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Sample records for dna extraction step

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

    PubMed

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

    2012-01-01

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

  2. The modified method of two-step differential extraction of sperm and vaginal epithelial cell DNA from vaginal fluid mixed with semen.

    PubMed

    Yoshida, K; Sekiguchi, K; Mizuno, N; Kasai, K; Sakai, I; Sato, H; Seta, S

    1995-03-21

    This investigation was undertaken as an efficient method for isolating sperm DNA from a mixed fluid sample which contains vaginal epithelial cells in a greater amount. The modified method of the two-step differential extraction procedure was found to be suitable for separating sperm DNA and vaginal epithelial cell DNA from the mixed stains. As the first step of digestion, vaginal epithelial cells in the mixed stains were lysed with Proteinase K and SDS, and sperm heads remaining in the lysed solution were collected by centrifugation. As the second step digestion, the sperm heads were lysed with the buffer containing Proteinase K, SDS and DTT as reducing agent. DNA fractions extracted from the two lysed solutions were enriched, one with sperm DNA and the other with vaginal epithelial cell DNA. MCT118(D1S80), ApoB VNTR and HLADQ alpha types of sperm DNA were detected and were confirmed by matching with corresponding male blood DNA. In the case of vaginal secretion mixed with semen of two males, the mixture of MCT118 types of the two males was detected in sperm DNA fraction.

  3. A rapid TRIzol-based two-step method for DNA-free RNA extraction from Arabidopsis siliques and dry seeds.

    PubMed

    Meng, Ling; Feldman, Lewis

    2010-02-01

    Extraction of high-quality RNA from Arabidopsis seeds has been a challenge. Here we report a two-step TRIzol-based procedure for RNA extraction from Arabidopsis siliques and dry seeds. This procedure employs a modified, high pH (pH 9.5) extraction buffer. High pH plus the addition of either DTT or beta-mercaptoethanol in the extraction buffer effectively inhibits RNase activity during the extraction, and removes most polysaccharides, polyphenols and other insoluble material. TRIzol reagent was subsequently used to purify the RNA. Using this procedure we isolated high-quality DNA-free RNA samples without DNase I treatment from Arabidopsis seeds or siliques in less than 3 h.

  4. DNA extraction from formalin-fixed, paraffin-embedded tissues: protein digestion as a limiting step for retrieval of high-quality DNA.

    PubMed

    Díaz-Cano, S J; Brady, S P

    1997-12-01

    Several DNA extraction methods have been used for formalin-fixed, paraffin-embedded tissues, with variable results being reported regarding the suitability of DNA obtained from such sources to serve as template in polymerase chain reaction (PCR)-based genetic analyses. We present a method routinely used for archival material in our laboratory that reliably yields DNA of sufficient quality for PCR studies. This method is based on extended proteinase K digestion (250 micrograms/ml in an EDTA-free calcium-containing buffer supplemented with mussel glycogen) followed by phenol-chloroform extraction. Agarose gel electrophoresis of both digestion buffer aliquots and PCR amplification of the beta-globin gene tested the suitability of the retrieved DNA for PCR amplification.

  5. 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.

  6. Comparison and optimization of ancient DNA extraction.

    PubMed

    Rohland, Nadin; Hofreiter, Michael

    2007-03-01

    Ancient DNA analyses rely on the extraction of the tiny amounts of DNA remaining in samples that are hundreds to tens of thousands of years old. Despite the critical role extraction efficiency plays in this field of research, no study has comprehensively compared ancient DNA extraction techniques to date. There are a wide range of methods currently in use, which rely on such disparate principles as spin columns, alcohol precipitation, or binding to silica. We have compared a number of these methods using quantitative PCR and then optimized each step of the most promising method. We found that most chemicals routinely added to ancient DNA extraction buffers do not increase, and sometimes even decrease, DNA yields. Consequently, our optimized method uses a buffer consisting solely of EDTA and proteinase K for bone digestion and binding DNA to silica via guanidinium thiocyanate for DNA purification. In a comparison with published methods, this minimalist approach, on average, outperforms all other methods in terms of DNA yields as measured using quantitative PCR. We also found that the addition of bovine serum albumin (BSA) to the PCR helps to overcome inhibitors in ancient DNA extracts. Finally, we observed a marked difference in the performance between different types of DNA polymerases, as measured by amplification success.

  7. Extraction of Chromosomal DNA from Schizosaccharomyces pombe.

    PubMed

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

    2016-05-02

    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.

  8. DNA extraction, preservation, and amplification.

    PubMed

    Knebelsberger, Thomas; Stöger, Isabella

    2012-01-01

    The effectiveness of DNA barcoding as a routine practice in biodiversity research is strongly dependent on the quality of the source material, DNA extraction method, and selection of adequate primers in combination with optimized polymerase chain reaction (PCR) conditions. For the isolation of nucleic acids, silica-gel membrane methods are to be favored because they are easy to handle, applicable for high sample throughput, relatively inexpensive, and provide high DNA quality, quantity, and purity which are pre-requisites for successful PCR amplification and long-term storage of nucleic acids in biorepositories, such as DNA banks. In this section, standard protocols and workflow schemes for sample preparation, DNA isolation, DNA storage, PCR amplification, PCR product quality control, and PCR product cleanup are proposed and described in detail. A PCR troubleshooting and primer design section may help to solve problems that hinder successful amplification of the desired barcoding gene region.

  9. DNA extraction from formalin-fixed material.

    PubMed

    Campos, Paula F; Gilbert, Thomas M P

    2012-01-01

    The principal challenges facing PCR-based analyses of DNA extracted from formalin-fixed materials are fragmentation of the DNA and cross-linked protein-DNA complexes. Here, we present an efficient protocol to extract DNA from formalin-fixed or paraffin-embedded tissues (FFPE). In this protocol, protein-DNA cross-links are reversed using heat and alkali treatment, yielding significantly longer fragments and larger amounts of PCR-amplifiable DNA than standard DNA extraction protocols.

  10. DNA extraction from herbarium specimens.

    PubMed

    Drábková, Lenka Záveská

    2014-01-01

    With the expansion of molecular techniques, the historical collections have become widely used. Studying plant DNA using modern molecular techniques such as DNA sequencing plays an important role in understanding evolutionary relationships, identification through DNA barcoding, conservation status, and many other aspects of plant biology. Enormous herbarium collections are an important source of material especially for specimens from areas difficult to access or from taxa that are now extinct. The ability to utilize these specimens greatly enhances the research. However, the process of extracting DNA from herbarium specimens is often fraught with difficulty related to such variables as plant chemistry, drying method of the specimen, and chemical treatment of the specimen. Although many methods have been developed for extraction of DNA from herbarium specimens, the most frequently used are modified CTAB and DNeasy Plant Mini Kit protocols. Nine selected protocols in this chapter have been successfully used for high-quality DNA extraction from different kinds of plant herbarium tissues. These methods differ primarily with respect to their requirements for input material (from algae to vascular plants), type of the plant tissue (leaves with incrustations, sclerenchyma strands, mucilaginous tissues, needles, seeds), and further possible applications (PCR-based methods or microsatellites, AFLP). PMID:24415470

  11. 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.

  12. 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.

  13. 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…

  14. Evaluation of DNA and RNA extraction methods.

    PubMed

    Edwin Shiaw, C S; Shiran, M S; Cheah, Y K; Tan, G C; Sabariah, A R

    2010-06-01

    This study was done to evaluate various DNA and RNA extractions from archival FFPE tissues. A total of 30 FFPE blocks from the years of 2004 to 2006 were assessed with each modified and adapted method. Extraction protocols evaluated include the modified enzymatic extraction method (Method A), Chelex-100 extraction method (Method B), heat-induced retrieval in alkaline solution extraction method (Methods C and D) and one commercial FFPE DNA Extraction kit (Qiagen, Crawley, UK). For RNA extraction, 2 extraction protocols were evaluated including the enzymatic extraction method (Method 1), and Chelex-100 RNA extraction method (Method 2). Results show that the modified enzymatic extraction method (Method A) is an efficient DNA extraction protocol, while for RNA extraction, the enzymatic method (Method 1) and the Chelex-100 RNA extraction method (Method 2) are equally efficient RNA extraction protocols.

  15. 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.

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

    PubMed

    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

    2013-02-01

    Simple and rapid extraction of human genomic DNA remains a bottleneck 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 onto 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 four 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 1 month was demonstrated for captured DNA, facilitating straightforward collection, delivery, and handling of genomic DNA in an environment-friendly protocol.

  17. Residual soil DNA extraction increases the discriminatory power between samples.

    PubMed

    Young, Jennifer M; Weyrich, Laura S; Clarke, Laurence J; Cooper, Alan

    2015-06-01

    Forensic soil analysis relies on capturing an accurate and reproducible representation of the diversity from limited quantities of soil; however, inefficient DNA extraction can markedly alter the taxonomic abundance. The performance of a standard commercial DNA extraction kit (MOBIO PowerSoil DNA Isolation kit) and three modified protocols of this kit: soil pellet re-extraction (RE); an additional 24-h lysis incubation step at room temperature (RT); and 24-h lysis incubation step at 55°C (55) were compared using high-throughput sequencing of the internal transcribed spacer I ribosomal DNA. DNA yield was not correlated with fungal diversity and the four DNA extraction methods displayed distinct fungal community profiles for individual samples, with some phyla detected exclusively using the modified methods. Application of a 24 h lysis step will provide a more complete inventory of fungal biodiversity, and re-extraction of the residual soil pellet offers a novel tool for increasing discriminatory power between forensic soil samples.

  18. Evaluation of DNA extraction methods from complex phototrophic biofilms.

    PubMed

    Ferrera, Isabel; Massana, Ramon; Balagué, Vanessa; Pedrós-Alió, Carles; Sánchez, Olga; Mas, Jordi

    2010-04-01

    Phototrophic biofilms are used in a variety of biotechnological and industrial processes. Understanding their structure, ie microbial composition, is a necessary step for understanding their function and, ultimately, for the success of their application. DNA analysis methods can be used to obtain information on the taxonomic composition and relative abundance of the biofilm members. The potential bias introduced by DNA extraction methods in the study of the diversity of a complex phototrophic sulfide-oxidizing biofilm was examined. The efficiency of eight different DNA extraction methods combining physical, mechanical and chemical procedures was assessed. Methods were compared in terms of extraction efficiency, measured by DNA quantification, and detectable diversity (16S rRNA genes recovered), evaluated by denaturing gradient gel electrophoresis (DGGE). Significant differences were found in DNA yields ranging from 116 +/- 12 to 1893 +/- 96 ng of DNA. The different DGGE fingerprints ranged from 7 to 12 bands. Methods including phenol-chloroform extraction after enzymatic lysis resulted in the greatest DNA yields and detectable diversity. Additionally, two methods showing similar yields and retrieved diversity were compared by cloning and sequencing. Clones belonging to members of the Alpha-, Beta- and Gamma- proteobacteria, Bacteroidetes, Cyanobacteria and to the Firmicutes were recovered from both libraries. However, when bead-beating was applied, clones belonging to the Deltaproteobacteria were also recovered, as well as plastid signatures. Phenol-chloroform extraction after bead-beating and enzymatic lysis was therefore considered to be the most suitable method for DNA extraction from such highly diverse phototrophic biofilms.

  19. 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

  20. 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.

  1. 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

  2. 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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  6. Monitoring of four DNA extraction methods upstream of high-throughput sequencing of Anisakidae nematodes.

    PubMed

    Seesao, Y; Audebert, C; Verrez-Bagnis, V; Merlin, S; Jérôme, M; Viscogliosi, E; Dei-Cas, E; Aliouat-Denis, C M; Gay, M

    2014-07-01

    Different methods were evaluated to extract DNA from pooled nematodes belonging to Anisakis, Contracaecum, Pseudoterranova and Hysterothylacium genera isolated from edible fish. Pooled DNA extraction is the first and compulsory step to allow the identification of a large number of samples through high-throughput DNA sequencing with drastic time and cost reductions. PMID:24845469

  7. 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

  8. 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.

  9. Extracting DNA from submerged pine wood.

    PubMed

    Reynolds, M Megan; Williams, Claire G

    2004-10-01

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

  10. DNA extraction from keratin and chitin.

    PubMed

    Campos, Paula F; Gilbert, Thomas M P

    2012-01-01

    DNA extracted from keratinous and chitinous materials can be a useful source of genetic information. To effectively liberate the DNA from these materials, buffers containing relatively high levels of DTT, proteinase K, and detergent are recommended, followed by purification using either silica-column or organic methods.

  11. 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

  12. 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

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

    PubMed

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

    2011-08-01

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

  14. An efficient method for genomic DNA extraction from different molluscs species.

    PubMed

    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.

  15. 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.

  16. 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

  17. 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)

  18. Comparison of DNA and RNA extraction methods for mummified tissues.

    PubMed

    Konomi, Nami; Lebwohl, Eve; Zhang, David

    2002-12-01

    Nucleic acids extracted from mummified tissues are valuable materials for the study of ancient human beings. Significant difficulty in extracting nucleic acids from mummified tissues has been reported due to chemical modification and degradation. The goal of this study was to determine a method that is more efficient for DNA and RNA extraction from mummified tissues. Twelve mummy specimens were analyzed with 9 different nucleic acid extraction methods, including guanidium thiocyanate (GTC) and proteinase K/detergent based methods prepared in our laboratory or purchased. Glyceraldehyde 3-phosphate dehydrogenase DNA and beta-actin RNA were used as markers for the presence of adequate DNA and RNA, respectively, for PCR and RT-PCR amplification. Our results show that 5 M GTC is more efficient of releasing nucleic acids from mummified tissue than proteinase K/detergent, and phenol/chloroform extraction with an additional chloroform step is more efficient than phenol/chloroform along. We were able to isolate DNAs from all 12 specimens and RNAs from 8 of 12 specimens, and the nucleic acids were sufficient for PCR and RT-PCR analysis. We further tested hepatitis viruses including hepatitis B virus, hepatitis C virus, hepatitis G virus, and TT virus DNA, and fail to detect these viruses in all 12 specimens.

  19. 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.

  20. 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.

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

    PubMed

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

    2016-09-01

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

  2. 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.

  3. DNA damage-induced replication arrest in Xenopus egg extracts

    PubMed Central

    Stokes, Matthew P.; Michael, W. Matthew

    2003-01-01

    Chromosomal replication is sensitive to the presence of DNA-damaging alkylating agents, such as methyl methanesulfonate (MMS). MMS is known to inhibit replication though activation of the DNA damage checkpoint and through checkpoint-independent slowing of replication fork progression. Using Xenopus egg extracts, we now report an additional pathway that is stimulated by MMS-induced damage. We show that, upon incubation in egg extracts, MMS-treated DNA activates a diffusible inhibitor that blocks, in trans, chromosomal replication. The downstream effect of the inhibitor is a failure to recruit proliferating cell nuclear antigen, but not DNA polymerase α, to the nascent replication fork. Thus, alkylation damage activates an inhibitor that intercepts the replication pathway at a point between the polymerase α and proliferating cell nuclear antigen execution steps. We also show that activation of the inhibitor does not require the DNA damage checkpoint; rather, stimulation of the pathway described here results in checkpoint activation. These data describe a novel replication arrest pathway, and they also provide an example of how subpathways within the DNA damage response network are integrated to promote efficient cell cycle arrest in response to damaged DNA. PMID:14581453

  4. Extraction of DNA from forensic-type sexual assault specimens using simple, rapid sonication procedures.

    PubMed

    Crouse, C A; Ban, J D; D'Alessio, J K

    1993-10-01

    Sonication procedures for the extraction of DNA from forensic-type semen specimens have been developed, which, when compared to currently utilized sperm DNA extraction techniques, are simple, rapid and result in comparable DNA yields. Sperm DNA extraction by sonication was performed on whole semen, seminal stains, buccal swabs and post-coital specimens. Ultrasound disruption of sperm cells and their ultimate release of cellular DNA has been conducted in the presence of sperm wash buffers followed by organic extraction or Chelex 100 with little or no compromise to DNA quality, quantity or amplifiability. Two advantages of sonication over currently used forensic techniques to extract sperm DNA include 1) sperm DNA extraction that occurs within five minutes of sonication compared with an hour or greater for water bath incubations in classic enzyme digestion DNA extractions and 2) one less preparatory step with the Chelex/sonication protocol and three less steps with the sonication/organic protocol compared with other procedures thus eliminating potential sample-to-sample cross-contamination. Sperm DNA extracted by optimum sonication procedures was used for forensic HLA DQ alpha typing and restriction fragment length polymorphisms analysis without any adverse effects on typing results.

  5. DNA extraction from liquid blood using QIAamp.

    PubMed

    Scherczinger, C A; Bourke, M T; Ladd, C; Lee, H C

    1997-09-01

    The implementation of convicted felon DNA databases by increasing numbers of forensic science laboratories has engendered the need for a quick, efficient, and cost-effective method for the isolation of DNA from liquid blood samples. Because of the large numbers of samples involved, the ideal method would combine high throughput capability with maximal yield, high quality, and minimal time. We have found that the QIAGEN QIAamp Blood Kit/Tissue Kit satisfy all of these requirements. This simple, low cost spin column procedure yields purified DNA of approximately 20-30 kb that can be used directly in PCR or other enzymatic reactions without further purification. We compared the QIAamp isolation procedure to the standard SDS-Proteinase K/organic extraction/microcon purification procedure currently used by many forensic laboratories. The QIAamp procedure consistently gave a two- to four-fold increased yield relative to the organic extraction procedure. The DNA obtained was of high molecular weight, exhibited little degradation, and was suitable for RFLP and PCR analyses. We have found QIAGEN's QIAamp DNA isolation procedure to be ideally suited for preparation of samples for DNA databasing.

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

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

    PubMed

    Wang, Xiao-Ying; Yu, Chen-Xi

    2014-10-01

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

  8. [Rapid extraction of DNA from Chinese medicinal products by alkaline lysis].

    PubMed

    Zheng, Qi; Jiang, Chao; Huang, Lu-Qi; Zhang, Zhi-Jie; Li, Rao-Rao; Chen, Kang; Yuan, Yuan; Jin, Yan

    2014-10-01

    The study is aimed to explore a rapid method to extract DNA from fried Chinese medicinal products. The alkaline lysis buffer was made of sodium hydroxide, 1% PVP and 1% TritonX-100 and Tris-HCl solution was neutralized, through heat cracking and neutralization two step to extract DNA from processed and prepared products of traditional Chinese medicine. Then universal primes were used to amplify PCR products for fired Chinese medicinal materials. The results indicated the optimized alkaline lysis method for extracting DNA is quick and easy. Extracting of the different processed Sophora japonica of DNA concentration was (420.61 ± 123.91) g x L(-1). Using 5% Chelex-100 resin purification can improve the DNA concentration. Our results showed that the optimized alkaline lysis method is suitable for Chinese medicinal materials for quickly DNA extraction. PMID:25612420

  9. A comparison of five methods for extracting DNA from paucicellular clinical samples.

    PubMed

    Cler, Leslie; Bu, Dawei; Lewis, Cheryl; Euhus, David

    2006-01-01

    Translational protocols in cancer and carcinogenesis often require isolation of genomic DNA from paucicellular clinical samples. DNA extraction methods for PCR-based applications should optimize the recovery of amplifiable DNA. We compared five methods for DNA extraction in paucicellular epithelial and lymphocyte samples using proportion of extractions producing amplifiable DNA and mean real-time PCR Ct values for GAPDH as the endpoint measures. The methods included solid-phase DNA adsorption (QIAamp), sequential protein and DNA precipitation (Puregene), magnetic bead adsorption (Dynabeads), phenol-chloroform extraction, and single-step proteinase K digestion. In general, the performance of the three commercial kits was superior to either phenol-chloroform extraction or single-step proteinase K digestion. However, QIAamp and Puregene produced amplifiable DNA more frequently than Dynabeads for starting cell numbers <50,000. GAPDH Ct values for QIAamp extractions showed the greatest dynamic range and the best linearity across the range of starting cell numbers, but QIAamp was not statistically significantly superior to Puregene. Of the three commercial kits, Puregene is the least expensive. QIAamp and Puregene DNA extraction methods are well-suited for the preparation of paucicellular clinical samples for PCR-based assays.

  10. Rapid DNA extraction of pig ear tissues.

    PubMed

    Kunhareang, S; Zhou, H; Hickford, J G H

    2010-07-01

    A single-step DNA isolation procedure from pig tissues was developed and the product used directly for polymerase chain reaction (PCR) amplification, single-strand conformational polymorphism (SSCP) analysis and sequencing. The procedure consists of proteinase K digestion of 2-10mg of fresh tissue, at 55 degrees C for 1h, followed by application of the products of digestion to filter paper. A 1.2mm-diameter punch of that paper has sufficient DNA to act as a template for PCR amplification. The quality of the genomic DNA appeared to be high as the PCR amplicons produced sharp banding patterns on both agarose gel electrophoresis and on SSCP analysis, and they could be used for DNA sequencing following cloning. The dried genomic DNA on filter paper can be kept at room temperature. The procedure is considered effective as it is simple, fast and inexpensive. It would be useful for large-scale genotyping and could be used to obtain genomic DNA from various tissues.

  11. Extraction of DNA from Human Skeletal Material.

    PubMed

    Pajnič, Irena Zupanič

    2016-01-01

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

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

    PubMed

    Verma, Digvijay; Satyanarayana, T

    2011-09-01

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

  13. Composite system mediates two-step DNA uptake into Helicobacter pylori

    PubMed Central

    Stingl, Kerstin; Müller, Stephanie; Scheidgen-Kleyboldt, Gerda; Clausen, Martin; Maier, Berenike

    2009-01-01

    The Gram-negative gastric pathogen Helicobacter pylori depends on natural transformation for genomic plasticity, which leads to host adaptation and spread of resistances. Here, we show that H. pylori takes up covalently labeled fluorescent DNA preferentially at the cell poles and that uptake is dependent on the type IV secretion system ComB. By titration of external pH and detection of accessibility of the fluorophor by protons, we localized imported fluorescent DNA in the periplasm. Single molecule analysis revealed that outer membrane DNA transport occurred at a velocity of 1.3 kbp·s−1 and that previously imported DNA was reversibly extracted from the bacterium at pulling forces exceeding 23 pN. Thus, transport velocities were 10-fold higher than in Bacillus subtilis, and stalling forces were substantially lower. dsDNA stained with the intercalator YOYO-1 was transiently detected in the periplasm in wild-type H. pylori but was periplasmatically trapped in a mutant lacking the B. subtilis membrane-channel homolog ComEC. We conclude that H. pylori uses a two-step DNA uptake mechanism in which ComB transports dsDNA across the outer membrane at low force and poor specificity for DNA structure. Subsequently, Hp-ComEC mediates transport into the cytoplasm, leading to the release of the noncovalently bound DNA dye. Our findings fill the gap to propose a model for composite DNA uptake machineries in competent bacteria, all comprising the conserved ComEC channel for cytoplasmic membrane transport in combination with various transporters for access of external DNA to the cytoplasmic membrane. PMID:20080542

  14. 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

  15. 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.

  16. 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.

  17. [DNA extraction using Chelex resin for oncogenic amplification analysis in head and neck tumours].

    PubMed

    García González, L A; Rodrigo Tapia, J P; Sánchez Lazo, P; Ramos, S; Suárez Nieto, C

    2004-03-01

    DNA extraction from tissues can be the most laborious and complex step in amplifying DNA by PCR when phenol-choroform procedure is used. We compare this lengthy, slow and expensive extraction method with other two based in the use of Chelex-100 resin. This chelating resin has been applied for extracting DNA from different tissues to use with the PCR. These procedures are simple, rapid and do not require multiple steps. In this study we compared DNA extraction from 30 head and neck squamous cell carcinomas (HNSCC) using organic solvent precipitation, Chelex 100 resin with and without proteinase K pretreatment. The results show that proteinase K-Chelex 100 procedure is as efficient as the phenol-chloroform one.

  18. 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

  19. Thermodynamics of the DNA damage repair steps of human 8-oxoguanine DNA glycosylase.

    PubMed

    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

  20. Forensic animal DNA analysis using economical two-step direct PCR.

    PubMed

    Kitpipit, Thitika; Chotigeat, Wilaiwan; Linacre, Adrian; Thanakiatkrai, Phuvadol

    2014-03-01

    Wildlife forensic DNA analysis by amplification of a mitochondrial locus followed by DNA sequencing is routine, yet suffers from being costly and time-consuming. To address these disadvantages we report on a low-cost two-step direct PCR assay to efficiently analyze 12 forensically relevant mammalian sample types without DNA extraction. A cytochrome oxidase I degenerate-universal primer pair was designed and validated for the developed assay. The 12 sample types, which included bone, horn, feces, and urine, were amplified successfully by the assay using a pre-direct PCR dilution protocol. The average amplification success rate was as high as 92.5 % (n = 350), with an average PCR product concentration of 220.71 ± 180.84 ng/μL. Differences in amplification success rate and PCR product quantity between sample types were observed; however, most samples provided high quality sequences, permitting a 100 % nucleotide similarity to their respective species via BLAST database queries. The combination of PBS and Phire(®) Hot Start II DNA polymerase gave comparable amplification success rate and amplicon quantity with the proprietary commercial kits (P > 0.05, n = 350) but at considerably lower cost. The stability of the assay was tested by successfully amplifying samples that had been stored for up to 12 months. Our data indicate that this low-cost two-step direct amplification assay has the potential to be a valuable tool for the forensic DNA community.

  1. Viral DNA Packaging: One Step at a Time

    NASA Astrophysics Data System (ADS)

    Bustamante, Carlos; Moffitt, Jeffrey R.

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

  2. Strategy for the extraction of yeast DNA from artisan agave must for quantitative PCR analysis.

    PubMed

    Kirchmayr, Manuel Reinhart; Segura-Garcia, Luis Eduardo; Flores-Berrios, Ericka Patricia; Gschaedler, Anne

    2011-11-01

    An efficient method for the direct extraction of yeast genomic DNA from agave must was developed. The optimized protocol, which was based on silica-adsorption of DNA on microcolumns, included an enzymatic cell wall degradation step followed by prolonged lysis with hot detergent. The resulting extracts were suitable templates for subsequent qPCR assays that quantified mixed yeast populations in artisan Mexican mezcal fermentations.

  3. Strategy for the extraction of yeast DNA from artisan agave must for quantitative PCR analysis.

    PubMed

    Kirchmayr, Manuel Reinhart; Segura-Garcia, Luis Eduardo; Flores-Berrios, Ericka Patricia; Gschaedler, Anne

    2011-11-01

    An efficient method for the direct extraction of yeast genomic DNA from agave must was developed. The optimized protocol, which was based on silica-adsorption of DNA on microcolumns, included an enzymatic cell wall degradation step followed by prolonged lysis with hot detergent. The resulting extracts were suitable templates for subsequent qPCR assays that quantified mixed yeast populations in artisan Mexican mezcal fermentations. PMID:21820955

  4. 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.

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

    PubMed

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

    2011-01-01

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

  6. [Comparative analysis of 2 methods of DNA extraction].

    PubMed

    Sánchez García, I; Martín Seisdedos, C; Pérez Grande, R; Corral de la Calle, J; Rodríguez Arias, C; González-Sarmiento, R

    1990-10-01

    The study of the organization of immunoglobulin and T cell receptor genes in leukaemias and lymphomas depends on our ability to obtain high molecular weight DNA. We have compared two different methods of DNA extraction: one being enzymatic, using the enzyme proteinase K, and the other chemical, using urea as a substance purifying DNA. Since the amount and purity of the DNA obtained are similar with each of the two methods of DNA extraction, both are valid to make such a genetic analysis.

  7. 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,…

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

    PubMed

    Bjorn-Mortensen, K; Zallet, J; Lillebaek, T; Andersen, A B; Niemann, S; Rasmussen, E M; Kohl, T A

    2015-08-01

    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.

  9. Evaluation of methods for the extraction of DNA from drinking water distribution system biofilms.

    PubMed

    Hwang, Chiachi; Ling, Fangqiong; Andersen, Gary L; LeChevallier, Mark W; Liu, Wen-Tso

    2012-01-01

    While drinking water biofilms have been characterized in various drinking water distribution systems (DWDS), little is known about the impact of different DNA extraction methods on the subsequent analysis of microbial communities in drinking water biofilms. Since different DNA extraction methods have been shown to affect the outcome of microbial community analysis in other environments, it is necessary to select a DNA extraction method prior to the application of molecular tools to characterize the complex microbial ecology of the DWDS. This study compared the quantity and quality of DNA yields from selected DWDS bacteria with different cell wall properties using five widely used DNA extraction methods. These were further selected and evaluated for their efficiency and reproducibility of DNA extraction from DWDS samples. Terminal restriction fragment length analysis and the 454 pyrosequencing technique were used to interpret the differences in microbial community structure and composition, respectively, from extracted DNA. Such assessments serve as a concrete step towards the determination of an optimal DNA extraction method for drinking water biofilms, which can then provide a reliable comparison of the meta-analysis results obtained in different laboratories.

