62 +/- 5%, respectively. The method was applied to evaluate O6BuG levels in DNA butylated in vitro with 10 mM N-nitroso-Nr-butylurea or isolated from rats given an i.p. dose of 185 mg/kg N-nitroso-N-butylurea or N-nitrosodibutylamine. In the first case the level of modifications present in calf thymus DNA was 104 mumol O6BuG/mol guanine, and in the second case O6BuG in liver DNA was about 6 times higher after N-nitroso-N-butylurea (2.11 mumol O6BuG/mol guanine) than after N-nitrosodibutylamine (0.34 mumol O6BuG/mol guanine) treatment. These results indicate that O6BuG formed in vivo can be isolated and quantitated by this method, which may also be useful for studying DNA damage and repair mechanisms.

  3. 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

  4. One-step immunoaffinity purification of human beta 1 thyroid hormone receptor with DNA and hormone binding activity.

    PubMed

    Park, J B; Ashizawa, K; Parkison, C; Cheng, S Y

    1993-09-01

    An efficient and versatile method to purify large amounts of active human beta 1 thyroid hormone receptor (h-TR beta 1) was developed. Using a T7 expression system, h-TR beta 1 was overexpressed in Escherichia coli. Approx. 80% of the expressed receptor protein was concentrated in the insoluble inclusion bodies and approximately 20% was in the soluble form (h-TR beta 1-S). h-TR beta 1-S was conveniently purified by one immunoaffinity chromatographic step. From 1 l of cell culture, approx. 0.1 mg of purified h-TR beta 1-S was obtained. The purified h-TR beta 1-S binds to 3,3',5-triiodo-L-thyronine with a Ka = 2 x 10(9) M-1 and exhibits analog specificity. The purified h-TR beta 1-S also binds to T3 response elements (TRE) with different orientation in the half-sites with differential activity. In addition, binding of h-TR beta 1-S to TREs was enhanced by retinoid X receptor. These results indicate that the purified h-TR beta 1-S retains its hormone and DNA binding activity. The purified h-TR beta 1-S is suitable for structural and functional studies. This method could be used to purify h-TR beta 1 or rat TR beta 1 expressed in insect cells or yeast. PMID:8227948

  5. Cyclin B-Cdk1 Kinase Stimulates ORC- and Cdc6-Independent Steps of Semiconservative Plasmid Replication in Yeast Nuclear Extracts

    PubMed Central

    Duncker, Bernard P.; Pasero, Philippe; Braguglia, Diego; Heun, Patrick; Weinreich, Michael; Gasser, Susan M.

    1999-01-01

    Nuclear extracts from Saccharomyces cerevisiae cells synchronized in S phase support the semiconservative replication of supercoiled plasmids in vitro. We examined the dependence of this reaction on the prereplicative complex that assembles at yeast origins and on S-phase kinases that trigger initiation in vivo. We found that replication in nuclear extracts initiates independently of the origin recognition complex (ORC), Cdc6p, and an autonomously replicating sequence (ARS) consensus. Nonetheless, quantitative density gradient analysis showed that S- and M-phase nuclear extracts consistently promote semiconservative DNA replication more efficiently than G1-phase extracts. The observed semiconservative replication is compromised in S-phase nuclear extracts deficient for the Cdk1 kinase (Cdc28p) but not in extracts deficient for the Cdc7p kinase. In a cdc4-1 G1-phase extract, which accumulates high levels of the specific Clb-Cdk1 inhibitor p40SIC1, very low levels of semiconservative DNA replication were detected. Recombinant Clb5-Cdc28 restores replication in a cdc28-4 S-phase extract yet fails to do so in the cdc4-1 G1-phase extract. In contrast, the addition of recombinant Xenopus CycB-Cdc2, which is not sensitive to inhibition by p40SIC1, restores efficient replication to both extracts. Our results suggest that in addition to its well-characterized role in regulating the origin-specific prereplication complex, the Clb-Cdk1 complex modulates the efficiency of the replication machinery itself. PMID:9891057

  6. Antioxidant activity of herbaceous plant extracts protect against hydrogen peroxide-induced DNA damage in human lymphocytes

    PubMed Central

    2013-01-01

    Background Herbaceous plants containing antioxidants can protect against DNA damage. The purpose of this study was to evaluate the antioxidant substances, antioxidant activity, and protection of DNA from oxidative damage in human lymphocytes induced by hydrogen peroxide (H2O2). Our methods used acidic methanol and water extractions from six herbaceous plants, including Bidens alba (BA), Lycium chinense (LC), Mentha arvensis (MA), Plantago asiatica (PA), Houttuynia cordata (HC), and Centella asiatica (CA). Methods Antioxidant compounds such as flavonol and polyphenol were analyzed. Antioxidant activity was determined by the inhibition percentage of conjugated diene formation in a linoleic acid emulsion system and by trolox-equivalent antioxidant capacity (TEAC) assay. Their antioxidative capacities for protecting human lymphocyte DNA from H2O2-induced strand breaks was evaluated by comet assay. Results The studied plants were found to be rich in flavonols, especially myricetin in BA, morin in MA, quercetin in HC, and kaemperol in CA. In addition, polyphenol abounded in BA and CA. The best conjugated diene formation inhibition percentage was found in the acidic methanolic extract of PA. Regarding TEAC, the best antioxidant activity was generated from the acidic methanolic extract of HC. Water and acidic methanolic extracts of MA and HC both had better inhibition percentages of tail DNA% and tail moment as compared to the rest of the tested extracts, and significantly suppressed oxidative damage to lymphocyte DNA. Conclusion Quercetin and morin are important for preventing peroxidation and oxidative damage to DNA, and the leaves of MA and HC extracts may have excellent potential as functional ingredients representing potential sources of natural antioxidants. PMID:24279749

  7. Double-stranded DNA induces the phosphorylation of several proteins including the 90 000 mol. wt. heat-shock protein in animal cell extracts.

    PubMed Central

    Walker, A I; Hunt, T; Jackson, R J; Anderson, C W

    1985-01-01

    Double-stranded DNA (dsDNA) induces the transfer of phosphate from ATP to several proteins in extracts of widely divergent eukaryotic cells. Extracts of HeLa cells, rabbit reticulocytes, Xenopus eggs and Arbacia eggs all show dsDNA-dependent protein phosphorylation. The mechanism is specific for dsDNA and will not respond to either RNA or single-stranded DNA. One of the proteins which is phosphorylated in response to dsDNA has a subunit mol. wt. of 90 000 and has been identified as a heat-shock protein (hsp90). Although mouse cell extracts were shown to contain hsp90, they failed to show a dsDNA-dependent protein phosphorylation. The observation that dsDNA can modulate the phosphorylation of a set of proteins raises the possibility that dsDNA may play a role as a cellular regulatory signal. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:4018025

  8. 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.

  9. High Abundance of Ammonia-Oxidizing Archaea in Coastal Waters, Determined Using a Modified DNA Extraction Method▿ †

    PubMed Central

    Urakawa, Hidetoshi; Martens-Habbena, Willm; Stahl, David A.

