Sensitive electrochemical assaying of DNA methyltransferase activity based on mimic-hybridization chain reaction amplified strategy.

PubMed

Zhang, Linqun; Liu, Yuanjian; Li, Ying; Zhao, Yuewu; Wei, Wei; Liu, Songqin

2016-08-24

A mimic-hybridization chain reaction (mimic-HCR) amplified strategy was proposed for sensitive electrochemically detection of DNA methylation and methyltransferase (MTase) activity In the presence of methylated DNA, DNA-gold nanoparticles (DNA-AuNPs) were captured on the electrode by sandwich-type assembly. It then triggered mimic-HCR of two hairpin probes to produce many long double-helix chains for numerous hexaammineruthenium (III) chloride ([Ru(NH3)6](3+), RuHex) inserting. As a result, the signal for electrochemically detection of DNA MTase activity could be amplified. If DNA was non-methylated, however, the sandwich-type assembly would not form because the short double-stranded DNAs (dsDNA) on the Au electrode could be cleaved and digested by restriction endonuclease HpaII (HapII) and exonuclease III (Exo III), resulting in the signal decrement. Based on this, an electrochemical approach for detection of M.SssI MTase activity with high sensitivity was developed. The linear range for M.SssI MTase activity was from 0.05 U mL(-1) to 10 U mL(-1), with a detection limit down to 0.03 U mL(-1). Moreover, this detecting strategy held great promise as an easy-to-use and highly sensitive method for other MTase activity and inhibition detection by exchanging the corresponding DNA sequence. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of whole genome amplified DNA to decrease material expenditure and increase quality.

PubMed

Bækvad-Hansen, Marie; Bybjerg-Grauholm, Jonas; Poulsen, Jesper B; Hansen, Christine S; Hougaard, David M; Hollegaard, Mads V

2017-06-01

The overall aim of this study is to evaluate whole genome amplification of DNA extracted from dried blood spot samples. We wish to explore ways of optimizing the amplification process, while decreasing the amount of input material and inherently the cost. Our primary focus of optimization is on the amount of input material, the amplification reaction volume, the number of replicates and amplification time and temperature. Increasing the quality of the amplified DNA and the subsequent results of array genotyping is a secondary aim of this project. This study is based on DNA extracted from dried blood spot samples. The extracted DNA was subsequently whole genome amplified using the REPLIg kit and genotyped on the PsychArray BeadChip (assessing > 570,000 SNPs genome wide). We used Genome Studio to evaluate the quality of the genotype data by call rates and log R ratios. The whole genome amplification process is robust and does not vary between replicates. Altering amplification time, temperature or number of replicates did not affect our results. We found that spot size i.e. amount of input material could be reduced without compromising the quality of the array genotyping data. We also showed that whole genome amplification reaction volumes can be reduced by a factor of 4, without compromising the DNA quality. Whole genome amplified DNA samples from dried blood spots is well suited for array genotyping and produces robust and reliable genotype data. However, the amplification process introduces additional noise to the data, making detection of structural variants such as copy number variants difficult. With this study, we explore ways of optimizing the amplification protocol in order to reduce noise and increase data quality. We found, that the amplification process was very robust, and that changes in amplification time or temperature did not alter the genotyping calls or quality of the array data. Adding additional replicates of each sample also lead to

A superstructure-based electrochemical assay for signal-amplified detection of DNA methyltransferase activity.

PubMed

Zhang, Hui; Yang, Yin; Dong, Huilei; Cai, Chenxin

2016-12-15

DNA methyltransferase (MTase) activity is highly correlated with the occurrence and development of cancer. This work reports a superstructure-based electrochemical assay for signal-amplified detection of DNA MTase activity using M.SssI as an example. First, low-density coverage of DNA duplexes on the surface of the gold electrode was achieved by immobilized mercaptohexanol, followed by immobilization of DNA duplexes. The duplex can be cleaved by BstUI endonuclease in the absence of DNA superstructures. However, the cleavage is blocked after the DNA is methylated by M.SssI. The DNA superstructures are formed with the addition of helper DNA. By using an electroactive complex, RuHex, which can bind to DNA double strands, the activity of M.SssI can be quantitatively detected by differential pulse voltammetry. Due to the high site-specific cleavage by BstUI and signal amplification by the DNA superstructure, the biosensor can achieve ultrasensitive detection of DNA MTase activity down to 0.025U/mL. The method can be used for evaluation and screening of the inhibitors of MTase, and thus has potential in the discovery of methylation-related anticancer drugs. Copyright © 2016 Elsevier B.V. All rights reserved.

DNA cards: determinants of DNA yield and quality in collecting genetic samples for pharmacogenetic studies.

PubMed

Mas, Sergi; Crescenti, Anna; Gassó, Patricia; Vidal-Taboada, Jose M; Lafuente, Amalia

2007-08-01

As pharmacogenetic studies frequently require establishment of DNA banks containing large cohorts with multi-centric designs, inexpensive methods for collecting and storing high-quality DNA are needed. The aims of this study were two-fold: to compare the amount and quality of DNA obtained from two different DNA cards (IsoCode Cards or FTA Classic Cards, Whatman plc, Brentford, Middlesex, UK); and to evaluate the effects of time and storage temperature, as well as the influence of anticoagulant ethylenediaminetetraacetic acid on the DNA elution procedure. The samples were genotyped by several methods typically used in pharmacogenetic studies: multiplex PCR, PCR-restriction fragment length polymorphism, single nucleotide primer extension, and allelic discrimination assay. In addition, they were amplified by whole genome amplification to increase genomic DNA mass. Time, storage temperature and ethylenediaminetetraacetic acid had no significant effects on either DNA card. This study reveals the importance of drying blood spots prior to isolation to avoid haemoglobin interference. Moreover, our results demonstrate that re-isolation protocols could be applied to increase the amount of DNA recovered. The samples analysed were accurately genotyped with all the methods examined herein. In conclusion, our study shows that both DNA cards, IsoCode Cards and FTA Classic Cards, facilitate genetic and pharmacogenetic testing for routine clinical practice.

Improving the yield and quality of DNA isolated from white-rot fungi.

PubMed

Kuhad, R C; Kapoor, R K; Lal, R

2004-01-01

A new simple method used to eliminate polysaccharides that cause problems during DNA isolation was established for 6 different white-rot fungi using 1% hexadecyltrimethylammonium bromide (CTAB) as wash buffer and followed by centrifugation. Variation in the DNA yield and quality was ascertained using precipitating agents, detergents and cell-wall-hydrolyzing chitinase. Considerable amount of exopolysaccharides from fungal biomass was removed with the use of 1% CTAB wash buffer followed by centrifugation. The DNA varied in terms of yield and quality. For the DNA extraction use of 2% SDS in extraction buffer worked best for Pycnoporus cinnabarinus, Cyathus bulleri, Cyathus striatus and Cyathus stercoreus, while 2% CTAB worked best for Phanerochaete chrysosporium and Pleurotus ostreatus. Elimination of phenol and use of absolute ethanol for precipitating DNA resulted in good yield and quality of DNA. This DNA was amenable to restriction endonuclease digestion.

TECHNICAL BRIEF: Isolation of total DNA from postmortem human eye tissues and quality comparison between iris and retina

PubMed Central

Wang, Jay Ching Chieh; Wang, Aikun; Gao, Jiangyuan; Cao, Sijia; Samad, Idris; Zhang, Dean; Ritland, Carol; Cui, Jing Z.

2012-01-01

Background Recent genomic technologies have propelled our understanding of the mechanisms underlying complex eye diseases such as age-related macular degeneration (AMD). Genotyping postmortem eye tissues for known single nucleotide polymorphisms (SNPs) associated with AMD may prove valuable, especially when combined with information obtained through other methods such as immunohistochemistry, western blot, enzyme-linked immunosorbent assay (ELISA), and proteomics. Initially intending to genotype postmortem eye tissues for AMD-related SNPs, our group became interested in isolating and comparing the quality of DNA from the iris and retina of postmortem donor eyes. Since there is no previously published protocol in the literature on this topic, we present a protocol suitable for isolating high-quality DNA from postmortem eye tissues for genomic studies. Methods DNA from 33 retinal samples and 35 iris samples was extracted using the phenol-chloroform-isoamyl method from postmortem donor eye tissues. The quantity of DNA was measured with a spectrophotometer while the quality was checked using gel electrophoresis. The DNA samples were then amplified with PCR for the complement factor H (CFH) gene. The purified amplified products were then genotyped for the SNPs in the CFH gene. Results Regarding concentration, the retina yielded 936 ng/μl of DNA, while the iris yielded 78 ng/μl of DNA. Retinal DNA was also purer than iris DNA (260/280=1.78 vs. 1.46, respectively), and produced superior PCR results. Retinal tissue yielded significantly more DNA than the iris tissue per mg of sample (21.7 ng/μl/mg vs. 7.42 ng/μl/mg). Retinal DNA can be readily amplified with PCR, while iris DNA can also be amplified by adding bovine serum albumin. Overall, retinal tissues yielded DNA of superior quality, quantity, and suitability for genotyping and genomic studies. Conclusions The protocol presented here provides a clear and reliable method for isolating total DNA from postmortem eye

Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples.

PubMed

Kresse, Stine H; Namløs, Heidi M; Lorenz, Susanne; Berner, Jeanne-Marie; Myklebost, Ola; Bjerkehagen, Bodil; Meza-Zepeda, Leonardo A

2018-01-01

Nucleic acid material of adequate quality is crucial for successful high-throughput sequencing (HTS) analysis. DNA and RNA isolated from archival FFPE material are frequently degraded and not readily amplifiable due to chemical damage introduced during fixation. To identify optimal nucleic acid extraction kits, DNA and RNA quantity, quality and performance in HTS applications were evaluated. DNA and RNA were isolated from five sarcoma archival FFPE blocks, using eight extraction protocols from seven kits from three different commercial vendors. For DNA extraction, the truXTRAC FFPE DNA kit from Covaris gave higher yields and better amplifiable DNA, but all protocols gave comparable HTS library yields using Agilent SureSelect XT and performed well in downstream variant calling. For RNA extraction, all protocols gave comparable yields and amplifiable RNA. However, for fusion gene detection using the Archer FusionPlex Sarcoma Assay, the truXTRAC FFPE RNA kit from Covaris and Agencourt FormaPure kit from Beckman Coulter showed the highest percentage of unique read-pairs, providing higher complexity of HTS data and more frequent detection of recurrent fusion genes. truXTRAC simultaneous DNA and RNA extraction gave similar outputs as individual protocols. These findings show that although successful HTS libraries could be generated in most cases, the different protocols gave variable quantity and quality for FFPE nucleic acid extraction. Selecting the optimal procedure is highly valuable and may generate results in borderline quality specimens.

Genomic relations among 31 species of Mammillaria haworth (Cactaceae) using random amplified polymorphic DNA.

PubMed

Mattagajasingh, Ilwola; Mukherjee, Arup Kumar; Das, Premananda

2006-01-01

Thirty-one species of Mammillaria were selected to study the molecular phylogeny using random amplified polymorphic DNA (RAPD) markers. High amount of mucilage (gelling polysaccharides) present in Mammillaria was a major obstacle in isolating good quality genomic DNA. The CTAB (cetyl trimethyl ammonium bromide) method was modified to obtain good quality genomic DNA. Twenty-two random decamer primers resulted in 621 bands, all of which were polymorphic. The similarity matrix value varied from 0.109 to 0.622 indicating wide variability among the studied species. The dendrogram obtained from the unweighted pair group method using arithmetic averages (UPGMA) analysis revealed that some of the species did not follow the conventional classification. The present work shows the usefulness of RAPD markers for genetic characterization to establish phylogenetic relations among Mammillaria species.

Assessing DNA recovery from chewing gum.

PubMed

Eychner, Alison M; Schott, Kelly M; Elkins, Kelly M

2017-01-01

The purpose of this study was to evaluate which DNA extraction method yields the highest quantity of DNA from chewing gum. In this study, several popular extraction methods were tested, including Chelex-100, phenol-chloroform-isoamyl alcohol (PCIA), DNA IQ, PrepFiler, and QIAamp Investigator, and the quantity of DNA recovered from chewing gum was determined using real-time polymerase chain reaction with Quantifiler. Chewed gum control samples were submitted by anonymous healthy adult donors, and discarded environmental chewing gum samples simulating forensic evidence were collected from outside public areas (e.g., campus bus stops, streets, and sidewalks). As expected, results indicate that all methods tested yielded sufficient amplifiable human DNA from chewing gum using the wet-swab method. The QIAamp performed best when DNA was extracted from whole pieces of control gum (142.7 ng on average), and the DNA IQ method performed best on the environmental whole gum samples (29.0 ng on average). On average, the QIAamp kit also recovered the most DNA from saliva swabs. The PCIA method demonstrated the highest yield with wet swabs of the environmental gum (26.4 ng of DNA on average). However, this method should be avoided with whole gum samples (no DNA yield) due to the action of the organic reagents in dissolving and softening the gum and inhibiting DNA recovery during the extraction.

Inter- and Intraspecific Identification of the New World Screwworm Using Random Amplified Polymorphic DNA-Polymerase Chain Reaction

USDA-ARS?s Scientific Manuscript database

New World screwworms (NWS), Cochliomyia hominivorax (Coquerel), are one of the most important arthropod pests of livestock in the Western Hemisphere. Early instars are very difficult to distinguish morphologically from several closely related blow fly species. Random amplified polymorphic DNA polyme...

Evaluation of genetic diversity in Chinese kale (Brassica oleracea L. var. alboglabra Bailey) by using rapid amplified polymorphic DNA and sequence-related amplified polymorphism markers.

PubMed

Zhang, J; Zhang, L G

2014-02-14

Chinese kale is an original Chinese vegetable of the Cruciferae family. To select suitable parents for hybrid breeding, we thoroughly analyzed the genetic diversity of Chinese kale. Random amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) molecular markers were used to evaluate the genetic diversity across 21 Chinese kale accessions from AVRDC and Guangzhou in China. A total of 104 bands were detected by 11 RAPD primers, of which 66 (63.5%) were polymorphic, and 229 polymorphic bands (68.4%) were observed in 335 bands amplified by 17 SRAP primer combinations. The dendrogram showed the grouping of the 21 accessions into 4 main clusters based on RAPD data, and into 6 clusters based on SRAP and combined data (RAPD + SRAP). The clustering of accessions based on SRAP data was consistent with petal colors. The Mantel test indicated a poor fit for the RAPD and SRAP data (r = 0.16). These results have an important implication for Chinese kale germplasm characterization and improvement.

Use of random amplified polymorphic DNA (RAPD) for generating specific DNA probes for oxyuroid species (Nematoda).

PubMed

Jobet, E; Bougnoux, M E; Morand, S; Rivault, C; Cloarec, A; Hugot, J P

1998-03-01

Random amplified DNA markers (RAPD; Williams et al., 1990) were used to obtained specific RAPD fragments characterising different species of oxyuroids. We tested six species of worms parasitizing vertebrates or invertebrates: Passalurus ambiguus Rudolphi, 1819, parasite of Leporids; Syphacia obvelata (Rudolphi, 1802) Seurat, 1916, a parasite of rodents; Blatticola blattae (Graeffe, 1860) Chitwood, 1932 parasite of the cockroach Blattella germanica; Hammerschmidtiella diesingi (Hammerschmidt, 1838) Chitwood, 1932 and Thelastoma bulhoesi (Magalhaes, 1990) Travassos, 1929, parasites of the cockroach Periplaneta americana, and an undescribed parasite species of a passalid insect from New Caledonia. Among 15 oligonucleotides tested, nine produced several specific bands allowing the interspecific discrimination.

Amplifying genetic logic gates.

PubMed

Bonnet, Jerome; Yin, Peter; Ortiz, Monica E; Subsoontorn, Pakpoom; Endy, Drew

2013-05-03

Organisms must process information encoded via developmental and environmental signals to survive and reproduce. Researchers have also engineered synthetic genetic logic to realize simpler, independent control of biological processes. We developed a three-terminal device architecture, termed the transcriptor, that uses bacteriophage serine integrases to control the flow of RNA polymerase along DNA. Integrase-mediated inversion or deletion of DNA encoding transcription terminators or a promoter modulates transcription rates. We realized permanent amplifying AND, NAND, OR, XOR, NOR, and XNOR gates actuated across common control signal ranges and sequential logic supporting autonomous cell-cell communication of DNA encoding distinct logic-gate states. The single-layer digital logic architecture developed here enables engineering of amplifying logic gates to control transcription rates within and across diverse organisms.

DNA-Encoded Chemical Libraries: A Selection System Based on Endowing Organic Compounds with Amplifiable Information.

PubMed

Neri, Dario; Lerner, Richard A

2018-06-20

The discovery of organic ligands that bind specifically to proteins is a central problem in chemistry, biology, and the biomedical sciences. The encoding of individual organic molecules with distinctive DNA tags, serving as amplifiable identification bar codes, allows the construction and screening of combinatorial libraries of unprecedented size, thus facilitating the discovery of ligands to many different protein targets. Fundamentally, one links powers of genetics and chemical synthesis. After the initial description of DNA-encoded chemical libraries in 1992, several experimental embodiments of the technology have been reduced to practice. This review provides a historical account of important milestones in the development of DNA-encoded chemical libraries, a survey of relevant ongoing research activities, and a glimpse into the future.

Assessing the germplasm of Laminaria (phaeophyceae) with random amplified polymorphic DNA (RAPD) method

NASA Astrophysics Data System (ADS)

He, Yingjun; Zou, Yuping; Wang, Xiaodong; Zheng, Zhiguo; Zhang, Daming; Duan, Delin

2003-06-01

Eighteen gametophytes including L. japonica, L. ochotensis and L. longissima, were verified with random amplified polymorphic DNA (RAPD) technique. Eighteen ten-base primers were chosen from 100 primers selected for final amplification test. Among the total of 205 bands amplified, 181 (88.3%) were polymorphic. The genetic distance among different strains ranged from 0.072 to 0.391. The dendrogram constructed by unweighted pair-group method with arithmetic (UPGMA) method showed that the female and male gametophytes of the same cell lines could be grouped in pairs respectively. It indicated that RAPD analysis could be used not only to distinguish different strains of Laminaria, but also to distinguish male and female gametophyte within the same cell lines. There is ambiguous systematic relationship if judged merely by the present data. It seems that the use of RAPD marker is limited to elucidation of the phylogenetic relationship among the species of Laminaria.

Kr-86 Ion-Beam Irradiation of Hydrated DNA: Free Radical and Unaltered Base Yields

PubMed Central

Becker, David; Adhikary, Amitava; Tetteh, Smedley T.; Bull, Arthur W.; Sevilla, Michael D.

2012-01-01

This work reports an ESR and product analysis investigation of Kr-86 ion-beam irradiation of hydrated DNA at 77 K. The irradiation results in the formation and trapping of both base radicals and sugar phosphate radicals (DNA backbone radicals). The absolute yields (G, μmol/J) of the base radicals are smaller than the yields found in similarly prepared γ-irradiated DNA samples, and the relative yields of backbone radicals relative to base radicals are much higher than that found in γ-irradiated samples. From these results, we have elaborated our radiation chemical model of the track structure for ion-beam irradiated DNA as it applies to krypton ion-beams. The base radicals, which are trapped as ion radicals or reversibly protonated or deprotonated ion radicals, are formed almost entirely in the track penumbra, a region in which radiation chemical effects are similar to those found in γ-irradiated samples. By comparing the yields of base radicals in ion-beam samples to the yields of the same radicals in γ-irradiated samples, the partition of energy between the low-LET region (penumbra) and the core is experimentally determined. The neutral sugar and other backbone radicals, which are not as susceptible to recombination as are ion radicals, are formed largely in the track core. The backbone radicals show a linear dose response up to very high doses. Unaltered base release yields in Kr-86 irradiated hydrated DNA are equal to sugar radical yields within experimental error limits, consistent with radiation-chemical processes in which all base release originates with sugar radicals. Two phosphorus-centered radicals from fragmentation of the DNA backbone are found in low yields. PMID:23106211

Kr-86 ion-beam irradiation of hydrated DNA: free radical and unaltered base yields.