  10. 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

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

    PubMed

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

    2016-04-07

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

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

    PubMed

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

    2016-01-01

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

  13. Further studies of infectious DNA extracted from mycobacteriophages.

    PubMed

    Sellers, M I; Tokunaga, T

    1966-02-01

    Results of the previous investigation in which it was found that DNA extracted from D29 mycobacteriophage was infectious for Mycobacterium smegmatis 607, have been extended. DNA extracted from mycobacteriophage D4 and D32 produced plaques when plated on their respective hosts; D28 DNA, extracted in the same manner and tested under similar conditions, failed to show infectivity. Species barriers were not crossed by mycobacteriophage DNA; bacteria resistant to intact phage were not infected with the phage DNA. The efficiency of plating of the DNA is very much lower than that of intact phage; infection of a given host was not accomplished by DNA when titration for plaque formation by the intact phage was less than 10(9) PFU. The base composition of DNA extracted from the four mycobacteriophages and the three propagating hosts was very similar. The bases were paired, adenine with thymine and guanine with cytosine. A relatively higher per cent of guanine-cytosine than of adenine-thymine, was found. The buoyant density of each DNA in CsCl was linearly related to its guanine-cytosine content whereas with the exception of D28 DNA, thermal denaturation temperatures failed to show this relationship. However, the thermal transition profiles were characteristic of double stranded DNA. Additional evidence that D29 DNA forms complexes with basic proteins was obtained. Binding between calf thymus histone and between RNAase and D29 DNA readily occurs with a resultant loss in DNA infectivity. Trypsin and D29 DNA are only weakly reactive.

  14. 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.

  15. Rapid non-enzymatic extraction method for isolating PCR-quality camelpox virus DNA from skin.

    PubMed

    Yousif, A Ausama; Al-Naeem, A Abdelmohsen; Al-Ali, M Ahmad

    2010-10-01

    Molecular diagnostic investigations of orthopoxvirus (OPV) infections are performed using a variety of clinical samples including skin lesions, tissues from internal organs, blood and secretions. Skin samples are particularly convenient for rapid diagnosis and molecular epidemiological investigations of camelpox virus (CMLV). Classical extraction procedures and commercial spin-column-based kits are time consuming, relatively expensive, and require multiple extraction and purification steps in addition to proteinase K digestion. A rapid non-enzymatic procedure for extracting CMLV DNA from dried scabs or pox lesions was developed to overcome some of the limitations of the available DNA extraction techniques. The procedure requires as little as 10mg of tissue and produces highly purified DNA [OD(260)/OD(280) ratios between 1.47 and 1.79] with concentrations ranging from 6.5 to 16 microg/ml. The extracted CMLV DNA was proven suitable for virus-specific qualitative and, semi-quantitative PCR applications. Compared to spin-column and conventional viral DNA extraction techniques, the two-step extraction procedure saves money and time, and retains the potential for automation without compromising CMLV PCR sensitivity.

  16. One-stop genomic DNA extraction by salicylic acid-coated magnetic nanoparticles.

    PubMed

    Zhou, Zhongwu; Kadam, Ulhas S; Irudayaraj, Joseph

    2013-11-15

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

  17. 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

  18. 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.

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

    PubMed

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

    2006-08-01

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

  20. Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses

    PubMed Central

    Lim, Natalie Y. N.; Roco, Constance A.; Frostegård, Åsa

    2016-01-01

    Adequate comparisons of DNA and cDNA libraries from complex environments require methods for co-extraction of DNA and RNA due to the inherent heterogeneity of such samples, or risk bias caused by variations in lysis and extraction efficiencies. Still, there are few methods and kits allowing simultaneous extraction of DNA and RNA from the same sample, and the existing ones generally require optimization. The proprietary nature of kit components, however, makes modifications of individual steps in the manufacturer’s recommended procedure difficult. Surprisingly, enzymatic treatments are often performed before purification procedures are complete, which we have identified here as a major problem when seeking efficient genomic DNA removal from RNA extracts. Here, we tested several DNA/RNA co-extraction commercial kits on inhibitor-rich soils, and compared them to a commonly used phenol-chloroform co-extraction method. Since none of the kits/methods co-extracted high-quality nucleic acid material, we optimized the extraction workflow by introducing small but important improvements. In particular, we illustrate the need for extensive purification prior to all enzymatic procedures, with special focus on the DNase digestion step in RNA extraction. These adjustments led to the removal of enzymatic inhibition in RNA extracts and made it possible to reduce genomic DNA to below detectable levels as determined by quantitative PCR. Notably, we confirmed that DNase digestion may not be uniform in replicate extraction reactions, thus the analysis of “representative samples” is insufficient. The modular nature of our workflow protocol allows optimization of individual steps. It also increases focus on additional purification procedures prior to enzymatic processes, in particular DNases, yielding genomic DNA-free RNA extracts suitable for metatranscriptomic analysis. PMID:27803690

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

    PubMed

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

    2016-10-01

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

  2. 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.

  3. 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.

  4. Method for improved extraction of DNA from Nocardia asteroides.

    PubMed Central

    Loeffelholz, M J; Scholl, D R

    1989-01-01

    In a variation of standard DNA extraction methods, Nocardia asteroides was repeatedly exposed to sodium dodecyl sulfate at 60 degrees C for 30 min; each extraction was followed by centrifugation, removal of the nucleic acid-rich supernatant, and suspension of the cell pellet in fresh sodium dodecyl sulfate. The pooled supernatants contained a substantially higher amount of DNA than the first supernatant alone. The possible implications of this procedure on the development of DNA probes are discussed. Images PMID:2671036

  5. A PCR amplification method without DNA extraction.

    PubMed

    Li, Hongwei; Xu, Haiyue; Zhao, Chunjiang; Sulaiman, Yiming; Wu, Changxin

    2011-02-01

    To develop a simple and inexpensive method for direct PCR amplification of animal DNA from tissues, we optimized different components and their concentration in lysis buffer systems. Finally, we acquired the optimized buffer system composed of 10 mmol tris(hydroxymethyl)aminomethane (Tris)-Cl (pH 8.0), 2 mmol ethylene diamine tetraacetic (EDTA) (pH 8.0), 0.2 mol NaCl and 200 μg/mL Proteinase K. Interestingly, the optimized buffer is also very effective when working with common human sample types, including blood, buccal cells and hair. The direct PCR method requires fewer reagents (Tris-Cl, EDTA, Protease K and NaCl) and less incubation time (only 35 min). The cost of treating every sample is less than $0.02, and all steps can be completed on a thermal cycler in a 96-well format. So, the proposed method will significantly improve high-throughput PCR-based molecular assays in animal systems and in common human sample types.

  6. 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

  7. Novel extraction strategy of ribosomal RNA and genomic DNA from cheese for PCR-based investigations.

    PubMed

    Bonaïti, Catherine; Parayre, Sandrine; Irlinger, Françoise

    2006-03-15

    Cheese microorganisms, such as bacteria and fungi, constitute a complex ecosystem that plays a central role in cheeses ripening. The molecular study of cheese microbial diversity and activity is essential but the extraction of high quality nucleic acid may be problematic: the cheese samples are characterised by a strong buffering capacity which negatively influenced the yield of the extracted rRNA. The objective of this study is to develop an effective method for the direct and simultaneous isolation of yeast and bacterial ribosomal RNA and genomic DNA from the same cheese samples. DNA isolation was based on a protocol used for nucleic acids isolation from anaerobic digestor, without preliminary washing step with the combined use of the action of chaotropic agent (acid guanidinium thiocyanate), detergents (SDS, N-lauroylsarcosine), chelating agent (EDTA) and a mechanical method (bead beating system). The DNA purification was carried out by two washing steps of phenol-chloroform. RNA was isolated successfully after the second acid extraction step by recovering it from the phenolic phase of the first acid extraction. The novel method yielded pure preparation of undegraded RNA accessible for reverse transcription-PCR. The extraction protocol of genomic DNA and rRNA was applicable to complex ecosystem of different cheese matrices.

  8. Evaluation of six commercial DNA extraction kits for recovery of Burkholderia pseudomallei DNA.

    PubMed

    Marques, Maria Angela de Mello; Zimmermann, Pia; Messelhäußer, Ute; Sing, Andreas

    2012-12-01

    Six commercially available DNA extraction kits, as well as thermal lysis and proteinase K DNA extraction were evaluated regarding bacterial inactivation, DNA yield and purity, and their use in a Burkholderia pseudomallei real-time PCR. While all methods successfully inactivated the bacteria, by measuring DNA purity and the level of detection by real-time PCR, the proteinase K method was the most sensitive.

  9. 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.

  10. Study of the DNA damage checkpoint using Xenopus egg extracts.

    PubMed

    Willis, Jeremy; DeStephanis, Darla; 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 group. 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. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures. DNA damage can be induced by ultraviolet (UV) irradiation, γ-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin. MMS is an alkylating agent that inhibits DNA replication and activates the ATR

  11. 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.

  12. Single step purification of lactoperoxidase from whey involving reverse micelles-assisted extraction and its comparison with reverse micellar extraction.

    PubMed

    Nandini, K E; Rastogi, Navin K

    2010-01-01

    The extraction of lactoperoxidase (EC 1.11.1.7) from whey was studied using single step reverse micelles-assisted extraction and compared with reverse micellar extraction. The reverse micelles-assisted extraction resulted in extraction of contaminating proteins and recovery of lactoperoxidase in the aqueous phase leading to its purification. Reverse micellar extraction at the optimized condition after forward and backward steps resulted in activity recovery of lactoperoxidase and purification factor of the order of 86.60% and 3.25-fold, respectively. Whereas reverse micelles-assisted extraction resulted in higher activity recovery of lactoperoxidase (127.35%) and purification factor (3.39-fold). The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) profiles also evidenced that higher purification was obtained in reverse micelles-assisted extraction as compared of reverse micellar extracted lactoperoxidase.

  13. Optimization of DNA extraction for RAPD and ISSR analysis of Arbutus unedo L. Leaves.

    PubMed

    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 A(260)/A(280), 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

  14. 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

  15. 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.

  16. Extraction of high quality DNA from bloodstains using diatoms.

    PubMed

    Günther, S; Herold, J; Patzelt, D

    1995-01-01

    A simple method is described for the extraction of high quality DNA for PCR amplification. The DNA was extracted by using Chelex-100 ion exchange resin or a special cell lysis buffer containing proteinase K. For further purification the DNA was bound to silica in the presence of a chaotrophic agent. Hence it is possible to unlimitedly wash the bound DNA and inhibitory substances are removed. By using diatoms as a source of silicates, this method is very economical and can therefore be used as a routine method.

  17. Comparison of the DNA extraction methods for polymerase chain reaction amplification from formalin-fixed and paraffin-embedded tissues.

    PubMed

    Sato, Y; Sugie, R; Tsuchiya, B; Kameya, T; Natori, M; Mukai, K

    2001-12-01

    To obtain an adequate quality and quantity of DNA from formalin-fixed and paraffin-embedded tissue, six different DNA extraction methods were compared. Four methods used deparaffinization by xylene followed by proteinase K digestion and phenol-chloroform extraction. The temperature of the different steps was changed to obtain higher yields and improved quality of extracted DNA. The remaining two methods used microwave heating for deparaffinization. The best DNA extraction method consisted of deparaffinization by microwave irradiation, protein digestion with proteinase K at 48 degrees C overnight, and no further purification steps. By this method, the highest DNA yield was obtained and the amplification of a 989-base pair beta-globin gene fragment was achieved. Furthermore, DNA extracted by means of this procedure from five gastric carcinomas was successfully used for single strand conformation polymorphism and direct sequencing assays of the beta-catenin gene. Because the microwave-based DNA extraction method presented here is simple, has a lower contamination risk, and results in a higher yield of DNA compared with the ordinary organic chemical reagent-based extraction method, it is considered applicable to various clinical and basic fields.

  18. 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

  19. 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

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

    NASA Astrophysics Data System (ADS)

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

    2004-09-01

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

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

    PubMed

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

    2015-04-01

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

  2. Comparison of DNA extraction methods in analysis of salivary bacterial communities.

    PubMed

    Lazarevic, Vladimir; Gaïa, Nadia; Girard, Myriam; François, Patrice; Schrenzel, Jacques

    2013-01-01

    Culture-independent high-throughput sequencing-based methods are widely used to study bacterial communities. Although these approaches are superior to traditional culture-based methods, they introduce bias at the experimental and bioinformatics levels. We assessed the diversity of the human salivary microbiome by pyrosequencing of the 16S rDNA V1-3 amplicons using metagenomic DNA extracted by two different protocols: a simple proteinase K digestion without a subsequent DNA clean-up step, and a bead-beating mechanical lysis protocol followed by column DNA purification. A high degree of congruence was found between the two extraction methods, most notably in regard to the microbial community composition. The results showed that for a given bioinformatics pipeline, all the taxa with an average proportion >0.12% in samples processed using one extraction method were also detected in samples extracted using the other method. The same taxa tended to be abundant and frequent for both extraction methods. The relative abundance of sequence reads assigned to the phyla Actinobacteria, Spirochaetes, TM7, Synergistetes, and Tenericutes was significantly higher in the mechanically-treated samples than in the enzymatically-treated samples, whereas the phylum Firmicutes showed the opposite pattern. No significant differences in diversity indices were found between the extraction methods, although the mechanical lysis method revealed higher operational taxonomic unit richness. Differences between the extraction procedures outweighed the variations due to the bioinformatics analysis pipelines used.

  3. High efficiency DNA extraction from bone by total demineralization.

    PubMed

    Loreille, Odile M; Diegoli, Toni M; Irwin, Jodi A; Coble, Michael D; Parsons, Thomas J

    2007-06-01

    In historical cases, missing persons' identification, mass disasters, and ancient DNA investigations, bone and teeth samples are often the only, and almost always the best, biological material available for DNA typing. This is because of the physical and chemical barrier that the protein:mineral matrix of bone poses to environmental deterioration and biological attack. Most bone extraction protocols utilized in the forensic community involve an incubation period of bone powder in extraction buffer for proteinase digestion, followed by the collection of the supernatant, and the disposal of large quantities of undissolved bone powder. Here we present an extremely efficient protocol for recovery of DNA by complete demineralization, resulting in full physical dissolution of the bone sample. This is performed in a manner that retains and concentrates all the reagent volume, for complete DNA recovery. For our study, we selected 14 challenging bone samples. The bones were extracted side-by-side with our new demineralization protocol and the standard extraction protocol in use at AFDIL. A real-time quantification assay based on the amplification of a 143 bp mtDNA fragment showed that this new demineralization protocol significantly enhances the quantity of DNA that can be extracted and amplified from degraded skeletal remains. We have used this technique to successfully recover authentic DNA sequences from extremely challenging samples that failed repeatedly using the standard protocol.

  4. A rapid DNA extraction method from culture and clinical samples. Suitable for the detection of human cytomegalovirus by the polymerase chain reaction.

    PubMed

    Zandotti, C; De Lamballerie, X; Guignole-Vignoli, C; Bollet, C; De Micco, P

    1993-02-01

    We propose an one-step DNA extraction method suitable for the polymerase chain reaction. This procedure utilizes Chelex 100, a chelating in exchange resin. This technique was compared with a traditional technique (proteinase K lysis, phenol-chloroform extraction and ethanol precipitation) for isolation of human cytomegalovirus DNA from clinical samples. The procedure using Chelex 100 appeared to be a simple and fast extraction method for human cytomegalovirus DNA.

  5. 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

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

    PubMed

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

    2012-10-15

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

  7. Extraction of DNA from paraffin blocks for Southern blot analysis.

    PubMed

    Mies, C; Houldsworth, J; Chaganti, R S

    1991-02-01

    Tissues stored as paraffin blocks are a potential source of DNA for retrospective clinicogenetic analysis. To assess the feasibility of Southern blot analysis, DNA extracted from paraffin blocks was compared with DNA obtained from fresh-frozen controls of the same tissues. Sections 50-100 microns thick cut from paraffin blocks of 11 normal tissues, 18 lymphoid lesions, and 9 gastric carcinoma samples were deparaffinized and incubated at 45 degrees C for 48 to 72 h in a sodium dodecyl sulfate (SDS)/proteinase K solution. Following organic extraction, alcohol precipitation, restriction endonuclease digestion, and gel electrophoresis, DNA was transferred to nylon membranes. 32P-labelled DNA probes for the immunoglobulin heavy-chain locus and T-cell receptor beta-chain gene were hybridized to the normal tissue and lymphoid samples; the gastric cancers were probed for the HER-2/neu protooncogene. Intact DNA was obtained from the majority of formalin-fixed samples, yielding results qualitatively similar to those from fresh tissues. Degradation is the most significant problem in analyzing DNA extracted from paraffin blocks and compromises accurate quantitation. DNA analysis using paraffin-embedded tissue has potential clinical and research applications and may be a particularly useful way to study gene abnormalities in unusual tumors infrequently available as fresh specimens.

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

    PubMed

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

    2016-09-01

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

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

    PubMed

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

    2016-09-01

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

  10. Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities.

    PubMed

    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

  11. 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

  12. 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.

  13. [Analysis of different methods of extracting DNA from paraffin-embedded tissues and the application of nest PCR].

    PubMed

    Yan, Limin; Sun, Baocun; Zhao, Xiulan; Liu, Zenghui; Song, Wenjing

    2011-08-01

    The aim of this research was to explore the most optimal method of DNA extraction from formalin-fixed, paraffin-embedded (FFPE) tissues, and to improve the amplification of long fragments with the method. Three methods, one step method, phenol-chloroform extraction method, and genomic DNA purification kit method, were employed to extract DNA from twenty normal thyroid tissues which were fixed with formalin and embedded with paraffin. The highest proportionality of OD260/OD280 in the examples was obtained by phenol-chloroform extraction method, 1.703 +/- 0.086, compared to the results of the other two methods. As for the long DNA segments amplification, the achievement ratio of one step method, phenol-chloroform extraction method and genomic DNA purification kit method were 0%, 5% and 10%, respectively, by traditional PCR method, but 0%, 95% and 85% respectively by the nest PCR. We have found that the best process of extracting DNA from FFPE is digesting by proteinase K and purifying by phenol-chloroform, and it is effective to amplify long DNA segments from FFPE by nest PCR.

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

    PubMed

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

    2016-08-01

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

  15. 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

  16. An improved electroelution method for separation of DNA from humic substances in marine sediment DNA extracts.

    PubMed

    Kallmeyer, Jens; Smith, David C

    2009-07-01

    We present a method for the rapid and simple extraction of DNA from marine sediments using electroelution. It effectively separates DNA from compounds, including humic substances, that interfere with subsequent DNA quantification and amplification. After extraction of the DNA from the sediment into an aqueous solution, the crude sample is encased in 2% agarose gel and exposed to an electrical current, which draws the DNA out of the gel into a centrifugal filter vial. After electroelution, the sample is centrifuged to remove contaminants DNA using this method is quantitative and does not discriminate on the basis of size, as determined using DNA standards and DNA extracts from environmental samples. Amplification of DNA is considerably improved due to removal of PCR inhibitors. For Archaea, only these purified extracts yielded PCR products. This method allows for the use of relatively large volumes of sediment and is particularly useful for sediments containing low biomass such as deeply buried marine sediments. It works with both organic-rich and -poor sediment, as well as with sediment where calcium carbonate is abundant and sediment where it is limited; consequently, adjustment of protocols is unnecessary for samples with very different organic and mineral contents.

  17. 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.

  18. Automated DNA extraction of single dog hairs without roots for mitochondrial DNA analysis.

    PubMed

    Bekaert, Bram; Larmuseau, Maarten H D; Vanhove, Maarten P M; Opdekamp, Anouschka; Decorte, Ronny

    2012-03-01

    Dogs are intensely integrated in human social life and their shed hairs can play a major role in forensic investigations. The overall aim of this study was to validate a semi-automated extraction method for mitochondrial DNA analysis of telogenic dog hairs. Extracted DNA was amplified with a 95% success rate from 43 samples using two new experimental designs in which the mitochondrial control region was amplified as a single large (± 1260 bp) amplicon or as two individual amplicons (HV1 and HV2; ± 650 and 350 bp) with tailed-primers. The results prove that the extraction of dog hair mitochondrial DNA can easily be automated to provide sufficient DNA yield for the amplification of a forensically useful long mitochondrial DNA fragment or alternatively two short fragments with minimal loss of sequence in case of degraded samples.

  19. 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.; 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.

  20. Yeast Pif1 Helicase Exhibits a One-base-pair Stepping Mechanism for Unwinding Duplex DNA*

    PubMed Central

    Ramanagoudr-Bhojappa, Ramanagouda; Chib, Shubeena; Byrd, Alicia K.; Aarattuthodiyil, Suja; Pandey, Manjula; Patel, Smita S.; Raney, Kevin D.

    2013-01-01

    Kinetic analysis of the DNA unwinding and translocation activities of helicases is necessary for characterization of the biochemical mechanism(s) for this class of enzymes. Saccharomyces cerevisiae Pif1 helicase was characterized using presteady state kinetics to determine rates of DNA unwinding, displacement of streptavidin from biotinylated DNA, translocation on single-stranded DNA (ssDNA), and ATP hydrolysis activities. Unwinding of substrates containing varying duplex lengths was fit globally to a model for stepwise unwinding and resulted in an unwinding rate of ∼75 bp/s and a kinetic step size of 1 base pair. Pif1 is capable of displacing streptavidin from biotinylated oligonucleotides with a linear increase in the rates as the length of the oligonucleotides increased. The rate of translocation on ssDNA was determined by measuring dissociation from varying lengths of ssDNA and is essentially the same as the rate of unwinding of dsDNA, making Pif1 an active helicase. The ATPase activity of Pif1 on ssDNA was determined using fluorescently labeled phosphate-binding protein to measure the rate of phosphate release. The quantity of phosphate released corresponds to a chemical efficiency of 0.84 ATP/nucleotides translocated. Hence, when all of the kinetic data are considered, Pif1 appears to move along DNA in single nucleotide or base pair steps, powered by hydrolysis of 1 molecule of ATP. PMID:23596008

  1. Rapid and effective DNA extraction method with bead grinding for a large amount of fungal DNA.

    PubMed

    Watanabe, M; Lee, K; Goto, K; Kumagai, S; Sugita-Konishi, Y; Hara-Kudo, Y

    2010-06-01

    To identify a rapid method for extracting a large amount of DNA from fungi associated with food hygiene, extraction methods were compared using fungal pellets formed rapidly in liquid media. Combinations of physical and chemical methods or commercial kits were evaluated with 3 species of yeast, 10 species of ascomycetous molds, and 4 species of zygomycetous molds. Bead grinding was the physical method, followed by chemical methods involving sodium dodecyl sulfate (SDS), cetyl trimethyl ammonium bromide (CTAB), and benzyl chloride and two commercial kits. Quantity was calculated by UV absorbance at 260 nm, quality was determined by the ratio of UV absorbance at 260 and 280 nm, and gene amplifications and electrophoresis profiles of whole genomes were analyzed. Bead grinding with the SDS method was the most effective for DNA extraction for yeasts and ascomycetous molds, and bead grinding with the CTAB method was most effective with zygomycetous molds. For both groups of molds, bead grinding with the CTAB method was the best approach for DNA extraction. Because this combination also is relatively effective for yeasts, it can be used to extract a large amount of DNA from a wide range of fungi. The DNA extraction methods are useful for developing gene indexes to identify fungi with molecular techniques, such as DNA fingerprinting.

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

    PubMed

    Jeddi, Fakhri; Piarroux, Renaud; Mary, Charles

    2013-01-01

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

  3. Bacterial and fungal DNA extraction from blood samples: manual protocols.

    PubMed

    Lorenz, Michael G; Mühl, Helge; Disqué, Claudia

    2015-01-01

    A critical point of molecular diagnosis of systemic infections is the method employed for the extraction of microbial DNA from blood. A DNA isolation method has to be able to fulfill several fundamental requirements for optimal performance of diagnostic assays. First of all, low- and high-molecular-weight substances of the blood inhibitory to downstream analytical reactions like PCR amplification have to be removed. This includes human DNA which is a known source of false-positive results and factor decreasing the analytical sensitivity of PCR assays by unspecific primer binding. At the same time, even extremely low amounts of microbial DNA need to be supplied to molecular diagnostic assays in order to detect low pathogen loads in the blood. Further, considering the variety of microbial etiologies of sepsis, a method should be capable of lysing Gram-positive, Gram-negative, and fungal organisms. Last, extraction buffers, reagents, and consumables have to be free of microbial DNA which leads to false-positive results. Here, we describe manual methods which allow the extraction of microbial DNA from small- and large-volume blood samples for the direct molecular analysis of pathogen.

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

    PubMed

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

    2016-09-21

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

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

    PubMed

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

    2016-01-01

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

  6. Random amplified polymorphic DNA analysis of DNA extracted from Trichuris trichiura (Linnaeus, 1771) eggs and its prospective application to paleoparasitological studies.

    PubMed

    Martinez, Elaine Machado; Correia, Jorge Antonio Santos; Villela, Erika Verissimo; Duarte, Antonio Nascimento; Ferreira, Luiz Fernando; Bello, Alexandre Ribeiro

    2003-01-01

    Random amplified polymorphic DNA analysis was applied to DNAs extracted from Trichuris trichiura eggs recovered from human fecal samples. Four out of 6 primers tested displayed 18 distinct and well defined polymorphic patterns, ranging from 650 to 3200 base pairs. These results, upon retrieval and DNA sequencing of some of these bands from agarose gels, might help in establishing. T. trichiura specific genetic markers, not available yet, and an important step to design primers to be used in molecular diagnosis approaches.

  7. 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.

  8. [Adaptation of a sensitive DNA extraction method for detection of Entamoeba histolytica by real-time polymerase chain reaction].

    PubMed

    Pınar, Ahmet; Akyön, Yakut; Alp, Alpaslan; Ergüven, Sibel

    2010-07-01

    This study was aimed to adapt a sensitive DNA extraction protocol in stool samples for real-time polymerase chain reaction (PCR) detection of Entamoeba histolytica which causes important morbidity and mortality worldwide. Stool extraction is a problematic step and has direct effects on PCR sensitivity. In order to improve the sensitivity of E.histolytica detection by real-time PCR, "QIAamp DNA stool minikit (Qiagen, Germany)" was modified by adding an overnight incubation step with proteinase K and sodium dodecyl sulfate (SDS) in this study. Three different extraction methods [(1) original method, (2) cetyltrimethyl-ammonium bromide (CTAB) method, (3) modified method] were evaluated for effects on sensitivity in real-time quantitative PCR (Artus RealArt TM E.histolytica RG PCR Kit, Qiagen Diagnostics, Germany). For this purpose, several concentrations of standard E.histolytica DNA were spiked in parasite-free stool samples and three different extraction protocols were performed. Detection sensitivities of "QIAamp DNA stool minikit" was found 5000 copies/ml and of CTAB method was found 500 copies/ml. Detection sensitivity of the extraction was improved to 5 copies/mL by modified "QIAamp DNA stool minikit" protocol. Since detection sensitivities of nucleic acid extraction protocols from stool samples directly affect the sensitivity of PCR amplification, different extraction protocols for different microorganisms should be evaluated.

  9. 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.

  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. 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.

  12. Rapid DNA extraction methods and new primers for randomly amplified polymorphic DNA analysis of Giardia duodenalis.

    PubMed

    Deng, M Q; Cliver, D O

    1999-08-01

    A randomly amplified polymorphic DNA (RAPD) procedure using simple genomic DNA preparation methods and newly designed primers was optimized for analyzing Giardia duodenalis strains. Genomic DNA was extracted from in vitro cultivated trophozoites by five freezing-thawing cycles or by sonic treatment. Compared to a conventional method involving proteinase K digestion and phenol extraction, both freezing-thawing and sonication were equally efficient, yet with the advantage of being much less time- and labor-intensive. Five of the 10 tested RAPD primers produced reproducible polymorphisms among five human origin G. duodenalis strains, and grouping of these strains based on RAPD profiles was in agreement among these primers. The consistent classification of two standard laboratory reference strains, Portland-1 and WB, in the same group confirmed previous results using other fingerprinting methods, indicating that the reported simple DNA extraction methods and the selected primers are useful in RAPD for molecular characterization of G. duodenalis strains.

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

    PubMed

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

    2014-11-01

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

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

    PubMed

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

    2015-01-01

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

  15. 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

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

    PubMed

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

    2016-01-01

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

  17. Back to basics: an evaluation of NaOH and alternative rapid DNA extraction protocols for DNA barcoding, genotyping, and disease diagnostics from fungal and oomycete samples.