    2010-01-01

    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

  10. 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

  11. 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 criterion by which to evaluate the quality and performance on different matrixes and extraction techniques. The effect of PCR efficiency on the resulting GMO content is demonstrated. Conclusion The crucial influence of extraction technique and sample matrix properties on the results of GMO quantification is demonstrated. Appropriate extraction techniques for each matrix need to be determined to achieve accurate DNA quantification. Nevertheless, as it is shown that in the area of food and feed testing matrix with certain specificities is impossible to define strict quality controls need to be introduced to monitor PCR. The results of our study are also applicable to other fields of quantitative testing by real-time PCR. PMID:16907967

  12. 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

  13. Aqueous extract of Crataegus azarolus protects against DNA damage in human lymphoblast Cell K562 and enhances antioxidant activity.

    PubMed

    Mustapha, Nadia; Bouhlel, Inès; Chaabane, Fadwa; Bzéouich, Imèn Mokdad; Ghedira, Kamel; Hennebelle, Thierry; Chekir-Ghedira, Leila

    2014-02-01

    The present study was carried out to characterize the cellular antioxidant effect of the aqueous extract of Crataegus azarolus and its antigenotoxic potential using human myelogenous cells, K562. The antioxidant capacity of this extract was evaluated by determining its cellular antioxidant activity (CAA) in K562 cells. Also, preceding antigenotoxicity assessment, its eventual genotoxicity property was investigated by evaluating its capacity to induce the DNA degradation of treated cell nuclei. As no genotoxicity was detected at different exposure times, its ability to protect cell DNA against H2O2 oxidative effect was investigated, using the "comet assay." It appears that 800 μg/mL of extract inhibited the genotoxicity induced by H2O2 with a rate of 41.30 %, after 4 h of incubation. In addition, this extract revealed a significant cellular antioxidant capacity against the reactive oxygen species in K562 cells. PMID:24347159

  14. Ion-Channel Genosensor for the Detection of Specific DNA Sequences Derived from Plum Pox Virus in Plant Extracts

    PubMed Central

    Malecka, Kamila; Michalczuk, Lech; Radecka, Hanna; Radecki, Jerzy

    2014-01-01

    A DNA biosensor for detection of specific oligonucleotides sequences of Plum Pox Virus (PPV) in plant extracts and buffer is proposed. The working principles of a genosensor are based on the ion-channel mechanism. The NH2-ssDNA probe was deposited onto a glassy carbon electrode surface to form an amide bond between the carboxyl group of oxidized electrode surface and amino group from ssDNA probe. The analytical signals generated as a result of hybridization were registered in Osteryoung square wave voltammetry in the presence of [Fe(CN)6]3−/4− as a redox marker. The 22-mer and 42-mer complementary ssDNA sequences derived from PPV and DNA samples from plants infected with PPV were used as targets. Similar detection limits of 2.4 pM (31.0 pg/mL) and 2.3 pM (29.5 pg/mL) in the concentration range 1–8 pM were observed in the presence of the 22-mer ssDNA and 42-mer complementary ssDNA sequences of PPV, respectively. The genosensor was capable of discriminating between samples consisting of extracts from healthy plants and leaf extracts from infected plants in the concentration range 10–50 pg/mL. The detection limit was 12.8 pg/mL. The genosensor displayed good selectivity and sensitivity. The 20-mer partially complementary DNA sequences with four complementary bases and DNA samples from healthy plants used as negative controls generated low signal. PMID:25302809

  15. 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.

  16. Selective Single-Step Separation of a Mixture of Three Metal Ions by a Triphasic Ionic-Liquid-Water-Ionic-Liquid Solvent Extraction System.

    PubMed

    Vander Hoogerstraete, Tom; Blockx, Jonas; De Coster, Hendrik; Binnemans, Koen

    2015-08-10

    In a conventional solvent extraction system, metal ions are distributed between two immiscible phases, typically an aqueous and an organic phase. In this paper, the proof-of-principle is given for the distribution of metal ions between three immiscible phases, two ionic liquid phases with an aqueous phase in between them. Three-liquid-phase solvent extraction allows separation of a mixture of three metal ions in a single step, whereas at least two steps are required to separate three metals in the case of two-liquid-phase solvent extraction. In the triphasic system, the lower organic phase is comprised of the ionic liquid betainium- or choline bis(trifluoromethylsulfonyl)imide, whereas the upper organic phase is comprised of the ionic liquid trihexyl(tetradecyl)phosphonium bis(trifluoromethylsulfonyl)imide. The triphasic system was used for the separation of a mixture of tin(II), yttrium(III), and scandium(III) ions. PMID:26178665

  17. Extraction of transcription regulatory signals from genome-wide DNAprotein interaction data

    PubMed Central

    Garten, Yael; Kaplan, Shai; Pilpel, Yitzhak

    2005-01-01

    Deciphering gene regulatory network architecture amounts to the identification of the regulators, conditions in which they act, genes they regulate, cis-acting motifs they bind, expression profiles they dictate and more complex relationships between alternative regulatory partnerships and alternative regulatory motifs that give rise to sub-modalities of expression profiles. The location data in yeast is a comprehensive resource that provides transcription factorDNA interaction information in vivo. Here, we provide two contributions: first, we developed means to assess the extent of noise in the location data, and consequently for extracting signals from it. Second, we couple signal extraction with better characterization of the genetic network architecture. We apply two methods for the detection of combinatorial associations between transcription factors (TFs), the integration of which provides a global map of combinatorial regulatory interactions. We discover the capacity of regulatory motifs and TF partnerships to dictate fine-tuned expression patterns of subsets of genes, which are clearly distinct from those displayed by most genes assigned to the same TF. Our findings provide carefully prioritized, high-quality assignments between regulators and regulated genes and as such should prove useful for experimental and computational biologists alike. PMID:15684410

  18. Extracting Kinetics from Single-Molecule Force Spectroscopy: Nanopore Unzipping of DNA Hairpins

    PubMed Central

    Dudko, Olga K.; Mathé, Jérôme; Szabo, Attila; Meller, Amit; Hummer, Gerhard

    2007-01-01

    Single-molecule force experiments provide powerful new tools to explore biomolecular interactions. Here, we describe a systematic procedure for extracting kinetic information from force-spectroscopy experiments, and apply it to nanopore unzipping of individual DNA hairpins. Two types of measurements are considered: unzipping at constant voltage, and unzipping at constant voltage-ramp speeds. We perform a global maximum-likelihood analysis of the experimental data at low-to-intermediate ramp speeds. To validate the theoretical models, we compare their predictions with two independent sets of data, collected at high ramp speeds and at constant voltage, by using a quantitative relation between the two types of measurements. Microscopic approaches based on Kramers theory of diffusive barrier crossing allow us to estimate not only intrinsic rates and transition state locations, as in the widely used phenomenological approach based on Bell's formula, but also free energies of activation. The problem of extracting unique and accurate kinetic parameters of a molecular transition is discussed in light of the apparent success of the microscopic theories in reproducing the experimental data. PMID:17384066

  19. Short Communication An efficient method for simultaneous extraction of high-quality RNA and DNA from various plant tissues.