PubMed

Becker, David; Adhikary, Amitava; Tetteh, Smedley T; Bull, Arthur W; Sevilla, Michael D

2012-12-01

This work reports an ESR and product analysis investigation of Kr-86 ion-beam irradiation of hydrated DNA at 77 K. The irradiation results in the formation and trapping of both base radicals and sugar phosphate radicals (DNA backbone radicals). The absolute yields (G, μmol/J) of the base radicals are smaller than the yields found in similarly prepared γ-irradiated DNA samples, and the relative yields of backbone radicals relative to base radicals are much higher than that found in γ-irradiated samples. From these results, we have elaborated our radiation chemical model of the track structure for ion-beam irradiated DNA as it applies to krypton ion-beams. The base radicals, which are trapped as ion radicals or reversibly protonated or deprotonated ion radicals, are formed almost entirely in the track penumbra, a region in which radiation chemical effects are similar to those found in γ-irradiated samples. By comparing the yields of base radicals in ion-beam samples to the yields of the same radicals in γ-irradiated samples, the partition of energy between the low-LET region (penumbra) and the core is experimentally determined. The neutral sugar and other backbone radicals, which are not as susceptible to recombination as are ion radicals, are formed largely in the track core. The backbone radicals show a linear dose response up to very high doses. Unaltered base release yields in Kr-86 irradiated hydrated DNA are equal to sugar radical yields within experimental error limits, consistent with radiation-chemical processes in which all base release originates with sugar radicals. Two phosphorus-centered radicals from fragmentation of the DNA backbone are found in low yields.

SU-E-T-05: Comparing DNA Strand Break Yields for Photons under Different Irradiation Conditions with Geant4-DNA.

PubMed

Pater, P; Bernal, M; Naqa, I El; Seuntjens, J

2012-06-01

To validate and scrutinize published DNA strand break data with Geant4-DNA and a probabilistic model. To study the impact of source size, electronic equilibrium and secondary electron tracking cutoff on direct relative biological effectiveness (DRBE). Geant4 (v4.9.5) was used to simulate a cylindrical region of interest (ROI) with r = 15 nm and length = 1.05 mm, in a slab of liquid water of 1.06 g/cm 3 density. The ROI was irradiated with mono-energetic photons, with a uniformly distributed volumetric isotropic source (0.28, 1.5 keV) or a plane beam (0.662, 1.25 MeV), of variable size. Electrons were tracked down to 50 or 10 eV, with G4-DNA processes and energy transfer greater than 10.79 eV was scored. Based on volume ratios, each scored event had a 0.0388 probability of happening on either DNA helix (break). Clusters of at least one break on each DNA helix within 3.4 nm were found using a DBSCAN algorithm and categorized as double strand breaks (DSB). All other events were categorized as single strand breaks (SSB). Geant4-DNA is able to reproduce strand break yields previously published. Homogeneous irradiation conditions should be present throughout the ROI for DRBE comparisons. SSB yields seem slightly dependent on the primary photon energy. DRBEs show a significant increasing trend for lower energy incident photons. A lower electron cutoff produces higher SSB yields, but decreases the SSB/DSB yields ratio. The probabilistic and geometrical DNA models can predict equivalent results. Using Geant4, we were able to reproduce previously published results on the direct strand break yields of photon and study the importance of irradiation conditions. We also show an ascending trend for DRBE with lower incident photon energies. A probabilistic model coupled with track structure analysis can be used to simulate strand break yields. NSERC, CIHR. © 2012 American Association of Physicists in Medicine.

DNA Yield From Tissue Samples in Surgical Pathology and Minimum Tissue Requirements for Molecular Testing.

PubMed

Austin, Melissa C; Smith, Christina; Pritchard, Colin C; Tait, Jonathan F

2016-02-01

Complex molecular assays are increasingly used to direct therapy and provide diagnostic and prognostic information but can require relatively large amounts of DNA. To provide data to pathologists to help them assess tissue adequacy and provide prospective guidance on the amount of tissue that should be procured. We used slide-based measurements to establish a relationship between processed tissue volume and DNA yield by A260 from 366 formalin-fixed, paraffin-embedded tissue samples submitted for the 3 most common molecular assays performed in our laboratory (EGFR, KRAS, and BRAF). We determined the average DNA yield per unit of tissue volume, and we used the distribution of DNA yields to calculate the minimum volume of tissue that should yield sufficient DNA 99% of the time. All samples with a volume greater than 8 mm(3) yielded at least 1 μg of DNA, and more than 80% of samples producing less than 1 μg were extracted from less than 4 mm(3) of tissue. Nine square millimeters of tissue should produce more than 1 μg of DNA 99% of the time. We conclude that 2 tissue cores, each 1 cm long and obtained with an 18-gauge needle, will almost always provide enough DNA for complex multigene assays, and our methodology may be readily extrapolated to individual institutional practice.

Identification of Legionella Species by Random Amplified Polymorphic DNA Profiles

PubMed Central

Lo Presti, François; Riffard, Serge; Vandenesch, François; Etienne, Jerome

1998-01-01

Random amplified polymorphic DNA (RAPD) was used for the identification of Legionella species. Primer SK2 (5′-CGGCGGCGGCGG-3′) and standardized RAPD conditions gave the technique a reproducibility of 93 to 100%, depending on the species tested. Species-specific patterns corresponding to the 42 Legionella species were consequently defined by this method; the patterns were dependent on the recognition of a core of common bands for each species. This specificity was demonstrated by testing 65 type strains and 265 environmental and clinical isolates. No serogroup-specific profiles were obtained. A number of unidentified Legionella isolates potentially corresponding to new species were clustered in four groups. RAPD analysis appears to be a rapid and reproducible technique for identification of Legionella isolates to the species level without further restriction or hybridization. PMID:9774564

A pseudoautosomal random amplified polymorphic DNA marker for the sex chromosomes of Silene dioica.

PubMed Central

Di Stilio, V S; Kesseli, R V; Mulcahy, D L

1998-01-01

The segregation pattern of an 810-bp random amplified polymorphic DNA (RAPD) band in the F1 and backcross generations of a Silene dioica (L.) Clairv. family provides evidence that this molecular marker is located in the pseudoautosomal region (PAR) of the X and Y chromosomes. The marker was found through a combination of bulked segregant analysis (BSA) and RAPD techniques. Recombination rates between this pseudoautosomal marker and the differentiating portion of the Y chromosome are 15% in both generations. Alternative explanations involving nondisjunction or autosomal inheritance are presented and discussed. Chromosome counts provide evidence against the nondisjunction hypothesis, and probability calculations argue against the possibility of autosomal inheritance. This constitutes the first report of a pseudoautosomal DNA marker for plant sex chromosomes. PMID:9691057

Valency-Controlled Framework Nucleic Acid Signal Amplifiers.

PubMed

Liu, Qi; Ge, Zhilei; Mao, Xiuhai; Zhou, Guobao; Zuo, Xiaolei; Shen, Juwen; Shi, Jiye; Li, Jiang; Wang, Lihua; Chen, Xiaoqing; Fan, Chunhai

2018-06-11

Weak ligand-receptor recognition events are often amplified by recruiting multiple regulatory biomolecules to the action site in biological systems. However, signal amplification in in vitro biomimetic systems generally lack the spatiotemporal regulation in vivo. Herein we report a framework nucleic acid (FNA)-programmed strategy to develop valence-controlled signal amplifiers with high modularity for ultrasensitive biosensing. We demonstrated that the FNA-programmed signal amplifiers could recruit nucleic acids, proteins, and inorganic nanoparticles in a stoichiometric manner. The valence-controlled signal amplifier enhanced the quantification ability of electrochemical biosensors, and enabled ultrasensitive detection of tumor-relevant circulating free DNA (cfDNA) with sensitivity enhancement of 3-5 orders of magnitude and improved dynamic range. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Label-free technology for the amplified detection of microRNA based on the allosteric hairpin DNA switch and hybridization chain reaction.

PubMed

Cai, Sheng; Cao, Zhijuan; Lau, Choiwan; Lu, Jianzhong

2014-11-21

By using the allosteric hairpin DNA switch, a novel assay for the detection of microRNA (miRNA) let-7a via a hybridization chain reaction (HCR) was introduced. Briefly, the hairpin DNA switch probe is a single-stranded DNA consisting of a streptavidin (SA) aptamer sequence, a target binding sequence and a certain sequence that acts as a trigger of the HCR. In the presence of target let-7a, the hairpin DNA switch would open and expose the stem region sequences, where a part of this sequence acts as initiator sequence strands for the HCR and triggers a cascade of hybridization events that yields nicked double helices analogous to alternating copolymers, another part is the SA aptamer sequence which activates its binding affinity to SA on SA-coated magnetic particles. The hybridization event could be sensitively detected via an instantaneous derivatization reaction between a special chemiluminescence (CL) reagent, 3,4,5-trimethoxylphenylglyoxal (TMPG) and the guanine nucleotides within the target, the hairpin DNA switch probe, and HCR helices to form an unstable CL intermediate for the generation of light. Our results show that the coupling of the hairpin DNA switch probe and the HCR for the amplified detection of let-7a achieves a better performance (e.g. wide linear response range: 0.1-1000 fmol, low detection limit: 0.1 fmol, and high specificity). Furthermore, this approach could be easily applied to the detection of let-7a in human lung cells, and extended to detect other types of miRNA and proteins such as PDGF based on aptamers. We believe such advancements will represent a significant step towards improved diagnostics and more personalized medical treatment.

Extracting DNA from 'jaws': high yield and quality from archived tiger shark (Galeocerdo cuvier) skeletal material.

PubMed

Nielsen, E E; Morgan, J A T; Maher, S L; Edson, J; Gauthier, M; Pepperell, J; Holmes, B J; Bennett, M B; Ovenden, J R

2017-05-01

Archived specimens are highly valuable sources of DNA for retrospective genetic/genomic analysis. However, often limited effort has been made to evaluate and optimize extraction methods, which may be crucial for downstream applications. Here, we assessed and optimized the usefulness of abundant archived skeletal material from sharks as a source of DNA for temporal genomic studies. Six different methods for DNA extraction, encompassing two different commercial kits and three different protocols, were applied to material, so-called bio-swarf, from contemporary and archived jaws and vertebrae of tiger sharks (Galeocerdo cuvier). Protocols were compared for DNA yield and quality using a qPCR approach. For jaw swarf, all methods provided relatively high DNA yield and quality, while large differences in yield between protocols were observed for vertebrae. Similar results were obtained from samples of white shark (Carcharodon carcharias). Application of the optimized methods to 38 museum and private angler trophy specimens dating back to 1912 yielded sufficient DNA for downstream genomic analysis for 68% of the samples. No clear relationships between age of samples, DNA quality and quantity were observed, likely reflecting different preparation and storage methods for the trophies. Trial sequencing of DNA capture genomic libraries using 20 000 baits revealed that a significant proportion of captured sequences were derived from tiger sharks. This study demonstrates that archived shark jaws and vertebrae are potential high-yield sources of DNA for genomic-scale analysis. It also highlights that even for similar tissue types, a careful evaluation of extraction protocols can vastly improve DNA yield. © 2016 John Wiley & Sons Ltd.

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.

Detection of trypanosomatid Phytomonas parasitic in plants by polymerase chain reaction amplification of small subunit ribosomal DNA.

PubMed

Teixeira, M M; Campaner, M; Camargo, E P

1994-01-01

To improve the diagnosis of Phytomonas infections in plants, we developed a polymerase chain reaction (PCR) assay using synthetic oligonucleotides complementary to conserved sequences of the 18S small subunit ribosomal (SSU) gene. From 10 ng upward of DNA of cultures of Phytomonas isolated from plants, fruits, and insects, PCR amplified an 800-bp DNA band that, after restriction analysis and probe hybridization, proved to be of 18S rDNA Phytomonas origin. PCR was also done with sap samples of tomatoes experimentally infected with Phytomonas, yielding amplified 800-bp ribosomal DNA bands before any flagellate could be detected by microscopic examination of the fruit sap.

DNA-based identification of spices: DNA isolation, whole genome amplification, and polymerase chain reaction.

PubMed

Focke, Felix; Haase, Ilka; Fischer, Markus

2011-01-26

Usually spices are identified morphologically using simple methods like magnifying glasses or microscopic instruments. On the other hand, molecular biological methods like the polymerase chain reaction (PCR) enable an accurate and specific detection also in complex matrices. Generally, the origins of spices are plants with diverse genetic backgrounds and relationships. The processing methods used for the production of spices are complex and individual. Consequently, the development of a reliable DNA-based method for spice analysis is a challenging intention. However, once established, this method will be easily adapted to less difficult food matrices. In the current study, several alternative methods for the isolation of DNA from spices have been developed and evaluated in detail with regard to (i) its purity (photometric), (ii) yield (fluorimetric methods), and (iii) its amplifiability (PCR). Whole genome amplification methods were used to preamplify isolates to improve the ratio between amplifiable DNA and inhibiting substances. Specific primer sets were designed, and the PCR conditions were optimized to detect 18 spices selectively. Assays of self-made spice mixtures were performed to proof the applicability of the developed methods.

Extraction of inhibitor-free metagenomic DNA from polluted sediments, compatible with molecular diversity analysis using adsorption and ion-exchange treatments.

PubMed

Desai, Chirayu; Madamwar, Datta

2007-03-01

PCR inhibitor-free metagenomic DNA of high quality and high yield was extracted from highly polluted sediments using a simple remediation strategy of adsorption and ion-exchange chromatography. Extraction procedure was optimized with series of steps, which involved gentle mechanical lysis, treatment with powdered activated charcoal (PAC) and ion-exchange chromatography with amberlite resin. Quality of the extracted DNA for molecular diversity analysis was tested by amplifying bacterial 16S rDNA (16S rRNA gene) with eubacterial specific universal primers (8f and 1492r), cloning of the amplified 16S rDNA and ARDRA (amplified rDNA restriction analysis) of the 16S rDNA clones. The presence of discrete differences in ARDRA banding profiles provided evidence for expediency of the DNA extraction protocol in molecular diversity studies. A comparison of the optimized protocol with commercial Ultraclean Soil DNA isolation kit suggested that method described in this report would be more efficient in removing metallic and organic inhibitors, from polluted sediment samples.

An improved extraction method to increase DNA yield from molted feathers

Treesearch

Shelley Bayard De Volo; Richard T. Reynolds; Marlis R. Douglas; Michael F. Antolin

2008-01-01

To assess the value of molted feathers as a noninvasive source of DNA for genetic studies of Northern Goshawks (Accipiter gentilis), we isolated and quantified DNA from molted feathers and compared yields across five feather types. We also compared PCR success across the same five feather types using five microsatellite genetic markers of varying...

Use of Conserved Randomly Amplified Polymorphic DNA (RAPD) Fragments and RAPD Pattern for Characterization of Lactobacillus fermentum in Ghanaian Fermented Maize Dough

PubMed Central

Hayford, Alice E.; Petersen, Anne; Vogensen, Finn K.; Jakobsen, Mogens

1999-01-01

The present work describes the use of randomly amplified polymorphic DNA (RAPD) for the characterization of 172 dominant Lactobacillus isolates from present and previous studies of Ghanaian maize fermentation. Heterofermentative lactobacilli dominate the fermentation flora, since approximately 85% of the isolates belong to this group. Cluster analysis of the RAPD profiles obtained showed the presence of two main clusters. Cluster 1 included Lactobacillus fermentum, whereas cluster 2 comprised the remaining Lactobacillus spp. The two distinct clusters emerged at the similarity level of <50%. All isolates in cluster 1 showed similarity in their RAPD profile to the reference strains of L. fermentum included in the study. These isolates, yielding two distinct bands of approximately 695 and 773 bp with the primers used, were divided into four subclusters, indicating that several strains are involved in the fermentation and remain dominant throughout the process. The two distinct RAPD fragments were cloned, sequenced, and used as probes in Southern hybridization experiments. With one exception, Lactobacillus reuteri LMG 13045, the probes hybridized only to fragments of different sizes in EcoRI-digested chromosomal DNA of L. fermentum strains, thus indicating the specificity of the probes and variation within the L. fermentum isolates. PMID:10388723

A Comparison of DNA Extraction Methods using Petunia hybrida Tissues

PubMed Central

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

2013-01-01

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

Sources of Pre-Analytical Variations in Yield of DNA Extracted from Blood Samples: Analysis of 50,000 DNA Samples in EPIC

PubMed Central

Caboux, Elodie; Lallemand, Christophe; Ferro, Gilles; Hémon, Bertrand; Mendy, Maimuna; Biessy, Carine; Sims, Matt; Wareham, Nick; Britten, Abigail; Boland, Anne; Hutchinson, Amy; Siddiq, Afshan; Vineis, Paolo; Riboli, Elio; Romieu, Isabelle; Rinaldi, Sabina; Gunter, Marc J.; Peeters, Petra H. M.; van der Schouw, Yvonne T.; Travis, Ruth; Bueno-de-Mesquita, H. Bas; Canzian, Federico; Sánchez, Maria-José; Skeie, Guri; Olsen, Karina Standahl; Lund, Eiliv; Bilbao, Roberto; Sala, Núria; Barricarte, Aurelio; Palli, Domenico; Navarro, Carmen; Panico, Salvatore; Redondo, Maria Luisa; Polidoro, Silvia; Dossus, Laure; Boutron-Ruault, Marie Christine; Clavel-Chapelon, Françoise; Trichopoulou, Antonia; Trichopoulos, Dimitrios; Lagiou, Pagona; Boeing, Heiner; Fisher, Eva; Tumino, Rosario; Agnoli, Claudia; Hainaut, Pierre

2012-01-01

The European Prospective Investigation into Cancer and nutrition (EPIC) is a long-term, multi-centric prospective study in Europe investigating the relationships between cancer and nutrition. This study has served as a basis for a number of Genome-Wide Association Studies (GWAS) and other types of genetic analyses. Over a period of 5 years, 52,256 EPIC DNA samples have been extracted using an automated DNA extraction platform. Here we have evaluated the pre-analytical factors affecting DNA yield, including anthropometric, epidemiological and technical factors such as center of subject recruitment, age, gender, body-mass index, disease case or control status, tobacco consumption, number of aliquots of buffy coat used for DNA extraction, extraction machine or procedure, DNA quantification method, degree of haemolysis and variations in the timing of sample processing. We show that the largest significant variations in DNA yield were observed with degree of haemolysis and with center of subject recruitment. Age, gender, body-mass index, cancer case or control status and tobacco consumption also significantly impacted DNA yield. Feedback from laboratories which have analyzed DNA with different SNP genotyping technologies demonstrate that the vast majority of samples (approximately 88%) performed adequately in different types of assays. To our knowledge this study is the largest to date to evaluate the sources of pre-analytical variations in DNA extracted from peripheral leucocytes. The results provide a strong evidence-based rationale for standardized recommendations on blood collection and processing protocols for large-scale genetic studies. PMID:22808065

Chemical synthesis and characterization of branched oligodeoxyribonucleotides (bDNA) for use as signal amplifiers in nucleic acid quantification assays.

PubMed

Horn, T; Chang, C A; Urdea, M S

1997-12-01

The divergent synthesis of bDNA structures is described. This new type of branched DNA contains one unique oligonucleotide, the primary sequence, covalently attached through a comb-like branching network to many identical copies of a different oligonucleotide, the secondary sequence. The bDNA comb molecules were assembled on a solid support using parameters optimized for bDNA synthesis. The chemistry was used to synthesize bDNA comb molecules containing 15 secondary sequences. The bDNA comb molecules were elaborated by enzymatic ligation into branched amplification multimers, large bDNA molecules (a total of 1068 nt) containing an average of 36 repeated DNA oligomer sequences, each capable of hybridizing specifically to an alkaline phosphatase-labeled oligonucleotide. The bDNA comb molecules were characterized by electrophoretic methods and by controlled cleavage at periodate-cleavable moieties incorporated during synthesis. The branched amplification multimers have been used as signal amplifiers in nucleic acid quantification assays for detection of viral infection. It is possible to detect as few as 50 molecules with bDNA technology.

Chemical synthesis and characterization of branched oligodeoxyribonucleotides (bDNA) for use as signal amplifiers in nucleic acid quantification assays.