    PubMed

    Osmundson, Todd W; Eyre, Catherine A; Hayden, Katherine M; Dhillon, Jaskirn; Garbelotto, Matteo M

    2013-01-01

    The ubiquity, high diversity and often-cryptic manifestations of fungi and oomycetes frequently necessitate molecular tools for detecting and identifying them in the environment. In applications including DNA barcoding, pathogen detection from plant samples, and genotyping for population genetics and epidemiology, rapid and dependable DNA extraction methods scalable from one to hundreds of samples are desirable. We evaluated several rapid extraction methods (NaOH, Rapid one-step extraction (ROSE), Chelex 100, proteinase K) for their ability to obtain DNA of quantity and quality suitable for the following applications: PCR amplification of the multicopy barcoding locus ITS1/5.8S/ITS2 from various fungal cultures and sporocarps; single-copy microsatellite amplification from cultures of the phytopathogenic oomycete Phytophthora ramorum; probe-based P. ramorum detection from leaves. Several methods were effective for most of the applications, with NaOH extraction favored in terms of success rate, cost, speed and simplicity. Frozen dilutions of ROSE and NaOH extracts maintained PCR viability for over 32 months. DNA from rapid extractions performed poorly compared to CTAB/phenol-chloroform extracts for TaqMan diagnostics from tanoak leaves, suggesting that incomplete removal of PCR inhibitors is an issue for sensitive diagnostic procedures, especially from plants with recalcitrant leaf chemistry. NaOH extracts exhibited lower yield and size than CTAB/phenol-chloroform extracts; however, NaOH extraction facilitated obtaining clean sequence data from sporocarps contaminated by other fungi, perhaps due to dilution resulting from low DNA yield. We conclude that conventional extractions are often unnecessary for routine DNA sequencing or genotyping of fungi and oomycetes, and recommend simpler strategies where source materials and intended applications warrant such use.

  18. 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.

  19. 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)

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

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

    PubMed

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

    2016-08-19

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

  3. 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.

  4. Use of a rapid and simple method to extract proviral DNA in the identification of HIV-1 by PCR.

    PubMed

    Tagliaferro, L; Corbelli, M; Maietta, G; Pellegrino, V; Pignatelli, P

    1995-07-01

    DNA extraction is a critical step in PCR analysis and is closely related to its sensitivity. Traditional methods, based on phenol-chloroform extraction, require more time and the use of toxic reagents. GeneReleaser (Bio Ventures Inc.) is a commercial product which releases DNA from whole blood, cell cultures, bacterial colonies and the like. Cells lysis and DNA extraction are accomplished directly in the amplification tube on a thermocycler. We used GeneReleaser in the identification of HIV-1 proviral DNA by PCR on whole blood samples. All samples arrived at our laboratory for HIV-1 detection were treated with two different procedures. The classical one was based on the lysis of separated lymphocytes by proteinase K, while the other consisted in DNA extraction by GeneReleaser from 5 microliters of whole blood in sodium citrate. All samples were amplified for HIV-1 GAG region; to prevent carry-over contamination Uracil N-glycosylase (UNG) sterilization was performed. Amplified sequences were revealed using the DEIA commercial system (Sorin Biomedica, Italy). To verify the suitability both of cell lysates and GeneReleaser DNA-extracted samples for PCR, we amplified a specific sequence of HLA-DQ-alpha gene. Initial data indicate that this new method might reduce the performance time of PCR (DNA extraction time was around 15 minutes) and improve PCR sensitivity.

  5. 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

  6. Evaluation of DNA extraction methods of rumen microbial populations.

    PubMed

    Villegas-Rivera, Gabriela; Vargas-Cabrera, Yevani; González-Silva, Napoleón; Aguilera-García, Florentino; Gutiérrez-Vázquez, Ernestina; Bravo-Patiño, Alejandro; Cajero-Juárez, Marcos; Baizabal-Aguirre, Víctor Manuel; Valdez-Alarcón, Juan José

    2013-02-01

    The dynamism of microbial populations in the rumen has been studied with molecular methods that analyze single nucleotide polymorphisms of ribosomal RNA gene fragments (rDNA). Therefore DNA of good quality is needed for this kind of analysis. In this work we report the evaluation of four DNA extraction protocols (mechanical lysis or chemical lysis with CTAB, ethylxanthogenate or DNAzol(®)) from ruminal fluid. The suitability of two of these protocols (mechanical lysis and DNAzol(®)) was tested on single-strand conformation polymorphism (SSCP) of rDNA of rumen microbial populations. DNAzol(®) was a simple method that rendered good integrity, yield and purity. With this method, subtle changes in protozoan populations were detected in young bulls fed with slightly different formulations of a supplement of multinutritional blocks of molasses and urea. Sequences related to Eudiplodinium maggi and a non-cultured Entodiniomorphid similar to Entodinium caudatum, were related to major fluctuating populations in an SSCP assay.

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

    NASA Astrophysics Data System (ADS)

    Morales, Mercedes C.

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

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

    PubMed Central

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

    2006-01-01

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

  9. 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

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

    PubMed

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

    2007-11-01

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

  11. PCR-based typing of DNA extracted from cigarette butts.

    PubMed

    Hochmeister, M N; Budowle, B; Jung, J; Borer, U V; Comey, C T; Dirnhofer, R

    1991-01-01

    Limited genetic marker information can be obtained from saliva by typing by conventional serological means. Thus, the application of PCR-based DNA typing methods was investigated as a potential approach for typing genetic markers in saliva. DNA was isolated from 200 cigarettes smoked by 10 different individuals (20 cigarettes per individual) and from 3 cigarette butts recovered from 2 crime scenes (adjudicated cases) using a Chelex 100 extraction procedure. The amount of recovered human DNA was quantified by slot-blot analysis and ranged from approximately less than 2-160 ng DNA per cigarette butt for the 200 samples, and 8 ng, 50 ng, and 100 ng for the cigarette butts from the adjudicated cases. The DNA was successfully amplified by the polymerase chain reaction (PCR) for the HLA-DQ alpha locus (99 out of 100 samples) as well as for the variable number of tandem repeat (VNTR) locus D1S80 (99 out of 100 samples). Amplification and typing of DNA was successful on all samples recovered from the crime scenes. The results suggest that PCR-based typing of DNA offers a potential method for genetically characterizing traces of saliva on cigarette butts.

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

    PubMed

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

    2015-06-01

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

  13. 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…

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

    PubMed

    Hawash, Yousry

    2014-06-01

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

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

    NASA Astrophysics Data System (ADS)

    Fotouhi, Gareth

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2014-12-10

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

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

    PubMed

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

    2015-06-11

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

  19. DNA from microdissected tissues may be extracted and stored on microscopic slides.

    PubMed

    Korabecna, M; Steiner, P; Jirkovska, M

    2016-01-01

    With regard to complex structure of tissues, laser capture microdissection represents an important step in analytical workflow streaming to proper molecular characterization of different cell types in examined samples. Therefore the simple method for simultaneous processing of higher numbers of microdissected tissues leading not only to rapid and efficient DNA isolation but allowing also the repeated sampling and easy storage may be useful in the practice of histopathological laboratories. We elaborated such a methodology applicable downstream after the microdissection from formalin-fixed paraffin embedded tissues.The tissues for examination are microdissected directly into the circular areas having the diameter 2 mm and marked on the microscopic slide. In this way, one slide is able to accommodate multiple samples. The DNA extraction is performed in low volume of buffer with Proteinase K in a droplet covered by mineral oil just on the slide. Mineral oil in the quality for molecular biology not only avoids evaporation during DNA extraction, but it helps to position the microdisssected tissue, to control the level of cell lysis microscopically and to protect the DNA sample during subsequent manipulations. We provided the evidence that DNA isolated by our methodology remains in the positions on microscopic slide for months without any changes in the lengths of available fragments and that it may be removed from each position repetitively for different kinds of analysis. The new methodological approach presented by us can be practically applied in broad spectrum of laboratories performing routinely genetic analysis on microdissected tissues. PMID:27268914

  20. DNA from microdissected tissues may be extracted and stored on microscopic slides.

    PubMed

    Korabecna, M; Steiner, P; Jirkovska, M

    2016-01-01

    With regard to complex structure of tissues, laser capture microdissection represents an important step in analytical workflow streaming to proper molecular characterization of different cell types in examined samples. Therefore the simple method for simultaneous processing of higher numbers of microdissected tissues leading not only to rapid and efficient DNA isolation but allowing also the repeated sampling and easy storage may be useful in the practice of histopathological laboratories. We elaborated such a methodology applicable downstream after the microdissection from formalin-fixed paraffin embedded tissues.The tissues for examination are microdissected directly into the circular areas having the diameter 2 mm and marked on the microscopic slide. In this way, one slide is able to accommodate multiple samples. The DNA extraction is performed in low volume of buffer with Proteinase K in a droplet covered by mineral oil just on the slide. Mineral oil in the quality for molecular biology not only avoids evaporation during DNA extraction, but it helps to position the microdisssected tissue, to control the level of cell lysis microscopically and to protect the DNA sample during subsequent manipulations. We provided the evidence that DNA isolated by our methodology remains in the positions on microscopic slide for months without any changes in the lengths of available fragments and that it may be removed from each position repetitively for different kinds of analysis. The new methodological approach presented by us can be practically applied in broad spectrum of laboratories performing routinely genetic analysis on microdissected tissues.

  1. 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.

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

    PubMed

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

    2011-06-15

    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.

  3. 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

    2015-12-22

    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.

  4. 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

  5. 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.

  6. 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

  7. Evaluation of Automated and Manual Commercial DNA Extraction Methods for Recovery of Brucella DNA from Suspensions and Spiked Swabs ▿

    PubMed Central

    Dauphin, Leslie A.; Hutchins, Rebecca J.; Bost, Liberty A.; Bowen, Michael D.

    2009-01-01

    This study evaluated automated and manual commercial DNA extraction methods for their ability to recover DNA from Brucella species in phosphate-buffered saline (PBS) suspension and from spiked swab specimens. Six extraction methods, representing several of the methodologies which are commercially available for DNA extraction, as well as representing various throughput capacities, were evaluated: the MagNA Pure Compact and the MagNA Pure LC instruments, the IT 1-2-3 DNA sample purification kit, the MasterPure Complete DNA and RNA purification kit, the QIAamp DNA blood mini kit, and the UltraClean microbial DNA isolation kit. These six extraction methods were performed upon three pathogenic Brucella species: B. abortus, B. melitensis, and B. suis. Viability testing of the DNA extracts indicated that all six extraction methods were efficient at inactivating virulent Brucella spp. Real-time PCR analysis using Brucella genus- and species-specific TaqMan assays revealed that use of the MasterPure kit resulted in superior levels of detection from bacterial suspensions, while the MasterPure kit and MagNA Pure Compact performed equally well for extraction of spiked swab samples. This study demonstrated that DNA extraction methodologies differ in their ability to recover Brucella DNA from PBS bacterial suspensions and from swab specimens and, thus, that the extraction method used for a given type of sample matrix can influence the sensitivity of real-time PCR assays for Brucella. PMID:19846627

  8. Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification.

    PubMed Central

    Pääbo, S

    1989-01-01

    Several chemical and enzymatic properties were examined in the DNA extracted from dry remains of soft tissues that vary in age from 4 to 13,000 years and represent four species, including two extinct animals (the marsupial wolf and giant ground sloth). The DNA obtained was invariably of a low average molecular size and damaged by oxidative processes, which primarily manifest themselves as modifications of pyrimidines and sugar residues as well as baseless sites and intermolecular cross-links. This renders molecular cloning difficult. However, the polymerase chain reaction can be used to amplify and study short mitochondrial DNA sequences that are of anthropological and evolutionary significance. This opens up the prospect of performing diachronical studies of molecular evolutionary genetics. Images PMID:2928314

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. Evaluation of a new experimental kit for the extraction of DNA from bones and teeth using a non-powder method.

    PubMed

    Kitayama, Tetsushi; Ogawa, Yoshinori; Fujii, Koji; Nakahara, Hiroaki; Mizuno, Natsuko; Sekiguchi, Kazumasa; Kasai, Kentaro; Yurino, Noriko; Yokoi, Takahide; Fukuma, Yoshiya; Yamamoto, Kenji; Oki, Takahito; Asamura, Hideki; Fukushima, Hirofumi

    2010-03-01

    An experimental DNA extraction kit (new kit) was recently developed to extract DNA from degraded skeletal remains without the need for powdering the samples. We compared the utility of the new kit with the conventional phenol/chloroform method using real-time quantitative PCR and multiplex STR analysis. The new kit yielded large amounts of DNA from a compact bone fragment compared with the conventional phenol/chloroform method. We were able to extract sufficient DNA for STR analysis from 75% (3 of 4) and 60% (3 of 5) of the un-powdered tooth and bone samples, respectively, using the new kit. We were able to perform mini-STR analysis of the remaining samples using DNA extracted with the new kit. Furthermore, we successfully performed mitochondrial DNA sequencing of every sample. The new kit simplifies the DNA extraction procedure as it does not require powdering samples. Decreasing the number of procedural steps in DNA extraction will be beneficial in controlling DNA contamination in laboratories. Our results suggest that the new kit may be used for the simple, simultaneous extraction of DNA from multiple samples.

  14. 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

  15. 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.

  16. A two-step strategy for constructing specifically self-subtracted cDNA libraries

    PubMed Central

    Laveder, Paolo; De Pittà, Cristiano; Toppo, Stefano; Valle, Giorgio; Lanfranchi, Gerolamo

    2002-01-01

    We have developed a new strategy for producing subtracted cDNA libraries that is optimized for connective and epithelial tissues, where a few exceptionally abundant (super-prevalent) RNA species account for a large fraction of the total mRNA mass. Our method consists of a two-step subtraction of the most abundant mRNAs: the first step involves a novel use of oligo-directed RNase H digestion to lower the concentration of tissue-specific, super-prevalent RNAs. In the second step, a highly specific subtraction is achieved through hybridization with probes from a 3′-end ESTs collection. By applying this technique in skeletal muscle, we have constructed subtracted cDNA libraries that are effectively enriched for genes expressed at low levels. We further report on frequent premature termination of transcription in human muscle mitochondria and discuss the importance of this phenomenon in designing subtractive approaches. The tissue-specific collections of cDNA clones generated by our method are particularly well suited for expression profiling. PMID:11972353

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

    PubMed

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

    2016-01-01

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

  18. Clonetegration Using OSIP Plasmids: One-Step DNA Assembly and Site-Specific Genomic Integration in Bacteria.

    PubMed

    Cui, Lun; Shearwin, Keith E

    2017-01-01

    Clonetegration is a method for site-specific insertion of DNA into prokaryotic chromosomes, based on bacteriophage integrases. The method combines DNA cloning/assembly and chromosomal integration into a single step, providing a simple and rapid strategy for inserting DNA sequences into bacterial chromosomes. PMID:27671938

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-08-21

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

  1. DNA extraction and a cost-effective detection method for Echinococcus granulosus protoscoleces.

    PubMed

    Petrigh, R S; Fugassa, M H

    2013-12-01

    Most methods of DNA purification from protoscoleces of Echinococcus granulosus involve the use of expensive kits and may also require a second step after extraction for an effective purification. The present work describes an optimized cost-effective method that is fast and simple. This method is based on a chemical lysis with proteinase K with a subsequent one-step PCR detection. In this study we used already available primers and newly designed primers to amplify two fragments of different size corresponding to the mitochondrial cytochrome C oxidase subunit 1 gene. By one-step PCR, both fragments were successfully amplified from even a single protoscolex. This result demonstrates that this method of extraction is efficient even with small amounts of sample and that PCR is highly sensitive. The major advantage of this lysis-PCR method is that it avoids a second step of purification resulting in a simpler and more economical method. Our research will serve as a base for future studies on E. granulosus genotyping, mainly with wild mammals with a low number of cysts.

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

    PubMed Central

    2007-01-01

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

  3. 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.

  4. 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

  5. Extraction of DNA from malaria-infected erythrocytes using isotachophoresis.

    PubMed

    Marshall, Lewis A; Han, Crystal M; Santiago, Juan G

    2011-12-15

    We demonstrate a technique for purification of nucleic acids from malaria parasites infecting human erythrocytes using isotachophoresis (ITP). We release nucleic acids from malaria-infected erythrocytes by lysing with heat and proteinase K for 10 min and immediately, thereafter, load sample onto a capillary device. We study the effect of temperature on lysis efficiency. We also implement pressure-driven counterflow during ITP extraction to extend focusing time and increase nucleic acid yield. We show that the purified genomic DNA samples are compatible with polymerase chain reaction (PCR) and demonstrate a clinically relevant limit of detection of 0.5 parasites per nanoliter using quantitative PCR.

  6. Development of a real-world direct interface for integrated DNA extraction and amplification in a microfluidic device.

    PubMed

    Shaw, Kirsty J; Joyce, Domino A; Docker, Peter T; Dyer, Charlotte E; Greenway, Gillian M; Greenman, John; Haswell, Stephen J

    2011-02-01

    Integrated DNA extraction and amplification have been carried out in a microfluidic device using electro-osmotic pumping (EOP) for fluidic control. All the necessary reagents for performing both DNA extraction and polymerase chain reaction (PCR) amplification were pre-loaded into the microfluidic device following encapsulation in agarose gel. Buccal cells were collected using OmniSwabs [Whatman™, UK] and manually added to a chaotropic binding/lysis solution pre-loaded into the microfluidic device. The released DNA was then adsorbed onto a silica monolith contained within the DNA extraction chamber and the microfluidic device sealed using polymer electrodes. The washing and elution steps for DNA extraction were carried out using EOP, resulting in transfer of the eluted DNA into the PCR chamber. Thermal cycling, achieved using a Peltier element, resulted in amplification of the Amelogenin locus as confirmed using conventional capillary gel electrophoresis. It was demonstrated that the PCR reagents could be stored in the microfluidic device for at least 8 weeks at 4 °C with no significant loss of activity. Such methodology lends itself to the production of 'ready-to-use' microfluidic devices containing all the necessary reagents for sample processing, with many obvious applications in forensics and clinical medicine.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  9. '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

  10. Two approaches to the blind phase shift extraction for two-step electronic speckle pattern interferometry

    NASA Astrophysics Data System (ADS)

    Muravsky, Leonid; Kmet', Arkady; Voronyak, Taras

    2013-10-01

    A two-step electronic speckle pattern interferometry (ESPI) method with blind phase shift of a reference wave, in which the phase shift extraction is fulfilled by using both a correlation and a discrete Fourier transform (DFT) approaches, is presented here. In the correlation approach, the blind phase shift is calculated via the correlation coefficient between two similar speckle interferograms (SI) differing only by the reference wave phase shift. The DFT approach is based on the blind phase shift extraction from frequency components of SI Fourier spectra. Comparative analysis of these approaches has testified to their high performance. Moreover, it is shown that the correlation approach is more preferable for blind phase shift extraction from SIs with a rough surface than from interferograms with a smooth surface; hence, it is more convenient for ESPI than for phase-shifting interferometry. In addition, this approach provides a lesser level of systematic error of extracted phase shift in comparison with the DFT. The correlation approach was used for experimental definition of Poisson's ratio for duralumin constructional material by the two-step ESPI method.

  11. Single-step capture and sequencing of natural DNA for detection of BRCA1 mutations

    PubMed Central

    Thompson, John F.; Reifenberger, Jeffrey G.; Giladi, Eldar; Kerouac, Kristen; Gill, Jaime; Hansen, Erik; Kahvejian, Avak; Kapranov, Philipp; Knope, Travis; Lipson, Doron; Steinmann, Kathleen E.; Milos, Patrice M.

    2012-01-01

    Genetic testing for disease risk is an increasingly important component of medical care. However, testing can be expensive, which can lead to patients and physicians having limited access to the genetic information needed for medical decisions. To simplify DNA sample preparation and lower costs, we have developed a system in which any gene can be captured and sequenced directly from human genomic DNA without amplification, using no proteins or enzymes prior to sequencing. Extracted whole-genome DNA is acoustically sheared and loaded in a flow cell channel for single-molecule sequencing. Gene isolation, amplification, or ligation is not necessary. Accurate and low-cost detection of DNA sequence variants is demonstrated for the BRCA1 gene. Disease-causing mutations as well as common variants from well-characterized samples are identified. Single-molecule sequencing generates very reproducible coverage patterns, and these can be used to detect any size insertion or deletion directly, unlike PCR-based methods, which require additional assays. Because no gene isolation or amplification is required for sequencing, the exceptionally low costs of sample preparation and analysis could make genetic tests more accessible to those who wish to know their own disease susceptibility. Additionally, this approach has applications for sequencing integration sites for gene therapy vectors, transposons, retroviruses, and other mobile DNA elements in a more facile manner than possible with other methods. PMID:21765009

  12. Single-step capture and sequencing of natural DNA for detection of BRCA1 mutations.

    PubMed

    Thompson, John F; Reifenberger, Jeffrey G; Giladi, Eldar; Kerouac, Kristen; Gill, Jaime; Hansen, Erik; Kahvejian, Avak; Kapranov, Philipp; Knope, Travis; Lipson, Doron; Steinmann, Kathleen E; Milos, Patrice M

    2012-02-01

    Genetic testing for disease risk is an increasingly important component of medical care. However, testing can be expensive, which can lead to patients and physicians having limited access to the genetic information needed for medical decisions. To simplify DNA sample preparation and lower costs, we have developed a system in which any gene can be captured and sequenced directly from human genomic DNA without amplification, using no proteins or enzymes prior to sequencing. Extracted whole-genome DNA is acoustically sheared and loaded in a flow cell channel for single-molecule sequencing. Gene isolation, amplification, or ligation is not necessary. Accurate and low-cost detection of DNA sequence variants is demonstrated for the BRCA1 gene. Disease-causing mutations as well as common variants from well-characterized samples are identified. Single-molecule sequencing generates very reproducible coverage patterns, and these can be used to detect any size insertion or deletion directly, unlike PCR-based methods, which require additional assays. Because no gene isolation or amplification is required for sequencing, the exceptionally low costs of sample preparation and analysis could make genetic tests more accessible to those who wish to know their own disease susceptibility. Additionally, this approach has applications for sequencing integration sites for gene therapy vectors, transposons, retroviruses, and other mobile DNA elements in a more facile manner than possible with other methods.

  13. Towards a rapid molecular diagnostic for melioidosis: Comparison of DNA extraction methods from clinical specimens.

    PubMed

    Richardson, Leisha J; Kaestli, Mirjam; Mayo, Mark; Bowers, Jolene R; Tuanyok, Apichai; Schupp, Jim; Engelthaler, David; Wagner, David M; Keim, Paul S; Currie, Bart J

    2012-01-01

    Optimising DNA extraction from clinical samples for Burkholderia pseudomallei Type III secretion system real-time PCR in suspected melioidosis patients confirmed that urine and sputum are useful diagnostic samples. Direct testing on blood remains problematic; testing DNA extracted from plasma was superior to DNA from whole blood or buffy coat.

  14. 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

  15. Deparaffinization with mineral oil: a simple procedure for extraction of high-quality DNA from archival formalin-fixed paraffin-embedded samples.

    PubMed

    Heikal, Nahla; Nussenzveig, Roberto H; Agarwal, Archana M

    2014-09-01

    Extracting DNA from formalin-fixed paraffin-embedded (FFPE) archival samples remains difficult. Successful polymerase chain reactions (PCR) with DNA extracted from FFPE samples is still very low. We extracted DNA from 12 recent and old archival FFPE bone marrow trephine biopsies by use of a simple protocol on the basis of deparaffinization with molecular biology-grade mineral oil followed by DNA extraction with the Qiagen FFPE kit. Comparison of this deparaffinization method with standard protocols, for example, xylene or Hemo-D with subsequent rehydration using graded ethanols, was investigated. The quality and quantity of extracted DNA were tested by a combination of ultraviolet spectroscopy, analysis on a Caliper LabChip GX, and real-time PCR combined with high-resolution melt analysis. Highest quality PCR-amplifiable DNA was obtained by deparaffinization with mineral oil, whereas more variable results were obtained for the other 2 deparaffinization procedures. This result was confirmed by real-time PCR and high-resolution melt analysis. Besides improvements in the quality of extracted DNA, use of mineral oil for deparaffinization has the added benefit of decreased time (20 vs. 75 min) and a significant reduction of hands-on labor (1 step vs. multiple hands-on centrifugation and decanting steps).

  16. 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

  17. Case study: using a nondestructive DNA extraction method to generate mtDNA sequences from historical chimpanzee specimens.

    PubMed

    Mohandesan, Elmira; Prost, Stefan; Hofreiter, Michael

    2012-01-01

    A major challenge for ancient DNA (aDNA) studies using museum specimens is that sampling procedures usually involve at least the partial destruction of each specimen used, such as the removal of skin, pieces of bone, or a tooth. Recently, a nondestructive DNA extraction method was developed for the extraction of amplifiable DNA fragments from museum specimens without appreciable damage to the specimen. Here, we examine the utility of this method by attempting DNA extractions from historic (older than 70 years) chimpanzee specimens. Using this method, we PCR-amplified part of the mitochondrial HVR-I region from 65% (56/86) of the specimens from which we attempted DNA extraction. However, we found a high incidence of multiple sequences in individual samples, suggesting substantial cross-contamination among samples, most likely originating from storage and handling in the museums. Consequently, reproducible sequences could be reconstructed from only 79% (44/56) of the successfully extracted samples, even after multiple extractions and amplifications. This resulted in an overall success rate of just over half (44/86 of samples, or 51% success), from which 39 distinct HVR-I haplotypes were recovered. We found a high incidence of C to T changes, arguing for both low concentrations of and substantial damage to the endogenous DNA. This chapter highlights both the potential and the limitations of nondestructive DNA extraction from museum specimens.

  18. 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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    1990-06-01

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

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

    PubMed

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

    2013-09-27

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

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

  3. Advantage of a rapid extraction method of HIV1 DNA suitable for polymerase chain reaction.

    PubMed

    Vignoli, C; de Lamballerie, X; Zandotti, C; Tamalet, C; de Micco, P

    1995-01-01

    We describe a new protocol for extraction of DNA suitable for HIV1 gene amplification from clinical samples using "Chelex-100" chelating resin. Comparison was made with the classic proteinase K extraction method; 154 specimens were extracted with both methods and subjected to PCR (polymerase chain reaction). The Chelex-100 procedure optimized the yield of DNA recovery and minimized contamination due to sample manipulation. It decreased false negative results due to PCR inhibitors. A DNA sample suitable for use in PCR was obtained in 30 minutes. Chelex-100 treatment is a simple, rapid and low-cost method for DNA extraction in clinical laboratories.

  4. Feature Extraction of One-step Ahead Daily Maximum Load with Regression Tree

    NASA Astrophysics Data System (ADS)

    Mori, Hiroyuki; Sakatani, Yoshinori; Fujino, Tatsurou; Numa, Kazuyuki

    In this paper, a new efficient feature extraction method is proposed to handle the one-step ahead daily maximum load forecasting. In recent years, power systems become more complicated under the deregulated and competitive environment. As a result, it is not easy to understand the cause and effect of short-term load forecasting with a bunch of data. This paper analyzes load data from a standpoint of data mining. By it we mean a technique that finds out rules or knowledge through large database. As a data mining method for load forecasting, this paper focuses on the regression tree that handles continuous variables and expresses a knowledge rule as if-then rules. Investigating the variable importance of the regression tree gives information on the transition of the load forecasting models. This paper proposes a feature extraction method for examining the variable importance. The proposed method allows to classify the transition of the variable importance through actual data.

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

    PubMed

    Asher, Robert J; Hofreiter, Michael

    2006-04-01

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

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

    PubMed

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

    2013-07-01

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

  7. How to Show the Real Microbial Biodiversity? A Comparison of Seven DNA Extraction Methods for Bacterial Population Analyses in Matrices Containing Highly Charged Natural Nanoparticles

    PubMed Central

    Kaden, Rene; Krolla-Sidenstein, Peter

    2015-01-01

    A DNA extraction that comprises the DNA of all available taxa in an ecosystem is an essential step in population analysis, especially for next generation sequencing applications. Many nanoparticles as well as naturally occurring clay minerals contain charged surfaces or edges that capture negatively charged DNA molecules after cell lysis within DNA extraction. Depending on the methodology of DNA extraction, this phenomenon causes a shift in detection of microbial taxa in ecosystems and a possible misinterpretation of microbial interactions. With the aim to describe microbial interactions and the bio-geo-chemical reactions during a clay alteration experiment, several methods for the detection of a high number of microbial taxa were examined in this study. Altogether, 13 different methods of commercially available DNA extraction kits provided by seven companies as well as the classical phenol-chloroform DNA extraction were compared. The amount and the quality of nucleic acid extracts were determined and compared to the amplifiable amount of DNA. The 16S rRNA gene fragments of several taxa were separated using denaturing gradient gel electrophoresis (DGGE) to determine the number of different species and sequenced to get the information about what kind of species the microbial population consists of. A total number of 13 species was detected in the system. Up to nine taxa could be detected with commercially available DNA extraction kits while phenol-chloroform extraction lead to three detected species. In this paper, we describe how to combine several DNA extraction methods for the investigation of microbial community structures in clay. PMID:27682112

  8. How to Show the Real Microbial Biodiversity? A Comparison of Seven DNA Extraction Methods for Bacterial Population Analyses in Matrices Containing Highly Charged Natural Nanoparticles

    PubMed Central

    Kaden, Rene; Krolla-Sidenstein, Peter

    2015-01-01

    A DNA extraction that comprises the DNA of all available taxa in an ecosystem is an essential step in population analysis, especially for next generation sequencing applications. Many nanoparticles as well as naturally occurring clay minerals contain charged surfaces or edges that capture negatively charged DNA molecules after cell lysis within DNA extraction. Depending on the methodology of DNA extraction, this phenomenon causes a shift in detection of microbial taxa in ecosystems and a possible misinterpretation of microbial interactions. With the aim to describe microbial interactions and the bio-geo-chemical reactions during a clay alteration experiment, several methods for the detection of a high number of microbial taxa were examined in this study. Altogether, 13 different methods of commercially available DNA extraction kits provided by seven companies as well as the classical phenol-chloroform DNA extraction were compared. The amount and the quality of nucleic acid extracts were determined and compared to the amplifiable amount of DNA. The 16S rRNA gene fragments of several taxa were separated using denaturing gradient gel electrophoresis (DGGE) to determine the number of different species and sequenced to get the information about what kind of species the microbial population consists of. A total number of 13 species was detected in the system. Up to nine taxa could be detected with commercially available DNA extraction kits while phenol-chloroform extraction lead to three detected species. In this paper, we describe how to combine several DNA extraction methods for the investigation of microbial community structures in clay.