    PubMed

    Oliveira, R R; Viana, A J C; Reátegui, A C E; Vincentz, M G A

    2015-01-01

    Determination of gene expression is an important tool to study biological processes and relies on the quality of the extracted RNA. Changes in gene expression profiles may be directly related to mutations in regulatory DNA sequences or alterations in DNA cytosine methylation, which is an epigenetic mark. Correlation of gene expression with DNA sequence or epigenetic mark polymorphism is often desirable; for this, a robust protocol to isolate high-quality RNA and DNA simultaneously from the same sample is required. Although commercial kits and protocols are available, they are mainly optimized for animal tissues and, in general, restricted to RNA or DNA extraction, not both. In the present study, we describe an efficient and accessible method to extract both RNA and DNA simultaneously from the same sample of various plant tissues, using small amounts of starting material. The protocol was efficient in the extraction of high-quality nucleic acids from several Arabidopsis thaliana tissues (e.g., leaf, inflorescence stem, flower, fruit, cotyledon, seedlings, root, and embryo) and from other tissues of non-model plants, such as Avicennia schaueriana (Acanthaceae), Theobroma cacao (Malvaceae), Paspalum notatum (Poaceae), and Sorghum bicolor (Poaceae). The obtained nucleic acids were used as templates for downstream analyses, such as mRNA sequencing, quantitative real time-polymerase chain reaction, bisulfite treatment, and others; the results were comparable to those obtained with commercial kits. We believe that this protocol could be applied to a broad range of plant species, help avoid technical and sampling biases, and facilitate several RNA- and DNA-dependent analyses. PMID:26782533

  20. Detection of polychlorinated biphenyl degradation genes in polluted sediments by direct DNA extraction and polymerase chain reaction.

    PubMed Central

    Erb, R W; Wagner-Döbler, I

    1993-01-01

    It was the aim of this study to specifically detect the DNA sequences for the bphC gene, the meta-cleavage enzyme of the aerobic catabolic pathway for biphenyl and polychlorinated biphenyl degradation, in aquatic sediments without prior cultivation of microorganisms by using extraction of total DNA, PCR amplification of bphC sequences, and detection with specific gene probes. The direct DNA extraction protocol used was modified to enhance lysis efficiency. Crude extracts of DNA were further purified by gel filtration, which yielded DNA that could be used for the PCR. PCR primers were designed for conserved regions of the bphC gene from a sequence alignment of five known sequences. The specificity of PCR amplification was verified by using digoxigenin-labeled DNA probes which were located internal to the amplified gene sequence. The detection limit for the bphC gene of Pseudomonas paucimobilis Q1 and Pseudomonas sp. strain LB400 was 100 cells per g (wet weight) or approximately five copies of the target sequence per PCR reaction mixture. In total-DNA extracts of aerobic top layers of sediment samples obtained from three different sampling sites along the Elbe River, which has a long history of anthropogenic pollution, Pseudomonas sp. strain LB 400-like sequences for the bphC gene were detected, but P. paucimobilis Q1 sequences were not detected. No bphC sequences were detected in an unpolluted lake sediment. A restriction analysis did not reveal any heterogeneity in the PCR product, and the possibility that sequences highly related to the bphC gene (namely, nahC and todE) were present was excluded.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8285706

  1. Novel DNA Extraction Method Unveiled the Ancient Hot Deep Biosphere Concealed in Terrestrial Sedimentary Rocks

    NASA Astrophysics Data System (ADS)

    Kouduka, M.; Suko, T.; Okuzawa, K.; Fukuda, A.; Nanba, K.; Yamamoto, M.; Sakata, S.; Ito, K.; Suzuki, Y.

    2009-12-01

    It has been proposed that the hot deep biosphere is distributed deeply in the crust of the Earth. Below the upper limit of life, prokaryotic habitats extend at a depth of 4 km within a typical range of thermal gradients (2.5-3°C/100m). In contrary, large geothermal gradients allow the hot deep biosphere approaching to the Earth’s surface. We conducted aseptic and deoxygenated drilling targeting Miocene marine siliceous rocks in a tectonically stable inland fore-arc basin in central Japan. Although the in-situ groundwater temperature was 30.2°C at a maximum drilling depth of 352 mbgl, opal-CT and clinoptilolite, which commonly form as a result of progressive burial at 30-60°C and over 70°C, respectively, were detected by XRD analysis. However, burial-driven degradation and transformation of sterols (sterene to sterane) was not evident in the core samples. As presented by Y. Suzuki et al. in this meeting, extant microbial populations were dominated by Pseudomonas spp. and Flavobacterium spp. with cell numbers ranging ~107-108 cells/cm3 rock. Despite the abundance of microbial cells, DNA were not extracted from the core samples by conventional methods. We developed a DNA extraction method to avoid binding of DNA onto the siliceous mineral matrix by heating under alkaline conditions, which resulted in the successful retrieval of PCR-amplifiable DNA. Unexpectedly, 16S rRNA gene sequences closely related thermophilic Geobacillus stearothermophilus and Thermus thermophilus were dominant in the clone libraries from 300- and 350-m deep core samples, while those almost identical to the Pseudomonas spp. were minor. Based on the correlation between the GC contents of 16S rRNA gene sequences and growth temperatures of prokaryotes, the estimated growth temperatures were 90.0°C (G+C=66.3%) and 67.5°C (G+C=61.3%) for G. stearothermophilus and T. thermophilus, respectively. From these results, it is implied that temperature rise in the past led to the colonization of thermophilic bacteria and the transformation of silica minerals in the deep subsurface. As intensive erosion is unlikely around the drilling site, a short period of hydrothermal activities rather than long-term burial at great depth caused high temperature conditions, which might explain the lack of maturity in hydrocarbon. A novel DNA-based approach coupled mineralogical and organic geochemical analyses has the potential to reconstruct ancient biogeochemical processes mediated in the deep subsurface as well as geothermal history. This study was supported by grants from the Nuclear and Industrial Safety Agency (NISA) and Japan Nuclear Energy Safety Organization (JNES).

  2. High efficiency protocol of DNA extraction from Micromys minutus mandibles from owl pellets: a tool for molecular research of cryptic mammal species.

    PubMed

    Bu?, Magdalena M; Zmihorski, Micha?; Romanowski, Jerzy; Bal?iauskien?, Laima; Cichocki, Jan; Bal?iauskas, Linas

    2014-01-01

    Owl pellets have high potential as a source of DNA. However, this noninvasive method of collecting DNA is rarely used, and its methodological aspects are poorly understood. We investigated the methodology for DNA extraction and amplification from owl pellets containing the smallest European rodent-the Harvest mouse Micromys minutus-as an example. We used mandibles identified in owl pellets for mitochondrial and nuclear DNA amplification. For DNA extraction, we tested two commercial protocols and utilized a protocol being a combination of two commercial kits which ensured high efficiency of DNA extraction. Additionally, we recorded that the amount of DNA was five times higher in extracts from teeth as compared to DNA extracts from jawbones derived from the same mandible. The quantity of DNA was significantly positively correlated with biological sample weight; however, the age of the pellet remains had an impact on the level of inhibition. We recorded inhibition in 40% of mtDNA extracts derived from pellets older than 150months, whereas in DNA extracts from pellets younger than 80months, we did not observe a negative impact of inhibition on PCR efficiency. The amplification success rate was 89.9% for the mitochondrial fragment and 39.4% in the case of the nuclear fragment. We observed partial degradation of DNA evidenced by the fact that the longest fragments that we were able to amplify in the case of mtDNA were 450 and 200bp for nuDNA. The study shows that pellets can be considered as a source of DNA and have high potential for molecular research in the case of threatened species and species that are difficult to study using standard field techniques. PMID:24436494

  3. Protection of DNA and erythrocytes from free radical induced oxidative damage by black gram (Vigna mungo L.) husk extract.