PubMed Central

Horn, T; Chang, C A; Urdea, M S

1997-01-01

The divergent synthesis of bDNA structures is described. This new type of branched DNA contains one unique oligonucleotide, the primary sequence, covalently attached through a comb-like branching network to many identical copies of a different oligonucleotide, the secondary sequence. The bDNA comb molecules were assembled on a solid support using parameters optimized for bDNA synthesis. The chemistry was used to synthesize bDNA comb molecules containing 15 secondary sequences. The bDNA comb molecules were elaborated by enzymatic ligation into branched amplification multimers, large bDNA molecules (a total of 1068 nt) containing an average of 36 repeated DNA oligomer sequences, each capable of hybridizing specifically to an alkaline phosphatase-labeled oligonucleotide. The bDNA comb molecules were characterized by electrophoretic methods and by controlled cleavage at periodate-cleavable moieties incorporated during synthesis. The branched amplification multimers have been used as signal amplifiers in nucleic acid quantification assays for detection of viral infection. It is possible to detect as few as 50 molecules with bDNA technology. PMID:9365266

DNA methylation changes detected by methylation-sensitive amplified polymorphism in two contrasting rice genotypes under salt stress.

PubMed

Wang, Wensheng; Zhao, Xiuqin; Pan, Yajiao; Zhu, Linghua; Fu, Binying; Li, Zhikang

2011-09-20

DNA methylation, one of the most important epigenetic phenomena, plays a vital role in tuning gene expression during plant development as well as in response to environmental stimuli. In the present study, a methylation-sensitive amplified polymorphism (MSAP) analysis was performed to profile DNA methylation changes in two contrasting rice genotypes under salt stress. Consistent with visibly different phenotypes in response to salt stress, epigenetic markers classified as stable inter-cultivar DNA methylation differences were determined between salt-tolerant FL478 and salt-sensitive IR29. In addition, most tissue-specific DNA methylation loci were conserved, while many of the growth stage-dependent DNA methylation loci were dynamic between the two genotypes. Strikingly, salt stress induced a decrease in DNA methylation specifically in roots at the seedling stage that was more profound in IR29 than in the FL478. This result may indicate that demethylation of genes is an active epigenetic response to salt stress in roots at the seedling stage, and helps to further elucidate the implications of DNA methylation in crop growth and development. Copyright © 2011. Published by Elsevier Ltd.

DNA methylation levels analysis in four tissues of sea cucumber Apostichopus japonicus based on fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) during aestivation.

PubMed

Zhao, Ye; Chen, Muyan; Storey, Kenneth B; Sun, Lina; Yang, Hongsheng

2015-03-01

DNA methylation plays an important role in regulating transcriptional change in response to environmental stimuli. In the present study, DNA methylation levels of tissues of the sea cucumber Apostichopus japonicus were analyzed by the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) technique over three stages of the aestivation cycle. Overall, a total of 26,963 fragments were amplified including 9112 methylated fragments among four sea cucumber tissues using 18 pairs of selective primers. Results indicated an average DNA methylation level of 33.79% for A. japonicus. The incidence of DNA methylation was different across tissue types in the non-aestivation stage: intestine (30.16%), respiratory tree (27.61%), muscle (27.94%) and body wall (56.25%). Our results show that hypermethylation accompanied deep-aestivation in A. japonicus, which suggests that DNA methylation may have an important role in regulating global transcriptional suppression during aestivation. Further analysis indicated that the main DNA modification sites were focused on intestine and respiratory tree tissues and that full-methylation but not hemi-methylation levels exhibited significant increases in the deep-aestivation stage. Copyright © 2014 Elsevier Inc. All rights reserved.

Improving efficiency and reliability of environmental DNA analysis for silver carp

USGS Publications Warehouse

Amberg, Jon J.; McCalla, S. Grace; Monroe, Emy; Lance, Richard; Baerwaldt, Kelly; Gaikowski, Mark P.

2015-01-01

Natural resource agencies have established surveillance programs which use environmental DNA (eDNA) for the early detection of bighead carp Hypophthalmichthys nobilis and silver carp Hypophthalmichthys molitrix before they establish populations within the Great Lakes. This molecular monitoring technique must be highly accurate and precise for confident interpretation and also efficient, both in detection threshold and cost. Therefore, we compared two DNA extraction techniques and compared a new quantitative PCR (qPCR) assay with the conventional PCR (cPCR) assay used by monitoring programs. Both the qPCR and cPCR assays were able to amplify the DNA of silver carp present in environmental samples taken from locations where mixed populations of bigheaded carps existed. However, the qPCR assay had substantially fewer PCR positive samples which were subsequently determined not to contain DNA of bigheaded carps than the cPCR assay. Additionally, the qPCR assay was able to amplify the DNA of bigheaded carps even in the presence of inhibitors that blocked amplification with cPCR. Also, the selection of an appropriate DNA extraction method can significantly alter the efficiency of eDNA surveillance programs by lowering detection limits and by decreasing costs associated with sample processing. The results reported herein are presently being incorporated into eDNA surveillance programs to decrease the costs, increase DNA yield and increase the confidence that assays are amplifying the target DNA. These results are critical to enhancing our ability to accurately and confidently interpret the results reported from monitoring programs using eDNA for early detection of invasive species.

Optimal Ancient DNA Yields from the Inner Ear Part of the Human Petrous Bone.

PubMed

Pinhasi, Ron; Fernandes, Daniel; Sirak, Kendra; Novak, Mario; Connell, Sarah; Alpaslan-Roodenberg, Songül; Gerritsen, Fokke; Moiseyev, Vyacheslav; Gromov, Andrey; Raczky, Pál; Anders, Alexandra; Pietrusewsky, Michael; Rollefson, Gary; Jovanovic, Marija; Trinhhoang, Hiep; Bar-Oz, Guy; Oxenham, Marc; Matsumura, Hirofumi; Hofreiter, Michael

2015-01-01

The invention and development of next or second generation sequencing methods has resulted in a dramatic transformation of ancient DNA research and allowed shotgun sequencing of entire genomes from fossil specimens. However, although there are exceptions, most fossil specimens contain only low (~ 1% or less) percentages of endogenous DNA. The only skeletal element for which a systematically higher endogenous DNA content compared to other skeletal elements has been shown is the petrous part of the temporal bone. In this study we investigate whether (a) different parts of the petrous bone of archaeological human specimens give different percentages of endogenous DNA yields, (b) there are significant differences in average DNA read lengths, damage patterns and total DNA concentration, and (c) it is possible to obtain endogenous ancient DNA from petrous bones from hot environments. We carried out intra-petrous comparisons for ten petrous bones from specimens from Holocene archaeological contexts across Eurasia dated between 10,000-1,800 calibrated years before present (cal. BP). We obtained shotgun DNA sequences from three distinct areas within the petrous: a spongy part of trabecular bone (part A), the dense part of cortical bone encircling the osseous inner ear, or otic capsule (part B), and the dense part within the otic capsule (part C). Our results confirm that dense bone parts of the petrous bone can provide high endogenous aDNA yields and indicate that endogenous DNA fractions for part C can exceed those obtained for part B by up to 65-fold and those from part A by up to 177-fold, while total endogenous DNA concentrations are up to 126-fold and 109-fold higher for these comparisons. Our results also show that while endogenous yields from part C were lower than 1% for samples from hot (both arid and humid) parts, the DNA damage patterns indicate that at least some of the reads originate from ancient DNA molecules, potentially enabling ancient DNA analyses of

Broadband linearisation of high-efficiency power amplifiers

NASA Technical Reports Server (NTRS)

Kenington, Peter B.; Parsons, Kieran J.; Bennett, David W.

1993-01-01

A feedforward-based amplifier linearization technique is presented which is capable of yielding significant improvements in both linearity and power efficiency over conventional amplifier classes (e.g. class-A or class-AB). Theoretical and practical results are presented showing that class-C stages may be used for both the main and error amplifiers yielding practical efficiencies well in excess of 30 percent, with theoretical efficiencies of much greater than 40 percent being possible. The levels of linearity which may be achieved are required for most satellite systems, however if greater linearity is required, the technique may be used in addition to conventional pre-distortion techniques.

Randomly amplified polymorphic-DNA analysis for detecting genotoxic effects of Boron on maize (Zea mays L.).

PubMed

Sakcali, M Serdal; Kekec, Guzin; Uzonur, Irem; Alpsoy, Lokman; Tombuloglu, Huseyin

2015-08-01

This study was carried out to investigate the genotoxic effect of boron (B) on maize using randomly amplified polymorphic DNA (RAPD) method. Experimental design was conducted under 0, 5, 10, 25, 50, 100, 125, and 150 ppm B exposures, and physiological changes have revealed a sharp decrease in root growth rates from 28% to 85%, starting from 25 ppm to 150 ppm, respectively. RAPD-polymerase chain reaction (PCR) analysis shows that DNA alterations are clearly observed from beginning to 100 ppm. B-induced inhibition in root growth had a positive correlation with DNA alterations. Total soluble protein, root and stem lengths, and B content analysis in root and leaves encourage these results as a consequence. These preliminary findings reveal that B causes chromosomal aberration and genotoxic effects on maize. Meanwhile, usage of RAPD-PCR technique is a suitable biomarker to detect genotoxic effect of B on maize and other crops for the future. © The Author(s) 2013.

Qualitative and quantitative assessment of DNA quality of frozen beef based on DNA yield, gel electrophoresis and PCR amplification and their correlations to beef quality.

PubMed

Zhao, Jing; Zhang, Ting; Liu, Yongfeng; Wang, Xingyu; Zhang, Lan; Ku, Ting; Quek, Siew Young

2018-09-15

Freezing is a practical method for meat preservation but the quality of frozen meat can deteriorate with storage time. This research investigated the effect of frozen storage time (up to 66 months) on changes in DNA yield, purity and integrity in beef, and further analyzed the correlation between beef quality (moisture content, protein content, TVB-N value and pH value) and DNA quality in an attempt to establish a reliable, high-throughput method for meat quality control. Results showed that frozen storage time influenced the yield and integrity of DNA significantly (p < 0.05). The DNA yield decreased as frozen storage time increased due to DNA degradation. The half-life (t 1/2  = ln2/0.015) was calculated as 46 months. The DNA quality degraded dramatically with the increased storage time based on gel electrophoresis results. Polymerase chain reaction (PCR) products from both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) were observed in all frozen beef samples. Using real-time PCR for quantitative assessment of DNA and meat quality revealed that correlations could be established successfully with mathematical models to evaluate frozen beef quality. Copyright © 2018 Elsevier Ltd. All rights reserved.

Autonomous replication of nucleic acids by polymerization/nicking enzyme/DNAzyme cascades for the amplified detection of DNA and the aptamer-cocaine complex.

PubMed

Wang, Fuan; Freage, Lina; Orbach, Ron; Willner, Itamar

2013-09-03

The progressive development of amplified DNA sensors and aptasensors using replication/nicking enzymes/DNAzyme machineries is described. The sensing platforms are based on the tailoring of a DNA template on which the recognition of the target DNA or the formation of the aptamer-substrate complex trigger on the autonomous isothermal replication/nicking processes and the displacement of a Mg(2+)-dependent DNAzyme that catalyzes the generation of a fluorophore-labeled nucleic acid acting as readout signal for the analyses. Three different DNA sensing configurations are described, where in the ultimate configuration the target sequence is incorporated into a nucleic acid blocker structure associated with the sensing template. The target-triggered isothermal autonomous replication/nicking process on the modified template results in the formation of the Mg(2+)-dependent DNAzyme tethered to a free strand consisting of the target sequence. This activates additional template units for the nucleic acid self-replication process, resulting in the ultrasensitive detection of the target DNA (detection limit 1 aM). Similarly, amplified aptamer-based sensing platforms for cocaine are developed along these concepts. The modification of the cocaine-detection template by the addition of a nucleic acid sequence that enables the autonomous secondary coupled activation of a polymerization/nicking machinery and DNAzyme generation path leads to an improved analysis of cocaine (detection limit 10 nM).

Comparison of ribotyping, randomly amplified polymorphic DNA analysis, and pulsed-field gel electrophoresis in typing of Lactobacillus rhamnosus and L. casei strains.

PubMed

Tynkkynen, S; Satokari, R; Saarela, M; Mattila-Sandholm, T; Saxelin, M

1999-09-01

A total of 24 strains, biochemically identified as members of the Lactobacillus casei group, were identified by PCR with species-specific primers. The same set of strains was typed by randomly amplified polymorphic DNA (RAPD) analysis, ribotyping, and pulsed-field gel electrophoresis (PFGE) in order to compare the discriminatory power of the methods. Species-specific primers for L. rhamnosus and L. casei identified the type strain L. rhamnosus ATCC 7469 and the neotype strain L. casei ATCC 334, respectively, but did not give any signal with the recently revived species L. zeae, which contains the type strain ATCC 15820 and the strain ATCC 393, which was previously classified as L. casei. Our results are in accordance with the suggested new classification of the L. casei group. Altogether, 21 of the 24 strains studied were identified with the species-specific primers. In strain typing, PFGE was the most discriminatory method, revealing 17 genotypes for the 24 strains studied. Ribotyping and RAPD analysis yielded 15 and 12 genotypes, respectively.

Microdosimetry of DNA conformations: relation between direct effect of (60)Co gamma rays and topology of DNA geometrical models in the calculation of A-, B- and Z-DNA radiation-induced damage yields.

PubMed

Semsarha, Farid; Raisali, Gholamreza; Goliaei, Bahram; Khalafi, Hossein

2016-05-01

In order to obtain the energy deposition pattern of ionizing radiation in the nanometric scale of genetic material and to investigate the different sensitivities of the DNA conformations, direct effects of (60)Co gamma rays on the three A, B and Z conformations of DNA have been studied. For this purpose, single-strand breaks (SSB), double-strand breaks (DSB), base damage (BD), hit probabilities and three microdosimetry quantities (imparted energy, mean chord length and lineal energy) in the mentioned DNA conformations have been calculated and compared by using GEometry ANd Tracking 4 (Geant4) toolkit. The results show that A-, B- and Z-DNA conformations have the highest yields of DSB (1.2 Gy(-1) Gbp(-1)), SSB (25.2 Gy(-1) Gbp(-1)) and BD (4.81 Gy(-1) Gbp(-1)), respectively. Based on the investigation of direct effects of radiation, it can be concluded that the DSB yield is largely correlated to the topological characteristics of DNA models, although the SSB yield is not. Moreover, according to the comparative results of the present study, a reliable candidate parameter for describing the relationship between DNA damage yields and geometry of DNA models in the theoretical radiation biology research studies would be the mean chord length (4 V/S) of the models.

Composting oily sludges: Characterizing microflora using randomly amplified polymorphic DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Persson, A.; Quednau, M.; Ahrne, S.

1995-12-31

Laboratory-scale composts in which oily sludge was composted under mesophilic conditions with amendments such as peat, bark, and fresh or decomposed horse manure, were studied with respect to basic parameters such as oil degradation, respirometry, and bacterial numbers. Further, an attempt was made to characterize a part of the bacterial flora using randomly amplified polymorphic DNA (RAPD). The compost based on decomposed horse manure showed the greatest reduction of oil (85%). Comparison with a killed control indicated that microbial degradation actually had occurred. However, a substantial part of the oil was stabilized rather than totally broken down. Volatiles, on themore » contrary, accounted for a rather small percentage (5%) of the observed reduction. RAPD indicated that a selection had taken place and that the dominating microbial flora during the active degradation of oil were not the same as the ones dominating the different basic materials. The stabilized compost, on the other hand, had bacterial flora with similarities to the ones found in peat and bark.« less

A Simple Method for Amplifying RNA Targets (SMART)

PubMed Central

McCalla, Stephanie E.; Ong, Carmichael; Sarma, Aartik; Opal, Steven M.; Artenstein, Andrew W.; Tripathi, Anubhav

2012-01-01

We present a novel and simple method for amplifying RNA targets (named by its acronym, SMART), and for detection, using engineered amplification probes that overcome existing limitations of current RNA-based technologies. This system amplifies and detects optimal engineered ssDNA probes that hybridize to target RNA. The amplifiable probe-target RNA complex is captured on magnetic beads using a sequence-specific capture probe and is separated from unbound probe using a novel microfluidic technique. Hybridization sequences are not constrained as they are in conventional target-amplification reactions such as nucleic acid sequence amplification (NASBA). Our engineered ssDNA probe was amplified both off-chip and in a microchip reservoir at the end of the separation microchannel using isothermal NASBA. Optimal solution conditions for ssDNA amplification were investigated. Although KCl and MgCl2 are typically found in NASBA reactions, replacing 70 mmol/L of the 82 mmol/L total chloride ions with acetate resulted in optimal reaction conditions, particularly for low but clinically relevant probe concentrations (≤100 fmol/L). With the optimal probe design and solution conditions, we also successfully removed the initial heating step of NASBA, thus achieving a true isothermal reaction. The SMART assay using a synthetic model influenza DNA target sequence served as a fundamental demonstration of the efficacy of the capture and microfluidic separation system, thus bridging our system to a clinically relevant detection problem. PMID:22691910

Multiplex picoliter-droplet digital PCR for quantitative assessment of DNA integrity in clinical samples.

PubMed

Didelot, Audrey; Kotsopoulos, Steve K; Lupo, Audrey; Pekin, Deniz; Li, Xinyu; Atochin, Ivan; Srinivasan, Preethi; Zhong, Qun; Olson, Jeff; Link, Darren R; Laurent-Puig, Pierre; Blons, Hélène; Hutchison, J Brian; Taly, Valerie

2013-05-01

Assessment of DNA integrity and quantity remains a bottleneck for high-throughput molecular genotyping technologies, including next-generation sequencing. In particular, DNA extracted from paraffin-embedded tissues, a major potential source of tumor DNA, varies widely in quality, leading to unpredictable sequencing data. We describe a picoliter droplet-based digital PCR method that enables simultaneous detection of DNA integrity and the quantity of amplifiable DNA. Using a multiplex assay, we detected 4 different target lengths (78, 159, 197, and 550 bp). Assays were validated with human genomic DNA fragmented to sizes of 170 bp to 3000 bp. The technique was validated with DNA quantities as low as 1 ng. We evaluated 12 DNA samples extracted from paraffin-embedded lung adenocarcinoma tissues. One sample contained no amplifiable DNA. The fractions of amplifiable DNA for the 11 other samples were between 0.05% and 10.1% for 78-bp fragments and ≤1% for longer fragments. Four samples were chosen for enrichment and next-generation sequencing. The quality of the sequencing data was in agreement with the results of the DNA-integrity test. Specifically, DNA with low integrity yielded sequencing results with lower levels of coverage and uniformity and had higher levels of false-positive variants. The development of DNA-quality assays will enable researchers to downselect samples or process more DNA to achieve reliable genome sequencing with the highest possible efficiency of cost and effort, as well as minimize the waste of precious samples. © 2013 American Association for Clinical Chemistry.

Strip biosensor for amplified detection of nerve growth factor-beta based on a molecular translator and catalytic DNA circuit.

PubMed

Liu, Jun; Lai, Ting; Mu, Kejie; Zhou, Zheng

2014-10-07

We have demonstrated a new visual detection approach based on a molecular translator and a catalytic DNA circuit for the detection of nerve growth factor-beta (NGF-β). In this assay, a molecular translator based on the binding-induced DNA strand-displacement reaction was employed to convert the input protein to an output DNA signal. The molecular translator is composed of a target recognition element and a signal output element. Target recognition is achieved by the binding of the anti-NGF-β antibody to the target protein. Polyclonal anti-NGF-β antibody is conjugated to DNA1 and DNA2. The antibody conjugated DNA1 is initially hybridized to DNA3 to form a stable DNA1/DNA3 duplex. In the presence of NGF-β, the binding of the same target protein brings DNA1 and DNA2 into close proximity, resulting in an increase in their local effective concentration. This process triggers the strand-displacement reaction between DNA2 and DNA3 and releases the output DNA3. The released DNA3 is further amplified by a catalytic DNA circuit. The product of the catalytic DNA circuit is detected by a strip biosensor. This proposed assay has high sensitivity and selectivity with a dynamic response ranging from 10 fM to 10 pM, and its detection limit is 10 fM of NGF-β. This work provides a sensitive, enzyme-free, and universal strategy for the detection of other proteins.

ILF2 Is a Regulator of RNA Splicing and DNA Damage Response in 1q21-Amplified Multiple Myeloma.