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

    PubMed Central

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

    2013-01-01

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

  10. 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

  11. 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

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

    NASA Astrophysics Data System (ADS)

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

    2010-06-01

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

  13. 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

  14. RecFOR proteins load RecA protein onto gapped DNA to accelerate DNA strand exchange: a universal step of recombinational repair.

    PubMed

    Morimatsu, Katsumi; Kowalczykowski, Stephen C

    2003-05-01

    Genetic evidence suggests that the RecF, RecO, and RecR (RecFOR) proteins participate in a common step of DNA recombination and repair, yet the biochemical event requiring collaboration of all three proteins is unknown. Here, we show that the concerted action of the RecFOR complex directs the loading of RecA protein specifically onto gapped DNA that is coated with single-stranded DNA binding (SSB) protein, thereby accelerating DNA strand exchange. The RecFOR complex recognizes the junction between the ssDNA and dsDNA regions and requires a base-paired 5' terminus at the junction. Thus, the RecFOR complex is a structure-specific mediator that targets recombinational repair to ssDNA-dsDNA junctions. This reaction reconstitutes the initial steps of recombinational gapped DNA repair and uncovers an event also common to the repair of ssDNA-tailed intermediates of dsDNA-break repair. We propose that the behavior of the RecFOR proteins is mimicked by functional counterparts that exist in all organisms. PMID:12769856

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

    PubMed

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

    2013-01-01

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

  16. 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

  17. 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.

  18. DNA extraction from fresh-frozen and formalin-fixed, paraffin-embedded human brain tissue.

    PubMed

    Wang, Jian-Hua; Gouda-Vossos, Amany; Dzamko, Nicolas; Halliday, Glenda; Huang, Yue

    2013-10-01

    Both fresh-frozen and formalin-fixed, paraffin-embedded (FFPE) human brain tissues are invaluable resources for molecular genetic studies of central nervous system diseases, especially neurodegenerative disorders. To identify the optimal method for DNA extraction from human brain tissue, we compared methods on differently-processed tissues. Fragments of LRRK2 and MAPT (257 bp and 483 bp/245 bp) were amplified for evaluation. We found that for FFPE samples, the success rate of DNA extraction was greater when using a commercial kit than a laboratory-based method (successful DNA extraction from 76% versus 33% of samples). PCR amplicon size and storage period were key factors influencing the success rate of DNA extraction from FFPE samples. In the fresh-frozen samples, the DNA extraction success rate was 100% using either a commercial kit (QIAamp DNA Micro) or a laboratory-based method (sample boiling in 0.1 mol/L NaOH, followed by proteinase K digestion, and then DNA extraction using Chelex-100) regardless of PCR amplicon length or tissue storage time. Although the present results demonstrate that PCR-amplifiable genomic DNA can be extracted from both fresh-frozen and FFPE samples, fresh brain tissue is recommended for DNA extraction in future neuropathological studies.

  19. 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

  20. 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.

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

    PubMed

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

    2016-01-01

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

  2. An improved method for extracting bacteria from soil for high molecular weight DNA recovery and BAC library construction.

    PubMed

    Liu, Juan; Li, Jingquan; Feng, Li; Cao, Hui; Cui, Zhongli

    2010-12-01

    Separation of bacterial cells from soil is a key step in the construction of metagenomic BAC libraries with large DNA inserts. Our results showed that when combined with sodium pyro-phosphate and homogenization for soil dispersion, sucrose density gradient centrifugation (SDGC) was more effective at separating bacteria from soil than was low speed centrifugation (LSC). More than 70% of the cells, along with some soil colloids, were recovered with one round of centrifugation. A solution of 0.8% NaCl was used to resuspend these cell and soil pellets for purification with nycodenz density gradient centrifugation (NDGC). After purification, more than 30% of the bacterial cells in the primary soil were extracted. This procedure effectively removed soil contamination and yielded sufficient cells for high molecular weight (HMW) DNA isolation. Ribosomal intergenic spacer analysis (RISA) showed that the microbial community structure of the extracted cells was similar to that of the primary soil, suggesting that this extraction procedure did not significantly change the the soil bacteria community structure. HMW DNA was isolated from bacterial cells extracted from red soil for metagenomic BAC library construction. This library contained DNA inserts of more than 200 Mb with an average size of 75 kb.

  3. A method for the extraction of genomic DNA from human brain tissue fixed and stored in formalin for many years.

    PubMed

    Savioz, A; Blouin, J L; Guidi, S; Antonarakis, S E; Bouras, C

    1997-04-01

    We report a method providing access to high molecular weight, polymerase chain reaction (PCR)-amplifiable genomic DNA from brains stored in formalin for many years. It consists mainly of an intensive proteinase K treatment of ground tissue previously embedded in agarose plugs, followed by a washing and an elution step. The method was tested on brains fixed and stored in formalin for up to 46 years. All extracted DNA show an identical pattern of degradation ranging from well-preserved (more than 20 kb) to 400-bp-long fragments. This was demonstrated for DNA extracted from the cerebellums of elderly psychiatric and geriatric patients (of more than 60 years of age), male and female, demented or not, with postmortem delays longer than 1 h and shorter than 1 day. In all these cases PCR amplification of a 838-bp-long beta-actin product was successfully performed when proteinase K treatment was sufficiently effective to generate pure DNA. Thus, high molecular weight, PCR-amplifiable genomic DNA can be extracted from brains stored in formalin for almost half a century.

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

    PubMed

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

    2013-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

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

  6. Optimized Protocol for Simple Extraction of High-Quality Genomic DNA from Clostridium difficile for Whole-Genome Sequencing.

    PubMed

    Sim, James Heng Chiak; Anikst, Victoria; Lohith, Akshar; Pourmand, Nader; Banaei, Niaz

    2015-07-01

    Successful sequencing of the Clostridium difficile genome requires high-quality genomic DNA (gDNA) as the starting material. gDNA extraction using conventional methods is laborious. We describe here an optimized method for the simple extraction of C. difficile gDNA using the QIAamp DNA minikit, which yielded high-quality sequence reads on the Illumina MiSeq platform.

  7. NUC-1, a caenorhabditis elegans DNase II homolog, functions in an intermediate step of DNA degradation during apoptosis.

    PubMed

    Wu, Y C; Stanfield, G M; Horvitz, H R

    2000-03-01

    One hallmark of apoptosis is the degradation of chromosomal DNA. We cloned the Caenorhabditis elegans gene nuc-1, which is involved in the degradation of the DNA of apoptotic cells, and found that nuc-1 encodes a homolog of mammalian DNase II. We used the TUNEL technique to assay DNA degradation in nuc-1 and other mutants defective in programmed cell death and discovered that TUNEL labels apoptotic cells only during a transient intermediate stage. Mutations in nuc-1 allowed the generation of TUNEL-reactive DNA but blocked the conversion of TUNEL-reactive DNA to a subsequent TUNEL-unreactive state. Completion of DNA degradation did not occur in the absence of cell-corpse engulfment. Our data suggest that the process of degradation of the DNA of a cell corpse occurs in at least three distinct steps and requires activities provided by both the dying and the engulfing cell.

  8. 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...

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

    PubMed

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

    2015-12-28

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

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2015-12-28

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2014-08-25

    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.

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2013-01-01

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

  16. A comparative study of two methods for the isolation of human leucocytes for DNA extraction.

    PubMed

    Lim, L H; Ton, S H; Cheong, S K

    1990-06-01

    The 'Dextran' and the 'Buffy-coat' methods for isolation of human leucocytes for DNA extraction were compared on the basis of DNA yield from the same amounts (10 ml) of blood. Human leucocytes from a total of 11 samples were isolated using both methods for each sample after which DNA was extracted. Extracted DNA samples were treated with ribonucleases and proteinase K after which the yields were quantitated by measuring absorbance at 260 nm. The 'Buffy-coat' method yielded a mean concentration of DNA of 476.7 micrograms/ml (range: 212 to 700 micrograms/ml) while the 'Dextran' method yielded 188.4 micrograms/ml (range: 64 to 340 micrograms/ml). The difference was confirmed by subjecting the extracted DNA samples to agarose gel electrophoresis.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-11-01

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

  19. Simultaneous Non-invasive Analysis of DNA Condensation and Stability by Two-step QD-FRET

    PubMed Central

    Chen, Hunter H.; Ho, Yi-Ping; Jiang, Xuan; Mao, Hai-Quan; Wang, Tza-Huei; Leong, Kam W.

    2009-01-01

    Summary Nanoscale vectors comprised of cationic polymers that condense DNA to form nanocomplexes are promising options for gene transfer. The rational design of more efficient nonviral gene carriers will be possible only with better mechanistic understanding of the critical rate-limiting steps, such as nanocomplex unpacking to release DNA and degradation by nucleases. We present a two-step quantum dot fluorescence resonance energy transfer (two-step QD-FRET) approach to simultaneously and non-invasively analyze DNA condensation and stability. Plasmid DNA, double-labeled with QD (525 nm emission) and nucleic acid dyes, were complexed with Cy5-labeled cationic gene carriers. The QD donor drives energy transfer stepwise through the intermediate nucleic acid dye to the final acceptor Cy5. At least three distinct states of DNA condensation and integrity were distinguished in single particle manner and within cells by quantitative ratiometric analysis of energy transfer efficiencies. This novel two-step QD-FRET method allows for more detailed assessment of the onset of DNA release and degradation simultaneously. PMID:20161048

  20. Fast-Track, One-Step E. coli Detection: A Miniaturized Hydrogel Array Permits Specific Direct PCR and DNA Hybridization while Amplification.

    PubMed

    Beyer, Antje; Pollok, Sibyll; Rudloff, Anne; Cialla-May, Dana; Weber, Karina; Popp, Jürgen

    2016-09-01

    A timesaving and convenient method for bacterial detection based on one-step, one-tube deoxyribonucleic acid (DNA) hybridization on hydrogel array while target gene amplification is described. The hydrogel array is generated by a fast one-pot synthesis, where N,N'-dimethylacrylamide/polyethyleneglycol(PEG1900 )-bisacrylamide mixture polymerizes via radical photoinitiation by visible light within 20 min concomitant with in situ capture probe immobilization. These DNA-functionalized hydrogel droplets arrayed on a planar glass surface are placed in the polymerase chain reaction (PCR) mixture during the thermal amplification cycles. The bacterial cells can be implemented in a direct PCR reaction, omitting the need for prior template DNA extraction. The resulting fluorescence signal is immediately detectable after the end of the PCR (1 h) following one short washing step by microscopy. Therefore a valid signal can be reached within 1.5 h including 10 min for pipetting and placement of the tubes and chips. The performance of this novel hydrogel DNA array was successfully proven with varying cell numbers down to a limit of 10(1) Escherichia coli cells. PMID:27220309

  1. Fast-Track, One-Step E. coli Detection: A Miniaturized Hydrogel Array Permits Specific Direct PCR and DNA Hybridization while Amplification.

    PubMed

    Beyer, Antje; Pollok, Sibyll; Rudloff, Anne; Cialla-May, Dana; Weber, Karina; Popp, Jürgen

    2016-09-01

    A timesaving and convenient method for bacterial detection based on one-step, one-tube deoxyribonucleic acid (DNA) hybridization on hydrogel array while target gene amplification is described. The hydrogel array is generated by a fast one-pot synthesis, where N,N'-dimethylacrylamide/polyethyleneglycol(PEG1900 )-bisacrylamide mixture polymerizes via radical photoinitiation by visible light within 20 min concomitant with in situ capture probe immobilization. These DNA-functionalized hydrogel droplets arrayed on a planar glass surface are placed in the polymerase chain reaction (PCR) mixture during the thermal amplification cycles. The bacterial cells can be implemented in a direct PCR reaction, omitting the need for prior template DNA extraction. The resulting fluorescence signal is immediately detectable after the end of the PCR (1 h) following one short washing step by microscopy. Therefore a valid signal can be reached within 1.5 h including 10 min for pipetting and placement of the tubes and chips. The performance of this novel hydrogel DNA array was successfully proven with varying cell numbers down to a limit of 10(1) Escherichia coli cells.

  2. Extraction of human genomic DNA from whole blood using a magnetic microsphere method.

    PubMed

    Gong, Rui; Li, Shengying

    2014-01-01

    With the rapid development of molecular biology and the life sciences, magnetic extraction is a simple, automatic, and highly efficient method for separating biological molecules, performing immunoassays, and other applications. Human blood is an ideal source of human genomic DNA. Extracting genomic DNA by traditional methods is time-consuming, and phenol and chloroform are toxic reagents that endanger health. Therefore, it is necessary to find a more convenient and efficient method for obtaining human genomic DNA. In this study, we developed urea-formaldehyde resin magnetic microspheres and magnetic silica microspheres for extraction of human genomic DNA. First, a magnetic microsphere suspension was prepared and used to extract genomic DNA from fresh whole blood, frozen blood, dried blood, and trace blood. Second, DNA content and purity were measured by agarose electrophoresis and ultraviolet spectrophotometry. The human genomic DNA extracted from whole blood was then subjected to polymerase chain reaction analysis to further confirm its quality. The results of this study lay a good foundation for future research and development of a high-throughput and rapid extraction method for extracting genomic DNA from various types of blood samples.

  3. 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.

  4. 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

  5. Repair synthesis step involving ERCC1-XPF participates in DNA repair of the Top1-DNA damage complex.

    PubMed

    Takahata, Chiaki; Masuda, Yuji; Takedachi, Arato; Tanaka, Kiyoji; Iwai, Shigenori; Kuraoka, Isao

    2015-08-01

    Topoisomerase 1 (Top1) is the intercellular target of camptothecins (CPTs). CPT blocks DNA religation in the Top1-DNA complex and induces Top1-attached nick DNA lesions. In this study, we demonstrate that excision repair cross complementing 1 protein-xeroderma pigmentosum group F (ERCC1-XPF) endonuclease and replication protein A (RPA) participate in the repair of Top1-attached nick DNA lesions together with other nucleotide excision repair (NER) factors. ERCC1-XPF shows nuclease activity in the presence of RPA on a 3'-phosphotyrosyl bond nick-containing DNA (Tyr-nick DNA) substrate, which mimics a Top1-attached nick DNA lesion. In addition, ERCC1-XPF and RPA form a DNA/protein complex on the nick DNA substrate in vitro, and co-localize in CPT-treated cells in vivo. Moreover, the DNA repair synthesis of Tyr-nick DNA lesions occurred in the presence of NER factors, including ERCC1-XPF, RPA, DNA polymerase delta, flap endonuclease 1 and DNA ligase 1. Therefore, some of the NER repair machinery might be an alternative repair pathway for Top1-attached nick DNA lesions. Clinically, these data provide insights into the potential of ERCC1 as a biomarker during CPT regimens.

  6. 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 ...

  7. Relaxase DNA binding and cleavage are two distinguishable steps in conjugative DNA processing that involve different sequence elements of the nic site.

    PubMed

    Lucas, María; González-Pérez, Blanca; Cabezas, Matilde; Moncalian, Gabriel; Rivas, Germán; de la Cruz, Fernando

    2010-03-19

    TrwC, the relaxase of plasmid R388, catalyzes a series of concerted DNA cleavage and strand transfer reactions on a specific site (nic) of its origin of transfer (oriT). nic contains the cleavage site and an adjacent inverted repeat (IR(2)). Mutation analysis in the nic region indicated that recognition of the IR(2) proximal arm and the nucleotides located between IR(2) and the cleavage site were essential for supercoiled DNA processing, as judged either by in vitro nic cleavage or by mobilization of a plasmid containing oriT. Formation of the IR(2) cruciform and recognition of the distal IR(2) arm and loop were not necessary for these reactions to take place. On the other hand, IR(2) was not involved in TrwC single-stranded DNA processing in vitro. For single-stranded DNA nic cleavage, TrwC recognized a sequence embracing six nucleotides upstream of the cleavage site and two nucleotides downstream. This suggests that TrwC DNA binding and cleavage are two distinguishable steps in conjugative DNA processing and that different sequence elements are recognized by TrwC in each step. IR(2)-proximal arm recognition was crucial for the initial supercoiled DNA binding. Subsequent recognition of the adjacent single-stranded DNA binding site was required to position the cleavage site in the active center of the protein so that the nic cleavage reaction could take place.

  8. DNA extraction from archival Giemsa-stained bone-marrow slides: comparison of six rapid methods.

    PubMed

    Vince, A; Poljak, M; Seme, K

    1998-05-01

    The ability of six rapid DNA extraction procedures to provide DNA for the polymerase chain reaction from archival Giemsa-stained bone marrow slides was tested on 120 samples. Boiling in distilled water, freeze-thaw method, boiling in 10% Chelex-100 resin solution, proteinase K/Tween 20/NP-40 method coupled with simplified phenol/ chloroform/isoamyl alcohol protocol or salting-out procedure using saturated NaCl and modification of commercial QIAamp procedure (Qiagen. Chatsworth, Calif.) gave DNA extraction efficiencies of 50%, 70%, 85%, 95%, 100% and 100%, respectively. Our results demonstrate that rough DNA extraction methods have decreased efficiencies compared to complete DNA extraction protocols and that the latter are required to ensure highly reproducible results from archival Giemsa-stained bone marrow slides.

  9. Rapid and reliable method of extracting DNA and RNA from sweetpotato, Ipomoea batatas (L). Lam.

    PubMed

    Kim, Sun-Hyung; Hamada, Tatsuro

    2005-12-01

    A quick, simple and reliable method of extracting DNA from sweetpotato (Ipomoea batatas (L.) Lam.) has been developed. The method was applied successfully for extraction of total DNA from leaves and total RNA from leaves and various tissues. The yield of DNA extracted by this procedure was high (about 1 mg/g leaf tissue). The extracted DNA was completely digested by restriction endonucleases indicating the absence of common contaminating compounds. The absorbancy ratios of A260/A230 and A260/A280 of isolated RNA were approx. 2 and the yield was about 0.2 mg/g fresh wt. CIPK and tublin genes were successfully amplified by RT-PCR, suggesting the integrity of isolated RNA. The total DNA and RNA isolated by this method was of sufficient quality for subsequent molecular analysis.

  10. 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

  11. Evaluation of commercial kits for extraction of DNA and RNA from Clostridium difficile.

    PubMed

    Metcalf, Devon; Weese, J Scott

    2012-12-01

    Commercial nucleic acid extraction kits are a cost effective, efficient and convenient way to isolate DNA and RNA from bacteria. Despite the increasing importance of the gastrointestinal pathogen, Clostridium difficile, and the increased use of nucleic acids in its identification, characterization, and investigation of virulence factors, no standardized or recommended methods for nucleic acid isolation exist. Here, we sought to evaluate 4 commercial DNA extraction kits and 3 commercial RNA extraction kits assessing cost, labor intensity, purity, quantity and quality of nucleic acid preparations. The DNA extraction kits produced a range of concentrations (20.9-546 ng/ml) and A(260/280) ratios (1.92-2.11). All kits were suitable for DNA extraction with the exception of the Roche MagNA pure LC DNA isolation kit III which produced DNA of high yield but with substantial shearing, but that did not affect downstream PCR amplifications. For RNA extraction, the Qiagen RNeasy mini kit stood out producing preparations of consistently higher concentrations and higher RNA integrity numbers (RIN). The Roche MagNA pure LC RNA isolation kit produced preparations that could not be properly assigned RINs due to a failure to remove small RNAs which were interpreted as degradation. Good DNA and RNA yield are critical but methods are often overlooked. This study highlights the potential for critical variation between established commercial systems and the need for assessment of any extraction methods that are used.

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

    PubMed Central

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

    1999-01-01

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

  13. A Rapid and Cost-Effective Method for DNA Extraction from Archival Herbarium Specimens.

    PubMed

    Krinitsina, A A; Sizova, T V; Zaika, M A; Speranskaya, A S; Sukhorukov, A P

    2015-11-01

    Here we report a rapid and cost-effective method for the extraction of total DNA from herbarium specimens up to 50-90-year-old. The method takes about 2 h, uses AMPure XP magnetic beads diluted by PEG-8000- containing buffer, and does not require use of traditional volatile components like chloroform, phenol, and liquid nitrogen. It yields up to 4 µg of total nucleic acid with high purity from about 30 mg of dry material. The quality of the extracted DNA was tested by PCR amplification of 5S rRNA and rbcL genes (nuclear and chloroplast DNA markers) and compared against the traditional chloroform/isoamyl alcohol method. Our results demonstrate that the use of the magnetic beads is crucial for extraction of DNA suitable for subsequent PCR from herbarium samples due to the decreasing inhibitor concentrations, reducing short fragments of degraded DNA, and increasing median DNA fragment sizes.

  14. A filter paper-based microdevice for low-cost, rapid, and automated DNA extraction and amplification from diverse sample types.

    PubMed

    Gan, Wupeng; Zhuang, Bin; Zhang, Pengfei; Han, Junping; Li, Cai-Xia; Liu, Peng

    2014-10-01

    A plastic microfluidic device that integrates a filter disc as a DNA capture phase was successfully developed for low-cost, rapid and automated DNA extraction and PCR amplification from various raw samples. The microdevice was constructed by sandwiching a piece of Fusion 5 filter, as well as a PDMS (polydimethylsiloxane) membrane, between two PMMA (poly(methyl methacrylate)) layers. An automated DNA extraction from 1 μL of human whole blood can be finished on the chip in 7 minutes by sequentially aspirating NaOH, HCl, and water through the filter. The filter disc containing extracted DNA was then taken out directly for PCR. On-chip DNA purification from 0.25-1 μL of human whole blood yielded 8.1-21.8 ng of DNA, higher than those obtained using QIAamp® DNA Micro kits. To realize DNA extraction from raw samples, an additional sample loading chamber containing a filter net with an 80 μm mesh size was designed in front of the extraction chamber to accommodate sample materials. Real-world samples, including whole blood, dried blood stains on Whatman® 903 paper, dried blood stains on FTA™ cards, buccal swabs, saliva, and cigarette butts, can all be processed in the system in 8 minutes. In addition, multiplex amplification of 15 STR (short tandem repeat) loci and Sanger-based DNA sequencing of the 520 bp GJB2 gene were accomplished from the filters that contained extracted DNA from blood. To further prove the feasibility of integrating this extraction method with downstream analyses, "in situ" PCR amplifications were successfully performed in the DNA extraction chamber following DNA purification from blood and blood stains without DNA elution. Using a modified protocol to bond the PDMS and PMMA, our plastic PDMS devices withstood the PCR process without any leakage. This study represents a significant step towards the practical application of on-chip DNA extraction methods, as well as the development of fully integrated genetic analytical systems.

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

    PubMed

    Chomczynski, Piotr; Sacchi, Nicoletta

    2006-01-01

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

  16. Simple practical approach for sample loading prior to DNA extraction using a silica monolith in a microfluidic device.

    PubMed

    Shaw, Kirsty J; Joyce, Domino A; Docker, Peter T; Dyer, Charlotte E; Greenman, John; Greenway, Gillian M; Haswell, Stephen J

    2009-12-01

    A novel DNA loading methodology is presented for performing DNA extraction on a microfluidic system. DNA in a chaotropic salt solution was manually loaded onto a silica monolith orthogonal to the subsequent flow of wash and elution solutions. DNA was successfully extracted from buccal swabs using electro-osmotic pumping (EOP) coupled with in situ reagents contained within a 1.5% agarose gel matrix. The extracted DNA was of sufficient quantity and purity for polymerase chain reaction (PCR) amplification.

  17. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

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

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

    PubMed

    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.

  20. 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.

  1. Extraction of total DNA and optimization of the RAPD reaction system in Dioscorea opposita Thunb.

    PubMed

    Wen, G Q; Li, J; Liu, X H; Zhang, Y S; Wen, S S

    2014-02-28

    Dioscorea opposita Thunb. has been used as health food and herbal medicinal ingredients in traditional Chinese medicine. In this study, the total DNA of D. opposita Thunb. was extracted using an improved cetyltrimethylammonium bromide (CTAB) method, and the extracted DNA was further used for random amplified polymorphic DNA (RAPD) reaction system by design of the L16 (4(4)) orthogonal diagram. The results showed that the improved CTAB method can be used to isolate high-quality and high-concentration DNA, and the optimized protocol can overcome the instability of RAPD reaction system. The knowledge stated here can be used to study the genetic diversity of D. opposita Thunb.

  2. 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

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

    PubMed

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

    2013-03-01

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

  4. 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

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

    PubMed

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

    2011-06-15

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

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

    PubMed

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

    2016-05-01

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

  7. 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.

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

    PubMed

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

    2012-01-01

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

  9. DNA extraction methods and multiple sampling to improve molecular diagnosis of Sarcocystis spp. in cattle hearts.

    PubMed

    Bräunig, Patrícia; Portella, Luiza Pires; Cezar, Alfredo Skrebsky; Libardoni, Felipe; Sangioni, Luis Antonio; Vogel, Fernanda Silveira Flores; Gonçalves, Paulo Bayard Dias

    2016-10-01

    Molecular detection of Sarcocystis spp. in tissue samples can be useful for experimental and diagnostic purposes. However, the parasite spreads unevenly through tissues, forming tissue cysts, and the cystic wall is an obstacle in DNA extraction protocols. Therefore, adequate sampling and effective disruption of the cysts are essential to improve the accuracy of DNA detection by PCR. The aims of this study were to evaluate the suitability of four protocols for DNA extraction from cysts of Sarcocystis spp. present in bovine myocardium samples or after their harvest in phosphate-buffered saline (PBS) solution as well as determine the effects of single or multiple sampling on the accuracy of molecular diagnosis of sarcocystosis in cattle hearts. Cysts and myocardium samples from nine bovine hearts were randomly distributed to four DNA extraction protocols: kit, kit with modification, DNAzol, and cetyl-trimethyl ammonium bromide (CTAB). Samples were submitted to DNA extraction and PCR as replicates of each heart (simplicate, duplicate, and triplicate), and the probability of a true positive diagnostic was calculated. Among the protocols tested, the kit with modification was determined to be the most suitable for DNA extraction from cysts in PBS solution (92.6 % of DNA detection by PCR); DNAzol resulted in higher DNA detection frequency from bovine myocardium samples (48.1 %). Multiple sampling improved the molecular diagnosis of Sarcocystis spp. infection in cattle hearts, increasing at 22.2 % the rate of true positive diagnostic.

  10. [Evaluation of 3 methods of DNA extraction from paraffin-embedded material for the amplification of genomic DNA using PCR].

    PubMed

    Mesquita, R A; Anzai, E K; Oliveira, R N; Nunes, F D

    2001-01-01

    There are several protocols reported in the literature for the extraction of genomic DNA from formalin-fixed paraffin-embedded samples. Genomic DNA is utilized in molecular analyses, including PCR. This study compares three different methods for the extraction of genomic DNA from formalin-fixed paraffin-embedded (inflammatory fibrous hyperplasia) and non-formalin-fixed (normal oral mucosa) samples: phenol with enzymatic digestion, and silica with and without enzymatic digestion. The amplification of DNA by means of the PCR technique was carried out with primers for the exon 7 of human keratin type 14. Amplicons were analyzed by means of electrophoresis in an 8% polyacrylamide gel with 5% glycerol, followed by silver-staining visualization. The phenol/enzymatic digestion and the silica/enzymatic digestion methods provided amplicons from both tissue samples. The method described is a potential aid in the establishment of the histopathologic diagnosis and in retrospective studies with archival paraffin-embedded samples.

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

    NASA Astrophysics Data System (ADS)

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

    2011-02-01

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

  12. Increasing DNA extraction yield from saliva stains with a modified Chelex method.

    PubMed

    Sweet, D; Lorente, M; Valenzuela, A; Lorente, J A; Alvarez, J C

    1996-12-27

    Recovery, preservation and analysis of body fluid stains is an important aspect of forensic science. PCR-based typing of DNA extracted from recovered stains is often a crucial method to identify a perpetrator or exclude an innocent suspect. This paper reports an improved method of extracting genomic DNA from saliva stains deposited on human skin in simulated bite mark situations. Results of organic (phenol-chloroform) extraction and Chelex extraction were compared to a modified Chelex method developed by the authors. Modifications include pre-extraction preparation with proteinase K and incubations at 56 degrees C and 100 degrees C plus microconcentration of the solution. Quantification results using the classical Chelex extraction method showed that 31.9 +/- 4.22% of the deposited DNA was recovered, but using the modified Chelex extraction method DNA recovery was increased to 47.7 +/- 6.90%. The quantity and quality of extracted DNA was shown to be adequate for PCR-based typing at two STR loci.