    PubMed

    Girish, Talakatta K; Vasudevaraju, Padmaraju; Prasada Rao, Ummiti J S

    2012-05-01

    Antioxidants present in various plant tissues exhibit health benefits by scavenging reactive oxygen species generated under various pathophysiological conditions. In the present study, bioactive compounds from black gram husk were extracted with water and the protection of black gram husk (BGH) extract against oxidative damage in DNA and erythrocytes were studied. BGH extract had total polyphenol content of 59 mg of gallic acid equivalents (GAE). The phenolic acids identified in the extract using RP-HPLC were gallic, protocatechuic, gentisic and ferulic acids. The extract showed good antioxidant properties. The IC(50) value for DPPH radical scavenging activity was found to be 3.92 μg of GAE. The BGH extract also showed α-glucosidase inhibition and the IC(50) value was found to be 2.78 μg of GAE. The oxidative hemolysis caused by hydrogen peroxide in rat erythrocytes was inhibited by BGH extract in a dose dependent manner. The IC(50) values for BGH extract and BHA for hemolysis were 11.5 and 14 μg of GAE, respectively. Morphological changes in erythrocyte membrane caused by hydrogen peroxide were protected by BGH extract. As BGH extract exhibited various antioxidant properties in different systems, it could be used as a functional food or nutraceutical product for health benefits. PMID:22330200

  4. Improved procedure for nucleus extraction for DNA measurements by flow cytometry of red beet (Beta vulgaris L.) hairy roots.

    PubMed

    Georgiev, Vasil; Weber, Jost; Bley, Thomas; Pavlov, Atanas

    2009-04-01

    Three often cited systems for the extraction of plant nuclei for flow cytometric measurement (CyStain PI, Partec GmbH, Münster, Germany, the method of Arumuganathan and Earle, and LB01 buffer) failed, when applied to the hairy roots of red beet (Beta vulgaris). By combining these systems and introducing a centrifugation step, the extraction, staining, and analysis of nuclei from this tissue were performed successfully. PMID:19332305

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 th...

  6. A Case of loiasis in a patient returning to China diagnosed by nested PCR using DNA extracted from tissue.

    PubMed

    Wang, Xiaohui; Zhang, Xiaoke; Zong, Zhiyong

    2012-01-01

    Recognition and diagnosis of loiasis is problematic in areas where this infection is not endemic but may be imported. A previously healthy Chinese male returned from Equatorial Guinea presenting with migratory masses. He was diagnosed with loiasis following detection of Loa loa by nested polymerase chain reaction using DNA extracted from tissue. PMID:22943273

  7. Extraction of the metagenomic DNA and assessment of the bacterial diversity from the petroleum-polluted sites.

    PubMed

    Akbari, Viral G; Pandya, Rupal D; Singh, Satya P

    2014-10-01

    The assessment of the microbial diversity of the entire community of a given habitat requires the extraction of the total environmental DNA. Metagenomic investigations of a petroleum-polluted habitat have its unique challenges. The specific methods were developed for the extraction of high-quality metagenome in good quantity from the petroleum-polluted saline and non-saline sites in Gujarat (India). The soil samples were washed to remove the toxic, hazardous organic pollutants which might interfere with the recovery of the metagenomic DNA. The metagenomic DNA extraction results were encouraging with the mechanical bead beating, soft lysis, and combination of both. The extracted DNA was assessed for its purity and yield followed by its application in the amplification of the 16S rRNA region. The amplicons were used for judging the molecular diversity by the denaturing gradient gel electrophoresis (DGGE). The microbial diversity was also analyzed statistically by calculating various diversity indices and principal component analysis (PCA). The results on the metagenomic diversity of the bacterial population among the three cohorts based on the culture-independent technique exhibited significant difference among the PAH sites and Okha-Madhi and Porbandar Madhavpur habitats. PMID:24869956

  8. Phenolic composition, DNA damage protective activity and hepatoprotective effect of free phenolic extract from Sphallerocarpus gracilis seeds.

    PubMed

    Gao, Chun-yan; Tian, Cheng-rui; Zhou, Rui; Zhang, Run-guang; Lu, Yue-hong

    2014-05-01

    The phenolic composition of the free phenolic extract from Sphallerocarpus gracilis seeds was analyzed by HPLC-MS and predominant compounds were chlorogenic acid, di-caffeoylquinic acid glucoside and luteolin-7-O-glucoside. The free phenolic extract was evaluated for DNA damage protective activity induced by ROO and OH radicals and hepatoprotective effect in vivo and in vitro. Results revealed that the free phenolic extract exhibited significant protective activity against both ROO and OH radical-induced DNA damage and the phenolic extract exerted more potent inhibitory activity against OH radical-induced damage than against that induced by ROO radicals. In vivo experimental results showed that the phenolic extract significantly prevented the increase of serum alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase activities and hepatic malondialdehyde level caused by CCl4 in rats, and markedly increased hepatic superoxide dismutase, catalase and glutathione peroxidase levels. Histopathological examinations further confirmed that the phenolic extract could protect the liver from CCl4-induced damage. In vitro experimental results showed that the phenolic extract could reduce BRL hepatocyte apoptosis and damage induced by CCl4. These findings indicate that the S. gracilis seed could be developed as a medicinal herb for the therapy and prevention of hepatic injury. PMID:24657314

  9. DNA extraction techniques compared for accurate detection of genetically modified organisms (GMOs) in maize food and feed products.

    PubMed

    Turkec, Aydin; Kazan, Hande; Karacanli, Burçin; Lucas, Stuart J

    2015-08-01

    In this paper, DNA extraction methods have been evaluated to detect the presence of genetically modified organisms (GMOs) in maize food and feed products commercialised in Turkey. All the extraction methods tested performed well for the majority of maize foods and feed products analysed. However, the highest DNA content was achieved by the Wizard, Genespin or the CTAB method, all of which produced optimal DNA yield and purity for different maize food and feed products. The samples were then screened for the presence of GM elements, along with certified reference materials. Of the food and feed samples, 8 % tested positive for the presence of one GM element (NOS terminator), of which half (4 % of the total) also contained a second element (the Cauliflower Mosaic Virus 35S promoter). The results obtained herein clearly demonstrate the presence of GM maize in the Turkish market, and that the Foodproof GMO Screening Kit provides reliable screening of maize food and feed products. PMID:26243938

  10. Wheat bran biorefinery: an investigation on the starch derived glucose extraction accompanied by pre- and post-treatment steps.

    PubMed

    Tirpanalan, Özge; Reisinger, Michael; Huber, Florian; Kneifel, Wolfgang; Novalin, Senad

    2014-07-01

    Wheat bran, a side product of the milling industry, can be considered as a feedstock for biorefineries. Unlike other lignocellulosic feedstock, wheat bran contains a reasonable amount of starch, which is not of recalcitrant nature. Therefore, it can be extracted without a costly pretreatment process. The present work evaluates the extraction of starch derived glucose in relation to a wheat bran biorefinery. The purity of free glucose extracted quantitatively was 44%. The extract was concentrated by threefold via nanofiltration, thereby reaching a glucose concentration of 49 g/L. Hydrothermal treatment (180°C - 20 min) of the starch-free bran did not induce the formation of hydroxymethylfurfural and levulinic acid. Interestingly, the furfural level increased compared to the process, in which bran was treated hydrothermally without a preceding starch extraction. By separation of water-extractables prior to enzymatic hydrolysis, the free glucose purity was increased to 58%, however the yield of glucose decreased to 61%. PMID:24835741

  11. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

    The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based ...