PubMed

Marchesini, Matteo; Ogoti, Yamini; Fiorini, Elena; Aktas Samur, Anil; Nezi, Luigi; D'Anca, Marianna; Storti, Paola; Samur, Mehmet Kemal; Ganan-Gomez, Irene; Fulciniti, Maria Teresa; Mistry, Nipun; Jiang, Shan; Bao, Naran; Marchica, Valentina; Neri, Antonino; Bueso-Ramos, Carlos; Wu, Chang-Jiun; Zhang, Li; Liang, Han; Peng, Xinxin; Giuliani, Nicola; Draetta, Giulio; Clise-Dwyer, Karen; Kantarjian, Hagop; Munshi, Nikhil; Orlowski, Robert; Garcia-Manero, Guillermo; DePinho, Ronald A; Colla, Simona

2017-07-10

Amplification of 1q21 occurs in approximately 30% of de novo and 70% of relapsed multiple myeloma (MM) and is correlated with disease progression and drug resistance. Here, we provide evidence that the 1q21 amplification-driven overexpression of ILF2 in MM promotes tolerance of genomic instability and drives resistance to DNA-damaging agents. Mechanistically, elevated ILF2 expression exerts resistance to genotoxic agents by modulating YB-1 nuclear localization and interaction with the splicing factor U2AF65, which promotes mRNA processing and the stabilization of transcripts involved in homologous recombination in response to DNA damage. The intimate link between 1q21-amplified ILF2 and the regulation of RNA splicing of DNA repair genes may be exploited to optimize the use of DNA-damaging agents in patients with high-risk MM. Copyright © 2017 Elsevier Inc. All rights reserved.

Experimental design of laminar proportional amplifiers

NASA Technical Reports Server (NTRS)

Hellbaum, R. F.

1976-01-01

An experimental program was initiated at Langley Research Center to study the effects of various parameters on the design of laminar proportional beam deflection amplifiers. Matching and staging of amplifiers to obtain high-pressure gain was also studied. Variable parameters were aspect ratio, setback, control length, receiver distance, receiver width, width of center vent, and bias pressure levels. Usable pressure gains from 4 to 19 per stage can now be achieved, and five amplifiers were staged together to yield pressure gains up to 2,000,000.

Identification of Species Related to Anopheles (Nyssorhynchus) albitarsis by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (Diptera: Culicidae)

DTIC Science & Technology

1995-11-01

Instituto de Biologia do ExCrcito, Rua Francisco Manuel 102, 2091 l-270 Rio de Janeiro, RJ, Brasil Species-specific Random Amplified Polymorphic DNA...da Panela Manaus Ilha Comprida 6 km SW Registro Ponte Melo Peixoto Capanema Ilha de Marajo Santa Helena nr. Guaira Aguia Branca Rio Socuavo...Brazil; 11, Ponte Melo Peixoto, Brazil. Fig. 3: RAPD amplifications of Albitarsis Complex species A with primer B05. Arrow on left indicates fragment

Rapid Amplification of Plasmid and Phage DNA Using Phi29 DNA Polymerase and Multiply-Primed Rolling Circle Amplification

PubMed Central

Dean, Frank B.; Nelson, John R.; Giesler, Theresa L.; Lasken, Roger S.

2001-01-01

We describe a simple method of using rolling circle amplification to amplify vector DNA such as M13 or plasmid DNA from single colonies or plaques. Using random primers and φ29 DNA polymerase, circular DNA templates can be amplified 10,000-fold in a few hours. This procedure removes the need for lengthy growth periods and traditional DNA isolation methods. Reaction products can be used directly for DNA sequencing after phosphatase treatment to inactivate unincorporated nucleotides. Amplified products can also be used for in vitro cloning, library construction, and other molecular biology applications. PMID:11381035

Identification of three randomly amplified polymorphic DNA-polymerase chain reaction markers for distinguishing Asian and North American Gypsy Moths (Lepidoptera: Lymantriidae)

Treesearch

David E. Schreiber; Karen J. Garner; James M. Slavicek

1997-01-01

Gypsy moths originating in Asia have recently been introduced into North America, making it necessary to develop markers for distinguishing the Asian strain from the established North American population. We have identified 3 randomly amplified polymorphic DNA-polymerase chain reaction generated (RAPD-PCR) markers which are specific for either Asian or North American...

AC instrumentation amplifier for bioimpedance measurements.

PubMed

Pallás-Areny, R; Webster, J G

1993-08-01

We analyze the input impedance and CMRR requirements for an amplifier for bioimpedance measurements when considering the capacitive components of the electrode-skin contact impedance. We describe an ac-coupled instrumentation amplifier (IA) that, in addition to fulfilling those requirements, both provides interference and noise reduction, and yields a zero phase shift over a wide frequency band without using broadband op amps.

Evaluating manta ray mucus as an alternative DNA source for population genetics study: underwater-sampling, dry-storage and PCR success.

PubMed

Kashiwagi, Tom; Maxwell, Elisabeth A; Marshall, Andrea D; Christensen, Ana B

2015-01-01

Sharks and rays are increasingly being identified as high-risk species for extinction, prompting urgent assessments of their local or regional populations. Advanced genetic analyses can contribute relevant information on effective population size and connectivity among populations although acquiring sufficient regional sample sizes can be challenging. DNA is typically amplified from tissue samples which are collected by hand spears with modified biopsy punch tips. This technique is not always popular due mainly to a perception that invasive sampling might harm the rays, change their behaviour, or have a negative impact on tourism. To explore alternative methods, we evaluated the yields and PCR success of DNA template prepared from the manta ray mucus collected underwater and captured and stored on a Whatman FTA™ Elute card. The pilot study demonstrated that mucus can be effectively collected underwater using toothbrush. DNA stored on cards was found to be reliable for PCR-based population genetics studies. We successfully amplified mtDNA ND5, nuclear DNA RAG1, and microsatellite loci for all samples and confirmed sequences and genotypes being those of target species. As the yields of DNA with the tested method were low, further improvements are desirable for assays that may require larger amounts of DNA, such as population genomic studies using emerging next-gen sequencing.

Evaluating manta ray mucus as an alternative DNA source for population genetics study: underwater-sampling, dry-storage and PCR success

PubMed Central

Maxwell, Elisabeth A.; Marshall, Andrea D.; Christensen, Ana B.

2015-01-01

Sharks and rays are increasingly being identified as high-risk species for extinction, prompting urgent assessments of their local or regional populations. Advanced genetic analyses can contribute relevant information on effective population size and connectivity among populations although acquiring sufficient regional sample sizes can be challenging. DNA is typically amplified from tissue samples which are collected by hand spears with modified biopsy punch tips. This technique is not always popular due mainly to a perception that invasive sampling might harm the rays, change their behaviour, or have a negative impact on tourism. To explore alternative methods, we evaluated the yields and PCR success of DNA template prepared from the manta ray mucus collected underwater and captured and stored on a Whatman FTA™ Elute card. The pilot study demonstrated that mucus can be effectively collected underwater using toothbrush. DNA stored on cards was found to be reliable for PCR-based population genetics studies. We successfully amplified mtDNA ND5, nuclear DNA RAG1, and microsatellite loci for all samples and confirmed sequences and genotypes being those of target species. As the yields of DNA with the tested method were low, further improvements are desirable for assays that may require larger amounts of DNA, such as population genomic studies using emerging next-gen sequencing. PMID:26413431

Fluorescence-labeled methylation-sensitive amplified fragment length polymorphism (FL-MS-AFLP) analysis for quantitative determination of DNA methylation and demethylation status.

PubMed

Kageyama, Shinji; Shinmura, Kazuya; Yamamoto, Hiroko; Goto, Masanori; Suzuki, Koichi; Tanioka, Fumihiko; Tsuneyoshi, Toshihiro; Sugimura, Haruhiko

2008-04-01

The PCR-based DNA fingerprinting method called the methylation-sensitive amplified fragment length polymorphism (MS-AFLP) analysis is used for genome-wide scanning of methylation status. In this study, we developed a method of fluorescence-labeled MS-AFLP (FL-MS-AFLP) analysis by applying a fluorescence-labeled primer and fluorescence-detecting electrophoresis apparatus to the existing method of MS-AFLP analysis. The FL-MS-AFLP analysis enables quantitative evaluation of more than 350 random CpG loci per run. It was shown to allow evaluation of the differences in methylation level of blood DNA of gastric cancer patients and evaluation of hypermethylation and hypomethylation in DNA from gastric cancer tissue in comparison with adjacent non-cancerous tissue.

Higher-order organisation of extremely amplified, potentially functional and massively methylated 5S rDNA in European pikes (Esox sp.).

PubMed

Symonová, Radka; Ocalewicz, Konrad; Kirtiklis, Lech; Delmastro, Giovanni Battista; Pelikánová, Šárka; Garcia, Sonia; Kovařík, Aleš

2017-05-18

Pikes represent an important genus (Esox) harbouring a pre-duplication karyotype (2n = 2x = 50) of economically important salmonid pseudopolyploids. Here, we have characterized the 5S ribosomal RNA genes (rDNA) in Esox lucius and its closely related E. cisalpinus using cytogenetic, molecular and genomic approaches. Intragenomic homogeneity and copy number estimation was carried out using Illumina reads. The higher-order structure of rDNA arrays was investigated by the analysis of long PacBio reads. Position of loci on chromosomes was determined by FISH. DNA methylation was analysed by methylation-sensitive restriction enzymes. The 5S rDNA loci occupy exclusively (peri)centromeric regions on 30-38 acrocentric chromosomes in both E. lucius and E. cisalpinus. The large number of loci is accompanied by extreme amplification of genes (>20,000 copies), which is to the best of our knowledge one of the highest copy number of rRNA genes in animals ever reported. Conserved secondary structures of predicted 5S rRNAs indicate that most of the amplified genes are potentially functional. Only few SNPs were found in genic regions indicating their high homogeneity while intergenic spacers were more heterogeneous and several families were identified. Analysis of 10-30 kb-long molecules sequenced by the PacBio technology (containing about 40% of total 5S rDNA) revealed that the vast majority (96%) of genes are organised in large several kilobase-long blocks. Dispersed genes or short tandems were less common (4%). The adjacent 5S blocks were directly linked, separated by intervening DNA and even inverted. The 5S units differing in the intergenic spacers formed both homogeneous and heterogeneous (mixed) blocks indicating variable degree of homogenisation between the loci. Both E. lucius and E. cisalpinus 5S rDNA was heavily methylated at CG dinucleotides. Extreme amplification of 5S rRNA genes in the Esox genome occurred in the absence of significant pseudogenisation

Use of Multiple Displacement Amplification as Pre-polymerase Chain Reaction (Pre-PCR) to amplify genomic DNA of siphonapterids preserved for long periods in scientific collections.

PubMed

Avelar, Daniel M; Linardi, Pedro M

2010-09-15

The recently developed Multiple Displacement Amplification technique (MDA) allows for the production of a large quantity of high quality genomic DNA from low amounts of the original DNA. The goal of this study was to evaluate the performance of the MDA technique to amplify genomic DNA of siphonapterids that have been stored for long periods in 70% ethanol at room temperature. We subjected each DNA sample to two different methodologies: (1) amplification of mitochondrial 16S sequences without MDA; (2) amplification of 16S after MDA. All the samples obtained from these procedures were then sequenced. Only 4 samples (15.4%) subjected to method 1 showed amplification. In contrast, the application of MDA (method 2) improved the performance substantially, with 24 samples (92.3%) showing amplification, with significant difference. Interestingly, one of the samples successfully amplified with this method was originally collected in 1909. All of the sequenced samples displayed satisfactory results in quality evaluations (Phred ≥ 20) and good similarities, as identified with the BLASTn tool. Our results demonstrate that the use of MDA may be an effective tool in molecular studies involving specimens of fleas that have traditionally been considered inadequately preserved for such purposes.

Use of Multiple Displacement Amplification as Pre-polymerase Chain Reaction (Pre-PCR) to amplify genomic DNA of siphonapterids preserved for long periods in scientific collections

PubMed Central

2010-01-01

The recently developed Multiple Displacement Amplification technique (MDA) allows for the production of a large quantity of high quality genomic DNA from low amounts of the original DNA. The goal of this study was to evaluate the performance of the MDA technique to amplify genomic DNA of siphonapterids that have been stored for long periods in 70% ethanol at room temperature. We subjected each DNA sample to two different methodologies: (1) amplification of mitochondrial 16S sequences without MDA; (2) amplification of 16S after MDA. All the samples obtained from these procedures were then sequenced. Only 4 samples (15.4%) subjected to method 1 showed amplification. In contrast, the application of MDA (method 2) improved the performance substantially, with 24 samples (92.3%) showing amplification, with significant difference. Interestingly, one of the samples successfully amplified with this method was originally collected in 1909. All of the sequenced samples displayed satisfactory results in quality evaluations (Phred ≥ 20) and good similarities, as identified with the BLASTn tool. Our results demonstrate that the use of MDA may be an effective tool in molecular studies involving specimens of fleas that have traditionally been considered inadequately preserved for such purposes. PMID:20840790

A microcantilever-based silver ion sensor using DNA-functionalized gold nanoparticles as a mass amplifier

NASA Astrophysics Data System (ADS)

You, Juneseok; Song, Yeongjin; Park, Chanho; Jang, Kuewhan; Na, Sungsoo

2017-06-01

Silver ions have been used to sterilize many products, however, it has recently been demonstrated that silver ions can be toxic. This toxicity has been studied over many years with the lethal concentration at 10 μM. Silver ions can accumulate through the food chain, causing serious health problems in many species. Hence, there is a need for a commercially available silver ion sensor, with high detection sensitivity. In this work, we develop an ultra-sensitive silver ion sensor platform, using cytosine based DNA and gold nanoparticles as the mass amplifier. We achieve a lower detection limit for silver ions of 10 pM; this detection limit is one million times lower than the toxic concentration. Using our sensor platform we examine highly selective characteristics of other typical ions in water from natural sources. Furthermore, our sensor platform is able to detect silver ions in a real practical sample of commercially available drinking water. Our sensor platform, which we have termed a ‘MAIS’ (mass amplifier ion sensor), with a simple detection procedure, high sensitivity, selectivity and real practical applicability has shown potential as an early toxicity assessment of silver ions in the environment.

Comparison of Ribotyping, Randomly Amplified Polymorphic DNA Analysis, and Pulsed-Field Gel Electrophoresis in Typing of Lactobacillus rhamnosus and L. casei Strains

PubMed Central

Tynkkynen, Soile; Satokari, Reetta; Saarela, Maria; Mattila-Sandholm, Tiina; Saxelin, Maija

1999-01-01

A total of 24 strains, biochemically identified as members of the Lactobacillus casei group, were identified by PCR with species-specific primers. The same set of strains was typed by randomly amplified polymorphic DNA (RAPD) analysis, ribotyping, and pulsed-field gel electrophoresis (PFGE) in order to compare the discriminatory power of the methods. Species-specific primers for L. rhamnosus and L. casei identified the type strain L. rhamnosus ATCC 7469 and the neotype strain L. casei ATCC 334, respectively, but did not give any signal with the recently revived species L. zeae, which contains the type strain ATCC 15820 and the strain ATCC 393, which was previously classified as L. casei. Our results are in accordance with the suggested new classification of the L. casei group. Altogether, 21 of the 24 strains studied were identified with the species-specific primers. In strain typing, PFGE was the most discriminatory method, revealing 17 genotypes for the 24 strains studied. Ribotyping and RAPD analysis yielded 15 and 12 genotypes, respectively. PMID:10473394

Determining Phylogenetic Relationships Among Date Palm Cultivars Using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) Markers.

PubMed

Haider, Nadia

2017-01-01

Investigation of genetic variation and phylogenetic relationships among date palm (Phoenix dactylifera L.) cultivars is useful for their conservation and genetic improvement. Various molecular markers such as restriction fragment length polymorphisms (RFLPs), simple sequence repeat (SSR), representational difference analysis (RDA), and amplified fragment length polymorphism (AFLP) have been developed to molecularly characterize date palm cultivars. PCR-based markers random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) are powerful tools to determine the relatedness of date palm cultivars that are difficult to distinguish morphologically. In this chapter, the principles, materials, and methods of RAPD and ISSR techniques are presented. Analysis of data generated from these two techniques and the use of these data to reveal phylogenetic relationships among date palm cultivars are also discussed.

Molecular Analysis of Date Palm Genetic Diversity Using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeats (ISSRs).

PubMed

El Sharabasy, Sherif F; Soliman, Khaled A

2017-01-01

The date palm is an ancient domesticated plant with great diversity and has been cultivated in the Middle East and North Africa for at last 5000 years. Date palm cultivars are classified based on the fruit moisture content, as dry, semidry, and soft dates. There are a number of biochemical and molecular techniques available for characterization of the date palm variation. This chapter focuses on the DNA-based markers random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) techniques, in addition to biochemical markers based on isozyme analysis. These techniques coupled with appropriate statistical tools proved useful for determining phylogenetic relationships among date palm cultivars and provide information resources for date palm gene banks.

Analysis of ploidy and the patterns of amplified fragment length polymorphism and methylation sensitive amplified polymorphism in strawberry plants recovered from cryopreservation.

PubMed

Hao, Yu-Jin; You, Chun-Xiang; Deng, Xui-Xin

2002-01-01

Shoot-tips of 10 strawberry genotypes were successfully cryopreserved using a modified encapsulation-dehydration method. All genotypes survived cryopreservation with high survival and regeneration rates. Eight Joho single-bud sibling lines were established as a model system for genetic analysis. Although cytological examination found chromosomal variation in both non-cryopreserved and cryopreserved samples, the ploidy constitution remained relatively stable after cryopreservation. DNA samples digested with MseI and PstI were used for amplified fragmentation length polymorphism (AFLP) assay. In 16 primer combinations, only one, namely, PCCA-MCAG, detected one site where band pattern changed after cryopreservation, which might be contributed to the change in DNA methylation status at PstI recognition site. Methylation sensitive amplified polymorphism (MSAP) assay was carried out for further investigation on the influence of cryopreservation on DNA methylation status. It was found that cryopreservation induced a significant change in DNA methylation status.

Genetic relatedness between oral and intestinal isolates of Porphyromonas endodontalis by analysis of random amplified polymorphic DNA.

PubMed

Gonçalves, R B; Väisänen, M L; Van Steenbergen, T J; Sundqvist, G; Mouton, C

1999-01-01

Genomic fingerprints from the DNA of 27 strains of Porphyromonas endodontalis from diverse clinical and geographic origins were generated as random amplified polymorphic DNA (RAPD) using the technique of PCR amplification with a single primer of arbitrary sequence. Cluster analysis of the combined RAPD data obtained with three selected 9- or 10-mer-long primers identified 25 distinct RAPD types which clustered as three main groups identifying three genogroups. Genogroups I and II included exclusively P. endodontalis isolates of oral origin, while 7/9 human intestinal strains of genogroup III which linked at a similarity level of 52% constituted the most homogeneous group in our study. Genotypic diversity within P. endodontalis, as shown by RAPD analysis, suggests that the taxon is composed of two oral genogroups and one intestinal genogroup. This hypothesis remains to be confirmed.

Multiple Differential-Amplifier MMICs Embedded in Waveguides

NASA Technical Reports Server (NTRS)

Kangaslahti, Pekka; Schlecht, Erich

2010-01-01

Compact amplifier assemblies of a type now being developed for operation at frequencies of hundreds of gigahertz comprise multiple amplifier units in parallel arrangements to increase power and/or cascade arrangements to increase gains. Each amplifier unit is a monolithic microwave integrated circuit (MMIC) implementation of a pair of amplifiers in differential (in contradistinction to single-ended) configuration. Heretofore, in cascading amplifiers to increase gain, it has been common practice to interconnect the amplifiers by use of wires and/or thin films on substrates. This practice has not yielded satisfactory results at frequencies greater than 200 Hz, in each case, for either or both of two reasons: Wire bonds introduce large discontinuities. Because the interconnections are typically tens of wavelengths long, any impedance mismatches give rise to ripples in the gain-vs.-frequency response, which degrade the performance of the cascade.

Molecular discrimination of lactobacilli used as starter and probiotic cultures by amplified ribosomal DNA restriction analysis.

PubMed

Roy, D; Sirois, S; Vincent, D

2001-04-01

Lactic acid bacteria such as Lactobacillus helveticus, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, L. acidophilus, and L. casei related taxa which are widely used as starter or probiotic cultures can be identified by amplified ribosomal DNA restriction analysis (ARDRA). The genetic discrimination of the related species belonging to these groups was first obtained by PCR amplifications by using group-specific or species-specific 16S rDNA primers. The numerical analysis of the ARDRA patterns obtained by using CfoI, HinfI, Tru9I, and ScrFI was an efficient typing tool for identification of species of the L. acidophilus and L. casei complex. ARDRA by using CfoI was a reliable method for differentiation of L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. Finally, strains ATCC 393 and ATCC 15820 exhibited unique ARDRA patterns with CfoI and Tru9I restriction enzymes as compared with the other strains of L. casei, L. paracasei, and L. rhamnosus.