  13. The use of carrier RNA to enhance DNA extraction from microfluidic-based silica monoliths.

    PubMed

    Shaw, Kirsty J; Thain, Lauren; Docker, Peter T; Dyer, Charlotte E; Greenman, John; Greenway, Gillian M; Haswell, Stephen J

    2009-10-12

    DNA extraction was carried out on silica-based monoliths within a microfluidic device. Solid-phase DNA extraction methodology was applied in which the DNA binds to silica in the presence of a chaotropic salt, such as guanidine hydrochloride, and is eluted in a low ionic strength solution, such as water. The addition of poly-A carrier RNA to the chaotropic salt solution resulted in a marked increase in the effective amount of DNA that could be recovered (25ng) compared to the absence of RNA (5ng) using the silica-based monolith. These findings confirm that techniques utilising nucleic acid carrier molecules can enhance DNA extraction methodologies in microfluidic applications.

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

    PubMed

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

    2016-09-01

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

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

    PubMed

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

    2016-09-01

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

  16. 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.

  17. A new, fast, and simple DNA extraction method for HLA and VNTR genotyping by PCR amplification.

    PubMed

    Planelles, D; Llopis, F; Puig, N; Montoro, J A

    1996-01-01

    In the present study a new DNA extraction method is described. The new protocol, which uses caprylic acid for isolating DNA, is technically simple and very fast, as it enables us to obtain DNA from peripheral blood in only 10 minutes. Moreover, DNA preparations obtained with this procedure can be effectively used for HLA class II and variable number tandem repeat genotyping by polymerase chain reaction, so the new method is well suited for routine clinical use in any type of analysis requiring DNA typing for individual characterization.

  18. 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

  19. 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.

  20. 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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2015-12-01

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

  3. Water extracts of tree Hypericum sps. protect DNA from oxidative and alkylating damage and enhance DNA repair in colon cells.

    PubMed

    Ramos, Alice A; Marques, Filipe; Fernandes-Ferreira, Manuel; Pereira-Wilson, Cristina

    2013-01-01

    Diet may induce colon carcinogenesis through oxidative or alkylating DNA damage. However, diet may also contain anticarcinogenic compounds that contribute to cancer prevention. DNA damage prevention and/or induction of repair are two important mechanisms involved in cancer chemoprevention by dietary compounds. Hypericum sps. are widely used in traditional medicine to prepare infusions due to their beneficial digestive and neurologic effects. In this study, we investigated the potential of water extracts from three Hypericum sps. and some of their main phenolic compounds to prevent and repair oxidative and alkylating DNA damage in colon cells. The results showed that water extracts of Hypericum perforatum, Hypericum androsaemum, Hypericum undulatum, quercetin and rutin have protective effect against oxidative DNA damage in HT29 cells. Protective effect was also observed against alkylating DNA damage induced by methyl-methanesulfonate, except for H. androsaemum. With regard to alkylating damage repair H. perforatum, H. androsaemum and chlorogenic acid increased repair of alkylating DNA damage by base excision repair pathway. No effect was observed on nucleotide excision repair pathway. Antigenotoxic effects of Hypericum sps. may contribute to colon cancer prevention and the high amount of phenolic compounds present in Hypericum sps. play an important role in DNA protective effects.

  4. A one step multiplex PCR assay for rapid screening of East Asian mtDNA haplogroups on forensic samples.

    PubMed

    Lee, Hwan Young; Yoon, Jung Ah; Yang, Woo Ick; Shin, Kyoung-Jin

    2013-01-01

    The mitochondrial DNA (mtDNA) haplogroup typing has become an essential tool to study human evolutionary history and to infer the matrilineal bio-geographic ancestry. In forensic field, the screening of mtDNA haplogroups by genotyping of mtDNA single nucleotide polymorphisms (SNPs) can help guarantee the quality of mtDNA sequence data as well as can reduce the need to sequence samples that do not match. Here, a multiplex mutagenically separated (MS) polymerase chain reaction (PCR) system was developed for simultaneous rapid detection of 14 coding region SNPs and one deletion motif representing common mtDNA haplogroups of East Asia. The multiplex MS PCR system we developed has the advantage of being a one step procedure that requires only a single PCR amplification with allele-specific primers and allowing straightforward designation of haplogroups along the branches of the phylogenetic tree. Therefore, it would be a simple, rapid, and reliable detection method useful for large-scale screening of mtDNA variations to determine East Asian mtDNA haplogroups.

  5. Reconstitution of initial steps of dsDNA break repair by the RecF pathway of E. coli.

    PubMed

    Handa, Naofumi; Morimatsu, Katsumi; Lovett, Susan T; Kowalczykowski, Stephen C

    2009-05-15

    The RecF pathway of Escherichia coli is important for recombinational repair of DNA breaks and gaps. Here ;we reconstitute in vitro a seven-protein reaction that recapitulates early steps of dsDNA break repair using purified RecA, RecF, RecO, RecR, RecQ, RecJ, and SSB proteins, components of the RecF system. Their combined action results in processing of linear dsDNA and its homologous pairing with supercoiled DNA. RecA, RecO, RecR, and RecJ are essential for joint molecule formation, whereas SSB and RecF are stimulatory. This reconstituted system reveals an unexpected essential function for RecJ exonuclease: the capability to resect duplex DNA. RecQ helicase stimulates this processing, but also disrupts joint molecules. RecO and RecR have two indispensable functions: They mediate exchange of RecA for SSB to form the RecA nucleoprotein filament, and act with RecF to load RecA onto the SSB-ssDNA complex at processed ssDNA-dsDNA junctions. The RecF pathway has many parallels with recombinational repair in eukaryotes. PMID:19451222

  6. 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).

  7. 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

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

    PubMed

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

    2002-11-29

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

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

    PubMed

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

    2002-11-29

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

  11. 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

  12. High performance aptamer affinity chromatography for single-step selective extraction and screening of basic protein lysozyme.

    PubMed

    Han, Bin; Zhao, Chao; Yin, Junfa; Wang, Hailin

    2012-08-15

    A DNA aptamer based high-performance affinity chromatography is developed for selective extraction and screening of a basic protein lysozyme. First, a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolithic column was synthesized in situ by thermally initiated radical polymerization, and then an anti-lysozyme DNA aptamer was covalently immobilized on the surface of the monolith through a 16-atom spacer arm. The target protein lysozyme but non-target proteins can be trapped by the immobilized anti-lysozyme DNA aptamer. In contrast, lysozyme cannot be trapped by the immobilized oligodeoxynucleotide that does not contain the sequence of the anti-lysozyme DNA aptamer. The study clearly demonstrates the trapping of lysozyme by the immobilized anti-lysozyme DNA aptamer is mainly due to specific recognition rather than simple electrostatic interaction of positively charged protein and the negatively charged DNA. The inter-day precision was determined as 0.8% for migration time and 4.2% for peak area, respectively. By the use of aptamer affinity monolith, a screening strategy is developed to selectively extract lysozyme from chicken egg white, showing the advantages of high efficiency, low cost and ease-of-operation.

  13. [Application of DNA extraction kit, 'GM quicker' for detection of genetically modified soybeans].

    PubMed

    Sato, Noriko; Sugiura, Yoshitsugu; Tanaka, Toshitsugu

    2012-01-01

    Several DNA extraction methods have been officially introduced to detect genetically modified soybeans, but the choice of DNA extraction kits depend on the nature of the samples, such as grains or processed foods. To overcome this disadvantage, we examined whether the GM quicker kit is available for both grains and processed foods. We compared GM quicker with four approved DNA extraction kits in respect of DNA purity, copy numbers of lectin gene, and working time. We found that the DNA quality of GM quicker was superior to that of the other kits for grains, and the procedure was faster. However, in the case of processed foods, GM quicker was not superior to the other kits. We therefore investigated an unapproved GM quicker 3 kit, which is available for DNA extraction from processed foods, such as tofu and boiled soybeans. The GM quicker 3 kit provided good DNA quality from both grains and processed foods, so we made a minor modification of the GM quicker-based protocol that was suitable for processed foods, using GM quicker and its reagents. The modified method enhanced the performance of GM quicker with processed foods. We believe that GM quicker with the modified protocol is an excellent tool to obtain high-quality DNA from grains and processed foods for detection of genetically modified soybeans.

  14. Practical method for extraction of PCR-quality DNA from environmental soil samples.

    PubMed

    Fitzpatrick, Kelly A; Kersh, Gilbert J; Massung, Robert F

    2010-07-01

    Methods for the extraction of PCR-quality DNA from environmental soil samples by using pairs of commercially available kits were evaluated. Coxiella burnetii DNA was detected in spiked soil samples at <1,000 genome equivalents per gram of soil and in 12 (16.4%) of 73 environmental soil samples.

  15. Quantitative and qualitative assessment of DNA extracted from saliva for its use in forensic identification

    PubMed Central

    Khare, Parul; Raj, Vineet; Chandra, Shaleen; Agarwal, Suraksha

    2014-01-01

    Saliva has long been known for its diagnostic value in several diseases. It also has a potential to be used in forensic science. Objective: The objective of this study is to compare the quantity and quality of DNA samples extracted from saliva with those extracted from blood in order to assess the feasibility of extracting sufficient DNA from saliva for its possible use in forensic identification. Materials and Methods: Blood and saliva samples were collected from 20 volunteers and DNA extraction was performed through Phenol Chloroform technique. The quantity and quality of isolated DNA was analyzed by spectrophotometery and the samples were then used to amplify short tandem repeat (STR) F13 using the polymerase chain reaction. Results: Mean quantity of DNA obtained in saliva was 48.4 ± 8.2 μg/ml and in blood was 142.5 ± 45.9 μg/ml. Purity of DNA obtained as assessed by the ratio of optical density 260/280, was found to be optimal in 45% salivary samples while remaining showed minor contamination. Despite this positive F13 STR amplification was achieved in 75% of salivary DNA samples. Conclusion: Results of this study showed that saliva may prove to be a useful source of DNA for forensic purpose. PMID:25125913

  16. Evaluation of six methods for extraction and purification of viral DNA from urine and serum samples.

    PubMed

    Bergallo, Massimiliano; Costa, Cristina; Gribaudo, Giorgio; Tarallo, Sonia; Baro, Sara; Negro Ponzi, Alessandro; Cavallo, Rossana

    2006-04-01

    The sensitivity and reliability of PCR for diagnostic and research purposes require efficient unbiased procedures of extraction and purification of nucleic acids. One of the major limitations of PCR-based tests is the inhibition of the amplification process by substances present in clinical samples. This study used specimens spiked with a known amount of plasmid pBKV (ATCC 33-1) to compare six methods for extraction and purification of viral DNA from urine and serum samples based on recovery efficiency in terms of yield of DNA and percentage of plasmid pBKV recovered, purity of extracted DNA, and percentage of inhibition. The most effective extraction methods were the phenol/chloroform technique and the silica gel extraction procedure for urine and serum samples, respectively. Considering DNA purity, the silica gel extraction procedure and the phenol/chloroform method produced the most satisfactory results in urine and serum samples, respectively. The presence of inhibitors was overcome by all DNA extraction techniques in urine samples, as evidenced by semiquantitative PCR amplification. In serum samples, the lysis method and the proteinase K procedure did not completely overcome the presence of inhibitors.

  17. Extraction of DNA suitable for PCR applications from mature leaves of Mangifera indica L.

    PubMed

    Azmat, Muhammad Abubakkar; Khan, Iqrar Ahmad; Cheema, Hafiza Masooma Naseer; Rajwana, Ishtiaq Ahmad; Khan, Ahmad Sattar; Khan, Asif Ali

    2012-04-01

    Good quality deoxyribonucleic acid (DNA) is the pre-requisite for its downstream applications. The presence of high concentrations of polysaccharides, polyphenols, proteins, and other secondary metabolites in mango leaves poses problem in getting good quality DNA fit for polymerase chain reaction (PCR) applications. The problem is exacerbated when DNA is extracted from mature mango leaves. A reliable and modified protocol based on the cetyltrimethylammonium bromide (CTAB) method for DNA extraction from mature mango leaves is described here. High concentrations of inert salt were used to remove polysaccharides; Polyvinylpyrrolidone (PVP) and β-mercaptoethanol were employed to manage phenolic compounds. Extended chloroform-isoamyl alcohol treatment followed by RNase treatment yielded 950-1050 µg of good quality DNA, free of protein and RNA. The problems of DNA degradation, contamination, and low yield due to irreversible binding of phenolic compounds and coprecipitation of polysaccharides with DNA were avoided by this method. The DNA isolated by the modified method showed good PCR amplification using simple sequence repeat (SSR) primers. This modified protocol can also be used to extract DNA from other woody plants having similar problems.

  18. Highly efficient automated extraction of DNA from old and contemporary skeletal remains.

    PubMed

    Zupanič Pajnič, Irena; Debska, Magdalena; Gornjak Pogorelc, Barbara; Vodopivec Mohorčič, Katja; Balažic, Jože; Zupanc, Tomaž; Štefanič, Borut; Geršak, Ksenija

    2016-01-01

    We optimised the automated extraction of DNA from old and contemporary skeletal remains using the AutoMate Express system and the PrepFiler BTA kit. 24 Contemporary and 25 old skeletal remains from WWII were analysed. For each skeleton, extraction using only 0.05 g of powder was performed according to the manufacturer's recommendations (no demineralisation - ND method). Since only 32% of full profiles were obtained from aged and 58% from contemporary casework skeletons, the extraction protocol was modified to acquire higher quality DNA and genomic DNA was obtained after full demineralisation (FD method). The nuclear DNA of the samples was quantified using the Investigator Quantiplex kit and STR typing was performed using the NGM kit to evaluate the performance of tested extraction methods. In the aged DNA samples, 64% of full profiles were obtained using the FD method. For the contemporary skeletal remains the performance of the ND method was closer to the FD method compared to the old skeletons, giving 58% of full profiles with the ND method and 71% of full profiles using the FD method. The extraction of DNA from only 0.05 g of bone or tooth powder using the AutoMate Express has proven highly successful in the recovery of DNA from old and contemporary skeletons, especially with the modified FD method. We believe that the results obtained will contribute to the possibilities of using automated devices for extracting DNA from skeletal remains, which would shorten the procedures for obtaining high-quality DNA from skeletons in forensic laboratories.

  19. Highly efficient automated extraction of DNA from old and contemporary skeletal remains.

    PubMed

    Zupanič Pajnič, Irena; Debska, Magdalena; Gornjak Pogorelc, Barbara; Vodopivec Mohorčič, Katja; Balažic, Jože; Zupanc, Tomaž; Štefanič, Borut; Geršak, Ksenija

    2016-01-01

    We optimised the automated extraction of DNA from old and contemporary skeletal remains using the AutoMate Express system and the PrepFiler BTA kit. 24 Contemporary and 25 old skeletal remains from WWII were analysed. For each skeleton, extraction using only 0.05 g of powder was performed according to the manufacturer's recommendations (no demineralisation - ND method). Since only 32% of full profiles were obtained from aged and 58% from contemporary casework skeletons, the extraction protocol was modified to acquire higher quality DNA and genomic DNA was obtained after full demineralisation (FD method). The nuclear DNA of the samples was quantified using the Investigator Quantiplex kit and STR typing was performed using the NGM kit to evaluate the performance of tested extraction methods. In the aged DNA samples, 64% of full profiles were obtained using the FD method. For the contemporary skeletal remains the performance of the ND method was closer to the FD method compared to the old skeletons, giving 58% of full profiles with the ND method and 71% of full profiles using the FD method. The extraction of DNA from only 0.05 g of bone or tooth powder using the AutoMate Express has proven highly successful in the recovery of DNA from old and contemporary skeletons, especially with the modified FD method. We believe that the results obtained will contribute to the possibilities of using automated devices for extracting DNA from skeletal remains, which would shorten the procedures for obtaining high-quality DNA from skeletons in forensic laboratories. PMID:26615474

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. Escherichia coli RecO protein anneals ssDNA complexed with its cognate ssDNA-binding protein: A common step in genetic recombination.

    PubMed

    Kantake, Noriko; Madiraju, Murty V V M; Sugiyama, Tomohiko; Kowalczykowski, Stephen C

    2002-11-26

    We present biochemical evidence for the functional similarity of Escherichia coli RecO protein and bacteriophage T4 UvsY protein to eukaryotic Rad52 protein. Although Rad52 protein is conserved in eukaryotes, no sequence homologue has been found in prokaryotes or archeabacteria. Rad52 protein has two unique activities: facilitation of replication protein-A (RPA) displacement by Rad51 protein and annealing of RPA-single-stranded DNA (ssDNA) complexes. Both activities require species-specific interaction between Rad52 protein and RPA. Both RecO and UvsY proteins also possess the former property with regard to their cognate ssDNA-binding protein. Here, we report that RecO protein anneals ssDNA that is complexed with only its cognate ssDNA-binding protein, suggesting the involvement of species-specific interactions. Optimal activity for RecO protein occurs after formation of a 1:1 complex with SSB protein. RecR protein, which is known to stimulate RecO protein to facilitate SSB protein displacement by RecA protein, inhibits annealing by RecO protein, suggesting that RecR protein may regulate the choice between the DNA strand invasion versus annealing pathways. In addition, we show that UvsY protein anneals ssDNA; furthermore, ssDNA, which is complexed only with its cognate ssDNA-binding protein, is annealed in the presence of UvsY protein. These results indicate that RecO and possibly UvsY proteins are functional counterparts of Rad52 protein. Based on the conservation of these functions, we propose a modified double-strand break repair model that includes DNA annealing as an important intermediate step. PMID:12438681

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

    PubMed

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

    1985-10-11

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

  6. Release of Bacterial DNA by Marine Nanoflagellates, an Intermediate Step in Phosphorus Regeneration

    PubMed Central

    Turk, Valentina; Rehnstam, Ann-Sofi; Lundberg, Erik; Hagström, Åke

    1992-01-01

    The concentrations of dissolved DNA and nanoflagellates were found to covary during a study of diel dynamics of the microbial food web in the Adriatic Sea. This observation was further investigated in a continuous seawater culture when nanoflagellates were fed bacteria grown in filtered seawater. Analysis of dissolved organic phosphorus and dissolved DNA showed a sixfold increase of dissolved DNA in the presence of the nanoflagellates (Ochromonas sp.). The amount of DNA released suggested that the majority of the consumed bacterial DNA was ejected. Phagotrophic nanoflagellates thus represent an important source of origin for dissolved DNA. The rate of breakdown of dissolved DNA and release of inorganic phosphorus in the pelagic ecosystem is suggested to be dependent on the ambient phosphate pool. In the P-limited northern Adriatic Sea, rapid degradation of the labelled DNA could be demonstrated, whereas the N-limited southern California bight water showed a much lower rate. Phosphorus originating from dissolved DNA was shown to be transferred mainly to organisms in the <3-μm-size fractions. On the basis of the C/P ratios, we suggest that a significant fraction of the phosphorus demand by the autotrophs may be sustained by the released DNA during stratified conditions. Thus, the nucleic acid-rich bacterial biomass grazed by protozoa plays an important role in the biogeochemical cycling of phosphorus in the marine environment. PMID:16348813

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

    PubMed

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

    2015-05-01

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

  8. New efficient DNA extraction method to access the microbiome of Ricinus communis seeds.

    PubMed

    Santos, C D; Dias, A C C; Amaral, I M R; Bonetti, A M; Campos, T A

    2013-02-28

    Ricinus communis (castor bean) seeds are used to produce an alcohol-soluble oil that is used in more than 400 industrial processes. Despite its economic importance, there has been little research on the endophytic microbiota of castor bean seeds. This microbiota is important for plant metabolic processes and may have considerable biotechnological potential, such as production of lipases and plant growth promoter agents. We evaluated several DNA extraction methodologies in order to access the microbial diversity of castor bean through a metagenomic approach. Based on our observations, we developed a new methodology that takes advantage of the low solubility of calcium phosphates and the high affinity of these phosphates for proteins and polysaccharides. The extracted DNA quality was evaluated by PCR, using a selective primer pair for bacterial and mitochondrial 16S rDNA genes (799F and 1492R). We found this methodology quantitatively and qualitatively more efficient than the other approaches. In evaluating this new extraction methodology, we found that the difficulties of DNA extraction from castor bean seeds, such as abundant oil, polysaccharides, phenolic compounds, and plant enzymes, could be overcome. The resulting extracts had high concentration and purity, and they were obtained faster than with previous methods. The samples contained virtually all of the DNA, including the microbial DNA; this was validated by PCR analysis.

  9. Cell-free Xenopus egg extracts for studying DNA damage response pathways

    PubMed Central

    CUPELLO, STEVEN; RICHARDSON, CHRISTINE; YAN, SHAN

    2016-01-01

    In response to a variety of DNA replication stress or DNA damaging agents, the DNA damage response (DDR) pathways are triggered for cells to coordinate DNA repair, cell cycle checkpoints, apoptosis, and senescence. Cell-free Xenopus egg extracts, derived from the eggs of African clawed frogs (Xenopus laevis), have been widely used for studies concerning DDR pathways. In this review we focus on how different experimental systems have been established using Xenopus egg extracts to investigate the DDR pathways that are activated in response to DNA replication stress, double-strand breaks (DSBs), inter-strand crosslinks (ICLs), and oxidative stress. We summarize how molecular details of DDR pathways are dissected by the mechanistic studies with Xenopus egg extracts. We also provide an update on the regulation of translesion DNA synthesis (TLS) polymerases (Pol κ and REV1) in the DDR pathways. A better understanding of DDR pathways using Xenopus egg extracts has opened new avenues for future cancer therapeutics. Finally, we offer our perspectives of future directions for studies of DDR pathways with Xenopus egg extracts. PMID:27160070

  10. 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

  11. Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques.

    PubMed

    Aljanabi, S M; Martinez, I

    1997-11-15

    A very simple, fast, universally applicable and reproducible method to extract high quality megabase genomic DNA from different organisms is described. We applied the same method to extract high quality complex genomic DNA from different tissues (wheat, barley, potato, beans, pear and almond leaves as well as fungi, insects and shrimps' fresh tissue) without any modification. The method does not require expensive and environmentally hazardous reagents and equipment. It can be performed even in low technology laboratories. The amount of tissue required by this method is approximately 50-100 mg. The quantity and the quality of the DNA extracted by this method is high enough to perform hundreds of PCR-based reactions and also to be used in other DNA manipulation techniques such as restriction digestion, Southern blot and cloning.

  12. A simple and sensitive method to extract bacterial, yeast and fungal DNA from blood culture material.

    PubMed

    Millar, B C; Jiru, X; Moore, J E; Earle, J A

    2000-10-01

    This study investigated the various commercially available kits and 'in-house' methods to extract DNA from Gram-negative and Gram-positive bacteria, yeast and fungal agents in commonly employed blood culture material. The main methods investigated were as follows; Qiagen QIAmp Blood kit, Roche high PCR template preparation kit, Puregene DNA extraction kit, boiling, glass beads/sonication and wash/alkali/heat lysis. The results indicated that a simple wash/alkali/heat lysis method was the most sensitive, reproducible, simple and cost-effective extraction method. This was the only method which removed any PCR inhibitors and inherent DNA which existed in virgin BacT/Alert aerobic, anaerobic and paediatric blood culture material. Contaminating microbial DNA from Lactococcus lactis or Bacillus coagulans was identified in all batches of BacT/Alert FAN aerobic blood culture material examined.

  13. 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

  14. [Extraction and analysis of nuclear DNA from free margin of nail material].

    PubMed

    Nie, Sheng-Jie; Yang, Yan-Mei; Tang, Wen-Ru; Xu, Bing-Ying; Jing, Qiang; Xiao, Chun-Jie

    2007-11-01

    To investigate the feasibility of DNA analysis from free margin of the nail, genomic DNA was extracted from the free margin of nail clipping of 10 volunteers using the proteinase K/SDS -based organic method, the Chelex-100 method, or a combined method. Target DNA was simultaneously amplified using a fluorescent multiplex AmpFlSTR Identifier kit. The PCR products were analyzed on the ABI PRISM 3130 Genetic Analyzer. The results showed that, compared with profiles achieved by genotyping of blood samples from each volunteer as reference, 100% concordance was achieved using the combined method. The STR genotype profiles obtained through the organic method were acceptable, despite preferential amplification at some loci. In contrast, no readable profiles could be determined when DNA was extracted by the Chelex-100 method, and there were a large number of alleles missing. Our data suggest that free margin of nail can be used for nuclear DNA analysis, but the type of DNA isolation method used is critical. The traditional organic extraction method works reasonably well for free margin nail DNA isolation, and combination of organic extraction and the Chelex-100 method works best.

  15. Extraction of DNA from soil for analysis of bacterial diversity in transgenic and nontransgenic papaya sites.

    PubMed

    Sheu, Ceshing; Wu, Chung-Yi; Chen, Shu-Chuan; Lo, Chi-Chu

    2008-12-24

    The influence of transgenic crops on the soil diversity of microorganisms is one of the major risk assessments being conducted in Taiwan since 2007, and a reliable soil DNA extraction method for denaturing gradient gel electrophoresis (DGGE) is required. Six soils of different type, organic matter content, cation exchange capacity, and pH were tested, and four previously reported soil DNA extraction methods were applied to these soils. Soil DNA extracts by Zhou's CS method plus QIAquick gel was recommended in our laboratory for DGGE to monitor the microbial diversity in soil. There were some differences on the bacterial diversity based on DGGE patterns at the beginning of planting, and the difference decreased after six months. The results also indicated that clay content (10.8-25.0%) and pH (4.4-6.9) of different soil samples we tested did not affect the DNA extraction efficiencies, but positive correlations were found between the organic matter content (1.2-3.9%) of soils and the DNA yields in Widmer's GS method (r = 0.93, p = 0.005) and the MoBio UC method (r = 0.92, p = 0.007). Coefficient of determinations between organic matter content and DNA yield were higher than those between clay content, CEC, and pH, indicating that organic matter content was more correlated with DNA yield than that clay content, CEC, and pH in our soil samples tested.

  16. 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

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

    PubMed Central

    2013-01-01

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

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

    PubMed

    Rothe, Jessica; Nagy, Marion

    2016-09-01

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

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

    PubMed

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

    2010-01-01

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

  20. 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

  1. 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

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

    PubMed

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

    2016-09-01

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

  3. 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.

  4. 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

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

    PubMed

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

    2009-12-15

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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2012-01-20

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

  8. Extraction of high quality genomic DNA from microsamples of human blood.

    PubMed

    Ma, H W; Cheng, J; Caddy, B

    1994-01-01

    A simple and efficient method for extracting human genomic DNA from microsamples of blood has been developed. This method used sodium perchlorate, chloroform, polymerised silica gel and a dumbbell-shape tube, instead of proteinase K and phenol. The entire process took less than two hours, and high molecular weight DNA, in high yield and purity, was obtained from a few microlitres of human blood. DNA prepared in this way can be easily digested with restriction endonucleases and has been employed for DNA profiling and the polymerase chain reaction.

  9. Microbes on building materials--evaluation of DNA extraction protocols as common basis for molecular analysis.

    PubMed

    Ettenauer, Jörg D; Piñar, Guadalupe; Lopandic, Ksenija; Spangl, Bernhard; Ellersdorfer, Günther; Voitl, Christian; Sterflinger, Katja

    2012-11-15

    The study of microbial life in building materials is an emerging topic concerning biodeterioration of materials as well as health risks in houses and at working places. Biodegradation and potential health implications associated with microbial growth in our residues claim for more precise methods for quantification and identification. To date, cultivation experiments are commonly used to gain insight into the microbial diversity. Nowadays, molecular techniques for the identification of microorganisms provide efficient methods that can be applied in this field. The efficiency of DNA extraction is decisive in order to perform a reliable and reproducible quantification of the microorganisms by qPCR or to characterize the structure of the microbial community. In this study we tested thirteen DNA extraction methods and evaluated their efficiency for identifying (1) the quantity of DNA, (2) the quality and purity of DNA and (3) the ability of the DNA to be amplified in a PCR reaction using three universal primer sets for the ITS region of fungi as well as one primer pair targeting the 16S rRNA of bacteria with three typical building materials - common plaster, red brick and gypsum cardboard. DNA concentration measurements showed strong variations among the tested methods and materials. Measurement of the DNA yield showed up to three orders of magnitude variation from the same samples, whereas A260/A280 ratios often prognosticated biases in the PCR amplifications. Visualization of the crude DNA extracts and the comparison of DGGE fingerprints showed additional drawbacks of some methods. The FastDNA Spin kit for soil showed to be the best DNA extraction method and could provide positive results for all tests with the three building materials. Therefore, we suggest this method as a gold standard for quantification of indoor fungi and bacteria in building materials.