  12. DNA Extraction Method Affects the Detection of a Fungal Pathogen in Formalin-Fixed Specimens Using qPCR.

    PubMed

    Adams, Andrea J; LaBonte, John P; Ball, Morgan L; Richards-Hrdlicka, Kathryn L; Toothman, Mary H; Briggs, Cheryl J

    2015-01-01

    Museum collections provide indispensable repositories for obtaining information about the historical presence of disease in wildlife populations. The pathogenic amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has played a significant role in global amphibian declines, and examining preserved specimens for Bd can improve our understanding of its emergence and spread. Quantitative PCR (qPCR) enables Bd detection with minimal disturbance to amphibian skin and is significantly more sensitive to detecting Bd than histology; therefore, developing effective qPCR methodologies for detecting Bd DNA in formalin-fixed specimens can provide an efficient and effective approach to examining historical Bd emergence and prevalence. Techniques for detecting Bd in museum specimens have not been evaluated for their effectiveness in control specimens that mimic the conditions of animals most likely to be encountered in museums, including those with low pathogen loads. We used American bullfrogs (Lithobates catesbeianus) of known infection status to evaluate the success of qPCR to detect Bd in formalin-fixed specimens after three years of ethanol storage. Our objectives were to compare the most commonly used DNA extraction method for Bd (PrepMan, PM) to Macherey-Nagel DNA FFPE (MN), test optimizations for Bd detection with PM, and provide recommendations for maximizing Bd detection. We found that successful detection is relatively high (80-90%) when Bd loads before formalin fixation are high, regardless of the extraction method used; however, at lower infection levels, detection probabilities were significantly reduced. The MN DNA extraction method increased Bd detection by as much as 50% at moderate infection levels. Our results indicate that, for animals characterized by lower pathogen loads (i.e., those most commonly encountered in museum collections), current methods may underestimate the proportion of Bd-infected amphibians. Those extracting DNA from archived museum specimens should ensure that the techniques they are using are known to provide high-quality throughput DNA for later analysis. PMID:26291624

  13. DNA Extraction Method Affects the Detection of a Fungal Pathogen in Formalin-Fixed Specimens Using qPCR

    PubMed Central

    Adams, Andrea J.; LaBonte, John P.; Ball, Morgan L.; Richards-Hrdlicka, Kathryn L.; Toothman, Mary H.; Briggs, Cheryl J.

    2015-01-01

    Museum collections provide indispensable repositories for obtaining information about the historical presence of disease in wildlife populations. The pathogenic amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has played a significant role in global amphibian declines, and examining preserved specimens for Bd can improve our understanding of its emergence and spread. Quantitative PCR (qPCR) enables Bd detection with minimal disturbance to amphibian skin and is significantly more sensitive to detecting Bd than histology; therefore, developing effective qPCR methodologies for detecting Bd DNA in formalin-fixed specimens can provide an efficient and effective approach to examining historical Bd emergence and prevalence. Techniques for detecting Bd in museum specimens have not been evaluated for their effectiveness in control specimens that mimic the conditions of animals most likely to be encountered in museums, including those with low pathogen loads. We used American bullfrogs (Lithobates catesbeianus) of known infection status to evaluate the success of qPCR to detect Bd in formalin-fixed specimens after three years of ethanol storage. Our objectives were to compare the most commonly used DNA extraction method for Bd (PrepMan, PM) to Macherey-Nagel DNA FFPE (MN), test optimizations for Bd detection with PM, and provide recommendations for maximizing Bd detection. We found that successful detection is relatively high (80–90%) when Bd loads before formalin fixation are high, regardless of the extraction method used; however, at lower infection levels, detection probabilities were significantly reduced. The MN DNA extraction method increased Bd detection by as much as 50% at moderate infection levels. Our results indicate that, for animals characterized by lower pathogen loads (i.e., those most commonly encountered in museum collections), current methods may underestimate the proportion of Bd-infected amphibians. Those extracting DNA from archived museum specimens should ensure that the techniques they are using are known to provide high-quality throughput DNA for later analysis. PMID:26291624

  14. One-step extraction and concentration of estrogens for an adequate monitoring of wastewater using ionic-liquid-based aqueous biphasic systems†

    PubMed Central

    Lima, Diana L. D.; Esteves, Valdemar I.; Coutinho, João A. P.; Freire, Mara G.

    2015-01-01

    Ethinylestradiol (EE2) is a synthetic hormone that has been recognized as one of the most prominent endocrine disruptors found in the aqueous environment. Nevertheless, the low content of EE2 in wastewater makes its identification/quantification unfeasible – a major drawback for the evaluation of its persistence and environmental impact. In this context, a novel extraction/concentration method for EE2 from wastewater is proposed here based on aqueous biphasic systems composed of ionic liquids (ILs). Aqueous biphasic systems formed by several hydrophilic ILs and KNaC4H4O6 were initially screened and optimized, with extraction efficiencies of EE2 for the IL-rich phase ranging between 92 and 100%. Remarkable results were obtained with systems that allow the complete extraction of EE2 in a single-step, and without loss of EE2 or the saturation of the extractive phase. Further, the concentration factors of EE2 attainable with these systems were investigated by a suitable manipulation of the composition of the phase-forming components and the corresponding volumes of the coexisting phases. An outstanding concentration of EE2 up to 1000-fold (from ng L−1 to μg L−1) in a single extraction and concentration step was achieved for the first time with IL-based aqueous biphasic systems. These systems are straightforwardly envisaged for the monitoring of wastewater as one-step extraction and concentration routes for a wide array of endocrine disrupting chemicals while allowing an adequate evaluation of their environmental impact. PMID:26379470

  15. Genotoxic Evaluation of Mikania laevigata Extract on DNA Damage Caused by Acute Coal Dust Exposure

    SciTech Connect

    Freitas, T.P.; Heuser, V.D.; Tavares, P.; Leffa, D.D.; da Silva, G.A.; Citadini-Zanette, V.; Romao, P.R.T.; Pinho, R.A.; Streck, E.L.; Andrade,V.M.

    2009-06-15

    We report data on the possible antigenotoxic activity of Mikania laevigata extract (MLE) after acute intratracheal instillation of coal dust using the comet assay in peripheral blood, bone marrow, and liver cells and the micronucleus test in peripheral blood of Wistar rats. The animals were pretreated for 2 weeks with saline solution (groups 1 and 2) or MLE (100 mg/kg) (groups 3 and 4). On day 15, the animals were anesthetized with ketamine (80 mg/kg) and xylazine (20 mg/kg), and gross mineral coal dust (3 mg/0.3 mL saline) (groups 2 and 4) or saline solution (0.3 mL) (groups 1 and 3) was administered directly in the lung by intratracheal administration. Fifteen days after coal dust or saline instillation, the animals were sacrificed, and the femur, liver, and peripheral blood were removed. The results showed a general increase in the DNA damage values at 8 hours for all treatment groups, probably related to surgical procedures that had stressed the animals. Also, liver cells from rats treated with coal dust, pretreated or not with MLE, showed statistically higher comet assay values compared to the control group at 14 days after exposure. These results could be expected because the liver metabolizes a variety of organic compounds to more polar by-products. On the other hand, the micronucleus assay results did not show significant differences among groups. Therefore, our data do not support the antimutagenic activity of M. laevigata as a modulator of DNA damage after acute coal dust instillation.

  16. Development of a one-step integrated pressurized liquid extraction and cleanup method for determining polycyclic aromatic hydrocarbons in marine sediments.