[Applying competitive polymerase chain reaction to the detection of hepatitis B virus DNA].

PubMed

Wang, Ling; Yang, Peng; Li, Shuang-qing; Xu, Shu-hui; Cao, Gui-qun; Zhang, Fa-qiang; Zhang, Mei-xia; Chen, Qing-ying; Xia, Qing-jie; Liu, Kai; Tang, Fang; Zhang, Yuan-zheng

2004-11-01

To reduce the rate of accidental false negative result in the HBV DNA PCR test on clinical serum samples. A competitive polymerase chain reaction (C-PCR) was used to decrease the false negative ratio. In the C-PCR, a constructed inner control DNA was added for co-amplification with the HBV target DNA. In a 20 microl C-PCR system, about 60 to 200 copies of inner control DNA could give apparent co-amplification signal band after electrophoresis on a 2% agarose gel. Five of 120 samples of clinical serum (4.2%) could not be amplified. C-PCR has the advantage of yielding information on false negative in the HBV DNA PCR assay of clinical serum samples.

Comparison of methods for the extraction of DNA from formalin-fixed, paraffin-embedded archival tissues.

PubMed

Sengüven, Burcu; Baris, Emre; Oygur, Tulin; Berktas, Mehmet

2014-01-01

Discussing a protocol involving xylene-ethanol deparaffinization on slides followed by a kit-based extraction that allows for the extraction of high quality DNA from FFPE tissues. DNA was extracted from the FFPE tissues of 16 randomly selected blocks. Methods involving deparaffinization on slides or tubes, enzyme digestion overnight or for 72 hours and isolation using phenol chloroform method or a silica-based commercial kit were compared in terms of yields, concentrations and the amplifiability. The highest yield of DNA was produced from the samples that were deparaffinized on slides, digested for 72 hours and isolated with a commercial kit. Samples isolated with the phenol-chloroform method produced DNA of lower purity than the samples that were purified with kit. The samples isolated with the commercial kit resulted in better PCR amplification. Silica-based commercial kits and deparaffinized on slides should be considered for DNA extraction from FFPE.

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. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Effects of microbial DNA on human DNA profiles generated using the PowerPlex® 16 HS system.

PubMed

Dembinski, Gina M; Picard, Christine J

2017-11-01

Most crime scenes are not sterile and therefore may be contaminated with environmental DNA, especially if a decomposing body is found. Collecting biological evidence from this individual will yield DNA samples mixed with microbial DNA. This also becomes important if postmortem swabs are collected from sexually assaulted victims. Although genotyping kits undergo validation tests, including bacterial screens, they do not account for the diverse microbial load during decomposition. We investigated the effect of spiking human DNA samples with known concentrations of DNA from 17 microbe species associated with decomposition on DNA profiles produced using the Promega PowerPlex ® HS system. Two species, Bacillus subtilis and Mycobacterium smegmatis, produced an extraneous allele at the TPOX locus. When repeated with the PowerPlex ® Fusion kit, the extra allele no longer amplified with these two species. This experiment demonstrates that caution should be exhibited if microbial load is high and the PowerPlex ® 16HS system is used. Copyright © 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

High precision and high yield fabrication of dense nanoparticle arrays onto DNA origami at statistically independent binding sites

NASA Astrophysics Data System (ADS)

Takabayashi, Sadao; Klein, William P.; Onodera, Craig; Rapp, Blake; Flores-Estrada, Juan; Lindau, Elias; Snowball, Lejmarc; Sam, Joseph T.; Padilla, Jennifer E.; Lee, Jeunghoon; Knowlton, William B.; Graugnard, Elton; Yurke, Bernard; Kuang, Wan; Hughes, William L.

2014-10-01

High precision, high yield, and high density self-assembly of nanoparticles into arrays is essential for nanophotonics. Spatial deviations as small as a few nanometers can alter the properties of near-field coupled optical nanostructures. Several studies have reported assemblies of few nanoparticle structures with controlled spacing using DNA nanostructures with variable yield. Here, we report multi-tether design strategies and attachment yields for homo- and hetero-nanoparticle arrays templated by DNA origami nanotubes. Nanoparticle attachment yield via DNA hybridization is comparable with streptavidin-biotin binding. Independent of the number of binding sites, >97% site-occupation was achieved with four tethers and 99.2% site-occupation is theoretically possible with five tethers. The interparticle distance was within 2 nm of all design specifications and the nanoparticle spatial deviations decreased with interparticle spacing. Modified geometric, binomial, and trinomial distributions indicate that site-bridging, steric hindrance, and electrostatic repulsion were not dominant barriers to self-assembly and both tethers and binding sites were statistically independent at high particle densities.High precision, high yield, and high density self-assembly of nanoparticles into arrays is essential for nanophotonics. Spatial deviations as small as a few nanometers can alter the properties of near-field coupled optical nanostructures. Several studies have reported assemblies of few nanoparticle structures with controlled spacing using DNA nanostructures with variable yield. Here, we report multi-tether design strategies and attachment yields for homo- and hetero-nanoparticle arrays templated by DNA origami nanotubes. Nanoparticle attachment yield via DNA hybridization is comparable with streptavidin-biotin binding. Independent of the number of binding sites, >97% site-occupation was achieved with four tethers and 99.2% site-occupation is theoretically possible with five

Identification of Clinical Isolates of Actinomyces Species by Amplified 16S Ribosomal DNA Restriction Analysis

PubMed Central

Hall, Val; Talbot, P. R.; Stubbs, S. L.; Duerden, B. I.

2001-01-01

Amplified 16S ribosomal DNA (rDNA) restriction analysis (ARDRA), using enzymes HaeIII and HpaII, was applied to 176 fresh and 299 stored clinical isolates of putative Actinomyces spp. referred to the Anaerobe Reference Unit of the Public Health Laboratory Service for confirmation of identity. Results were compared with ARDRA results obtained previously for reference strains and with conventional phenotypic reactions. Identities of some strains were confirmed by analysis of partial 16S rDNA sequences. Of the 475 isolates, 331 (70%) were clearly assigned to recognized Actinomyces species, including 94 isolates assigned to six recently described species. A further 52 isolates in 12 ARDRA profiles were designated as apparently resembling recognized species, and 44 isolates, in 18 novel profiles, were confirmed as members of genera other than Actinomyces. The identities of 48 isolates in nine profiles remain uncertain, and they may represent novel species of Actinomyces. For the majority of species, phenotypic results, published reactions for the species, and ARDRA profiles concurred. However, of 113 stored isolates originally identified as A. meyeri or resembling A. meyeri by phenotypic tests, only 21 were confirmed as A. meyeri by ARDRA; 63 were reassigned as A. turicensis, 7 as other recognized species, and 22 as unidentified actinomycetes. Analyses of incidence and clinical associations of Actinomyces spp. add to the currently sparse knowledge of some recently described species. PMID:11574572

Evaluation of the applicability of amplified rDNA-restriction analysis (ARDRA) to identification of species of the genus Corynebacterium.

PubMed

Vaneechoutte, M; Riegel, P; de Briel, D; Monteil, H; Verschraegen, G; De Rouck, A; Claeys, G

1995-10-01

The 16S rRNA genes (rDNA) of 50 strains belonging to 26 different coryneform bacterial species and genomospecies and of the type strain of Rhodococcus equi were enzymatically amplified. Amplified rDNA restriction analysis (ARDRA) with the enzymes AluI, CfoI and RsaI was carried out. The combination of the ARDRA patterns obtained after restriction with these three different enzymes enabled the differentiation between the following species: Corynebacterium accolens (number of strains = 2), C. afermentans subsp. afermentans (2), C. afermentans subsp. lipophilum (2), C. amycolatum (3), CDC coryneform group ANF-1-like (1), CDC coryneform group ANF-3-like (1), C. cystitidis (1), C. diphtheriae (4), C. jeikeium (3), C. macginleyi (2), C. minutissimum (1), C. pilosum (1), C. pseudotuberculosis (2), C. renale (2), C. striatum (2), C. urealyticum (3), C. xerosis (1), CDC coryneform groups B-1 (2), B-3 (2), F-1, genomospecies 1 and 2 (6), G, genomospecies 1 (1) and G, genomospecies 2 (2). The following strains or species could not be differentiated from each other: C. pseudodiphtheriticum (2) from C. propinquum (former CDC coryneform group ANF-3) (2), CDC coryneform group F-1, genomospecies 1 (4) from genomospecies 2 (2) and C. jeikeium genomospecies A (1) from genomospecies C (2). ARDRA may represent a possible alternative for identification of coryneforms, since this technique enabled the identification of most coryneforms tested and since DNA extraction (i.e. cell lysis by boiling), amplification, restriction and electrophoresis can be carried out within 8 hours. This might allow quick identification of C. diphtheriae and other possible pathogens of the genus Corynebacterium.

A signal-amplified electrochemical DNA biosensor incorporated with a colorimetric internal control for Vibrio cholerae detection using shelf-ready reagents.

PubMed

Low, Kim-Fatt; Zain, Zainiharyati Mohd; Yean, Chan Yean

2017-01-15

A novel enzyme/nanoparticle-based DNA biosensing platform with dual colorimetric/electrochemical approach has been developed for the sequence-specific detection of the bacterium Vibrio cholerae, the causative agent of acute diarrheal disease in cholera. This assay platform exploits the use of shelf-stable and ready-to-use (shelf-ready) reagents to greatly simplify the bioanalysis procedures, allowing the assay platform to be more amenable to point-of-care applications. To assure maximum diagnosis reliability, an internal control (IC) capable of providing instant validation of results was incorporated into the assay. The microbial target, single-stranded DNA amplified with asymmetric PCR, was quantitatively detected via electrochemical stripping analysis of gold nanoparticle-loaded latex microspheres as a signal-amplified hybridization tag, while the incorporated IC was analyzed using a simplified horseradish peroxidase enzyme-based colorimetric scheme by simple visual observation of enzymatic color development. The platform showed excellent diagnostic sensitivity and specificity (100%) when challenged with 145 clinical isolate-spiked fecal specimens. The limits of detection were 0.5ng/ml of genomic DNA and 10 colony-forming units (CFU)/ml of bacterial cells with dynamic ranges of 0-100ng/ml (R 2 =0.992) and log 10 (1-10 4 CFU/ml) (R 2 =0.9918), respectively. An accelerated stability test revealed that the assay reagents were stable at temperatures of 4-37°C, with an estimated ambient shelf life of 200 days. The versatility of the biosensing platform makes it easily adaptable for quantitative detection of other microbial pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.

Predictors of mother and child DNA yields in buccal cell samples collected in pediatric cancer epidemiologic studies: a report from the Children's Oncology group.

PubMed

Poynter, Jenny N; Ross, Julie A; Hooten, Anthony J; Langer, Erica; Blommer, Crystal; Spector, Logan G

2013-08-12

Collection of high-quality DNA is essential for molecular epidemiology studies. Methods have been evaluated for optimal DNA collection in studies of adults; however, DNA collection in young children poses additional challenges. Here, we have evaluated predictors of DNA quantity in buccal cells collected for population-based studies of infant leukemia (N = 489 mothers and 392 children) and hepatoblastoma (HB; N = 446 mothers and 412 children) conducted through the Children's Oncology Group. DNA samples were collected by mail using mouthwash (for mothers and some children) and buccal brush (for children) collection kits and quantified using quantitative real-time PCR. Multivariable linear regression models were used to identify predictors of DNA yield. Median DNA yield was higher for mothers in both studies compared with their children (14 μg vs. <1 μg). Significant predictors of DNA yield in children included case-control status (β = -0.69, 50% reduction, P = 0.01 for case vs. control children), brush collection type, and season of sample collection. Demographic factors were not strong predictors of DNA yield in mothers or children in this analysis. The association with seasonality suggests that conditions during transport may influence DNA yield. The low yields observed in most children in these studies highlight the importance of developing alternative methods for DNA collection in younger age groups.

A Genetic Linkage Map of Longleaf Pine (Pinus palustris Mill.) Based on Random Amplified Polymorphic DNAs

Treesearch

C.D. Nelson; Thomas L. Kubisiak; M. Stine; W.L. Nance

1994-01-01

Eight megagametophyte DNA samples from a single longleaf pine (Pinus palustris Mill.) tree were used to screen 576 oligonucleotide primers for random amplified polymorphic DNA (RAPD) fragments. Primers amplifying repeatable polymorphic fragments were further characterized within a sample of 72 megagametophytes from the same tree. Fragments...

Enhanced Delivery and Potency of Self-Amplifying mRNA Vaccines by Electroporation in Situ

PubMed Central

Cu, Yen; Broderick, Kate E.; Banerjee, Kaustuv; Hickman, Julie; Otten, Gillis; Barnett, Susan; Kichaev, Gleb; Sardesai, Niranjan Y.; Ulmer, Jeffrey B.; Geall, Andrew

2013-01-01

Nucleic acid-based vaccines such as viral vectors, plasmid DNA (pDNA), and mRNA are being developed as a means to address limitations of both live-attenuated and subunit vaccines. DNA vaccines have been shown to be potent in a wide variety of animal species and several products are now licensed for commercial veterinary but not human use. Electroporation delivery technologies have been shown to improve the generation of T and B cell responses from synthetic DNA vaccines in many animal species and now in humans. However, parallel RNA approaches have lagged due to potential issues of potency and production. Many of the obstacles to mRNA vaccine development have recently been addressed, resulting in a revival in the use of non-amplifying and self-amplifying mRNA for vaccine and gene therapy applications. In this paper, we explore the utility of EP for the in vivo delivery of large, self-amplifying mRNA, as measured by reporter gene expression and immunogenicity of genes encoding HIV envelope protein. These studies demonstrated that EP delivery of self-amplifying mRNA elicited strong and broad immune responses in mice, which were comparable to those induced by EP delivery of pDNA. PMID:26344119

Effect of berberine on the yield of pyrimidine dimers in uv-irradiated DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klimek, M.; Sevcikova, P.; Pidra, M.

1973-01-01

From international conference on the bases of the biological effects of ultraviolet radiation; Brno, Czechoslovakia (2 Oct The effect of berberine on the yield of thymine dimers produced by uv light in DNA isolated from mouse leukemic cells and in DNA within irradiated cells was investigated. In solutions of isolated DNA the complete inhibition of thynnine dimerization was found at the concentration of berberine equal to 2 x 10/sup -3M/. However, in the cells inhibition of dimerization by berberine was never complete. In L cells a pronounced decrease in the intensity of DNA synthesis was found in cells treated withmore » berberine, dependent on berberine concentration used. But despite the presence of berberine in cell nuclei, no inhibition of pyrimidine dimerization in uv irradiated cells could be established. (auth)« less

Extracting DNA from FFPE Tissue Biospecimens Using User-Friendly Automated Technology: Is There an Impact on Yield or Quality?

PubMed

Mathieson, William; Guljar, Nafia; Sanchez, Ignacio; Sroya, Manveer; Thomas, Gerry A

2018-05-03

DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue blocks is amenable to analytical techniques, including sequencing. DNA extraction protocols are typically long and complex, often involving an overnight proteinase K digest. Automated platforms that shorten and simplify the process are therefore an attractive proposition for users wanting a faster turn-around or to process large numbers of biospecimens. It is, however, unclear whether automated extraction systems return poorer DNA yields or quality than manual extractions performed by experienced technicians. We extracted DNA from 42 FFPE clinical tissue biospecimens using the QiaCube (Qiagen) and ExScale (ExScale Biospecimen Solutions) automated platforms, comparing DNA yields and integrities with those from manual extractions. The QIAamp DNA FFPE Spin Column Kit was used for manual and QiaCube DNA extractions and the ExScale extractions were performed using two of the manufacturer's magnetic bead kits: one extracting DNA only and the other simultaneously extracting DNA and RNA. In all automated extraction methods, DNA yields and integrities (assayed using DNA Integrity Numbers from a 4200 TapeStation and the qPCR-based Illumina FFPE QC Assay) were poorer than in the manual method, with the QiaCube system performing better than the ExScale system. However, ExScale was fastest, offered the highest reproducibility when extracting DNA only, and required the least intervention or technician experience. Thus, the extraction methods have different strengths and weaknesses, would appeal to different users with different requirements, and therefore, we cannot recommend one method over another.

Comparison of Methods for the Extraction of DNA from Formalin-Fixed, Paraffin-Embedded Archival Tissues

PubMed Central

Sengüven, Burcu; Baris, Emre; Oygur, Tulin; Berktas, Mehmet

2014-01-01

Aim: Discussing a protocol involving xylene-ethanol deparaffinization on slides followed by a kit-based extraction that allows for the extraction of high quality DNA from FFPE tissues. Methods: DNA was extracted from the FFPE tissues of 16 randomly selected blocks. Methods involving deparaffinization on slides or tubes, enzyme digestion overnight or for 72 hours and isolation using phenol chloroform method or a silica-based commercial kit were compared in terms of yields, concentrations and the amplifiability. Results: The highest yield of DNA was produced from the samples that were deparaffinized on slides, digested for 72 hours and isolated with a commercial kit. Samples isolated with the phenol-chloroform method produced DNA of lower purity than the samples that were purified with kit. The samples isolated with the commercial kit resulted in better PCR amplification. Conclusion: Silica-based commercial kits and deparaffinized on slides should be considered for DNA extraction from FFPE. PMID:24688314

Evaluating differential nuclear DNA yield rates and osteocyte numbers among human bone tissue types: A synchrotron radiation micro-CT approach.

PubMed

Andronowski, Janna M; Mundorff, Amy Z; Pratt, Isaac V; Davoren, Jon M; Cooper, David M L

2017-05-01

Molecular human identification has conventionally focused on DNA sampling from dense, weight-bearing cortical bone tissue, typically from femora or tibiae. A comparison of skeletal elements from three contemporary individuals demonstrated that elements with high quantities of cancellous bone yielded nuclear DNA at the highest rates, suggesting that preferentially sampling cortical bone may be suboptimal (Mundorff & Davoren, 2014). Despite these findings, the reason for the differential DNA yields between cortical and cancellous bone tissues remains unknown. The primary goal of this work is to ascertain whether differences in bone microstructure can be used to explain differential nuclear DNA yield among bone tissue types observed by Mundorff and Davoren (2014), with a focus on osteocytes and the three-dimensional (3D) quantification of their associated lacunae. Osteocytes and other bone cells are recognized to house DNA in bone tissue, thus examining the density of their lacunae may explain why nuclear DNA yield rates differ among bone tissue types. Lacunae were visualized and quantified using synchrotron radiation-based micro-Computed Tomographic imaging (SR micro-CT). Volumes of interest (VOIs) from cortical and cancellous bone tissues (n=129) were comparatively analyzed from the three skeletons sampled for Mundorff and Davoren's (2014) study. Analyses tested the primary hypothesis that the abundance and density of osteocytes (inferred from their lacunar spaces) vary between cortical and cancellous bone tissue types. Results demonstrated that osteocyte lacunar abundance and density vary between cortical and cancellous bone tissue types, with cortical bone VOIs containing a higher lacunar abundance and density. We found that the osteocyte lacunar density values are independent of nuclear DNA yield, suggesting an alternative explanation for the higher nuclear DNA yields from bones with greater quantities of cancellous bone tissue. The use of SR micro-CT allowed for

[Analysis of genomic DNA methylation level in radish under cadmium stress by methylation-sensitive amplified polymorphism technique].

PubMed

Yang, Jin-Lan; Liu, Li-Wang; Gong, Yi-Qin; Huang, Dan-Qiong; Wang, Feng; He, Ling-Li

2007-06-01

The level of cytosine methylation induced by cadmium in radish (Raphanus sativus L.) genome was analysed using the technique of methylation-sensitive amplified polymorphism (MSAP). The MSAP ratios in radish seedling exposed to cadmium chloride at the concentration of 50, 250 and 500 mg/L were 37%, 43% and 51%, respectively, and the control was 34%; the full methylation levels (C(m)CGG in double strands) were at 23%, 25% and 27%, respectively, while the control was 22%. The level of increase in MSAP and full methylation indicated that de novo methylation occurred in some 5'-CCGG sites under Cd stress. There was significant positive correlation between increase of total DNA methylation level and CdCl(2) concentration. Four types of MSAP patterns: de novo methylation, de-methylation, atypical pattern and no changes of methylation pattern were identified among CdCl(2) treatments and the control. DNA methylation alteration in plants treated with CdCl(2) was mainly through de novo methylation.