  10. Automated DNA extraction, quantification, dilution, and PCR preparation for genotyping by high-resolution melting.

    PubMed

    Seipp, Michael T; Herrmann, Mark; Wittwer, Carl T

    2010-12-01

    Genotyping by high-resolution amplicon melting uses only two PCR primers per locus and a generic, saturating DNA dye that detects heteroduplexes as well as homoduplexes. Heterozygous genotypes have a characteristic melting curve shape and a broader width than homozygous genotypes, which are usually differentiated by their melting temperature (T(m)). The H63D mutation, associated with hemochromatosis, is a single nucleotide polymorphism, which is impossible to genotype based on T(m), as the homozygous WT and mutant amplicons melt at the same temperature. To distinguish such homozygous variants, WT DNA can be added to controls and unknown samples to create artificial heterozygotes with all genotypes distinguished by quantitative heteroduplex analysis. By automating DNA extraction, quantification, and PCR preparation, a hands-off integrated solution for genotyping is possible. A custom Biomek® NX robot with an onboard spectrophotometer and custom programming was used to extract DNA from whole blood, dilute the DNA to appropriate concentrations, and add the sample DNA to preprepared PCR plates. Agencourt® Genfind™ v.2 chemistry was used for DNA extraction. PCR was performed on a plate thermocycler, high-resolution melting data collected on a LightScanner-96, followed by analysis and automatic genotyping using custom software. In a blinded study of 42 H63D samples, 41 of the 42 sample genotypes were concordant with dual hybridization probe genotyping. The incorrectly assigned genotype was a heterozygote that appeared to be a homozygous mutant as a result of a low sample DNA concentration. Automated DNA extraction from whole blood with quantification, dilution, and PCR preparation was demonstrated using quantitative heteroduplex analysis. Accuracy is critically dependent on DNA quantification.

  11. A practical and novel method to extract genomic DNA from blood collection kits for plasma protein preservation.

    PubMed

    Waters, Jon; Dhere, Vishal; Benjamin, Adam; Sekar, Arvind; Kumar, Archana; Prahalad, Sampath; Okou, David T; Kugathasan, Subra

    2013-05-18

    Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources. The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes(1), to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes(2-4). Challenges from these methods were mainly

  12. A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

    PubMed Central

    Waters, Jon; Dhere, Vishal; Benjamin, Adam; Sekar, Arvind; Kumar, Archana; Prahalad, Sampath; Okou, David T.; Kugathasan, Subra

    2013-01-01

    Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources. The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly

  13. 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...

  14. Nuclear extracts of chicken embryos promote an active demethylation of DNA by excision repair of 5-methyldeoxycytidine.

    PubMed Central

    Jost, J P

    1993-01-01

    Here I show that nuclear extracts of chicken embryos can promote the active demethylation of DNA. The evidence shows that in hemimethylated DNA (i.e., methylated on one strand only) demethylation of 5mCpG occurs through nucleotide excision repair. The first step of demethylation is the formation of specific nicks 5' from 5-methyldeoxycytidine. Nicks are also observed in vitro on symmetrically methylated CpGs (i.e., methylated on both strands) but they result in breakage of the oligonucleotide with no repair. No specific nicks are observed on the nonmethylated CpG. Nicks are strictly 5mCpG specific and do not occur on 5mCpC, 5mCpT, 5mCpA, or 6mApT. The effect of nonspecific nuclease(s) has been ruled out. The nicking of mCpG takes place in the presence of 20 mM EDTA irrespective of the nature of the sequence surrounding the 5mCpG. No methylcytosine glycosylase activity could be detected. The repair is aphidicolin and N-ethylmaleimide resistant, suggesting a repair action by DNA polymerase beta. In extracts of chicken embryos, the excision repair of mCpG is highest between the 6th and the 12th day of development, whereas it is barely detectable in nuclear extracts from different organs of adults. The possible implications of 5mCpG endonuclease activity in active demethylation of DNA during differentiation is discussed. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 PMID:8506318

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

    PubMed

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

    2011-01-01

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

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

    PubMed

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

    2011-03-26

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

  17. Microfluidic extraction, stretching and analysis of human chromosomal DNA from single cells†

    PubMed Central

    Benítez, Jaime J.; Topolancik, Juraj; Tian, Harvey C.; Wallin, Christopher B.; Latulippe, David R.; Szeto, Kylan; Murphy, Patrick J.; Cipriany, Benjamin R.; Levy, Stephen L.; Soloway, Paul D.; Craighead, Harold G.

    2014-01-01

    We describe a microfluidic device for the extraction, purification and stretching of human chromosomal DNA from single cells. A two-dimensional array of micropillars in a microfluidic polydimethylsiloxane channel was designed to capture a single human cell. Megabase-long DNA strands released from the cell upon lysis are trapped in the micropillar array and stretched under optimal hydrodynamic flow conditions. Intact chromosomal DNA is entangled in the array, while other cellular components are washed from the channel. To demonstrate the entrapment principle, a single chromosome was hybridized to whole chromosome paints, and imaged by fluorescence microscopy. DNA extracted from a single cell and small cell populations (less than 100) was released from the device by restriction endonuclease digestion under continuous flow and collected for offchip analysis. Quantification of the extracted material reveals that the microdevice efficiently extracts essentially all chromosomal DNA. The device described represents a novel platform to perform a variety of analyses on chromosomal DNA at the single cell level. PMID:23018789

  18. Microfluidic extraction, stretching and analysis of human chromosomal DNA from single cells.

    PubMed

    Benítez, Jaime J; Topolancik, Juraj; Tian, Harvey C; Wallin, Christopher B; Latulippe, David R; Szeto, Kylan; Murphy, Patrick J; Cipriany, Benjamin R; Levy, Stephen L; Soloway, Paul D; Craighead, Harold G

    2012-11-21

    We describe a microfluidic device for the extraction, purification and stretching of human chromosomal DNA from single cells. A two-dimensional array of micropillars in a microfluidic polydimethylsiloxane channel was designed to capture a single human cell. Megabase-long DNA strands released from the cell upon lysis are trapped in the micropillar array and stretched under optimal hydrodynamic flow conditions. Intact chromosomal DNA is entangled in the array, while other cellular components are washed from the channel. To demonstrate the entrapment principle, a single chromosome was hybridized to whole chromosome paints, and imaged by fluorescence microscopy. DNA extracted from a single cell and small cell populations (less than 100) was released from the device by restriction endonuclease digestion under continuous flow and collected for off-chip analysis. Quantification of the extracted material reveals that the microdevice efficiently extracts essentially all chromosomal DNA. The device described represents a novel platform to perform a variety of analyses on chromosomal DNA at the single cell level.

  19. 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

    2015-09-30

    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.

  20. 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

  1. 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.

  2. Efficient DNA extraction from nail clippings using the protease solution from Cucumis melo.

    PubMed

    Yoshida-Yamamoto, Shumi; Nishimura, Sayaka; Okuno, Teruko; Rakuman, Miki; Takii, Yukio

    2010-09-01

    Owing to the increasing importance of genomic information, obtaining genomic DNA easily from biological specimens has become more and more important. This article proposes an efficient method for obtaining genomic DNA from nail clippings. Nail clippings can be easily obtained, are thermostable and easy to transport, and have low infectivity. The drawback of their use, however, has been the difficulty of extracting genomic material from them. We have overcome this obstacle using the protease solution obtained from Cucumis melo. The keratinolytic activity of the protease solution was 1.78-fold higher than that of proteinase K, which is commonly used to degrade keratin. With the protease solution, three times more DNA was extracted than when proteinase K was used. In order to verify the integrity of the extracted DNA, genotype analysis on 170 subjects was performed by both PCR-RFLP and Real Time PCR. The results of the genotyping showed that the extracted DNA was suitable for genotyping analysis. In conclusion, we have developed an efficient extraction method for using nail clippings as a genome source and a research tool in molecular epidemiology, medical diagnostics, and forensic science.

  3. DNA extraction from hair shafts of wild Brazilian felids and canids.

    PubMed

    Alberts, C C; Ribeiro-Paes, J T; Aranda-Selverio, G; Cursino-Santos, J R; Moreno-Cotulio, V R; Oliveira, A L D; Porchia, B F M M; Santos, W F; Souza, E B

    2010-12-21

    Wild felids and canids are usually the main predators in the food chains where they dwell and are almost invisible to behavior and ecology researchers. Due to their grooming behavior, they tend to swallow shed hair, which shows up in the feces. DNA found in hair shafts can be used in molecular studies that can unravel, for instance, genetic variability, reproductive mode and family structure, and in some species, it is even possible to estimate migration and dispersion rates in given populations. First, however, DNA must be extracted from hair. We extracted successfully and dependably hair shaft DNA from eight wild Brazilian felids, ocelot, margay, oncilla, Geoffroy's cat, pampas cat, jaguarundi, puma, and jaguar, as well as the domestic cat and from three wild Brazilian canids, maned wolf, crab-eating fox, and hoary fox, as well as the domestic dog. Hair samples came mostly from feces collected at the São Paulo Zoo and were also gathered from non-sedated pet or from recently dead wild animals and were also collected from museum specimens. Fractions of hair samples were stained before DNA extraction, while most samples were not. Our extraction protocol is based on a feather DNA extraction technique, based in the phenol:chloroform:isoamyl alcohol general method, with proteinase K as digestive enzyme.

  4. 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

  5. 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

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

    PubMed

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

    2010-04-01

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

  7. Effects of different methods of DNA extraction for activated sludge on the subsequent analysis of bacterial community profiles.

    PubMed

    Sun, Lianpeng; Ouyang, Xiong; Tang, Yueheng; Yang, Ying; Luo, Ying

    2012-02-01

    The effect of different DNA extraction protocols on activated sludge DNA yield and bacterial community composition was evaluated by temperature gradient gel electrophoresis (TGGE). Nine different procedures to extract DNA were compared-sonication (30s), sonication (40s), sonication (50s), freezing-thawing, bead milling, sodium dodecyl sulfate (SDS)-lysozyme, SDS-proteinase K, SDS-lysozyme-proteinase, and a commercial extraction kit. It was found that the TGGE profiles and the DNA band numbers made significant differences via various extraction methods. The yield and purity of DNA extracted by sonication and other physical methods were not satisfactory, while the DNA purity extracted by SDS and other chemical-biological methods were better. Crude DNA extracts isolated by sonication and other physical methods passed the polymerase chain reaction, despite the absence of purification and acquired affluent DNA bands in TGGE. The affluence of bands in TGGE was not consistent with the yield and purification of DNA, but was correlative with extraction protocols. To analyze the activated sludge bacterial community by TGGE fingerprint, it is necessary to make a synthesis of the TGGE fingerprint profiles of chemical and physical DNA extraction methods to overcome the representative bias.

  8. 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

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

    PubMed

    Lech, T

    2016-05-06

    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.

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

    PubMed Central

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

    2012-01-01

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

  11. Comparison of methods for extracting DNA from formalin-fixed paraffin sections for nonisotopic PCR.

    PubMed

    Frank, T S; Svoboda-Newman, S M; Hsi, E D

    1996-09-01

    DNA was extracted from unstained 5-microns sections of neutral buffered 10% formalin-fixed paraffin-embedded tissue by proteinase K digestion without detergents followed by boiling, proteinase K digestion with ionic detergents with and without phenol chloroform extraction and ethanol precipitation, sonication with proteinase K followed by boiling, or boiling alone. Serial 1:10 dilutions of the extracted DNA were subject to polymerase chain reaction (PCR) amplification of a 255-bp portion of the p53 gene. Digestion with proteinase K without ionic detergents followed by boiling (without phenol chloroform extraction) gave the best yield, enabling visualization of ethidium bromide-stained PCR product from a DNA dilution corresponding to 0.1 mm2 of tissue containing of the order of 10(3) nuclear profiles. Proteinase K digestion with detergents followed by phenol-chloroform extraction was no more effective than simple boiling. Although the success of PCR from preserved tissue will vary with the fixative and size of the amplified fragment, DNA extracted with this optimized method can be used for identification of viruses, loss of heterozygosity, and immunoglobulin gene rearrangements in paraffin-embedded tissue without radioisotopes.

  12. Black tea extract: a supplementary antioxidant in radiation-induced damage to DNA and normal lymphocytes.

    PubMed

    Ghosh, Debjani; Pal, Sandip; Saha, Chabita; Chakrabarti, Amit Kumar; Datta, Salil C; Dey, Subrata Kumar

    2012-01-01

    Myriad research has contributed significantly toward the understanding and identification of health benefits stemming from tea polyphenols and many other naturally occurring flavonoids present in fruits and vegetables. These flavonoids are known to mitigate reactive oxygen species-induced damage by scavenging them. In this study, hot-water black tea extract rich in flavonoids is evaluated as a supplementary antioxidant. The antioxidant efficacy of black tea extract was investigated by evaluating radioprotection conferred to pBR322 DNA, calf thymus DNA, and normal lymphocytes during gamma irradiation. The protection was measured by gel electrophoresis, fluorimetric study, cell viability assay, cytokinesis-blocked micronuclei assay, and comet assay. The 2,2-diphenyl-1-picrylhydrazyl scavenging ability of the tea extract used increased in a dose-dependent manner (IC50: 182.45 µg/mL). Positive correlation of radioprotection with antioxidant activity of black tea extract was observed in all systems. Maximum protection against radiation-induced damage was observed in pBR322 DNA and calf thymus DNA at ≥200 µg/mL of black tea extract. At a dose of black tea extract as low as 5 µg/mL, efficient radioprotection was observed in normal lymphocytes, which is encouraging and can be tested in the future as a natural antioxidant supplement during radiotherapy.

  13. 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

  14. 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.

  15. Extracting evidence from forensic DNA analyses: future molecular biology directions.

    PubMed

    Budowle, Bruce; van Daal, Angela

    2009-04-01

    Molecular biology tools have enhanced the capability of the forensic scientist to characterize biological evidence to the point where it is feasible to analyze minute samples and achieve high levels of individualization. Even with the forensic DNA field's maturity, there still are a number of areas where improvements can be made. These include: enabling the typing of samples of limited quantity and quality; using genetic information and novel markers to provide investigative leads; enhancing automation with robotics, different chemistries, and better software tools; employing alternate platforms for typing DNA samples; developing integrated microfluidic/microfabrication devices to process DNA samples with higher throughput, faster turnaround times, lower risk of contamination, reduced labor, and less consumption of evidentiary samples; and exploiting high-throughput sequencing, particularly for attribution in microbial forensics cases. Knowledge gaps and new directions have been identified where molecular biology will likely guide the field of forensics. This review aims to provide a roadmap to guide those interested in contributing to the further development of forensic genetics.

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

    DOE PAGES

    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

  17. 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.

  18. 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

  19. Method Specific Calibration Corrects for DNA Extraction Method Effects on Relative Telomere Length Measurements by Quantitative PCR

    PubMed Central

    Holland, Rebecca; Underwood, Sarah; Fairlie, Jennifer; Psifidi, Androniki; Ilska, Joanna J.; Bagnall, Ainsley; Whitelaw, Bruce; Coffey, Mike; Banos, Georgios; Nussey, Daniel H.

    2016-01-01

    Telomere length (TL) is increasingly being used as a biomarker in epidemiological, biomedical and ecological studies. A wide range of DNA extraction techniques have been used in telomere experiments and recent quantitative PCR (qPCR) based studies suggest that the choice of DNA extraction method may influence average relative TL (RTL) measurements. Such extraction method effects may limit the use of historically collected DNA samples extracted with different methods. However, if extraction method effects are systematic an extraction method specific (MS) calibrator might be able to correct for them, because systematic effects would influence the calibrator sample in the same way as all other samples. In the present study we tested whether leukocyte RTL in blood samples from Holstein Friesian cattle and Soay sheep measured by qPCR was influenced by DNA extraction method and whether MS calibration could account for any observed differences. We compared two silica membrane-based DNA extraction kits and a salting out method. All extraction methods were optimized to yield enough high quality DNA for TL measurement. In both species we found that silica membrane-based DNA extraction methods produced shorter RTL measurements than the non-membrane-based method when calibrated against an identical calibrator. However, these differences were not statistically detectable when a MS calibrator was used to calculate RTL. This approach produced RTL measurements that were highly correlated across extraction methods (r > 0.76) and had coefficients of variation lower than 10% across plates of identical samples extracted by different methods. Our results are consistent with previous findings that popular membrane-based DNA extraction methods may lead to shorter RTL measurements than non-membrane-based methods. However, we also demonstrate that these differences can be accounted for by using an extraction method-specific calibrator, offering researchers a simple means of accounting for

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

    PubMed

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

    2014-11-01

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

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

    PubMed

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

    2014-11-01

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

  2. One-step immobilization of aminated and thiolated DNA onto poly(methylmethacrylate) (PMMA) substrates.

    PubMed

    Fixe, F; Dufva, M; Telleman, P; Christensen, C B V

    2004-06-01

    Direct immobilisation of modified DNA oligonucleotides (aminated or thiolated) onto a plastic substrate, poly(methylmethacrylate), (PMMA) is described. Using the methyl esters present on non-modified PMMA, it was possible to establish a covalent bond between the electron donor of a DNA probe and the C terminal ester of the PMMA substrate. Since the procedure consists of a single brief wash in isopropanol or ethanol, the procedure is simple and environmentally friendly. The new immobilization strategy was characterized by analysing DNA microarray performance. The new procedure resulted in probe- and hybridization densities that were greater or equivalent to those obtained with commercially available surfaces and other procedures to immobilize DNA onto PMMA. The described chemistry selectively immobilized the DNA via terminal thiol or amine groups indicating that probe orientation could be controlled. Furthermore, the chemical bond between the immobilized DNA and the PMMA could endure repeated heat cycling with only 50% probe loss after 20 cycles, indicating that the chemistry could be used in integrated PCR/microarray devices. PMID:15159777

  3. Application of FTA technology to extraction of sperm DNA from mixed body fluids containing semen.

    PubMed

    Fujita, Yoshihiko; Kubo, Shin-ichi

    2006-01-01

    FTA technology is a novel method designed to simplify the collection, shipment, archiving and purification of nucleic acids from a wide variety of biological sources. In this study, we report a rapid and simple method of extracting DNA from sperm when body fluids mixed with semen were collected using FTA cards. After proteinase K digestion of the sperm and body fluid mixture, the washed pellet suspension as the sperm fraction and the concentrated supernatant as the epithelial cell fraction were respectively applied to FTA cards containing DTT. The FTA cards were dried, then directly added to a polymerase chain reaction (PCR) mix and processed by PCR. The time required from separation of the mixed fluid into sperm and epithelial origin DNA extractions was only about 2.5-3h. Furthermore, the procedure was extremely simple. It is considered that our designed DNA extraction procedure using an FTA card is available for application to routine work.

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

    PubMed

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

    2012-05-22

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  8. 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

  9. Extraction of toxic compounds from saliva by magnetic-stirring-assisted micro-solid-phase extraction step followed by headspace-gas chromatography-ion mobility spectrometry.

    PubMed

    Criado-García, Laura; Arce, Lourdes

    2016-09-01

    A new sample extraction procedure based on micro-solid-phase extraction (μSPE) using a mixture of sorbents of different polarities (polymeric reversed-phase sorbent HLB, silica-based sorbent C18, and multiwalled carbon nanotubes) was applied to extract benzene, toluene, butyraldehyde, benzaldehyde, and tolualdehyde present in saliva to avoid interference from moisture and matrix components and enhance sensitivity and selectivity of the ion mobility spectrometry (IMS) methodology proposed. The extraction of target analytes from saliva samples by using μSPE were followed by the desorption step carried out in the headspace vials placed in the autosampler of the IMS device. Then, 200 μL of headspace was injected into the GC column coupled to the IMS for its analysis. The method was fully validated in terms of sensitivity, precision, and recovery. The LODs and LOQs obtained, when analytes were dissolved in saliva samples to consider the matrix effect, were within the range of 0.38-0.49 and 1.26-1.66 μg mL(-1), respectively. The relative standard deviations were <3.5 % for retention time and drift time values, which indicate that the method proposed can be applied to determine toxic compounds in saliva samples. Graphical abstract Summary of steps followed in the experimental set up of this work. PMID:27481168

  10. 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.

  11. 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

  12. 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

  13. Technical note: improved DNA extraction from ancient bones using silica-based spin columns.

    PubMed

    Yang, D Y; Eng, B; Waye, J S; Dudar, J C; Saunders, S R

    1998-04-01

    We describe a simple method for extracting polymerase chain reaction-amplifiable DNA from ancient bones without the use of organic solvents. Bone powders are digested with proteinase K, and the DNA is purified directly using silica-based spin columns (QIAquick3, QIAGEN). The efficiency of this protocol is demonstrated using human bone samples ranging in age from 15 to 5,000 years old.

  14. Efficient extraction of canthaxanthin from Escherichia coli by a 2-step process with organic solvents.

    PubMed

    Scaife, Mark A; Ma, Cynthia A; Armenta, Roberto E

    2012-05-01

    Canthaxanthin has a substantial commercial market in aquaculture, poultry production, and cosmetic and nutraceutical industries. Commercial production is dominated by chemical synthesis; however, changing consumer demands fuel research into the development of biotechnology processes. Highly productive microbial systems to produce carotenoids can be limited by the efficiency of extraction methods. Extraction with hexane, acetone, methanol, 2-propanol, ethanol, 1-butanol, tetrahydrofuran and ethyl acetate was carried out with each solvent separately, and subsequently the most efficient solvents were tested in combination, both as mixtures and sequentially. Sequential application of methanol followed by acetone proved most efficient. Extraction efficiency remained stable over a solvent to biomass range of 100:1 to 55:1, but declined significantly at a ratio of 25:1. Application of this method to a canthaxanthin-producing Escherichia coli production system enabled efficient canthaxanthin extraction of up to 8.5 mg g(-1) dry biomass. PMID:22353211

  15. Improvement of the BCR three step sequential extraction procedure prior to the certification of new sediment and soil reference materials.

    PubMed

    Rauret, G; López-Sánchez, J F; Sahuquillo, A; Rubio, R; Davidson, C; Ure, A; Quevauviller, P

    1999-02-01

    The Standards, Measurements and Testing Programme (formerly BCR) of the European Commission proposed a three-step sequential extraction procedure for sediment analysis, following extensive expert consultations and two interlaboratory studies. This scheme was recently used to certify the extractable trace element contents of a sediment reference material (CRM 601). Although this procedure offers a means to ensure the comparability of data in this field, some difficulties concerning the interlaboratory reproducibility still remain, and a new project is currently being conducted to determine the causes of poor reproducibility in the extraction scheme. The final objective of the project is the certification of new sediment and soil reference materials for their extractable contents of Cd, Cr, Cu, Ni, Pb and Zn. This paper presents the results of a small-scale interlaboratory study, which aimed to test a revised version of the extraction schemes by comparing the original and the modified protocols using the CRM 601 sample. This work offers an improvement to the BCR sequential extraction procedure through intercomparison exercises. This improved procedure will allow the obtaining of CRMs to validate analytical data in the analysis of soils and sediments, and it will also facilitate comparability of data in the European Union.

  16. Ku-Dependent Nonhomologous DNA End Joining in Xenopus Egg Extracts

    PubMed Central

    Labhart, Paul

    1999-01-01

    An extract from activated Xenopus eggs joins both matching and nonmatching ends of exogenous linear DNA substrates with high efficiency and fidelity (P. Pfeiffer and W. Vielmetter, Nucleic Acids Res. 16:907–924, 1988). In mammalian cells, such nonhomologous end joining (NHEJ) is known to require the Ku heterodimer, a component of DNA-dependent protein kinase. Here I investigated whether Ku is also required for the in vitro reaction in the egg extract. Immunological assays indicate that Ku is very abundant in the extract. I found that all NHEJ was inhibited by autoantibodies against Ku and that NHEJ between certain combinations of DNA ends was also decreased after immunodepletion of Ku from the extract. The formation of a joint between a DNA end with a 5′-protruding single strand (PSS) and an end with a 3′-PSS, between two ends with 3′-PSS, and between two blunt ends was most Ku dependent. On the other hand, NHEJ between two DNA ends bearing 5′-PSS was Ku independent. These results show that the Xenopus cell-free system will be useful to biochemically dissect the role of Ku in eukaryotic NHEJ. PMID:10082524

  17. Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR

    NASA Astrophysics Data System (ADS)

    Ansari, Aseem Z.; Chael, Mark L.; O'Halloran, Thomas V.

    1992-01-01

    POSITIVE control of transcription often involves stimulatory protein-protein interactions between regulatory factors and RNA polymerase1. Critical steps in the activation process itself are seldom ascribed to protein-DNA distortions. Activator-induced DNA bending is typically assigned a role in binding-site recognition2, alterations in DNA loop structures3 or optimal positioning of the activator for interaction with polymerase4. Here we present a transcriptional activation mechanism that does not require a signal-induced DNA bend but rather a receptor-induced untwisting of duplex DNA. The allosterically modulated transcription factor MerR is a represser and an Hg(II)-responsive activator of bacterial mercury-resistance genes5-7.Escherichia coliRNA polymerase binds to the MerR-promoter complex but cannot proceed to a transcriptionally active open complex until Hg(II) binds to MerR (ref. 6). Chemical nuclease studies show that the activator form, but not the represser, induces a unique alteration of the helical structure localized at the centre of the DNA-binding site6. Data presented here indicate that this Hg-MerR-induced DNA distortion corresponds to a local underwinding of the spacer region of the promoter by about 33° relative to the MerR-operator complex. The magnitude and the direction of the Hg-MerR-induced change in twist angle are consistent with a positive control mechanism involving reorientation of conserved, but suboptimally phased, promoter elements and are consistent with a role for torsional stress in formation of an open complex.

  18. Comparison of methods for high quantity and quality genomic DNA extraction from raw cow milk.

    PubMed

    Usman, T; Yu, Y; Liu, C; Fan, Z; Wang, Y

    2014-01-01

    Isolation of sufficient quantities of high quality DNA is a prerequisite for molecular studies. Milk somatic cells can be used; however, inhibitors such as fats and proteins make milk a difficult medium for extracting large amounts of quality DNA. We optimized, evaluated and compared three methods, Modified Nucleospin Blood Kit method, Modified TianGen Kit method and Phenol-Chloroform method for genomic DNA extraction from bovine milk. Individual cows' milk and bulk milk samples were collected from a China agricultural university dairy farm. Genomic DNA extracted from each milk sample by the three methods was evaluated for quantity and purity by spectrophotometry and gel electrophoresis, as well as PCR and sequencing. All the three methods were found suitable for genomic DNA isolation from bovine milk, PCR applications, and sequencing. Comparing the three methods, we found that the Modified Nucleospin Blood Kit method was significantly better than the Phenol-Chloroform method in terms of quantity as well as quality (amount, concentration, 260/280 nm and 260/230 nm absorbance ratio), whereas, the Modified TianGen Kit method was more efficient than the Phenol-Chloroform method and cheaper than the Modified Nucleospine Blood Kit method; it yielded reasonably good quantities of good quality DNA and would be suitable for large-scale genotyping of lactating cows.

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

    PubMed

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

    2007-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  1. [Comparative assessment of DNA extraction methods for identification of glanders and melioidosis etiological agents by PCR].

    PubMed

    Zinchenko, O V; Antonov, V A; Tkachenko, G A; Altukhova, V V; Zamaraev, V S; Piven', N N; Goloseev, Iu A; Vasil'ev, V P; Lomova, L V; Alekseev, V V

    2008-01-01

    Pathogenic Burkholderia are considered as a cause of dangerous infections and potential agents of bioterrorism. Comparative assessment of different methods of extraction and purification of DNA for PCR analysis of pure cultures and samples contaminated by etiological agents of glanders and melioidosis was performed. Samples of soil and food artificially contaminated by pathogenic Burkholderia as well as organs of infected animals were tested. DNA was extracted by methods of boiling, nucleosorption with presence of guanidine thiocyanate, guanidine thiocyanatephenol extraction, guanidine thiocyanate-phenol extraction with additional purification of DNA by nucleosorption. Amplification was performed by "Flash" technique and detector of fluorescence was used for analysis of PCR products. Utilization of the recommended methods of preparation depending on the nature of sample let to detect by the "Flash" technique the etiological agents of glanders and melioidosis in concentration =10(3) microbial cells per ml. Choice of DNA extraction and purification methods is determined by type of a sample and presence in it of admixtures inhibiting PCR.