    PubMed

    Choi, Minkyu; Kim, Ye-Jung; Lee, In-Seok; Choi, Hee-Gu

    2014-05-01

    A rapid and accurate one-step integrated pressurized liquid extraction (PLE) and cleanup method was developed and validated for 34 polycyclic aromatic hydrocarbons in marine sediments, giving an extract that could be analyzed by gas chromatography-mass spectrometry without further cleanup. Marine sediment (5 g) was loaded into the stainless-steel extraction cell above activated copper (5 g) and activated silica gel (5 g). An extraction temperature of 100°C and two 5 min extraction cycles using a 4:1 (v/v) hexane-dichloromethane mixture gave a good extraction efficiency. The integrated method gave extracts that were as clean as those obtained using PLE, followed by separate activated copper and silica gel cleanups. The method was validated, in terms of its accuracy, precision, and application using a certified reference material (NIST SRM 1944), marine sediments spiked at low and high concentrations, and contaminated harbor sediments. The mean recoveries were 92% and 94% for the low and high spike concentrations, respectively, and the accuracy was good (giving a mean of 86% of the certified reference material concentrations). The method developed gave a precision and accuracy equal to or better than the precision and accuracy found using PLE with separate cleanups. The method developed gives a shorter sample preparation time and uses much less solvent than PLE and separate cleanups. PMID:24671040

  17. Evaluation of six different DNA extraction methods for detection of Mycobacterium tuberculosis by means of PCR-IS6110: preliminary study

    PubMed Central

    2013-01-01

    Background Developments in molecular detection and strain differentiation of members of Mycobacterium tuberculosis complex have proved to be useful. The DNA extraction method influences the amplification efficiency, causing interference on the sensitivity and respective inhibitors. The aim of this study was to standardize a simple and fast DNA extraction method, providing DNA amplification by IS6110-PCR effectively free from undue interferences. Findings The efficiency of the six different protocols tested in M. tuberculosis cultures has varied from 75% to 92.5%. This preliminary study evaluating the IS6110 PCR sensitivity and specificity was developed in DNA extracted from microscope slides, and achieved 100% of efficiency. Conclusions DNA extraction by Chelex + NP-40 method from both, cultures of M. tuberculosis and smear slides, resulted in good quantity of interference free DNA, especially in samples with low concentrations of genetic material; therefore, such technique may be used for the molecular diagnosis of tuberculosis. PMID:24373461

  18. A Simple and Effective Method for High Quality Co-Extraction of Genomic DNA and Total RNA from Low Biomass Ectocarpus siliculosus, the Model Brown Alga

    PubMed Central

    Greco, Maria; Sáez, Claudio A.; Brown, Murray T.; Bitonti, Maria Beatrice

    2014-01-01

    The brown seaweed Ectocarpus siliculosus is an emerging model species distributed worldwide in temperate coastal ecosystems. Over 1500 strains of E. siliculosus are available in culture from a broad range of geographic locations and ecological niches. To elucidate the molecular mechanisms underlying its capacity to cope with different environmental and biotic stressors, genomic and transcriptomic studies are necessary; this requires the co-isolation of genomic DNA and total RNA. In brown algae, extraction of nucleic acids is hindered by high concentrations of secondary metabolites that co-precipitate with nucleic acids. Here, we propose a reliable, rapid and cost-effective procedure for the co-isolation of high-quality nucleic acids using small quantities of biomass (25-, 50- and 100 mg) from strains of E. siliculosus (RHO12; LIA4A; EC524 and REP10–11) isolated from sites with different environmental conditions. The procedure employs a high pH extraction buffer (pH 9.5) which contains 100 mM Tris-HCl and 150 mM NaCl, with the addition of 5 mM DTT and 1% sarkosyl to ensure maximum solubility of nucleic acids, effective inhibition of nuclease activity and removal of interfering contaminants (e.g. polysaccharides, polyphenols). The use of sodium acetate together with isopropanol shortened precipitation time and enhanced the yields of DNA/RNA. A phenol:chlorophorm:isoamyl alcohol step was subsequently used to purify the nucleic acids. The present protocol produces high yields of nucleic acids from only 25 mg of fresh algal biomass (0.195 and 0.284 µg mg−1 fresh weigh of RNA and DNA, respectively) and the high quality of the extracted nucleic acids was confirmed through spectrophotometric and electrophoretic analyses. The isolated RNA can be used directly in downstream applications such as RT-PCR and the genomic DNA was suitable for PCR, producing reliable restriction enzyme digestion patterns. Co-isolation of DNA/RNA from different strains indicates that this method is likely to have wider applications for intra- and inter-specific studies on other brown algae. PMID:24867404

  19. A simple and effective method for high quality co-extraction of genomic DNA and total RNA from low biomass Ectocarpus siliculosus, the model brown alga.

    PubMed

    Greco, Maria; Sáez, Claudio A; Brown, Murray T; Bitonti, Maria Beatrice

    2014-01-01

    The brown seaweed Ectocarpus siliculosus is an emerging model species distributed worldwide in temperate coastal ecosystems. Over 1500 strains of E. siliculosus are available in culture from a broad range of geographic locations and ecological niches. To elucidate the molecular mechanisms underlying its capacity to cope with different environmental and biotic stressors, genomic and transcriptomic studies are necessary; this requires the co-isolation of genomic DNA and total RNA. In brown algae, extraction of nucleic acids is hindered by high concentrations of secondary metabolites that co-precipitate with nucleic acids. Here, we propose a reliable, rapid and cost-effective procedure for the co-isolation of high-quality nucleic acids using small quantities of biomass (25-, 50- and 100 mg) from strains of E. siliculosus (RHO12; LIA4A; EC524 and REP10-11) isolated from sites with different environmental conditions. The procedure employs a high pH extraction buffer (pH 9.5) which contains 100 mM Tris-HCl and 150 mM NaCl, with the addition of 5 mM DTT and 1% sarkosyl to ensure maximum solubility of nucleic acids, effective inhibition of nuclease activity and removal of interfering contaminants (e.g. polysaccharides, polyphenols). The use of sodium acetate together with isopropanol shortened precipitation time and enhanced the yields of DNA/RNA. A phenol:chlorophorm:isoamyl alcohol step was subsequently used to purify the nucleic acids. The present protocol produces high yields of nucleic acids from only 25 mg of fresh algal biomass (0.195 and 0.284 µg mg(-1) fresh weigh of RNA and DNA, respectively) and the high quality of the extracted nucleic acids was confirmed through spectrophotometric and electrophoretic analyses. The isolated RNA can be used directly in downstream applications such as RT-PCR and the genomic DNA was suitable for PCR, producing reliable restriction enzyme digestion patterns. Co-isolation of DNA/RNA from different strains indicates that this method is likely to have wider applications for intra- and inter-specific studies on other brown algae. PMID:24867404

  20. A simple and rapid DNA extraction method from whole blood for highly sensitive detection and quantitation of HIV-1 proviral DNA by real-time PCR.