Freshly excavated fossil bones are best for amplification of ancient DNA.

PubMed

Pruvost, Mélanie; Schwarz, Reinhard; Correia, Virginia Bessa; Champlot, Sophie; Braguier, Séverine; Morel, Nicolas; Fernandez-Jalvo, Yolanda; Grange, Thierry; Geigl, Eva-Maria

2007-01-16

Despite the enormous potential of analyses of ancient DNA for phylogeographic studies of past populations, the impact these analyses, most of which are performed with fossil samples from natural history museum collections, has been limited to some extent by the inefficient recovery of ancient genetic material. Here we show that the standard storage conditions and/or treatments of fossil bones in these collections can be detrimental to DNA survival. Using a quantitative paleogenetic analysis of 247 herbivore fossil bones up to 50,000 years old and originating from 60 different archeological and paleontological contexts, we demonstrate that freshly excavated and nontreated unwashed bones contain six times more DNA and yield twice as many authentic DNA sequences as bones treated with standard procedures. This effect was even more pronounced with bones from one Neolithic site, where only freshly excavated bones yielded results. Finally, we compared the DNA content in the fossil bones of one animal, a approximately 3,200-year-old aurochs, excavated in two separate seasons 57 years apart. Whereas the washed museum-stored fossil bones did not permit any DNA amplification, all recently excavated bones yielded authentic aurochs sequences. We established that during the 57 years when the aurochs bones were stored in a collection, at least as much amplifiable DNA was lost as during the previous 3,200 years of burial. This result calls for a revision of the postexcavation treatment of fossil bones to better preserve the genetic heritage of past life forms.

Ames and random amplified polymorphic DNA tests for the validation of the mutagenic and/or genotoxic potential of the drinking water disinfection by-products chloroform and bromoform.

PubMed

Khallef, Messaouda; Cenkci, Süleyman; Akyil, Dilek; Özkara, Arzu; Konuk, Muhsin; Benouareth, Djamel Eddine

2018-01-28

Chloroform and Bromoform are two abundant trihalomethanes found in Algerian drinking water. The investigation of the mutagenic hazard of these disinfection by-products was studied by Ames test as prokaryotic bioassay to show their mutagenic effects. For this, Salmonella typhimurium TA98 and TA100 strains were employed. Both chloroform and bromoform showed a direct mutagenic effect since the number of revertant colonies gradually increase in dose-dependent manner with all concentrations tested with the two bacterial strains and these were both in the absence and presence of S9 metabolic activation. The genotoxic hazard was also studied by random amplified polymorphic DNA test on the root cells of Allium cepa as eukaryotic bioassay. DNA extracted from the roots of the onion were incubated at different concentrations of chloroform and bromoform and then amplified by polymerase chain reaction. This was based on demonstrating a major effect of disappearance of bands compared to roots incubated in the negative control (distilled water). The results showed that these two compounds affected genomic DNA by breaks although by mutations.

Predictors of mother and child DNA yields in buccal cell samples collected in pediatric cancer epidemiologic studies: a report from the Children’s Oncology group

PubMed Central

2013-01-01

Background Collection of high-quality DNA is essential for molecular epidemiology studies. Methods have been evaluated for optimal DNA collection in studies of adults; however, DNA collection in young children poses additional challenges. Here, we have evaluated predictors of DNA quantity in buccal cells collected for population-based studies of infant leukemia (N = 489 mothers and 392 children) and hepatoblastoma (HB; N = 446 mothers and 412 children) conducted through the Children’s Oncology Group. DNA samples were collected by mail using mouthwash (for mothers and some children) and buccal brush (for children) collection kits and quantified using quantitative real-time PCR. Multivariable linear regression models were used to identify predictors of DNA yield. Results Median DNA yield was higher for mothers in both studies compared with their children (14 μg vs. <1 μg). Significant predictors of DNA yield in children included case–control status (β = −0.69, 50% reduction, P = 0.01 for case vs. control children), brush collection type, and season of sample collection. Demographic factors were not strong predictors of DNA yield in mothers or children in this analysis. Conclusions The association with seasonality suggests that conditions during transport may influence DNA yield. The low yields observed in most children in these studies highlight the importance of developing alternative methods for DNA collection in younger age groups. PMID:23937514

Evolution of finger millet: evidence from random amplified polymorphic DNA.

PubMed

Hilu, K W

1995-04-01

Finger millet (Eleusine coracana ssp. coracana) is an annual tetraploid member of a predominantly African genus. The crop is believed to have been domesticated from the tetraploid E. coracana ssp. africana. Cytogenetic and isozyme data point to the allopolyploid nature of the species and molecular information has shown E. indica to be one of the genomic donors. A recent isozyme study questioned the proposed phylogenetic relationship between finger millet and its direct ancestor subspecies africana. An approach using random amplified polymorphic DNA (RAPD) was employed in this study to examine genetic diversity and to evaluate hypotheses concerning the evolution of domesticated and wild annual species of Eleusine. Unlike previous molecular approaches, the RAPD study revealed genetic diversity in the crop. The pattern of genetic variation was loosely correlated to geographic distribution. The allotetraploid nature of the crop was confirmed and molecular markers that can possibly identify the other genomic donor were proposed. Genotypes of subspecies africana did not group closely with those of the crop but showed higher affinities to E. indica, reflecting the pattern of similarity revealed by the isozyme study. The multiple origin of subspecies africana could explain the discrepancy between the isozyme-RAPD evidence and previous information. The RAPD study showed the close genetic affinity of E. tristachya to the E. coracana--E. indica group and understood the distinctness of E. multiflora.

DNA biosensor combining single-wavelength colorimetry and a digital lock-in amplifier within a smartphone.

PubMed

Wu, Tzu-Heng; Chang, Chia-Chen; Vaillant, Julien; Bruyant, Aurélien; Lin, Chii-Wann

2016-11-15

Smartphone camera based gold nanoparticle colorimetry (SCB-AuNP colorimetry) has shown good potential for point-of-care applications. However, due to the use of a camera as a photo-detector, there are major limitations to this technique such as a low bit resolution (∼8 bits mainstream) and a low data acquisition rate. These issues have limited the ultimate sensitivity of smartphone based colorimetry as well as the possibility to integrate efficiently a more sensitive approach such as detection based on a lock-in amplifier (LIA). In this paper, we improve the metrological performance of the smartphone to overcome existing issues by adding the LIA capability to AuNP sensing. In this work, instead of using the camera as a photo-detector, the audio jack is used as a photo-detector reader and function generator for driving a laser diode in order to achieve a smartphone based digital lock-in amplifier AuNP colorimetric (SBLIA-AuNP colorimetry) system. A full investigation on the SBLIA design, parameters and performance is comprehensively provided. It is found that the SBLIA can reduce most of the noise and provides a detection noise-to-signal ratio down to -63 dB, which is much better than the -49 dB of the state-of-the-art SCB based method. A DNA detection experiment is demonstrated to reveal the efficacy of the proposed metrological method. The results are compared to UV-visible spectrometry, which is the gold standard for colorimetric measurement. Based on our results, the SBLIA-AuNP colorimetric system has a detection limit of 0.77 nM on short strand DNA detection, which is 5.7 times better than the 4.36 nM limit of a commercial UV-visible spectrometer. Judging from the results, we believe that the sensitive SBLIA would be further extended to other optical diagnostic tools in the near future.

Typing and Subtyping of 83 Clinical Isolates Purified from Surgically Implanted Silicone Feeding Tubes by Random Amplified Polymorphic DNA Amplification

PubMed Central

Dautle, Melanie P.; Ulrich, Ricky L.; Hughes, Thomas A.

2002-01-01

In this study, 83 clinical isolates purified from biofilms colonizing 18 silicone gastrostomy devices (12 “buttons” and six tubes converted to skin level devices) were selected for subtype characterization utilizing genetic analysis. The tubes, previously used for feeding, remained in place for 3 to 47 months (mean, 20.0 months) in children ranging in age from 6 months to 17 years. Classification of specific microbes using random amplified polymorphic DNA (RAPD) analysis revealed genetic similarities and differences among isolates belonging to the same genus. Both gram-positive and -negative bacteria were investigated, including 2 isolates of Bacillus brevis, 4 isolates of Bacillus licheniformis, 2 isolates of Bacillus pumilus, 3 isolates of Enterococcus durans, 19 isolates of Enterococcus faecalis, 8 isolates of Enterococcus faecium, 2 isolates of Enterococcus hirae, 7 isolates of Escherichia coli, 8 isolates of Lactobacillus plantarum, 19 isolates of Staphylococcus aureus, 2 isolates of Staphylococcus epidermidis, and 7 isolates of Staphylococcus saprophyticus. Amplified DNA fragments (amplicons) provided species-specific fingerprints for comparison by agarose gel electrophoresis. A total of 62 distinct RAPD types were categorized from the five genera studied. Typing analysis suggested cross acquisition of E. coli, E. faecalis, and S. aureus in three patient pairs. Genomic polymorphism detection proved efficient and reliable for classifying bacterial subtypes isolated from biofilms adhering to various portions of commonly employed enteral access tubes. PMID:11825951

Identification of the bacterial community of maple sap by using amplified ribosomal DNA (rDNA) restriction analysis and rDNA sequencing.

PubMed

Lagacé, L; Pitre, M; Jacques, M; Roy, D

2004-04-01

The bacterial community of maple sap was characterized by analysis of samples obtained at the taphole of maple trees for the 2001 and 2002 seasons. Among the 190 bacterial isolates, 32 groups were formed according to the similarity of the banding patterns obtained by amplified ribosomal DNA restriction analysis (ARDRA). A subset of representative isolates for each ARDRA group was identified by 16S rRNA gene fragment sequencing. Results showed a wide variety of organisms, with 22 different genera encountered. Pseudomonas and Ralstonia, of the gamma- and beta-Proteobacteria, respectively, were the most frequently encountered genera. Gram-positive bacteria were also observed, and Staphylococcus, Plantibacter, and Bacillus were the most highly represented genera. The sampling period corresponding to 50% of the cumulative sap flow percentage presented the greatest bacterial diversity according to its Shannon diversity index value (1.1). gamma-Proteobacteria were found to be dominant almost from the beginning of the season to the end. These results are providing interesting insights on maple sap microflora that will be useful for further investigation related to microbial contamination and quality of maple products and also for guiding new strategies on taphole contamination control.

Identification of the Bacterial Community of Maple Sap by Using Amplified Ribosomal DNA (rDNA) Restriction Analysis and rDNA Sequencing

PubMed Central

Lagacé, L.; Pitre, M.; Jacques, M.; Roy, D.

2004-01-01

The bacterial community of maple sap was characterized by analysis of samples obtained at the taphole of maple trees for the 2001 and 2002 seasons. Among the 190 bacterial isolates, 32 groups were formed according to the similarity of the banding patterns obtained by amplified ribosomal DNA restriction analysis (ARDRA). A subset of representative isolates for each ARDRA group was identified by 16S rRNA gene fragment sequencing. Results showed a wide variety of organisms, with 22 different genera encountered. Pseudomonas and Ralstonia, of the γ- and β-Proteobacteria, respectively, were the most frequently encountered genera. Gram-positive bacteria were also observed, and Staphylococcus, Plantibacter, and Bacillus were the most highly represented genera. The sampling period corresponding to 50% of the cumulative sap flow percentage presented the greatest bacterial diversity according to its Shannon diversity index value (1.1). γ-Proteobacteria were found to be dominant almost from the beginning of the season to the end. These results are providing interesting insights on maple sap microflora that will be useful for further investigation related to microbial contamination and quality of maple products and also for guiding new strategies on taphole contamination control. PMID:15066796

A new strategy for complete identification of sea buckthorn cultivars by using random amplified polymorphic DNA markers.

PubMed

Yang, G; Ding, J; Wu, L R; Duan, Y D; Li, A Y; Shan, J Y; Wu, Y X

2015-03-13

DNA fingerprinting is both a popular and important technique with several advantages in plant cultivar identification. However, this technique has not been used widely and efficiently in practical plant identification because the analysis and recording of data generated from fingerprinting and genotyping are tedious and difficult. We developed a novel approach known as a cultivar identification diagram (CID) strategy that uses DNA markers to separate plant individuals in a more efficient, practical, and referable manner. A CID was manually constructed and a polymorphic marker was generated from each polymerase chain reaction for sample separation. In this study, 67 important sea buckthorn cultivars cultivated in China were successfully separated with random amplified polymorphic DNA markers using the CID analysis strategy, with only seven 11-nucleotide primers employed. The utilization of the CID of these 67 sea buckthorn cultivars was verified by identifying 2 randomly chosen groups of cultivars among the 67 cultivars. The main advantages of this identification strategy include fewer primers used and separation of all cultivars using the corresponding primers. This sea buckthorn CID was able to separate any sea buckthorn cultivars among the 67 studied, which is useful for sea buckthorn cultivar identification, cultivar-right-protection, and for the sea buckthorn nursery industry in China.

[Assessment of two DNA extraction methods to amplify the pneumolysin gene (PLY) from blood culture samples of Streptococcus pneumoniae].

PubMed

Hernández, Carolina; Durán, Claudia; Ulloa, María Teresa; Prado, Valeria

2004-05-01

Streptococcus pneumoniae is a common etiologic agent of invasive respiratory infections among children under 5 years of age and older adults. Isolation rates of S. pneumoniae by traditional culture techniques are low. To study the sensitivity and specificity of two different DNA extraction methods to amplify the ply gene, applied to three different types of blood culture broths, experimentally inoculated with S. pneumoniae. DNA was extracted from the cultures using an organic method or a technique that consists in dilution, washing with NaOH and concentration of the sample. This was followed by PCR amplification of a 355 pb fragment of the pneumolysin gene (ply). The organic DNA extraction method inhibited the PCR reaction at all concentrations studied (0.6 to 10(6) colony forming units/mL). Using the NaOH extraction, ply gene amplification was positive in all three blood culture broths, but only at concentrations of 10(3) colony forming units/mL, or higher. Using the same DNA extraction method, PCR was negative when the broths were inoculated with seven other related bacterial species, which results in a 100% specificity. Detection of S. pneumoniae by amplification of ply gene from blood cultures using the protocol of NaOH for DNA extraction is specific and provides results in a short lapse. However, the diagnostic sensitivity is not optimal, which limits its clinical use.

Using long ssDNA polynucleotides to amplify STRs loci in degraded DNA samples

PubMed Central

Pérez Santángelo, Agustín; Corti Bielsa, Rodrigo M.; Sala, Andrea; Ginart, Santiago; Corach, Daniel

2017-01-01

Obtaining informative short tandem repeat (STR) profiles from degraded DNA samples is a challenging task usually undermined by locus or allele dropouts and peak-high imbalances observed in capillary electrophoresis (CE) electropherograms, especially for those markers with large amplicon sizes. We hereby show that the current STR assays may be greatly improved for the detection of genetic markers in degraded DNA samples by using long single stranded DNA polynucleotides (ssDNA polynucleotides) as surrogates for PCR primers. These long primers allow a closer annealing to the repeat sequences, thereby reducing the length of the template required for the amplification in fragmented DNA samples, while at the same time rendering amplicons of larger sizes suitable for multiplex assays. We also demonstrate that the annealing of long ssDNA polynucleotides does not need to be fully complementary in the 5’ region of the primers, thus allowing for the design of practically any long primer sequence for developing new multiplex assays. Furthermore, genotyping of intact DNA samples could also benefit from utilizing long primers since their close annealing to the target STR sequences may overcome wrong profiling generated by insertions/deletions present between the STR region and the annealing site of the primers. Additionally, long ssDNA polynucleotides might be utilized in multiplex PCR assays for other types of degraded or fragmented DNA, e.g. circulating, cell-free DNA (ccfDNA). PMID:29099837

ANIMAL DNA IN PCR REAGENTS PLAGUES ANCIENT DNA RESEARCH

EPA Science Inventory

Ancient DNA analysis is becoming widespread. These studies use polymerase chain reaction (PCR) to amplify minute quantities of heavily damaged template. Unusual steps are taken to achieve the sensitivity necessary to detect ancient DNA, including high-cycle PCR amplification targ...

Comparison of Haemophilus parasuis reference strains and field isolates by using random amplified polymorphic DNA and protein profiles

PubMed Central

2012-01-01

Background Haemophilus parasuis is the causative agent of Glässer’s disease and is a pathogen of swine in high-health status herds. Reports on serotyping of field strains from outbreaks describe that approximately 30% of them are nontypeable and therefore cannot be traced. Molecular typing methods have been used as alternatives to serotyping. This study was done to compare random amplified polymorphic DNA (RAPD) profiles and whole cell protein (WCP) lysate profiles as methods for distinguishing H. parasuis reference strains and field isolates. Results The DNA and WCP lysate profiles of 15 reference strains and 31 field isolates of H. parasuis were analyzed using the Dice and neighbor joining algorithms. The results revealed unique and reproducible DNA and protein profiles among the reference strains and field isolates studied. Simpson’s index of diversity showed significant discrimination between isolates when three 10mer primers were combined for the RAPD method and also when both the RAPD and WCP lysate typing methods were combined. Conclusions The RAPD profiles seen among the reference strains and field isolates did not appear to change over time which may reflect a lack of DNA mutations in the genes of the samples. The recent field isolates had different WCP lysate profiles than the reference strains, possibly because the number of passages of the type strains may affect their protein expression. PMID:22703293

Evaluation of the efficacy of constitutional array-based comparative genomic hybridization in the diagnosis of aneuploidy using genomic and amplified DNA.

PubMed

Tan, Niap H; Palmer, Rodger; Wang, Rubin

2010-02-01

Array-based comparative genomic hybridization (array CGH) is a new molecular technique that has the potential to revolutionize cytogenetics. However, use of high resolution array CGH in the clinical setting is plagued by the problem of widespread copy number variations (CNV) in the human genome. Constitutional microarray, containing only clones that interrogate regions of known constitutional syndromes, may circumvent the dilemma of detecting CNV of unknown clinical significance. The present study investigated the efficacy of constitutional microarray in the diagnosis of trisomy. Test samples included genomic DNA from trisomic cell lines, amplification products of 50 ng of genomic DNA and whole genome amplification products of single cells. DNA amplification was achieved by means of multiple displacement amplification (MDA) over 16 h. The trisomic and sex chromosomes copy number imbalances in the genomic DNA were correctly identified by the constitutional microarrays. However, there was a failure to detect the trisomy in the amplification products of 50 ng of genomic DNA and whole genome amplification products of single cells. Using carefully selected clones, Spectral Genomics constitutional microarray was able to detect the chromosomal copy number imbalances in genomic DNA without the confounding effects of CNV. The diagnostic failure in amplified DNA samples could be attributed to the amplification process. The MDA duration of 16 h generated excessive amount of biases and shortening the duration might minimize the problem.

Genetic relatedness of artichoke (Cynara scolymus L.) hybrids using random amplified polymorphic DNA (RAPD) fingerprinting.

PubMed

Sharaf-Eldin, M A; Al-Tamimi, A; Alam, P; Elkholy, S F; Jordan, J R

2015-12-28

The artichoke (Cynara scolymus L.) is an important food and medicinal crop that is cultivated in Mediterranean countries. Morphological characteristics, such as head shape and diameter, leaf shape, and bract shape, are mainly affected by environmental conditions. A molecular marker approach was used to analyze the degree of polymorphism between artichoke hybrid lines. The degree of genetic difference among three artichoke hybrids was evaluated using random amplified polymorphic DNA-PCR (RAPD-PCR). In this study, the DNA fingerprints of three artichoke lines (A13-010, A11-018, and A12-179) were generated, and a total of 10 decamer primers were applied for RAPD-PCR analyses. Polymorphism  (16.66 to 62.50%) was identified using eight arbitrary decamers and total genomic DNA extracted from the hybrids. Of the 59 loci detected, there were 25 polymorphic and 34 monomorphic loci. Jaccard's similarity index (JSI) ranged between 1.0 and 0.84. Based on the unweighted pair group method with arithmetic mean (UPGMA) similarity matrix and dendrogram, the results indicated that two hybrids (A13-010 and A11-018) were closely related to each other, and the A12-179 line showed more divergence. When identifying correct accessions, consideration of the genetic variation and genetic relationships among the genotypes are required. The RAPD-PCR fingerprinting of artichoke lines clearly showed that it is possible to analyze the RAPD patterns for correlation between genetic means and differences or resemblance between close accessions (A13-010 and A11- 018) at the genomic level.