  2. A comparison of methods for total community DNA preservation and extraction from various thermal environments.

    PubMed

    Mitchell, Kendra R; Takacs-Vesbach, Cristina D

    2008-10-01

    The widespread use of molecular techniques in studying microbial communities has greatly enhanced our understanding of microbial diversity and function in the natural environment and contributed to an explosion of novel commercially viable enzymes. One of the most promising environments for detecting novel processes, enzymes, and microbial diversity is hot springs. We examined potential biases introduced by DNA preservation and extraction methods by comparing the quality, quantity, and diversity of environmental DNA samples preserved and extracted by commonly used methods. We included samples from sites representing the spectrum of environmental conditions that are found in Yellowstone National Park thermal features. Samples preserved in a non-toxic sucrose lysis buffer (SLB), along with a variation of a standard DNA extraction method using CTAB resulted in higher quality and quantity DNA than the other preservation and extraction methods tested here. Richness determined using DGGE revealed that there was some variation within replicates of a sample, but no statistical difference among the methods. However, the sucrose lysis buffer preserved samples extracted by the CTAB method were 15-43% more diverse than the other treatments.

  3. Evaluation of MolYsis™ Complete5 DNA Extraction Method for Detecting Staphylococcus aureus DNA from Whole Blood in a Sepsis Model Using PCR/Pyrosequencing

    PubMed Central

    McCann, Chase D.; Jordan, Jeanne A.

    2014-01-01

    Bacterial bloodstream infections (BSI) and ensuing sepsis are important causes of morbidity and mortality. Early diagnosis and rapid treatment with appropriate antibiotics are vital for improving outcome. Nucleic acid amplification of bacteria directly from whole blood has the potential of providing a faster means of diagnosing BSI than automated blood culture. However, effective DNA extraction of commonly low levels of bacterial target from whole blood is critical for this approach to be successful. This study compared the Molzyme MolYsis™ Complete5 DNA extraction method to a previously described organic bead-based method for use with whole blood. A well-characterized S. aureus-induced pneumonia model of sepsis in canines was used to provide clinically relevant whole blood samples. DNA extracts were assessed for purity and concentration and analyzed for bacterial rRNA gene targets using PCR and sequence-based identification. Both extraction methods yielded relatively pure DNA with median A260/280 absorbance ratios of 1.71 (MolYsis™) and 1.97 (bead-based). The organic bead-based extraction method yielded significantly higher average DNA concentrations (P <0.05) at each time point throughout the experiment, closely correlating with changes observed in white blood cell (WBC) concentrations during this same time period, while DNA concentrations of the MolYsis™ extracts closely mirrored quantitative blood culture results. Overall, S. aureus DNA was detected from whole blood samples in 70.7% (58/82) of MolYsis™ DNA extracts, and in 59.8% (49/82) of organic bead-based extracts, with peak detection rates seen at 48 h for both MolYsis™ (87.0%) and organic bead-based (82.6%) methods. In summary, the MolYsis™ Complete5 DNA extraction kit proved to be the more effective method for isolating bacterial DNA directly from extracts made from whole blood. PMID:24503182

  4. Efficient DNA Extraction for HPV Genotyping in Formalin-Fixed, Paraffin-Embedded Tissues

    PubMed Central

    Steinau, Martin; Patel, Sonya S.; Unger, Elizabeth R.

    2011-01-01

    DNA from archived FFPE can be used for papillomavirus genotyping, but potential problems include paraffin as a physical barrier, DNA cross-linking, and PCR inhibitors. To address these complications, we combined a commercially available DNA isolation kit (Qiagen DNeasy) with a heat treatment and evaluated the resulting DNA with regards to HPV typing. DNA was extracted from 10-μm sections from 150 FFPE cancer samples. One protocol followed the manufacturer's recommendation, including paraffin removal by xylene and tissue lysis at 56°C. A second section was directly incubated at 120°C and subsequently lysed at 65°C. After spin-column purification, both extracts were tested with a linear array HPV genotyping assay. Additionally, cellular DNA yield, HPV16 DNA copies, and PCR inhibitors were assessed by real-time qPCR assays. Inadequate linear array HPV genotyping assay results were significantly more frequent (P = 0.0003) in xylene-treated (29/150, 19.3%) than in heat-treated extracts (8/150, 5.3%). HPV detection also differed, with 94/150 (62.7%) and 110/150 (73.3%) positive results, respectively (P = 0.0026). The heat method also yielded more PCR-amplifiable cellular DNA (8.2-fold; P < 0.001) and HPV16 copies (6.5-fold; P = 0.009), although PCR inhibitors also had a greater effect (P = 0.035). Aggressive heat treatment demonstrated an advantage over traditional xylene purification protocols, resulting in higher DNA yields and increased sensitivity for HPV testing. PMID:21704270

  5. Efficient DNA extraction for HPV genotyping in formalin-fixed, paraffin-embedded tissues.

    PubMed

    Steinau, Martin; Patel, Sonya S; Unger, Elizabeth R

    2011-07-01

    DNA from archived FFPE can be used for papillomavirus genotyping, but potential problems include paraffin as a physical barrier, DNA cross-linking, and PCR inhibitors. To address these complications, we combined a commercially available DNA isolation kit (Qiagen DNeasy) with a heat treatment and evaluated the resulting DNA with regards to HPV typing. DNA was extracted from 10-μm sections from 150 FFPE cancer samples. One protocol followed the manufacturer's recommendation, including paraffin removal by xylene and tissue lysis at 56°C. A second section was directly incubated at 120°C and subsequently lysed at 65°C. After spin-column purification, both extracts were tested with a linear array HPV genotyping assay. Additionally, cellular DNA yield, HPV16 DNA copies, and PCR inhibitors were assessed by real-time qPCR assays. Inadequate linear array HPV genotyping assay results were significantly more frequent (P = 0.0003) in xylene-treated (29/150, 19.3%) than in heat-treated extracts (8/150, 5.3%). HPV detection also differed, with 94/150 (62.7%) and 110/150 (73.3%) positive results, respectively (P = 0.0026). The heat method also yielded more PCR-amplifiable cellular DNA (8.2-fold; P < 0.001) and HPV16 copies (6.5-fold; P = 0.009), although PCR inhibitors also had a greater effect (P = 0.035). Aggressive heat treatment demonstrated an advantage over traditional xylene purification protocols, resulting in higher DNA yields and increased sensitivity for HPV testing.

  6. 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

  7. 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.

  8. DNA Barcoding Marine Biodiversity: Steps from Mere Cataloguing to Giving Reasons for Biological Differences.

    PubMed

    Nikinmaa, Mikko; Götting, Miriam

    2016-01-01

    DNA barcoding has become a useful tool in many contexts and has opened up a completely new avenue for taxonomy. DNA barcoding has its widest application in biodiversity and ecological research to detect and describe diversity whenever morphological discrimination is difficult or impossible (e.g., in the case of species lacking diagnostic characters, early life stages, or cryptic species). In this chapter, we outline the utility of including physiological parameters as part of species description in publicly available databases that catalog taxonomic information resulting from barcoding projects. Cryptic species or different life stages of a species often differ in their physiological traits. Thus, if physiological aspects were included in species definitions, the presently cryptic species could be distinguished. We furthermore give suggestions for physiological information that should be included in a species description and describe potential applications of DNA barcoding for research with physiological components. PMID:27460377

  9. Fully automated DNA extraction from blood using magnetic particles modified with a hyperbranched polyamidoamine dendrimer.

    PubMed

    Yoza, Brandon; Arakaki, Atsushi; Maruyama, Kohei; Takeyama, Haruko; Matsunaga, Tadashi

    2003-01-01

    Bacterial and artificial magnetic particles were modified using a polyamidoamine (PAMAM) dendrimer and outer shell amines determined. Bacterial magnetic particles were the most consistently modified. Transmission electron microscopic (TEM) analysis showed that the artificial magnetic particles were structurally damaged by the modification process including sonication. Furthermore, laser particle analysis of the magnetite also revealed damage. Small quantities of dendrimer-modified bacterial magnetic particles were used to extract DNA from blood. The efficiency of DNA recovery was consistently about 30 ng of DNA using 2-10 microg of dendrimer-modified bacterial magnetite. This technique was fully automated using newly developed liquid handling robots and bacterial magnetic particles.

  10. 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

  11. 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.

  12. Probabilistically determining the cellular source of DNA derived from differential extractions in sexual assault scenarios.

    PubMed

    Taylor, Duncan

    2016-09-01

    Sexual assault cases are the type of case that often produces questions about the cellular source of DNA. In these cases multiple findings of microscopy, DNA profiling and presumptive testing need to be considered when addressing source level propositions. In this work, I consider a line of questioning that has been raised a number of times in the recent past, where in court it was disputed that low levels of sperm seen on a microscope slide were the cellular source of the male DNA profile component generated from the sperm fraction of a differential DNA extraction. I demonstrate how the cell scoring results and DNA profiling results can be considered together, in helping address this source level question through the use of Bayesian Networks.

  13. Probabilistically determining the cellular source of DNA derived from differential extractions in sexual assault scenarios.

    PubMed

    Taylor, Duncan

    2016-09-01

    Sexual assault cases are the type of case that often produces questions about the cellular source of DNA. In these cases multiple findings of microscopy, DNA profiling and presumptive testing need to be considered when addressing source level propositions. In this work, I consider a line of questioning that has been raised a number of times in the recent past, where in court it was disputed that low levels of sperm seen on a microscope slide were the cellular source of the male DNA profile component generated from the sperm fraction of a differential DNA extraction. I demonstrate how the cell scoring results and DNA profiling results can be considered together, in helping address this source level question through the use of Bayesian Networks. PMID:27388428

  14. 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.

  15. 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.

  16. Extraction of DNA from exfoliative cytology specimens and its suitability for analysis by the polymerase chain reaction.

    PubMed

    Jackson, D P; Payne, J; Bell, S; Lewis, F A; Taylor, G R; Peel, K R; Sutton, J; Quirke, P

    1990-01-01

    The extraction of DNA from archival exfoliative cytology samples would allow the molecular biological analysis of this readily available material using the polymerase chain reaction (PCR). We have quantitatively and qualitatively studied the extraction of DNA from a variety of cytological preparations. For both fresh and archival cervical smears, overnight incubation with proteinase K produces high yields of high molecular weight DNA, but simply boiling the samples produces DNA suitable for PCR amplification of a single copy gene. Increasing the proteinase K incubation to several days allows the extraction of DNA from fixed and stained archival cytology slides from a variety of sites. The extracted DNA was again suitable for PCR analysis. Fresh and archival cytological material can be utilized for molecular biological study of disease processes using PCR. Archival cytological material is probably the best source of DNA and RNA after stored frozen tissue.

  17. 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

  18. Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis.

    PubMed

    Huang, Shichu; Do, Jaephil; Mahalanabis, Madhumita; Fan, Andy; Zhao, Lei; Jepeal, Lisa; Singh, Satish K; Klapperich, Catherine M

    2013-01-01

    In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile) DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA) to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65°C±2°C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.25×10(-2) pg of C. difficile DNA) while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health.

  19. Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis.

    PubMed

    Huang, Shichu; Do, Jaephil; Mahalanabis, Madhumita; Fan, Andy; Zhao, Lei; Jepeal, Lisa; Singh, Satish K; Klapperich, Catherine M

    2013-01-01

    In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile) DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA) to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65°C±2°C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.25×10(-2) pg of C. difficile DNA) while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health. PMID:23555883

  20. Comparison of hepatitis B virus DNA extractions from serum by the QIAamp blood kit, GeneReleaser, and the phenol-chloroform method.

    PubMed

    Kramvis, A; Bukofzer, S; Kew, M C

    1996-11-01

    The abilities of GeneReleaser and QIAamp to extract the hepatitis B virus (HBV) DNA template from serum for amplification by PCR were evaluated and compared with that of the standard phenol-chloroform method. Differences in the sensitivities of the three methods were revealed by nested PCR of HBV DNA extracted from serially diluted hepatitis B e antigen (HBeAg)-positive (high-titer) serum. Phenol-chloroform was found to be the most sensitive extraction method but was time-consuming and labor intensive, and the many steps required increased the possibility of contamination. In a titration of HBeAg-negative (low-titer) serum, all three methods coupled with nested PCR were capable of detecting low levels of HBV DNA. In the case of QIAamp and GeneReleaser, the extraction was relatively simple and rapid. The higher quantity of serum (200 microliters) used in the QIAamp extraction did not provide higher sensitivity, possibly because of incomplete removal of Taq polymerase inhibitors from the serum or inadequate disruption of the virion. GeneReleaser was more efficient because it gave the same detection limit in low-titer serum as phenol-chloroform even though it utilizes only 5 microliters of serum. However, it did not produce consistent amplifications of HBV DNA, giving false-negative results in 7 of the 50 cases (14%) in one experiment. Use of a larger volume of serum and replicate extractions may overcome this problem. Advantages thus exist in each of the extraction methods, and these should be weighed against the disadvantages when deciding which extraction method is appropriate.

  1. Microwave assisted extraction combined with solvent bar microextraction for one-step solvent-minimized extraction, cleanup and preconcentration of polycyclic aromatic hydrocarbons in soil samples.

    PubMed

    Guo, Liang; Lee, Hian Kee

    2013-04-19

    For the first time, a novel one-step sample preparation method that combines microwave assisted extraction and solvent bar microextraction (MAE-SBME) with analysis by gas chromatography-mass spectrometry (GC-MS), was developed for the fast and efficient determination of polycyclic aromatic hydrocarbons (PAHs) in environmental soil samples. An interesting feature of the new procedure is that SBME was conducted simultaneously with MAE. Thus, the extract from the SBME could be directly and immediately analyzed by GC-MS. A separate clean up and/or preconcentration process, such as time-consuming and tedious gel permeation chromatography, solid-phase extraction, filtration, or adsorption chromatography, normally associated with conventional MAE, was not necessary. It is also notable that the procedure was environmentally benign since water was used as the extraction solvent in MAE, and only several microliters of organic solvent were used in SBME. Some factors affecting the extraction were studied and optimized. Under the most favorable conditions, the method showed good linearities (between 0.2 and 500, 0.5 and 500, 1 and 500, and 2 and 500 ng/g, depending on the analytes), low limits of detection (from 0.03 to 0.25 ng/g), and satisfactory precision (with relative standard deviations below 9.8%). The MAE-SBME procedure provides a fast and simple sample preparation approach for the processing of environmental soil samples.

  2. One-step insertion of oligonucleotide linkers or adapters to DNA using unphosphorylated oligonucleotides.

    PubMed

    Kang, C; Inouye, M

    1993-10-01

    A simple and efficient method was developed for insertion of oligonucleotide sequences into plasmids. In this method, an unphosphorylated oligonucleotide was ligated to the restriction-digested phagemid DNA. Only the single strand of the oligonucleotide was ligated at the 5' end of the phagemid, and this resulted in the creation of a long self-complementary single-strand overhang. These single-strand overhang-possessing phagemids were used to transform XL-1 cells. This simple ligation and transformation reaction rendered approximately 7.5 x 10(4) to 5 x 10(5) of white colonies per microgram DNA from the isopropyl-beta-D-thiogalactopyranoside and 5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside plate. This number is almost the same or even higher than the number of blue colonies from the control reaction in which ligase was used without the oligonucleotide. By this method we could mutate one enzyme site to another or create ribozyme and substrate phagemid very easily. Fidelity of this method was checked by restriction digestion, DNA sequencing and ribozyme reaction. By DNA sequencing, we observed that 100% of the white colonies contained a single oligonucleotide sequence.

  3. Adsorption and desorption of DNA tuned by hydroxyl groups in graphite oxides-based solid extraction material.

    PubMed

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

    2015-12-01

    The extraction of DNA is the most crucial method used in molecular biology. Up to date silica matrices has been widely applied as solid support for selective DNA adsorption and extraction. However, since adsorption force of SiOH functional groups is much greater than that of desorption force, the DNA extraction efficiency of silica surfaces is limited. In order to increase the DNA extraction yield, a new surface with different functional groups which possess of greater desorption property is required. In this study, we proposed cellulose/graphite oxide (GO) composite as an alternative material for DNA adsorption and extraction. GO/Cellulose composite provides the major adsorption and desorption of DNA by COH, which belongs to alkyl or phenol type of OH functional group. Compared to SiOH, COH is less polarized and reactive, therefore the composite might provide a higher desorption of DNA during the elution process. The GO/cellulose composite were prepared in spherical structure by mixing urea, cellulose, NaOH, Graphite oxide and water. The concentration of GO within the composites were controlled to be 0-4.15 wt.%. The extraction yield of DNA increased with increasing weight percentage of GO. The highest yield was achieved at 4.15 wt.% GO, where the extraction efficiency was reported as 660.4 ng/μl when applying 2M GuHCl as the binding buffer. The absorbance ratios between 260 nm and 280 nm (A260/A280) of the DNA elution was demonstrated as 1.86, indicating the extracted DNA consisted of high purity. The results proved that GO/cellulose composite provides a simple method for selective DNA extraction with high extraction efficiency of pure DNA.

  4. Evaluation of antioxidant activity and preventing DNA damage effect of pomegranate extracts by chemiluminescence method.

    PubMed

    Guo, Shanshan; Deng, Qianchun; Xiao, Junsong; Xie, Bijun; Sun, Zhida

    2007-04-18

    The antioxidant activities of three parts (peel, juice, and seed) and extracts of three pomegranate varieties in China were investigated by using a chemiluminescence (CL) method in vitro. The scavenging ability of pomegranate extracts (PEs) on superoxide anion, hydroxide radical, and hydrogen peroxide was determined by the pyrogallol-luminol system, the CuSO4-Phen-Vc-H2O2 system, and the luminol-H2O2 system, respectively. DNA damage preventing the effect of PE was determined by the CuSO4-Phen-Vc-H2O2-DNA CL system. The results showed that the peel extract of red pomegranate had the best effect on the scavenging ability of superoxide anion because its IC50 value (4.01 +/- 0.09 microg/mL) was the lowest in all PEs. The seed extract of white pomegranate could scavenge hydroxide radical most effectively of the nine extracts (the IC50 value was 1.69 +/- 0.03 microg/mL). The peel extract of white pomegranate had the best scavenging ability on hydrogen peroxide, which had the lowest IC50 value (0.032 +/- 0.003 microg/mL) in the nine extracts. The seed extract of white pomegranate (the IC50 value was 3.67 +/- 0.03 microg/mL) was the most powerful on the DNA damage-preventing effect in all of the PEs. Also, the statistical analysis indicated that there were significant differences (at P < 0.05) among the extracts of the different varieties and parts in each system. PMID:17381116

  5. Automated DNA extraction platforms offer solutions to challenges of assessing microbial biofouling in oil production facilities.

    PubMed

    Oldham, Athenia L; Drilling, Heather S; Stamps, Blake W; Stevenson, Bradley S; Duncan, Kathleen E

    2012-11-20

    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.

  6. Automated DNA extraction platforms offer solutions to challenges of assessing microbial biofouling in oil production facilities.

    PubMed

    Oldham, Athenia L; Drilling, Heather S; Stamps, Blake W; Stevenson, Bradley S; Duncan, Kathleen E

    2012-01-01

    The analysis of microbial assemblages in industrial, marine, and medical systems can inform decisions regarding quality control or mitigation. Modern molecular approaches to detect, characterize, and quantify microorganisms provide rapid and thorough measures unbiased by the need for cultivation. The requirement of timely extraction of high quality nucleic acids for molecular analysis is faced with specific challenges when used to study the influence of microorganisms on oil production. Production facilities are often ill equipped for nucleic acid extraction techniques, making the preservation and transportation of samples off-site a priority. As a potential solution, the possibility of extracting nucleic acids on-site using automated platforms was tested. The performance of two such platforms, the Fujifilm QuickGene-Mini80™ and Promega Maxwell®16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm™ DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition. Three pipeline biofilm samples were chosen for these comparisons; two contained crude oil and corrosion products and the third transported seawater. Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach. DNA quality was evaluated for amplification by quantitative PCR (qPCR) and end-point PCR to generate 454 pyrosequencing libraries for 16S rRNA microbial community analysis. Microbial community structure, as assessed by DGGE analysis and pyrosequencing, was comparable among the three extraction methods. Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources. PMID:23168231

  7. A comparison of different pre-lysis methods and extraction kits for recovery of Streptococcus agalacticae (Lancefield group B Streptococcus) DNA from whole blood.

    PubMed

    Burke, Rachael M; McKenna, James P; Cox, Ciara; Coyle, Peter V; Shields, Michael D; Fairley, Derek J

    2016-10-01

    Sub-optimal recovery of bacterial DNA from whole blood samples can limit the sensitivity of molecular assays to detect pathogenic bacteria. We compared 3 different pre-lysis protocols (none, mechanical pre-lysis and achromopeptidase pre-lysis) and 5 commercially available DNA extraction platforms for direct detection of Group B Streptococcus (GBS) in spiked whole blood samples, without enrichment culture. DNA was extracted using the QIAamp Blood Mini kit (Qiagen), UCP Pathogen Mini kit (Qiagen), QuickGene DNA Whole Blood kit S (Fuji), Speed Xtract Nucleic Acid Kit 200 (Qiagen) and MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche Diagnostics Corp). Mechanical pre-lysis increased yields of bacterial genomic DNA by 51.3 fold (95% confidence interval; 31.6-85.1, p<0.001) and pre-lysis with achromopeptidase by 6.1 fold (95% CI; 4.2-8.9, p<0.001), compared with no pre-lysis. Differences in yield due to pre-lysis were 2-3 fold larger than differences in yield between extraction methods. Including a pre-lysis step can improve the limits of detection of GBS using PCR or other molecular methods without need for culture. PMID:27546716

  8. 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...

  9. Efficient and scalable serial extraction of DNA and RNA from frozen tissue samples.

    PubMed

    Mathot, Lucy; Lindman, Monica; Sjöblom, Tobias

    2011-01-01

    Advances in cancer genomics have created a demand for scalable sample processing. We here present a process for serial extraction of nucleic acids from the same frozen tissue sample based on magnetic silica particles. The process is automation friendly with high recoveries of pure DNA and RNA suitable for analysis.

  10. Comparison of DNA extraction protocols for the analysis of gut microbiota in fishes.

    PubMed

    Larsen, Andrea M; Mohammed, Haitham H; Arias, Covadonga R

    2015-03-01

    This study investigated the impacts of bacterial DNA extraction methodology on downstream analysis of fish gut microbiota. Feces and intestine samples were taken from three sympatric freshwater fish species with varying diets. Samples were processed immediately (approximately 4 h after capture; fresh), stored at -20 °C for 15 days or preserved in RNAlater® reagent for 15 days. DNA was then extracted using two commercial kits: one designed for animal tissues and one specifically formulated for stool samples. Microbial community fingerprints were generated using ribosomal intergenic spacer analysis. Factors including diversity as depicted by band number, band intensity, repeatability and practicalities such as cost and time were considered. Despite significant differences in microbiota structure, results were similar between feces and intestine samples. Frozen samples were consistently outperformed by other storage methods and the stool kit typically outperformed the tissue kit. Overall, we recommend extraction of bacterial DNA from fresh samples using the stool kit for both sample types. If samples cannot be processed immediately, preservation in RNAlater® is preferred to freezing. Choice of DNA extraction method significantly influences the results of downstream microbial community analysis and thus should be taken into consideration for metadata analysis. PMID:25757730

  11. 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.

  12. Nucleosome assembly in mammalian cell extracts before and after DNA replication.

    PubMed Central

    Gruss, C; Gutierrez, C; Burhans, W C; DePamphilis, M L; Koller, T; Sogo, J M

    1990-01-01

    Protein-free DNA in a cytosolic extract supplemented with SV40 large T-antigen (T-Ag), is assembled into chromatin structure when nuclear extract is added. This assembly was monitored by topoisomer formation, micrococcal nuclease digestion and psoralen crosslinking of the DNA. Plasmids containing SV40 sequences (ori- and ori+) were assembled into chromatin with similar efficiencies whether T-Ag was present or not. Approximately 50-80% of the number of nucleosomes in vivo could be assembled in vitro; however, the kinetics of assembly differed on replicated and unreplicated molecules. In replicative intermediates, nucleosomes were observed on both the pre-replicated and post-replicated portions. We conclude that the extent of nucleosome assembly in mammalian cell extracts is not dependent upon DNA replication, in contrast to previous suggestions. However, the highly sensitive psoralen assay revealed that DNA replication appears to facilitate precise folding of DNA in the nucleosome. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. PMID:2167837

  13. Identification of the traditional Tibetan medicine "Shaji" and their different extracts through tri-step infrared spectroscopy

    NASA Astrophysics Data System (ADS)

    Liu, Yue; Li, Jingyi; Fan, Gang; Sun, Suqin; Zhang, Yuxin; Zhang, Yi; Tu, Ya

    2016-11-01

    Hippophae rhamnoides subsp. sinensis Rousi, Hippophae gyantsensis (Rousi) Y. S. Lian, Hippophae neurocarpa S. W. Liu & T. N. He and Hippophae tibetana Schlechtendal are typically used under one name "Shaji", to treat cardiovascular diseases and lung disorders in Tibetan medicine (TM). A complete set of infrared (IR) macro-fingerprints of these four Hippophae species should be characterized and compared simply, accurately, and in detail for identification. In the present study, tri-step IR spectroscopy, which included Fourier transform IR (FT-IR) spectroscopy, second derivative IR (SD-IR) spectroscopy and two-dimensional correlation IR (2D-IR) spectroscopy, was employed to discriminate the four Hippophae species and their corresponding extracts using different solvents. The relevant spectra exhibited the holistic chemical compositions and variations. Flavonoids, fatty acids and sugars were found to be the main chemical components. Characteristic peak positions, intensities and shapes derived from FT-IR, SD-IR and 2D-IR spectra provided valuable information for sample discrimination. Principal component analysis (PCA) of spectral differences was performed to illustrate the objective identification. Results showed that the species and their extracts can be clearly distinguished. Thus, a quick, precise and effective tri-step IR spectroscopy combined with PCA can be applied to identify and discriminate medicinal materials and their extracts in TM research.

  14. 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.

  15. DNA degradation by aqueous extract of Aloe vera in the presence of copper ions.

    PubMed

    Naqvi, Shoa; Ullah, M F; Hadi, S M

    2010-06-01

    The plant Aloe vera has long been used in medicine, as dietary supplements and for cosmetic purposes. Aloe vera extracts are a rich source of polyphenols, such as aloin and aloe emodin and have shown a wide range of pharmacological properties, including anti-inflammatory and anti-cancer properties. The bioactive component aloe emodin has been reported to induce apoptosis in various cancer cell lines. Many of the biological activities of Aloe vera have been attributed to its antioxidant properties. However, most plant-derived polyphenols that are also present in Aloe vera may exhibit pro-oxidant properties either alone or in the presence of transition metals, such as copper. Previous reports from this laboratory have implicated the pro-oxidant action as one of the mechanisms for their anti-cancer properties. In the present paper, we show that aqueous extract of Aloe vera is also able to cause DNA degradation in the presence of copper ions. Further, the extract is also able to reduce Cu(II) to Cu(I) and generate reactive oxygen species, such as superoxide anion and hydroxyl radicals in a dose-dependent manner, which correlates with ability of the extract to cause DNA breakage. Thus, the study shows that in addition to antioxidant activity, Aloe vera extract also possess pro-oxidant properties, leading to oxidative DNA breakage.

  16. 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

  17. Hippophae leaf extract concentration regulates antioxidant and prooxidant effects on DNA.

    PubMed

    Saini, Manu; Tiwari, Sandhya; Prasad, Jagdish; Singh, Surender; Kumar, M S Yogendra; Bala, Madhu

    2010-03-01

    Extracts from Hippophae leaves constitute some commonly consumed beverages such as tea and wine. We had developed an extract of Hippophae leaves (SBL-1), which was rich in quercetin, had antimutagenic effects, radioprotective effects, and countered radiation-induced gene conversion in Saccharomyces cerevisiae. This study was designed to investigate the action of SBL-1 on guanine cytosine (GC)-rich nascent and mouse genomic DNA in vitro. The human and mouse liver DNA have about 43% GC content. Our results showed that at small concentration SBL-1 protected nascent as well as genomic DNA, while at large concentration SBL-1 damaged both types of DNA. The concentration of SBL-1 that protected DNA also demonstrated higher free radical scavenging activity. The reducing power of SBL-1 was greater than its free radical scavenging activity. The greater reducing power may have reduced the trace metals present in the SBL-1, leading to generation of hydroxyl radicals via Fenton reaction. The increased proportion of unscavenged hydroxyl radicals with increase in SBL-1 concentration may have been responsible for DNA damage or prooxidant effect of SBL-1 in vitro. This study suggests that the dietary supplements prepared from Hippophae should have low metal content. PMID:22435574

  18. An Improved Method for Soil DNA Extraction to Study the Microbial Assortment within Rhizospheric Region.

    PubMed

    Fatima, Faria; Pathak, Neelam; Rastogi Verma, Smita

    2014-01-01

    The need for identification of soil microbial community mainly depends on direct extraction of DNA from soil, a multifaceted environment that is a major pool for microbial genetic diversity. The soil DNA extraction procedures usually suffer from two major problems, namely, inappropriate rupturing of cells and contamination with humic substances. In the present study, five protocols for single type of rhizospheric soil were investigated and their comparison indicated that the inclusion of 120 mM phosphate buffered saline (PBS) for washing and mannitol in the lysis buffer allowed the processing of soil sample in minimal time with no specific equipment requirement. Furthermore, DNA purity and yield were also improved, which allowed the exploitation of genetic potential of soil microbes within soil sample thereby facilitating the amplification of metagenomic DNA. The effectiveness of methods was analyzed using random amplification of polymorphic DNA. The banding patterns revealed that both the abundance and the composition of indigenous microbial community depend on the DNA recovery method.