    PubMed

    McFall, Sally M; Wagner, Robin L; Jangam, Sujit R; Yamada, Douglas H; Hardie, Diana; Kelso, David M

    2015-03-01

    Early diagnosis and access to treatment for infants with human immunodeficiency virus-1 (HIV-1) is critical to reduce infant mortality. The lack of simple point-of-care tests impedes the timely initiation of antiretroviral therapy. The development of FINA, filtration isolation of nucleic acids, a novel DNA extraction method that can be performed by clinic personnel in less than 2 min has been reported previously. In this report, significant improvements in the DNA extraction and amplification methods are detailed that allow sensitive quantitation of as little as 10 copies of HIV-1 proviral DNA and detection of 3 copies extracted from 100 μl of whole blood. An internal control to detect PCR inhibition was also incorporated. In a preliminary field evaluation of 61 South African infants, the FINA test demonstrated 100% sensitivity and specificity. The proviral copy number of the infant specimens was quantified, and it was established that 100 microliters of whole blood is required for sensitive diagnosis of infants. PMID:25681524

  1. Exonuclease III-Assisted Target Recycling Amplification Coupled with Liposome-Assisted Amplification: One-Step and Dual-Amplification Strategy for Highly Sensitive Fluorescence Detection of DNA.

    PubMed

    Zhou, Fulin; Li, Baoxin

    2015-07-21

    Detection of ultralow concentration of specific DNA sequence is a central challenge in the early diagnosis of gene-related disease and biodefense application. Herein, we report a dual-amplification strategy for highly sensitive fluorescence detection of DNA. In this proposed strategy, a dumbbell-shaped DNA probe is designed to integrate target binding, magnetic separation, and signal response. In the presence of specific DNA target, the multifunctional dumbbell probe can initiate exonuclease III (Exo III)-aided target recycling amplification, and, in the meantime, generate a large number of fluorescein (FAM)-encapsulated liposomes. The developed method offers very high sensitivity due to primary amplification via numerous FAM from a liposome and secondary amplification via target recycling amplification. The detection limit of the proposed method can reach 4 aM, which is much lower than that of the Exo III-aided target recycling technique applied for DNA quantification without FAM-encapsulated liposomes amplification. Moreover, the dual-signal amplification process can be completed one-step in this system. Therefore, this method provides a simple, isothermal, and low-cost approach for sensitive detection of DNA and holds a great potential for early diagnosis in gene-related diseases. PMID:26111123

  2. Short-step chemical synthesis of DNA by use of MMTrS group for protection of 5'-hydroxyl group.

    PubMed

    Shiraishi, Miyuki; Utagawa, Eri; Ohkubo, Akihiro; Sekine, Mitsuo; Seio, Kohji

    2007-01-01

    4-methoxytrithylthio (MMTrS) group was applied for the appropriately protected four canonical nucleosides. We prepared the phosphoroamidite units by use of these nucleosides and developed the synthesis of oligodeoxynucleotides without any acidic treatment. Moreover, the new DNA synthesis protocol was applied to an automated DNA synthesizer for the synthesis of longer oligodeoxynucleotides. PMID:18029620

  3. Microwave heating of long-term formalin-fixed surgical pathology specimens improves quality of extracted DNA.

    PubMed

    Wu, Shanshui; Qian, Xin; Yu, Xinchun; Sheng, Hongqiang; L, Bingjian

    2012-10-01

    A large amount of DNA of high quality is essential for molecular analysis. The amount of DNA in routine paraffin sections is small. Surgical specimens retained in formalin for the long-term (several months) left over from the sampling required for wax embedding can be referred to as "long-term formalin-fixed" specimens, and clearly this material is a rich source of DNA; however, it is difficult to extract. In the current study, we designed a microwave-heating method for DNA extraction from these specimens. We found that the heating procedure achieved greater DNA yields than a common nonheating method used for comparison (DNA contents meanSD, heating 2.160.95 ?g/?L vs. common 1.750.90 ?g/?L, P<0.05). Fluorescence multiplex polymerase chain reaction-capillary electrophoresis successfully detected microsatellite instability (MSI) in the DNA samples derived from this heating procedure (98.4%, 689 of 700 sample tests), at significantly higher levels than from the conventional method (82.3%, 247 of 300 sample tests, P<0.05). We identified 10 (14.3%) MSI with high frequency and 6 (8.6%) MSI with low frequency colorectal cancers. MSI with high frequency cancers showed distinct clinicopathologic features including higher incidence of right-sided location, high histologic grade, mucin-production, and prominent intraepithelial lymphocyte infiltration. We concluded that the microwave-heating method was efficient for DNA isolation from long-term formalin-fixed tissue samples. The successful fluorescence multiplex polymerase chain reaction-capillary electrophoresis analysis in these samples might facilitate MSI detection in clinical practice. PMID:22495375

  4. Comparison of three different DNA extraction methods from a highly degraded biological material.

    PubMed

    Kuś, M; Ossowski, A; Zielińska, G

    2016-05-01

    The identification of unknown victims is one of the most challenging tasks faced by forensic medicine. This is due to the rapid decomposition of tissues, beginning at the moment of death and caused by released enzymes and microbial activity. Decay is directly associated with the decomposition of soft tissues and also the degradation of genetic material inside cells. Decomposition rates vary depending on a number of environmental factors, including temperature, humidity, season, and soil properties. Decomposition also differs between bodies left in the open air or buried. To date, forensic medicine has identified mainly people who were the victims of various types of criminal offences. However, with advances in identification methods, increasingly frequent attempts are made to identify the victims of armed conflicts, crimes of totalitarian regimes, or genocide. The aim of the study was to compare three different methods for the extraction of nuclear DNA from material considered in forensic medicine as difficult to handle, i.e. fragments of bones and teeth, and to determine the performance of these methods and their suitability for identification procedures. PMID:27016882

  5. Kinetic Parameter Extraction of Square Wave Voltammograms from DNA-Modified Gold Electrodes

    NASA Astrophysics Data System (ADS)

    McWilliams, Marc; Wohlgamuth, Chris; Slinker, Jason

    2012-10-01

    The field of surface bound electrochemistry is important in a variety of applications specifically sensing. A fundamental understanding of the processes involved could help to improve detection limits, optimize rates of detection and direct changes in device design. Accurate extraction of electrochemical kinetic parameters such as the rate constant k and charge transfer coefficient α from cyclic voltammograms can be challenging when confronted with large background currents and relatively weak signals. The commonly used technique of Laviron analysis is both time consuming and somewhat subjective. Square wave voltammetry (SWV) is therefore an ideal alternative method given that it maximizes signal while minimizing capacitive effects. In this experiment kinetic parameters of DNA-modified gold electrodes are obtained from SWV curves through background subtraction followed by nonlinear least squares fitting using a first order quasi-reversible surface process model. The fitting is accomplished using the Nelder-Mead simplex algorithm with standard parameters and a convergence condition of less than 0.0001%. General agreement with experimental data is shown with varying levels of confidence. Difficulties specific to this experiment are discussed as well as the possible benefits of utilizing the Bayesian statistical approach of nested sampling when confronted with multiple peaks of interest and the background source is well defined.