Freshly excavated fossil bones are best for amplification of ancient DNA

PubMed Central

Pruvost, Mélanie; Schwarz, Reinhard; Correia, Virginia Bessa; Champlot, Sophie; Braguier, Séverine; Morel, Nicolas; Fernandez-Jalvo, Yolanda; Grange, Thierry; Geigl, Eva-Maria

2007-01-01

Despite the enormous potential of analyses of ancient DNA for phylogeographic studies of past populations, the impact these analyses, most of which are performed with fossil samples from natural history museum collections, has been limited to some extent by the inefficient recovery of ancient genetic material. Here we show that the standard storage conditions and/or treatments of fossil bones in these collections can be detrimental to DNA survival. Using a quantitative paleogenetic analysis of 247 herbivore fossil bones up to 50,000 years old and originating from 60 different archeological and paleontological contexts, we demonstrate that freshly excavated and nontreated unwashed bones contain six times more DNA and yield twice as many authentic DNA sequences as bones treated with standard procedures. This effect was even more pronounced with bones from one Neolithic site, where only freshly excavated bones yielded results. Finally, we compared the DNA content in the fossil bones of one animal, a ≈3,200-year-old aurochs, excavated in two separate seasons 57 years apart. Whereas the washed museum-stored fossil bones did not permit any DNA amplification, all recently excavated bones yielded authentic aurochs sequences. We established that during the 57 years when the aurochs bones were stored in a collection, at least as much amplifiable DNA was lost as during the previous 3,200 years of burial. This result calls for a revision of the postexcavation treatment of fossil bones to better preserve the genetic heritage of past life forms. PMID:17210911

An ultrasensitive label-free biosensor for assaying of sequence-specific DNA-binding protein based on amplifying fluorescent conjugated polymer.

PubMed

Liu, Xingfen; Ouyang, Lan; Cai, Xiaohui; Huang, Yanqin; Feng, Xiaomiao; Fan, Quli; Huang, Wei

2013-03-15

Sensitive, reliable, and simple detection of sequence-specific DNA-binding proteins (DBP) is of paramount importance in the area of proteomics, genomics, and biomedicine. We describe herein a novel fluorescent-amplified strategy for ultrasensitive, visual, quantitative, and "turn-on" detection of DBP. A Förster resonance energy transfer (FRET) assay utilizing a cationic conjugated polymer (CCP) and an intercalating dye was designed to detect a key transcription factor, nuclear factor-kappa B (NF-κB), the model target. A series of label-free DNA probes bearing one or two protein-binding sites (PBS) were used to identify the target protein specifically. The binding DBP protects the probe from digestion by exonuclease III, resulting in high efficient FRET due to the high affinity between the intercalating dye and duplex DNA, as well as strong electrostatic interactions between the CCP and DNA probe. By using label-free hairpin DNA or double-stranded DNA containing two PBS as probe, we could detect as low as 1 pg/μL of NF-κB in HeLa nuclear extracts, which is 10000-fold more sensitive than the previously reported methods. The approach also allows naked-eye detection by observing fluorescent color of solutions with the assistance of a hand-held UV lamp. Additionally, a less than 10% relative standard deviation was obtained, which offers a new platform for superior precision, low-cost, and simple detection of DBP. The features of our optical biosensor shows promising potential for early diagnosis of many diseases and high-throughput screening of new drugs targeted to DNA-binding proteins. Copyright © 2012 Elsevier B.V. All rights reserved.

Sensitive detection of porcine DNA in processed animal proteins using a TaqMan real-time PCR assay.

PubMed

Pegels, N; González, I; Fernández, S; García, T; Martín, R

2012-01-01

A TaqMan real-time PCR method was developed for specific detection of porcine-prohibited material in industrial feeds. The assay combines the use of a porcine-specific primer pair, which amplifies a 79 bp fragment of the mitochondrial (mt) 12 S rRNA gene, and a locked nucleic acid (LNA) TaqMan probe complementary to a target sequence lying between the porcine-specific primers. The nuclear 18 S rRNA gene system, yielding a 77 bp amplicon, was employed as a positive amplification control to monitor the total content of amplifiable DNA in the samples. The specificity of the porcine primers-probe system was verified against different animal and plant species, including mammals, birds and fish. The applicability of the real-time PCR protocol to detect the presence of porcine mt DNA in feeds was determined through the analysis of 190 industrial feeds (19 known reference and 171 blind samples) subjected to stringent processing treatments. The performance of the method allows qualitative and highly sensitive detection of short fragments from porcine DNA in all the industrial feeds declared to contain porcine material. Although the method has quantitative potential, the real quantitative capability of the assay is limited by the existing variability in terms of composition and processing conditions of the feeds, which affect the amount and quality of amplifiable DNA.

A 1-W, 30-ghz, CPW Amplifier for ACTS Small Terminal Uplink

NASA Technical Reports Server (NTRS)

Taub, Susan R.; Simons, Rainee N.

1992-01-01

The progress is described of the development of a 1 W, 30 GHz, coplanar waveguide (CPW) amplifier for the Advanced Communication Technology Satellite (ACTS)Small Terminal Uplink. The amplifier is based on Texas Instruments' monolithic microwave integrated circuit (MMIC) amplifiers; a three stage, low power amplifier, and a single stage, high power amplifier. The amplifiers have a power output of 190 mW and 0.710 W, gain of 23 and 4.2 dB, and efficiencies of 30.2 and 24 percent for the three stage and one stage amplifiers, respectively. The chips are to be combined via a CPW power divider/combiner circuit to yield the desired 1 W of output power.

[The use of multilocus sequence typing (MLST) and randomly amplified polymorphic DNA (RAPD) for the differentiation between strains of Burkholderia mallei].

PubMed

Antonov, V A; Altukhova, V V; Savchenko, S S; Zamaraev, V S; Iliukhin, V I; Alekseev, V V

2007-01-01

Burkholderia mallei is highly pathogenic microorganism for both humans and animals. In this work, the possibility of the use of the genotyping method for differentiation between strains of B. mallei was studied. A collection of 14 isolates of B. mallei was characterized using randomly amplified polymorphic DNA (RAPD) and multilocus sequence typing (MLST). RAPD was the best method used for detecting strain differences of B. mallei. It was suggested that this method would be an increasingly useful molecular epidemiological tool.

Sex Determination from Fragmented and Degenerated DNA by Amplified Product-Length Polymorphism Bidirectional SNP Analysis of Amelogenin and SRY Genes.

PubMed

Masuyama, Kotoka; Shojo, Hideki; Nakanishi, Hiroaki; Inokuchi, Shota; Adachi, Noboru

2017-01-01

Sex determination is important in archeology and anthropology for the study of past societies, cultures, and human activities. Sex determination is also one of the most important components of individual identification in criminal investigations. We developed a new method of sex determination by detecting a single-nucleotide polymorphism in the amelogenin gene using amplified product-length polymorphisms in combination with sex-determining region Y analysis. We particularly focused on the most common types of postmortem DNA damage in ancient and forensic samples: fragmentation and nucleotide modification resulting from deamination. Amplicon size was designed to be less than 60 bp to make the method more useful for analyzing degraded DNA samples. All DNA samples collected from eight Japanese individuals (four male, four female) were evaluated correctly using our method. The detection limit for accurate sex determination was determined to be 20 pg of DNA. We compared our new method with commercial short tandem repeat analysis kits using DNA samples artificially fragmented by ultraviolet irradiation. Our novel method was the most robust for highly fragmented DNA samples. To deal with allelic dropout resulting from deamination, we adopted "bidirectional analysis," which analyzed samples from both sense and antisense strands. This new method was applied to 14 Jomon individuals (3500-year-old bone samples) whose sex had been identified morphologically. We could correctly identify the sex of 11 out of 14 individuals. These results show that our method is reliable for the sex determination of highly degenerated samples.

Magnetic bead/capture DNA/glucose-loaded nanoliposomes for amplifying the glucometer signal in the rapid screening of hepatitis C virus RNA.

PubMed

Tu, Haijian; Lin, Kun; Lun, Yongzhi; Yu, Liuming

2018-06-01

A digital detection strategy based on a portable personal glucometer (PGM) was developed for the simple, rapid, and sensitive detection of hepatitis C virus (HCV) RNA, involving the release of glucose-loaded nanoliposomes due to coupling-site-specific cleavage by the endonuclease BamHI. The glucose-loaded nanoliposomes were synthesized using a reversed-phase evaporation method and provided an amplified signal at the PGM in the presence of HCV RNA. Initially, a 21-mer oligonucleotide complementary to HCV RNA was covalently conjugated to a magnetic bead through the amino group at the 5' end of the oligonucleotide, and then bound to a glucose-loaded liposome by typical carbodiimide coupling at its 3' end. In the presence of the target HCV RNA, the target hybridized with the oligonucleotide to form double-stranded DNA. The symmetrical duplex sequence 5'-GGATCC-3' between guanines was then catalytically cleaved by BamHI, which detached the glucose-loaded liposome from the magnetic bead. Following magnetic separation of the bead, the detached glucose-loaded liposome was lysed using Triton X-100 to release the glucose molecules within it, which were then detected as an amplified signal at the digital PGM. Under optimal conditions, the PGM signal increased with increasing HCV RNA, and displayed a strongly linear dependence on the level of HCV RNA for concentrations ranging from 10 pM to 1.0 μM. The detection limit (LOD) of the system was 1.9 pM. Good reproducibility and favorable specificity were achieved in the analysis of the target HCV RNA. Human serum samples containing HCV RNA were analyzed using this strategy, and the developed sensing platform was observed to yield satisfactory results based on a comparison with the corresponding results from a Cobas ® Amplicor HCV Test Analyzer. Graphical abstract A digital detection strategy utilizing a personal glucometer was developed for the detection of hepatitis C virus RNA. The strategy involved the use of the

Evaluation of different sources of DNA for use in genome wide studies and forensic application.

PubMed

Al Safar, Habiba S; Abidi, Fatima H; Khazanehdari, Kamal A; Dadour, Ian R; Tay, Guan K

2011-02-01

In the field of epidemiology, Genome-Wide Association Studies (GWAS) are commonly used to identify genetic predispositions of many human diseases. Large repositories housing biological specimens for clinical and genetic investigations have been established to store material and data for these studies. The logistics of specimen collection and sample storage can be onerous, and new strategies have to be explored. This study examines three different DNA sources (namely, degraded genomic DNA, amplified degraded genomic DNA and amplified extracted DNA from FTA card) for GWAS using the Illumina platform. No significant difference in call rate was detected between amplified degraded genomic DNA extracted from whole blood and amplified DNA retrieved from FTA™ cards. However, using unamplified-degraded genomic DNA reduced the call rate to a mean of 42.6% compared to amplified DNA extracted from FTA card (mean of 96.6%). This study establishes the utility of FTA™ cards as a viable storage matrix for cells from which DNA can be extracted to perform GWAS analysis.

Assessment of cellularity, genomic DNA yields, and technical platforms for BRAF mutational testing in thyroid fine-needle aspirate samples.

PubMed

Dyhdalo, Kathryn; Macnamara, Stephen; Brainard, Jennifer; Underwood, Dawn; Tubbs, Raymond; Yang, Bin

2014-02-01

BRAF mutation V600E (substitution Val600Glu) is a molecular signature for papillary thyroid carcinoma (PTC). Testing for BRAF mutation is clinically useful in providing prognostic prediction and facilitating accurate diagnosis of PTC in thyroid fine-needle aspirate (FNA) samples. This study assessed the correlation of cellularity with DNA yield and compared 2 technical platforms with different sensitivities in detection of BRAF mutation in cytologic specimens. Cellularity was evaluated based on groups of 10+ cells on a ThinPrep slide: 1+ (1-5 groups), 2+ (6-10 groups), 3+ (11-20 groups), and 4+ (> 20 groups). Genomic DNA was extracted from residual materials of thyroid FNAs after cytologic diagnosis. Approximately 49% of thyroid FNA samples had low cellularity (1-2+). DNA yield is proportionate with increased cellularity and increased nearly 4-fold from 1+ to 4+ cellularity in cytologic samples. When applied to BRAF mutational assay, using a cutoff of 6 groups of follicular cells with 10+ cells per group, 96.7% of cases yielded enough DNA for at least one testing for BRAF mutation. Five specimens (11.6%) with lower cellularity did not yield sufficient DNA for duplicate testing. Comparison of Sanger sequencing to allele-specific polymerase chain reaction methods shows the latter confers better sensitivity in detection of BRAF mutation, especially in limited cytologic specimens with a lower percentage of malignant cells. This study demonstrates that by using 6 groups of 10+ follicular cells as a cutoff, nearly 97% of thyroid FNA samples contain enough DNA for BRAF mutational assay. Careful selection of a molecular testing system with high sensitivity facilitates the successful conduction of molecular testing in limited cytologic specimens. Cancer (Cancer Cytopathol) 2014;122:114-22 © 2013 American Cancer Society. © 2013 American Cancer Society.

Sequence independent amplification of DNA

DOEpatents

Bohlander, S.K.

1998-03-24

The present invention is a rapid sequence-independent amplification procedure (SIA). Even minute amounts of DNA from various sources can be amplified independent of any sequence requirements of the DNA or any a priori knowledge of any sequence characteristics of the DNA to be amplified. This method allows, for example, the sequence independent amplification of microdissected chromosomal material and the reliable construction of high quality fluorescent in situ hybridization (FISH) probes from YACs or from other sources. These probes can be used to localize YACs on metaphase chromosomes but also--with high efficiency--in interphase nuclei. 25 figs.

Sequence independent amplification of DNA

DOEpatents

Bohlander, Stefan K.

1998-01-01

The present invention is a rapid sequence-independent amplification procedure (SIA). Even minute amounts of DNA from various sources can be amplified independent of any sequence requirements of the DNA or any a priori knowledge of any sequence characteristics of the DNA to be amplified. This method allows, for example the sequence independent amplification of microdissected chromosomal material and the reliable construction of high quality fluorescent in situ hybridization (FISH) probes from YACs or from other sources. These probes can be used to localize YACs on metaphase chromosomes but also--with high efficiency--in interphase nuclei.

An enzyme-free catalytic DNA circuit for amplified detection of aflatoxin B1 using gold nanoparticles as colorimetric indicators

NASA Astrophysics Data System (ADS)

Chen, Junhua; Wen, Junlin; Zhuang, Li; Zhou, Shungui

2016-05-01

An enzyme-free biosensor for the amplified detection of aflatoxin B1 has been constructed based on a catalytic DNA circuit. Three biotinylated hairpin DNA probes (H1, H2, and H3) were designed as the assembly components to construct the sensing system (triplex H1-H2-H3 product). Cascaded signal amplification capability was obtained through toehold-mediated strand displacement reactions to open the hairpins and recycle the trigger DNA. By the use of streptavidin-functionalized gold nanoparticles as the signal indicators, the colorimetric readout can be observed by the naked eye. In the presence of a target, the individual nanoparticles (red) aggregate into a cross-linked network of nanoparticles (blue) via biotin-streptavidin coupling. The colorimetric assay is ultrasensitive, enabling the visual detection of trace levels of aflatoxin B1 (AFB1) as low as 10 pM without instrumentation. The calculated limit of detection (LOD) is 2 pM in terms of 3 times standard deviation over the blank response. The sensor is robust and works even when challenged with complex sample matrices such as rice samples. Our sensing platform is simple and convenient in operation, requiring only the mixing of several solutions at room temperature to achieve visible and intuitive results, and holds great promise for the point-of-use monitoring of AFB1 in environmental and food samples.An enzyme-free biosensor for the amplified detection of aflatoxin B1 has been constructed based on a catalytic DNA circuit. Three biotinylated hairpin DNA probes (H1, H2, and H3) were designed as the assembly components to construct the sensing system (triplex H1-H2-H3 product). Cascaded signal amplification capability was obtained through toehold-mediated strand displacement reactions to open the hairpins and recycle the trigger DNA. By the use of streptavidin-functionalized gold nanoparticles as the signal indicators, the colorimetric readout can be observed by the naked eye. In the presence of a target, the

Effects of different preservation methods on inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) molecular markers in botanic samples.

PubMed

Wang, Xiaolong; Li, Lin; Zhao, Jiaxin; Li, Fangliang; Guo, Wei; Chen, Xia

2017-04-01

To evaluate the effects of different preservation methods (stored in a -20°C ice chest, preserved in liquid nitrogen and dried in silica gel) on inter simple sequence repeat (ISSR) or random amplified polymorphic DNA (RAPD) analyses in various botanical specimens (including broad-leaved plants, needle-leaved plants and succulent plants) for different times (three weeks and three years), we used a statistical analysis based on the number of bands, genetic index and cluster analysis. The results demonstrate that methods used to preserve samples can provide sufficient amounts of genomic DNA for ISSR and RAPD analyses; however, the effect of different preservation methods on these analyses vary significantly, and the preservation time has little effect on these analyses. Our results provide a reference for researchers to select the most suitable preservation method depending on their study subject for the analysis of molecular markers based on genomic DNA. Copyright © 2017 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.

Isolation and characterization of DNA from archaeological bone.

PubMed

Hagelberg, E; Clegg, J B

1991-04-22

DNA was extracted from human and animal bones recovered from archaeological sites and mitochondrial DNA sequences were amplified from the extracts using the polymerase chain reaction. Evidence is presented that the amplified sequences are authentic and do not represent contamination by extraneous DNA. The results show that significant amounts of genetic information can survive for long periods in bone, and have important implications for evolutionary genetics, anthropology and forensic science.

Single-Cell-Based Platform for Copy Number Variation Profiling through Digital Counting of Amplified Genomic DNA Fragments.

PubMed

Li, Chunmei; Yu, Zhilong; Fu, Yusi; Pang, Yuhong; Huang, Yanyi

2017-04-26

We develop a novel single-cell-based platform through digital counting of amplified genomic DNA fragments, named multifraction amplification (mfA), to detect the copy number variations (CNVs) in a single cell. Amplification is required to acquire genomic information from a single cell, while introducing unavoidable bias. Unlike prevalent methods that directly infer CNV profiles from the pattern of sequencing depth, our mfA platform denatures and separates the DNA molecules from a single cell into multiple fractions of a reaction mix before amplification. By examining the sequencing result of each fraction for a specific fragment and applying a segment-merge maximum likelihood algorithm to the calculation of copy number, we digitize the sequencing-depth-based CNV identification and thus provide a method that is less sensitive to the amplification bias. In this paper, we demonstrate a mfA platform through multiple displacement amplification (MDA) chemistry. When performing the mfA platform, the noise of MDA is reduced; therefore, the resolution of single-cell CNV identification can be improved to 100 kb. We can also determine the genomic region free of allelic drop-out with mfA platform, which is impossible for conventional single-cell amplification methods.