  19. 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...

  20. Comparison of Eight Methods for the Extraction of Bacillus atrophaeus Spore DNA from Eleven Common Interferents and a Common Swab

    PubMed Central

    Rose, Helen L.; Dewey, Caroline A.; Ely, Morgan S.; Willoughby, Sarah L.; Parsons, Tanya M.; Cox, Victoria; Spencer, Phillippa M.; Weller, Simon A.

    2011-01-01

    Eight DNA extraction products or methods (Applied Biosystems PrepFiler Forensic DNA Extraction Kit; Bio-Rad Instagene Only, Bio-Rad Instagene & Spin Column Purification; EpiCentre MasterPure DNA & RNA Kit; FujiFilm QuickGene Mini80; Idaho Technologies 1-2-3 Q-Flow Kit; MoBio UltraClean Microbial DNA Isolation Kit; Sigma Extract-N-Amp Plant and Seed Kit) were adapted to facilitate extraction of DNA under BSL3 containment conditions. DNA was extracted from 12 common interferents or sample types, spiked with spores of Bacillus atropheaus. Resulting extracts were tested by real-time PCR. No one method was the best, in terms of DNA extraction, across all sample types. Statistical analysis indicated that the PrepFiler method was the best method from six dry powders (baking, biological washing, milk, plain flour, filler and talcum) and one solid (Underarm deodorant), the UltraClean method was the best from four liquids (aftershave, cola, nutrient broth, vinegar), and the MasterPure method was the best from the swab sample type. The best overall method, in terms of DNA extraction, across all sample types evaluated was the UltraClean method. PMID:21818364

  1. 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

  2. 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

  3. Two-Step Extraction of the Lower First Molar for Class III Treatment in Adult Patient

    PubMed Central

    Paulin, Ricardo Fabris; Raveli, Taísa Barnabé; Raveli, Dirceu Barnabé

    2016-01-01

    The aim of this article is to describe a case report of Class III malocclusion treatment with lower first molar extraction. The 27-year-old Caucasian male patient presented a symmetric face with a straight profile, hyperdivergent growth pattern, molar and cuspid Class III relation, and an anterior crossbite as well as a mild crowding on cuspids area, in both upper and lower arches and a tendency to posterior crossbite. The treatment was performed by the use of Haas expansion appliance followed by an initial alignment and leveling of the upper and lower arches with a fixed edgewise appliance, extraction of lower teeth aiming the correction of the incisors proclination and end the treatment with a Class I molar relationship. It resulted in a significant change in the patient's profile, dentoalveolar Class III correction, upper arch expansion, leveling and alignment of the upper and lower arches, and improvement of tipping of the upper and lowers incisors. In cases of a dentoalveolar compensation in well positioned bone bases the treatment with fixed appliances is an alternative and extraction of lower teeth is considered.

  4. Two-Step Extraction of the Lower First Molar for Class III Treatment in Adult Patient

    PubMed Central

    Paulin, Ricardo Fabris; Raveli, Taísa Barnabé; Raveli, Dirceu Barnabé

    2016-01-01

    The aim of this article is to describe a case report of Class III malocclusion treatment with lower first molar extraction. The 27-year-old Caucasian male patient presented a symmetric face with a straight profile, hyperdivergent growth pattern, molar and cuspid Class III relation, and an anterior crossbite as well as a mild crowding on cuspids area, in both upper and lower arches and a tendency to posterior crossbite. The treatment was performed by the use of Haas expansion appliance followed by an initial alignment and leveling of the upper and lower arches with a fixed edgewise appliance, extraction of lower teeth aiming the correction of the incisors proclination and end the treatment with a Class I molar relationship. It resulted in a significant change in the patient's profile, dentoalveolar Class III correction, upper arch expansion, leveling and alignment of the upper and lower arches, and improvement of tipping of the upper and lowers incisors. In cases of a dentoalveolar compensation in well positioned bone bases the treatment with fixed appliances is an alternative and extraction of lower teeth is considered. PMID:27699072

  5. 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

  6. Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR.

    PubMed

    Tatti, Enrico; McKew, Boyd A; Whitby, Corrine; Smith, Cindy J

    2016-01-01

    Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included. PMID:27341629

  7. Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

    PubMed Central

    Tatti, Enrico; McKew, Boyd A.; Whitby, Corrine; Smith, Cindy J.

    2016-01-01

    Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included. PMID:27341629

  8. Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR.

    PubMed

    Tatti, Enrico; McKew, Boyd A; Whitby, Corrine; Smith, Cindy J

    2016-06-11

    Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

  9. DNA stretch during electrophoresis due to a step change in mobility.

    PubMed

    Underhill, Patrick T; Doyle, Patrick S

    2007-07-01

    We investigate DNA stretching during electrophoresis when the mobility abruptly changes. This is a simplified geometry that produces a nonhomogeneous strain rate over the scale of a single molecule. An effective Weissenberg number (Wi) and Deborah number were identified, and the degree of stretching was examined as a function of these two parameters. The system does not undergo a coil-stretch transition. The finite extensibility of the chains only affects the response if the chain is stretched to a significant fraction of the contour length. The wormlike chain shows a characteristic approach to full extension of Wi(-1/2).

  10. Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor (L. Moench).

    PubMed

    Maropola, Mapula Kgomotso Annah; Ramond, Jean-Baptiste; Trindade, Marla

    2015-05-01

    Culture-independent studies rely on the quantity and quality of the extracted environmental metagenomic DNA (mDNA). To fully access the plant tissue microbiome, the extracted plant mDNA should allow optimal PCR applications and the genetic content must be representative of the total microbial diversity. In this study, we evaluated the endophytic bacterial diversity retrieved using different mDNA extraction procedures. Metagenomic DNA from sorghum (Sorghum bicolor L. Moench) stem and root tissues were extracted using two classical DNA extraction protocols (CTAB- and SDS-based) and five commercial kits. The mDNA yields and quality as well as the reproducibility were compared. 16S rRNA gene terminal restriction fragment length polymorphism (t-RFLP) was used to assess the impact on endophytic bacterial community structures observed. Generally, the classical protocols obtained high mDNA yields from sorghum tissues; however, they were less reproducible than the commercial kits. Commercial kits retrieved higher quality mDNA, but with lower endophytic bacterial diversities compared to classical protocols. The SDS-based protocol enabled access to the highest sorghum endophytic diversities. Therefore, "SDS-extracted" sorghum root and stem microbiome diversities were analysed via 454 pyrosequencing, and this revealed that the two tissues harbour significantly different endophytic communities. Nevertheless, both communities are dominated by agriculturally important genera such as Microbacterium, Agrobacterium, Sphingobacterium, Herbaspirillum, Erwinia, Pseudomonas and Stenotrophomonas; which have previously been shown to play a role in plant growth promotion. This study shows that DNA extraction protocols introduce biases in culture-independent studies of environmental microbial communities by influencing the mDNA quality, which impacts the microbial diversity analyses and evaluation. Using the broad-spectrum SDS-based DNA extraction protocol allows the recovery of the most

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. Determination of initiation of DNA replication before and after nuclear formation in Xenopus egg cell free extracts

    PubMed Central

    1993-01-01

    Xenopus egg extracts prepared before and after egg activation retain M- and S-phase specific activity, respectively. Staurosporine, a potent inhibitor of protein kinase, converted M-phase extracts into interphase- like extracts that were capable of forming nuclei upon the addition of sperm DNA. The nuclei formed in the staurosporine treated M-phase extract were incapable of replicating DNA, and they were unable to initiate replication upon the addition of S-phase extracts. Furthermore, replication was inhibited when the staurosporine-treated M- phase extract was added in excess to the staurosporine-treated S-phase extract before the addition of DNA. The membrane-depleted S-phase extract supported neither nuclear formation nor replication; however, preincubation of sperm DNA with these extracts allowed them to form replication-competent nuclei upon the addition of excess staurosporine- treated M-phase extract. These results demonstrate that positive factors in the S-phase extracts determined the initiation of DNA replication before nuclear formation, although these factors were unable to initiate replication after nuclear formation. PMID:8253833

  17. 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

  18. Genomic DNA extraction methods using formalin-fixed paraffin-embedded tissue.

    PubMed

    Potluri, Keerti; Mahas, Ahmed; Kent, Michael N; Naik, Sameep; Markey, Michael

    2015-10-01

    As new technologies come within reach for the average cytogenetic laboratory, the study of chromosome structure has become increasingly more sophisticated. Resolution has improved from karyotyping (in which whole chromosomes are discernible) to fluorescence in situ hybridization and comparative genomic hybridization (CGH, with which specific megabase regions are visualized), array-based CGH (aCGH, examining hundreds of base pairs), and next-generation sequencing (providing single base pair resolution). Whole genome next-generation sequencing remains a cost-prohibitive method for many investigators. Meanwhile, the cost of aCGH has been reduced during recent years, even as resolution has increased and protocols have simplified. However, aCGH presents its own set of unique challenges. DNA of sufficient quantity and quality to hybridize to arrays and provide meaningful results is required. This is especially difficult for DNA from formalin-fixed paraffin-embedded (FFPE) tissues. Here, we compare three different methods for acquiring DNA of sufficient length, purity, and "amplifiability" for aCGH and other downstream applications. Phenol-chloroform extraction and column-based commercial kits were compared with adaptive focused acoustics (AFA). Of the three extraction methods, AFA samples showed increased amplicon length and decreased polymerase chain reaction (PCR) failure rate. These findings support AFA as an improvement over previous DNA extraction methods for FFPE tissues.

  19. An optimized DNA extraction and multiplex PCR for the detection of Fasciola sp. in lymnaeid snails.

    PubMed

    Caron, Y; Righi, S; Lempereur, L; Saegerman, C; Losson, B

    2011-05-31

    This study deals with the development and validation of an original PCR protocol to assess the presence of Fasciola hepatica in Galba truncatula its main intermediate host in Western Europe. In the present study two DNA extraction techniques are compared and a new multiplex PCR is described. The Chelex(®) DNA extraction technique showed to be more appropriate than the classical Phenol/Chloroform/Proteinase K based method because of the absence of toxic organic solvent, shorter duration and lower cost, and a higher reproducibility regarding DNA concentrations and wavelength ratios. The multiplex PCR was set up to amplify the lymnaeid internal transcribed spacer 2 sequence (500-600 bp) that act as an internal control and a 124 bp Fasciola sp. sequence that is repeated more than 300,000 times in fluke whole genome. Ninety six snails were pooled and 6 snails (6.25%) found positive for Fasciola sp. The limit of detection is lower than the minimal biological infestation unit (one miracidium). DNA extracts from Paramphistomum daubneyi, Dicrocoelium lanceolatum, and Fascioloides magna did not cross react. PMID:21242033

  20. An optimized DNA extraction and multiplex PCR for the detection of Fasciola sp. in lymnaeid snails.

    PubMed

    Caron, Y; Righi, S; Lempereur, L; Saegerman, C; Losson, B

    2011-05-31

    This study deals with the development and validation of an original PCR protocol to assess the presence of Fasciola hepatica in Galba truncatula its main intermediate host in Western Europe. In the present study two DNA extraction techniques are compared and a new multiplex PCR is described. The Chelex(®) DNA extraction technique showed to be more appropriate than the classical Phenol/Chloroform/Proteinase K based method because of the absence of toxic organic solvent, shorter duration and lower cost, and a higher reproducibility regarding DNA concentrations and wavelength ratios. The multiplex PCR was set up to amplify the lymnaeid internal transcribed spacer 2 sequence (500-600 bp) that act as an internal control and a 124 bp Fasciola sp. sequence that is repeated more than 300,000 times in fluke whole genome. Ninety six snails were pooled and 6 snails (6.25%) found positive for Fasciola sp. The limit of detection is lower than the minimal biological infestation unit (one miracidium). DNA extracts from Paramphistomum daubneyi, Dicrocoelium lanceolatum, and Fascioloides magna did not cross react.

  1. Use of neuropathological tissue for molecular genetic studies: parameters affecting DNA extraction and polymerase chain reaction.

    PubMed

    Kösel, S; Graeber, M B

    1994-01-01

    Nuclear and mitochondrial DNA were extracted from gray matter of human cerebral cortex which had either been formalin-fixed and embedded into paraffin or stored in formalin for up to 26 years. Extraction conditions were optimized for proteinase K digestion, i.e., enzyme concentration, digestion temperature and incubation time. Using the polymerase chain reaction (PCR), DNA was successfully amplified from archival material and sequenced employing a direct nonradioactive cycle sequencing protocol. In general, tissue embedded into paraffin following brief fixation in formalin gave good quantitative results, i.e., up to 1 microgram DNA/mg tissue were extracted. This yield was at least one order of magnitude higher than that obtained with tissue stored in formalin. However, paraffin-embedded neuropathological material was found to contain an as-yet-unidentified PCR inhibitor, and a deleterious effect of long-term fixation in unbuffered low-grade formalin was clearly detectable. Importantly, both paraffin-embedded tissue blocks and human brain that had been stored in formalin for many years yielded DNA sufficient for qualitative analysis. The implications of these findings for the use of neuropathological material in molecular genetic studies are discussed.

  2. Genomic DNA extraction methods using formalin-fixed paraffin-embedded tissue.

    PubMed

    Potluri, Keerti; Mahas, Ahmed; Kent, Michael N; Naik, Sameep; Markey, Michael

    2015-10-01

    As new technologies come within reach for the average cytogenetic laboratory, the study of chromosome structure has become increasingly more sophisticated. Resolution has improved from karyotyping (in which whole chromosomes are discernible) to fluorescence in situ hybridization and comparative genomic hybridization (CGH, with which specific megabase regions are visualized), array-based CGH (aCGH, examining hundreds of base pairs), and next-generation sequencing (providing single base pair resolution). Whole genome next-generation sequencing remains a cost-prohibitive method for many investigators. Meanwhile, the cost of aCGH has been reduced during recent years, even as resolution has increased and protocols have simplified. However, aCGH presents its own set of unique challenges. DNA of sufficient quantity and quality to hybridize to arrays and provide meaningful results is required. This is especially difficult for DNA from formalin-fixed paraffin-embedded (FFPE) tissues. Here, we compare three different methods for acquiring DNA of sufficient length, purity, and "amplifiability" for aCGH and other downstream applications. Phenol-chloroform extraction and column-based commercial kits were compared with adaptive focused acoustics (AFA). Of the three extraction methods, AFA samples showed increased amplicon length and decreased polymerase chain reaction (PCR) failure rate. These findings support AFA as an improvement over previous DNA extraction methods for FFPE tissues. PMID:26126956

  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.

  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 μg/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.

  5. Development and validation of an inexpensive and efficient method for the extraction of Theileria orientalis DNA from blood.

    PubMed

    Bogema, D R; Fell, S A; O'Rourke, B A; Collins, D; Eamens, G J; Jenkins, C

    2015-09-15

    Theileria orientalis is an emerging bovine pathogen in Australasia. PCR-based detection methods for this parasite are sensitive but relatively expensive, partly due to costs associated with DNA extraction. An inexpensive and efficient technique was developed for the extraction of T. orientalis DNA from blood based on hypotonic erythrocyte lysis and detergent-proteinase K treatment (DPK method). The DPK method compares favourably to a commercial extraction kit when paired with a T. orientalis multiplex qPCR.

  6. Evaluation of RNA and DNA extraction from liquid-based cytology specimens.

    PubMed

    Fujii, Tomomi; Asano, Aya; Shimada, Keiji; Tatsumi, Yoshihiro; Obayashi, Chiho; Konishi, Noboru

    2016-10-01

    Molecular diagnosis using DNA and RNA derived from malignant tumors and molecular biological tools such as the quantitative polymerase-chain-reaction (qPCR) is a commonly used technique in clinical pathology. In this report, we compared the qualitative extraction of RNA and DNA from cancer cells fixed using several liquid-based cytology (LBC) kits. Ten to 1,000 cells from the T24 urinary bladder cancer cell line and SKG-II cervical cancer cell line were fixed with 55% methanol and three different methanol-based LBC solutions. The mRNA levels of CD44 in T24 cells and E7 in SKG-II cells and DNA levels of p53 in T24 cells and E7 in SKG-II cells were analyzed by qPCR. mRNA and DNA extracted from T24 and/or SKG-II cells fixed with methanol-based LBC solutions were efficiently detected, but to differing degrees, by qPCR. mRNA, and DNA from cells fixed with a formaldehyde-containing fixative liquid were detected at significantly low copy numbers by qPCR. Our results demonstrate that LBC systems are powerful tools for cytopathology and immunocytochemistry applications. However, the appropriate fixative must be selected for cell preservation when a small number of LBC samples is used for molecular testing, particularly in RNA-based molecular analyses. Diagn. Cytopathol. 2016;44:833-840. © 2016 Wiley Periodicals, Inc. PMID:27357064

  7. 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.

  8. DNA Extraction Protocols for Whole-Genome Sequencing in Marine Organisms.

    PubMed

    Panova, Marina; Aronsson, Henrik; Cameron, R Andrew; Dahl, Peter; Godhe, Anna; Lind, Ulrika; Ortega-Martinez, Olga; Pereyra, Ricardo; Tesson, Sylvie V M; Wrange, Anna-Lisa; Blomberg, Anders; Johannesson, Kerstin

    2016-01-01

    The marine environment harbors a large proportion of the total biodiversity on this planet, including the majority of the earths' different phyla and classes. Studying the genomes of marine organisms can bring interesting insights into genome evolution. Today, almost all marine organismal groups are understudied with respect to their genomes. One potential reason is that extraction of high-quality DNA in sufficient amounts is challenging for many marine species. This is due to high polysaccharide content, polyphenols and other secondary metabolites that will inhibit downstream DNA library preparations. Consequently, protocols developed for vertebrates and plants do not always perform well for invertebrates and algae. In addition, many marine species have large population sizes and, as a consequence, highly variable genomes. Thus, to facilitate the sequence read assembly process during genome sequencing, it is desirable to obtain enough DNA from a single individual, which is a challenge in many species of invertebrates and algae. Here, we present DNA extraction protocols for seven marine species (four invertebrates, two algae, and a marine yeast), optimized to provide sufficient DNA quality and yield for de novo genome sequencing projects.

  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. Evaluation of RNA and DNA extraction from liquid-based cytology specimens.

    PubMed

    Fujii, Tomomi; Asano, Aya; Shimada, Keiji; Tatsumi, Yoshihiro; Obayashi, Chiho; Konishi, Noboru

    2016-10-01

    Molecular diagnosis using DNA and RNA derived from malignant tumors and molecular biological tools such as the quantitative polymerase-chain-reaction (qPCR) is a commonly used technique in clinical pathology. In this report, we compared the qualitative extraction of RNA and DNA from cancer cells fixed using several liquid-based cytology (LBC) kits. Ten to 1,000 cells from the T24 urinary bladder cancer cell line and SKG-II cervical cancer cell line were fixed with 55% methanol and three different methanol-based LBC solutions. The mRNA levels of CD44 in T24 cells and E7 in SKG-II cells and DNA levels of p53 in T24 cells and E7 in SKG-II cells were analyzed by qPCR. mRNA and DNA extracted from T24 and/or SKG-II cells fixed with methanol-based LBC solutions were efficiently detected, but to differing degrees, by qPCR. mRNA, and DNA from cells fixed with a formaldehyde-containing fixative liquid were detected at significantly low copy numbers by qPCR. Our results demonstrate that LBC systems are powerful tools for cytopathology and immunocytochemistry applications. However, the appropriate fixative must be selected for cell preservation when a small number of LBC samples is used for molecular testing, particularly in RNA-based molecular analyses. Diagn. Cytopathol. 2016;44:833-840. © 2016 Wiley Periodicals, Inc.

  11. DNA Extraction Protocols for Whole-Genome Sequencing in Marine Organisms.

    PubMed

    Panova, Marina; Aronsson, Henrik; Cameron, R Andrew; Dahl, Peter; Godhe, Anna; Lind, Ulrika; Ortega-Martinez, Olga; Pereyra, Ricardo; Tesson, Sylvie V M; Wrange, Anna-Lisa; Blomberg, Anders; Johannesson, Kerstin

    2016-01-01

    The marine environment harbors a large proportion of the total biodiversity on this planet, including the majority of the earths' different phyla and classes. Studying the genomes of marine organisms can bring interesting insights into genome evolution. Today, almost all marine organismal groups are understudied with respect to their genomes. One potential reason is that extraction of high-quality DNA in sufficient amounts is challenging for many marine species. This is due to high polysaccharide content, polyphenols and other secondary metabolites that will inhibit downstream DNA library preparations. Consequently, protocols developed for vertebrates and plants do not always perform well for invertebrates and algae. In addition, many marine species have large population sizes and, as a consequence, highly variable genomes. Thus, to facilitate the sequence read assembly process during genome sequencing, it is desirable to obtain enough DNA from a single individual, which is a challenge in many species of invertebrates and algae. Here, we present DNA extraction protocols for seven marine species (four invertebrates, two algae, and a marine yeast), optimized to provide sufficient DNA quality and yield for de novo genome sequencing projects. PMID:27460368

  12. Comparison of STR profiling from low template DNA extracts with and without the consensus profiling method

    PubMed Central

    2012-01-01

    Background The consensus profiling method was introduced to overcome the exaggerated stochastic effects associated with low copy number DNA typing. However, little empirical evidence has been provided which shows that a consensus profile, derived from dividing a sample into separate aliquots and including only alleles seen at least twice, gives the most informative profile, compared to a profile obtained by amplifying the entire low template DNA extract in one reaction. Therefore, this study aimed to investigate the quality of consensus profiles compared to profiles obtained using the whole low template extract for amplification. Methods A total of 100 pg and 25 pg DNA samples were amplified with the PowerPlex® ESI 16 Kits using 30 or 34 PCR cycles. A total of 100 pg and 25 pg DNA samples were then divided into three aliquots for a 34-cycle PCR and a consensus profile derived that included alleles that appeared in at least two of the replicates. Profiles from the non-split samples were compared to the consensus profiles focusing on peak heights, allele drop out, locus drop out and allele drop in. Results Performing DNA profiling on non-split extracts produced profiles with a higher percentage of correct loci compared to the consensus profiling technique. Consensus profiling did eliminate any spurious alleles from the final profile. However, there was a notable increase in allele and locus drop out when a LTDNA sample was divided prior to amplification. Conclusions The loss of information that occurs when a sample is split for amplification indicates that consensus profiling may not be producing the most informative DNA profile for samples where the template amount is limited. PMID:22748106

  13. 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.

  14. 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.

  15. 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

  16. Comparative evaluation of three different extraction methods for rice (Oryza sativa L.) genomic DNA.

    PubMed

    Sagi, Naoki; Monma, Kimio; Ibe, Akihiro; Kamata, Kunihiro

    2009-04-01

    Recently, more immediate and precise cultivar-identifying methods targeting the specific and/or introduced gene(s) have been put into practical use for various rice cultivars. However trustworthy and innovative the biotechnological analyses may be, DNA purity and quality do have unpredictable influences upon the identification. Extraction methods of rice DNA have hardly ever been compared in a comprehensive manner. In this study, we investigated extraction characteristics of three methods by using 10 rice cultivars and then examined template availability of rice DNAs thereby extracted. An UV spectrophotometric study with a view toward methods revealed three different facts: The Isoplant II kit method with inhibitor absorption yielded the most DNAs, the Takara kit method with magnetic trapping produced the best DNAs free from contaminative proteins, and the enzymatic digestion method exclusively with enzymatic digestions prepared the best DNAs free from contaminative polysaccharides. Moreover, with a view toward cultivars, an insignificant difference in yield was not entirely bore out, and some difference in cultivar might cause significant difference in yield; however, no significant difference in purity was found among the cultivars used. On the other hand, electrophoretic images of the DNAs from the same cultivars showed considerable differences in quality among the methods. Furthermore, the DNA extracts from certain brands of rice proved really available for cultivar identification by using polymerase chain reaction (PCR) related to sequence-tagged sites. Therefore, this study suggested that these extraction methods may be used as the situation demands and that the DNAs thereby extracted might work successfully even in cultivar-identifying PCRs.

  17. 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.

  18. 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

  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. 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

  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 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.

  3. RecA-dependent programmable endonuclease Ref cleaves DNA in two distinct steps.

    PubMed

    Ronayne, Erin A; Cox, Michael M

    2014-04-01

    The bacteriophage P1 recombination enhancement function (Ref) protein is a RecA-dependent programmable endonuclease. Ref targets displacement loops formed when an oligonucleotide is bound by a RecA filament and invades homologous double-stranded DNA sequences. Mechanistic details of this reaction have been explored, revealing that (i) Ref is nickase, cleaving the two target strands of a displacement loop sequentially, (ii) the two strands are cleaved in a prescribed order, with the paired strand cut first and (iii) the two cleavage events have different requirements. Cutting the paired strand is rapid, does not require RecA-mediated ATP hydrolysis and is promoted even by Ref active site variant H153A. The displaced strand is cleaved much more slowly, requires RecA-mediated ATP hydrolysis and does not occur with Ref H153A. The two cleavage events are also affected differently by solution conditions. We postulate that the second cleavage (displaced strand) is limited by some activity of RecA protein.

  4. Seabuckthron (Hippophae rhamnoides L.) leaf extract ameliorates the gamma radiation mediated DNA damage and hepatic alterations.

    PubMed

    Khan, Amitava; Manna, Krishnendu; Chinchubose; Das, Dipesh Kr; Sinha, Mahuya; Kesh, Swaraj Bandhu; Das, Ujjal; Dey, Rakhi Sharma; Banerji, Asoke; Dey, Sanjit

    2014-10-01

    In vitro assessment showed that H. rhamnoides (HrLE) extract possessed free radical scavenging activities and can protect gamma (gamma) radiation induced supercoiled DNA damage. For in vivo study, Swiss albino mice were administered with HrLE (30 mg/kg body weight) for 15 consecutive days before exposing them to a single dose of 5 Gy of beta radiation. HrLE significantly prevented the radiation induced genomic DNA damage indicated as a significant reduction in the comet parameters. The lipid peroxidation, liver function enzymes, expression of phosphorylated NFkappaB (p65) and IkappaBalpha increased whereas the endogenous antioxidants diminished upon radiation exposure compared to control. Pretreatment of HrLE extract ameliorated these changes. Based on the present results it can be concluded that H. rhamnoides possess a potential preventive element in planned and accidental nuclear exposures. PMID:25345244

  5. DNA topoisomerase IIα controls replication origin cluster licensing and firing time in Xenopus egg extracts

    PubMed Central

    Gaggioli, Vincent; Le Viet, Barbara; Germe, Thomas; Hyrien, Olivier

    2013-01-01

    Sperm chromatin incubated in Xenopus egg extracts undergoes origin licensing and nuclear assembly before DNA replication. We found that depletion of DNA topoisomerase IIα (topo IIα), the sole topo II isozyme of eggs and its inhibition by ICRF-193, which clamps topo IIα around DNA have opposite effects on these processes. ICRF-193 slowed down replication origin cluster activation and fork progression in a checkpoint-independent manner, without altering replicon size. In contrast, topo IIα depletion accelerated origin cluster activation, and topo IIα add-back negated overinitiation. Therefore, topo IIα is not required for DNA replication, but topo IIα clamps slow replication, probably by forming roadblocks. ICRF-193 had no effect on DNA synthesis when added after nuclear assembly, confirming that topo IIα activity is dispensable for replication and revealing that topo IIα clamps formed on replicating DNA do not block replication, presumably because topo IIα acts behind and not in front of forks. Topo IIα depletion increased, and topo IIα addition reduced, chromatin loading of MCM2-7 replicative helicase, whereas ICRF-193 did not affect MCM2-7 loading. Therefore, topo IIα restrains MCM2-7 loading in an ICRF-193-resistant manner during origin licensing, suggesting a model for establishing the sequential firing of origin clusters. PMID:23757188

  6. 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...

  7. Cytotoxicity, apoptosis and DNA damage induced by Alpinia galanga rhizome extract.

    PubMed

    Muangnoi, P; Lu, M; Lee, J; Thepouyporn, A; Mirzayans, R; Le, X C; Weinfeld, M; Changbumrung, S

    2007-07-01

    Alpinia galanga, or galangal, has been a popular condiment used in Thai and Asian cuisine for many years. However, relatively little is known of the potential beneficial or adverse health effects of this spice. This study was conducted to analyze the capacity of galangal extract to induce cytotoxicity and DNA damage in six different human cell lines including normal and p53-inactive fibroblasts, normal epithelial and tumour mammary cells and a lung adenocarcinoma cell line. We deliberately focused on treatment with the crude aqueous extract of galangal rhizomes, rather than compounds extracted into an organic solvent, to more closely reflect the mode of dietary consumption of galangal. The cell lines displayed a broad range of cytotoxicity. There was no evidence for preferential cytotoxicity of tumour cells, but there was an indication that p53-active cell lines may be