  6. A Powerful DNA Extraction Method and PCR for Detection of Microsporidia in Clinical Stool Specimens

    PubMed Central

    Müller, Andreas; Stellermann, Kerstin; Hartmann, Pia; Schrappe, Matthias; Fätkenheuer, Gerd; Salzberger, Bernd; Diehl, Volker; Franzen, Caspar

    1999-01-01

    The diagnosis of intestinal microsporidiosis has traditionally depended on direct visualization of the parasite in stool specimens or intestinal biopsy samples by light and/or electron microscopy. Limited information about the specificity and sensitivity of PCR for the detection microsporidia in clinical stool specimens is available. To establish a sensitive and specific method for the detection of microsporidia in clinical samples, we studied clinical stool specimens of 104 randomly selected human immunodeficiency virus-infected patients with diarrhea to compare light microscopy and PCR. Fluorochrome Uvitex 2B staining was used for light microscopy. To raise the sensitivity of PCR, we used a powerful and fast DNA extraction method including stool sedimentation, glass bead disruption, and proteinase K and chitinase digestion. PCR was performed with primer pairs V1-PMP2, V1-EB450, and V1-SI500, and the nature of the PCR products was confirmed by Southern blot hybridization. Microsporidiosis was diagnosed by light microscopy in eight patients. Ten patients tested positive for microsporidiosis by PCR. Enterocytozoon bieneusi was found in seven cases, and Encephalitozoon intestinalis was found in four cases. In one case a double infection with E. bieneusi and E. intestinalis was diagnosed by PCR, whereas light microscopy showed only E. bieneusi infection. PCR testing of stool specimens is useful for diagnosis and species differentiation of intestinal microsporidiosis in HIV patients. PMID:10066661

  7. Preparation of magnetite-loaded silica microspheres for solid-phase extraction of genomic DNA from soy-based foodstuffs.

    PubMed

    Shi, Ruobing; Wang, Yucong; Hu, Yunli; Chen, Lei; Wan, Qian-Hong

    2009-09-01

    Solid-phase extraction has been widely employed for the preparation of DNA templates for polymerase chain reaction (PCR)-based analytical methods. Among the variety of adsorbents studied, magnetically responsive silica particles are particularly attractive due to their potential to simplify, expedite, and automate the extraction process. Here we report a facile method for the preparation of such magnetic particles, which entails impregnation of porous silica microspheres with iron salts, followed by calcination and reduction treatments. The samples were characterized using powder X-ray diffractometry (XRD), scanning electron microscopy (SEM), nitrogen adsorption/desorption isotherms, and vibrating sample magnetometry (VSM). XRD data show that magnetite nanocrystals of about 27.2 nm are produced within the pore channels of the silica support after reduction. SEM images show that the as-synthesized particles exhibit spherical shape and uniform particle size of about 3 microm as determined by the silica support. Nitrogen sorption data confirm that the magnetite-loaded silica particles possess typical mesopore structure with BET surface area of about 183 m(2)/g. VSM data show that the particles display paramagnetic behavior with saturation magnetization of 11.37 emu/g. The magnetic silica microspheres coated with silica shells were tested as adsorbents for rapid extraction of genomic DNA from soybean-derived products. The purified DNA templates were amplified by PCR for screening of genetically modified organisms (GMOs). The preliminary results confirm that the DNA extraction protocols using magnetite-loaded silica microspheres are capable of producing DNA templates which are inhibitor-free and ready for downstream analysis. PMID:19632684

  8. Back to Basics – The Influence of DNA Extraction and Primer Choice on Phylogenetic Analysis of Activated Sludge Communities

    PubMed Central

    Kirkegaard, Rasmus H.; Nielsen, Per H.

    2015-01-01

    DNA extraction and primer choice have a large effect on the observed community structure in all microbial amplicon sequencing analyses. Although the biases are well known, no comprehensive analysis has been conducted in activated sludge communities. In this study we systematically explored the impact of a number of parameters on the observed microbial community: bead beating intensity, primer choice, extracellular DNA removal, and various PCR settings. In total, 176 samples were subjected to 16S rRNA amplicon sequencing, and selected samples were investigated through metagenomics and metatranscriptomics. Quantitative fluorescence in situ hybridization was used as a DNA extraction-independent method for qualitative comparison. In general, an effect on the observed community was found on all parameters tested, although bead beating and primer choice had the largest effect. The effect of bead beating intensity correlated with cell-wall strength as seen by a large increase in DNA from Gram-positive bacteria (up to 400%). However, significant differences were present at lower phylogenetic levels within the same phylum, suggesting that additional factors are at play. The best primer set based on in silico analysis was found to underestimate a number of important bacterial groups. For 16S rRNA gene analysis in activated sludge we recommend using the FastDNA SPIN Kit for Soil with four times the normal bead beating and V1-3 primers. PMID:26182345

  9. Detection of polychlorinated biphenyl degradation genes in polluted sediments by direct DNA extraction and polymerase chain reaction

    SciTech Connect

    Erb, R.W.; Wagner-Doebler, I. )

    1993-12-01

    It was the aim of this study to specifically detect the DNA sequences for the bphC gene, the meta-cleavage enzyme of the aerobic catabolic pathway for biphenyl and polychlorinated biphenyl degradation, in aquatic sediments without prior cultivation of microorganisms by using extraction of total DNA, PCR amplification of bphC sequences, and detection with specific gene probes. The direct DNA extraction protocol used was modified to enhance lysis efficiency. Crude extracts of DNA were further purified by gel filtration, which yielded DNA that could be used for the PCR. PCR primers were designed for conserved regions of the bphC gene from a sequence alignment of five known sequences. The specificity of PCR amplification was verified by using digoxigenin-labeled DNA probes which were located internal to the amplified gene sequence. The detection limit for the bphC gene of Pseudomonas paucimobilis Q1 and Pseudomaonas sp. strain LB 400-like sequences for the bphC gene were detected, but P. paucimobilis Q1 sequences were not detected. No bphC sequences were detected in an unpolluted lake sediment. A restriction analysis did not reveal any heterogeneity in the PCR product, and the possibility that sequences highly related to the bphC gene (namel y, nahC and TodE) were present was excluded. Thus, for the first time it was possible to directly amplify and detect a chromosomally encoded, single-copy gene from a highly specialized subpopulation of the total microbial community in natural sediments.

  10. A molecular exploration of human DNA/RNA co-extracted from the palmar surface of the hands and fingers.

    PubMed

    Lacerenza, D; Aneli, S; Omedei, M; Gino, S; Pasino, S; Berchialla, P; Robino, C

    2016-05-01

    "Touch DNA" refers to the DNA that is left behind when a person touches or comes into contact with an item. However, the source of touch DNA is still debated and the large variability in DNA yield from casework samples suggests that, besides skin, various body fluids can be transferred through contact. Another important issue concerning touch DNA is the possible occurrence of secondary transfer, but the data published in the literature in relation to the background levels of foreign DNA present on the hand surfaces of the general population are very limited. As the present study aimed at better understanding the nature and characteristics of touch DNA, samples were collected from the palmar surface of the hands and fingers ("PHF" samples) of 30 male and 30 female donors by tape-lifting/swabbing and subjected to DNA/RNA co-extraction. Multiplex mRNA profiling showed that cellular material different from skin could be observed in 15% of the PHF samples. The total amount of DNA recovered from these samples (median 5.1ng) was significantly higher than that obtained from samples containing skin cells only (median 1.6ng). The integrity of the DNA isolated from the donors' hands and fingers as well as the prevalence of DNA mixtures were evaluated by STR typing and compared with reference STR profiles from buccal swabs. DNA integrity appeared significantly higher in the male rather than in the female subsample, as the average percentage of the donors' alleles effectively detected in PHF profiles was 75.1% and 60.1%, respectively. The prevalence of mixtures with a foreign DNA contribution ≥20% was 19.2% (30.0% in the female PHF samples and 8.3% in the male PHF samples). The obtained results support the hypothesis that transfer of cellular material different from skin may underlie the occasional recovery of quality STR profiles from handled items. These results also suggest that gender may represent an important factor influencing t