Josephson junction microwave amplifier in self-organized noise compression mode

PubMed Central

Lähteenmäki, Pasi; Vesterinen, Visa; Hassel, Juha; Seppä, Heikki; Hakonen, Pertti

2012-01-01

The fundamental noise limit of a phase-preserving amplifier at frequency is the standard quantum limit . In the microwave range, the best candidates have been amplifiers based on superconducting quantum interference devices (reaching the noise temperature at 700 MHz), and non-degenerate parametric amplifiers (reaching noise levels close to the quantum limit at 8 GHz). We introduce a new type of an amplifier based on the negative resistance of a selectively damped Josephson junction. Noise performance of our amplifier is limited by mixing of quantum noise from Josephson oscillation regime down to the signal frequency. Measurements yield nearly quantum-limited operation, at 2.8 GHz, owing to self-organization of the working point. Simulations describe the characteristics of our device well and indicate potential for wide bandwidth operation. PMID:22355788

Genetic Diversity of Pinus Roxburghii Sarg. Collected from Different Himalayan Regions of India Assessed by Random Amplified Polymorphic DNA Analysis

PubMed Central

Sinha, Dwaipayan; Singh, Jyotsna; Tandon, P. K.; Kakkar, Poonam

2013-01-01

Present study was aimed at molecular genetic fingerprint profile of 15 genotypes of three populations of Pinus roxburghii Sarg. from Himalayan regions of India using random amplified polymorphic DNA (RAPD) based markers. Needles of Pinus roxburghii Sarg. were collected from Dharamshala, Himachal Pradesh (HP), Nainital, Uttarakhand (UK) and Darjeeling, West Bengal (WB) regions of India. The samples were subjected to DNA extraction and RAPD analysis using oligonucleotide purification cartridge (OPC) primers. Out of 15 primers tested, nine primers gave scorable bands. Altogether 48 bands were obtained, out of which 43 were found to be polymorphic. Number of amplified fragments with RAPD primers ranged from four to eight with the size of amplicon ranging from 500 to 7,000bp. Investigation of natural diversity at intraspecies level was performed with 15 genotypes. Forty-eight amplification products were scored by RAPD and showed 89.58% polymorphism with a mean intrapopulation genetic diversity (Hpop) of 0.2754. A significant inter- and intrapopulation diversity was observed, with the percentage of polymorphic loci (Pp) ranging from 50.09 to 70.83%, Shannon's information index (I) from 0.3262 to 0.4689 and Nei's gene diversity (h) from 0.2032 to 0.3335 with mean Nei's gene diversity 0.377 and the overall estimate of gene flow being (Nm) 1.3555. Unweighted pair-group method with arithmetic average (UPGMA) analysis based Dendrogram showed single cluster. The variation amongst the samples of the three ecological regions can be attributed to varied climatic conditions and may help in conservation/future cultivation of these species. PMID:24403729

Sex Determination from Fragmented and Degenerated DNA by Amplified Product-Length Polymorphism Bidirectional SNP Analysis of Amelogenin and SRY Genes

PubMed Central

Masuyama, Kotoka; Shojo, Hideki; Nakanishi, Hiroaki; Inokuchi, Shota; Adachi, Noboru

2017-01-01

Sex determination is important in archeology and anthropology for the study of past societies, cultures, and human activities. Sex determination is also one of the most important components of individual identification in criminal investigations. We developed a new method of sex determination by detecting a single-nucleotide polymorphism in the amelogenin gene using amplified product-length polymorphisms in combination with sex-determining region Y analysis. We particularly focused on the most common types of postmortem DNA damage in ancient and forensic samples: fragmentation and nucleotide modification resulting from deamination. Amplicon size was designed to be less than 60 bp to make the method more useful for analyzing degraded DNA samples. All DNA samples collected from eight Japanese individuals (four male, four female) were evaluated correctly using our method. The detection limit for accurate sex determination was determined to be 20 pg of DNA. We compared our new method with commercial short tandem repeat analysis kits using DNA samples artificially fragmented by ultraviolet irradiation. Our novel method was the most robust for highly fragmented DNA samples. To deal with allelic dropout resulting from deamination, we adopted “bidirectional analysis,” which analyzed samples from both sense and antisense strands. This new method was applied to 14 Jomon individuals (3500-year-old bone samples) whose sex had been identified morphologically. We could correctly identify the sex of 11 out of 14 individuals. These results show that our method is reliable for the sex determination of highly degenerated samples. PMID:28052096

Linear nicking endonuclease-mediated strand-displacement DNA amplification.

PubMed

Joneja, Aric; Huang, Xiaohua

2011-07-01

We describe a method for linear isothermal DNA amplification using nicking endonuclease-mediated strand displacement by a DNA polymerase. The nicking of one strand of a DNA target by the endonuclease produces a primer for the polymerase to initiate synthesis. As the polymerization proceeds, the downstream strand is displaced into a single-stranded form while the nicking site is also regenerated. The combined continuous repetitive action of nicking by the endonuclease and strand-displacement synthesis by the polymerase results in linear amplification of one strand of the DNA molecule. We demonstrate that DNA templates up to 5000 nucleotides can be linearly amplified using a nicking endonuclease with 7-bp recognition sequence and Sequenase version 2.0 in the presence of single-stranded DNA binding proteins. We also show that a mixture of three templates of 500, 1000, and 5000 nucleotides in length is linearly amplified with the original molar ratios of the templates preserved. Moreover, we demonstrate that a complex library of hydrodynamically sheared genomic DNA from bacteriophage lambda can be amplified linearly. Copyright © 2011 Elsevier Inc. All rights reserved.

Linear nicking endonuclease-mediated strand displacement DNA amplification

PubMed Central

Joneja, Aric; Huang, Xiaohua

2011-01-01

We describe a method for linear isothermal DNA amplification using nicking endonuclease-mediated strand displacement by a DNA polymerase. The nicking of one strand of a DNA target by the endonuclease produces a primer for the polymerase to initiate synthesis. As the polymerization proceeds, the downstream strand is displaced into a single-stranded form while the nicking site is also regenerated. The combined continuous repetitive action of nicking by the endonuclease and strand displacement synthesis by the polymerase results in linear amplification of one strand of the DNA molecule. We demonstrate that DNA templates up to five thousand nucleotides can be linearly amplified using a nicking endonuclease with seven base-pair recognition sequence and Sequenase version 2.0 in the presence of single-stranded DNA binding proteins. We also show that a mixture of three templates of 500, 1000, and 5000 nucleotides in length are linearly amplified with the original molar ratios of the templates preserved. Moreover, we demonstrate that a complex library of hydrodynamically sheared genomic DNA from bacteriophage lambda can be amplified linearly. PMID:21342654

End Joining-Mediated Gene Expression in Mammalian Cells Using PCR-Amplified DNA Constructs that Contain Terminator in Front of Promoter.

PubMed

Nakamura, Mikiko; Suzuki, Ayako; Akada, Junko; Tomiyoshi, Keisuke; Hoshida, Hisashi; Akada, Rinji

2015-12-01

Mammalian gene expression constructs are generally prepared in a plasmid vector, in which a promoter and terminator are located upstream and downstream of a protein-coding sequence, respectively. In this study, we found that front terminator constructs-DNA constructs containing a terminator upstream of a promoter rather than downstream of a coding region-could sufficiently express proteins as a result of end joining of the introduced DNA fragment. By taking advantage of front terminator constructs, FLAG substitutions, and deletions were generated using mutagenesis primers to identify amino acids specifically recognized by commercial FLAG antibodies. A minimal epitope sequence for polyclonal FLAG antibody recognition was also identified. In addition, we analyzed the sequence of a C-terminal Ser-Lys-Leu peroxisome localization signal, and identified the key residues necessary for peroxisome targeting. Moreover, front terminator constructs of hepatitis B surface antigen were used for deletion analysis, leading to the identification of regions required for the particle formation. Collectively, these results indicate that front terminator constructs allow for easy manipulations of C-terminal protein-coding sequences, and suggest that direct gene expression with PCR-amplified DNA is useful for high-throughput protein analysis in mammalian cells.

DNA extraction from coral reef sediment bacteria for the polymerase chain reaction.

PubMed

Guthrie, J N; Moriarty, D J; Blackall, L L

2000-12-15

A rapid and effective method for the direct extraction of high molecular weight amplifiable DNA from two coral reef sediments was developed. DNA was amplified by the polymerase chain reaction (PCR) using 16S rDNA specific primers. The amplicons were digested with HaeIII, HinP1I and MspI and separated using polyacrylamide gel electrophoresis and silver staining. The resulting amplified ribosomal DNA restriction analysis (ARDRA) patterns were used as a fingerprint to discern differences between the coral reef sediment samples. Results indicated that ARDRA is an effective method for determining differences within the bacterial community amongst different environmental samples.

Comparison of six methods for the recovery of PCR-compatible microbial DNA from an agricultural biogas plant.

PubMed

Stagnati, L; Soffritti, G; Lanubile, A; Busconi, M

2017-05-01

Six different commercial methods were compared to evaluate their efficiency in recovering high quantity/quality PCR compatible microbial DNA from an agricultural biogas plant. Within the last two decades, biogas plants have been developed to produce energy from organic wastes and from devoted biomass. The complex biotransformations are performed by a diverse consortium of microorganisms that is an important reserve of genes and enzymatic activities with a huge range of applications in various commercial fields. In this respect, the ability to isolate DNA from a complex matrix is of high importance. Important parameters of the recovered DNA are good yield, purity, and quality. The methods examined showed considerable differences about quantity and quality of the recovered DNA and, usually, it was observed that a higher amount was accompanied by more degradation. DNA purity was determined by its PCR amplificability. Only two methods were able to provide DNA pure enough to be directly amplified. For the rest of the methods, a few intermediate steps such as dilution and/or the addition of polyvinylpyrrolidone were necessary to remove the inhibitors present and to amplify the DNA. Real-time PCR analysis evidenced that, as expected, prokaryotic DNA was much more abundant than eukaryotic DNA, but some methods were more suited to recovering prokaryotic or eukaryotic DNA. The digestion analysis of ribosomal DNA amplicons confirmed the influence of the methods on the final output, allowing the recovery of only a fraction of the present species as determined by sequencing a small prokaryotic and eukaryotic ribosomal library.

Authentication of medicinal herbs using PCR-amplified ITS2 with specific primers.

PubMed

Chiou, Shu-Jiau; Yen, Jui-Hung; Fang, Cheng-Li; Chen, Hui-Ling; Lin, Tsai-Yun

2007-10-01

Different parts of medicinal herbs have long been used as traditional Chinese drugs for treating many diseases, whereas materials of similar morphology and chemical fingerprints are often misidentified. Analyses of sequence variations in the nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) have become a valid method for authentication of medicinal herbs at the intergenic and interspecific levels. DNA extracted from processed materials is usually severely degraded or contaminated by microorganisms, thus generates no or unexpected PCR products. The goal of this study is to apply the ITS fragments selectively amplified with two designed primer sets for efficient and precise authentication of medicinal herbs. The designed primers led to an accurate PCR product of the specific region in ITS2, which was confirmed with DNA extracted from 55 processed medicinal herbs belonging to 48 families. Moreover, the selectively amplified ITS2 authenticated five sets of easily confusable Chinese herbal materials. The designed primers were proven to be suitable for a broad application in the authentication of herbal materials.

Mitochondrial DNA as a non-invasive biomarker: Accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malik, Afshan N., E-mail: afshan.malik@kcl.ac.uk; Shahni, Rojeen; Rodriguez-de-Ledesma, Ana

2011-08-19

Highlights: {yields} Mitochondrial dysfunction is central to many diseases of oxidative stress. {yields} 95% of the mitochondrial genome is duplicated in the nuclear genome. {yields} Dilution of untreated genomic DNA leads to dilution bias. {yields} Unique primers and template pretreatment are needed to accurately measure mitochondrial DNA content. -- Abstract: Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that themore » methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as {beta}-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a 'dilution bias' when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples.« less

PULSE AMPLIFIER

DOEpatents

Johnstone, C.W.

1958-06-17

The improvement of pulse amplifiers used with scintillation detectors is described. The pulse amplifier circuit has the advantage of reducing the harmful effects of overloading cause by large signal inputs. In general the pulse amplifier circuit comprises two amplifier tubes with the input pulses applied to one amplifier grid and coupled to the second amplifier tube through a common cathode load. The output of the second amplifier is coupled from the plate circuit to a cathode follower tube grid and a diode tube in connected from grid to cathode of the cathode follower tube. Degenerative feedback is provided in the second amplifier by coupling a signal from the cathode follower cathode to the second amplifier grid. The circuit proqides moderate gain stability, and overload protection for subsequent pulse circuits.

Genetic analysis of Apuleia leiocarpa as revealed by random amplified polymorphic DNA markers: prospects for population genetic studies.

PubMed

Lencina, K H; Konzen, E R; Tsai, S M; Bisognin, D A

2016-12-19

Apuleia leiocarpa (Vogel) J.F. MacBride is a hardwood species native to South America, which is at serious risk of extinction. Therefore, it is of prime importance to examine the genetic diversity of this species, information required for developing conservation, sustainable management, and breeding strategies. Although scarcely used in recent years, random amplified polymorphic DNA markers are useful resources for the analysis of genetic diversity and structure of tree species. This study represents the first genetic analysis based on DNA markers in A. leiocarpa that aimed to investigate the levels of polymorphism and to select markers for the precise characterization of its genetic structure. We adapted the original DNA extraction protocol based on cetyltrimethyl ammonium bromide, and describe a simple procedure that can be used to obtain high-quality samples from leaf tissues of this tree. Eighteen primers were selected, revealing 92 bands, from which 75 were polymorphic and 61 were sufficient to represent the overall genetic structure of the population without compromising the precision of the analysis. Some fragments were conserved among individuals, which can be sequenced and used to analyze nucleotide diversity parameters through a wider set of A. leiocarpa individuals and populations. The individuals were separated into 11 distinct groups with variable levels of genetic diversity, which is important for selecting desirable genotypes and for the development of a conservation and sustainable management program. Our results are of prime importance for further investigations concerning the genetic characterization of this important, but vulnerable species.

Salmonella enteritidis outbreak in Thailand: study by random amplified polymorphic DNA (RAPD) analysis.

PubMed

Kantama, L; Jayanetra, P

1996-03-01

An outbreak of Salmonella enteritidis in Thailand was reported in 1990. The majority of isolates were found in chicken and human throughout the country. The continuation of a high rate of spreading which is presently continuing prompted us to investigate possible clonal involvement in the outbreak. One hundred and twenty five isolates of S. enteritidis which were isolated between 1990-1993 were clonally identified by the technique of Random Amplified Polymorphic DNA (RAPD) analysis. Eight profiles were found indicating the presence of 8 clones, designated no. 1-8. The predominant clone was profile no. 4 which was encountered in 93.6% of tested isolates while the rest of the profile comprised only 0.8-1.6%. The predominant clone was distributed mainly in isolates from chickens and humans which is suggestive that the profile no. 4 is the major clone involved in this outbreak and that chickens were the source of S. enteritidis infection. The information from the Microbiology Laboratory at Ramathibodi Hospital revealed that nearly 40% of S. enteritidis were isolated from blood specimens. This may reflect the invasiveness of S. enteritidis in Thailand. We concluded that the outbreak involved the single clone, RAPD profile no. 4 which may disperse dominantly during the epidemic.

Forensic DNA Profiling and Database

PubMed Central

Panneerchelvam, S.; Norazmi, M.N.

2003-01-01

The incredible power of DNA technology as an identification tool had brought a tremendous change in crimnal justice . DNA data base is an information resource for the forensic DNA typing community with details on commonly used short tandem repeat (STR) DNA markers. This article discusses the essential steps in compilation of COmbined DNA Index System (CODIS) on validated polymerase chain amplified STRs and their use in crime detection. PMID:23386793

Experimental design studies and flow visualization of proportional laminar-flow fluidic amplifiers

NASA Technical Reports Server (NTRS)

Hellbaum, R. F.; Mcdermon, J. N.

1977-01-01

The effects of certain parameter variations on the performance characteristics of laminar, proportional, jet-deflection fluidic amplifiers were studied. The matching and staging of amplifiers to obtain high pressure gain was included, but dynamic effects were not. The parameter variations considered were aspect ratio, setback, control length, splitter distance, receiver-duct width, width of center-vent duct, and bias pressure. Usable pressure gains of 19 per stage were achieved, and 5 amplifier stages were integrated to yield an overall pressure gain of 2,000,000.

Determination of DNA methylation associated with Acer rubrum (red maple) adaptation to metals: analysis of global DNA modifications and methylation-sensitive amplified polymorphism.

PubMed

Kim, Nam-Soo; Im, Min-Ji; Nkongolo, Kabwe

2016-08-01

Red maple (Acer rubum), a common deciduous tree species in Northern Ontario, has shown resistance to soil metal contamination. Previous reports have indicated that this plant does not accumulate metals in its tissue. However, low level of nickel and copper corresponding to the bioavailable levels in contaminated soils in Northern Ontario causes severe physiological damages. No differentiation between metal-contaminated and uncontaminated populations has been reported based on genetic analyses. The main objective of this study was to assess whether DNA methylation is involved in A. rubrum adaptation to soil metal contamination. Global cytosine and methylation-sensitive amplified polymorphism (MSAP) analyses were carried out in A. rubrum populations from metal-contaminated and uncontaminated sites. The global modified cytosine ratios in genomic DNA revealed a significant decrease in cytosine methylation in genotypes from a metal-contaminated site compared to uncontaminated populations. Other genotypes from a different metal-contaminated site within the same region appear to be recalcitrant to metal-induced DNA alterations even ≥30 years of tree life exposure to nickel and copper. MSAP analysis showed a high level of polymorphisms in both uncontaminated (77%) and metal-contaminated (72%) populations. Overall, 205 CCGG loci were identified in which 127 were methylated in either outer or inner cytosine. No differentiation among populations was established based on several genetic parameters tested. The variations for nonmethylated and methylated loci were compared by analysis of molecular variance (AMOVA). For methylated loci, molecular variance among and within populations was 1.5% and 13.2%, respectively. These values were low (0.6% for among populations and 5.8% for within populations) for unmethylated loci. Metal contamination is seen to affect methylation of cytosine residues in CCGG motifs in the A. rubrum populations that were analyzed.

Comparing interfertility data with random amplified microsatellites DNA (RAMS) studies in Ganoderma Karst. Taxonomy.

PubMed

Nudin, Nur Fatihah Hasan; S, Siddiquee

2012-03-01

The taxonomy of the causal pathogen of basal stem rot of oil palms, Ganoderma is somewhat problematic at present. In order to determine the genetic distance relationship between G. boninense isolates and non-boninense isolates, a random amplified microsatellites DNA (RAMS) technique was carried out. The result was then compared with interfertility data of G. boninense that had been determined in previous mating studies to confirm the species of G. boninense. Dendrogram from cluster analysis based on UPGMA of RAMS data showed that two major clusters, I and II which separated at a genetic distance of 0.7935 were generated. Cluster I consisted of all the biological species G. boninense isolates namely CNLB, GSDK 3, PER 71, WD 814, GBL 3, GBL 6, OC, GH 02, 170 SL and 348781 while all non-boninense isolates namely G. ASAM, WRR, TFRI 129, G. RES, GJ, and CNLM were grouped together in cluster II. Although the RAMS markers showed polymorphisms in all the isolates tested, the results obtained were in agreement with the interfertility data. Therefore, the RAMS data could support the interfertility data for the identification of Ganoderma isolates.

Segmented amplifier configurations for laser amplifier

DOEpatents

Hagen, Wilhelm F.

1979-01-01

An amplifier system for high power lasers, the system comprising a compact array of segments which (1) preserves high, large signal gain with improved pumping efficiency and (2) allows the total amplifier length to be shortened by as much as one order of magnitude. The system uses a three dimensional array of segments, with the plane of each segment being oriented at substantially the amplifier medium Brewster angle relative to the incident laser beam and with one or more linear arrays of flashlamps positioned between adjacent rows of amplifier segments, with the plane of the linear array of flashlamps being substantially parallel to the beam propagation direction.

Measurement of locus copy number by hybridisation with amplifiable probes

PubMed Central

Armour, John A. L.; Sismani, Carolina; Patsalis, Philippos C.; Cross, Gareth

2000-01-01

Despite its fundamental importance in genome analysis, it is only recently that systematic approaches have been developed to assess copy number at specific genetic loci, or to examine genomic DNA for submicroscopic deletions of unknown location. In this report we show that short probes can be recovered and amplified quantitatively following hybridisation to genomic DNA. This simple observation forms the basis of a new approach to determining locus copy number in complex genomes. The power and specificity of multiplex amplifiable probe hybridisation is demonstrated by the simultaneous assessment of copy number at a set of 40 human loci, including detection of deletions causing Duchenne muscular dystrophy and Prader–Willi/Angelman syndromes. Assembly of other probe sets will allow novel, technically simple approaches to a wide variety of genetic analyses, including the potential for extension to high resolution genome-wide screens for deletions and amplifications. PMID:10606661

Measurement of locus copy number by hybridisation with amplifiable probes.

PubMed

Armour, J A; Sismani, C; Patsalis, P C; Cross, G

2000-01-15

Despite its fundamental importance in genome analysis, it is only recently that systematic approaches have been developed to assess copy number at specific genetic loci, or to examine genomic DNA for submicro-scopic deletions of unknown location. In this report we show that short probes can be recovered and amplified quantitatively following hybridisation to genomic DNA. This simple observation forms the basis of a new approach to determining locus copy number in complex genomes. The power and specificity of multiplex amplifiable probe hybridisation is demonstrated by the simultaneous assessment of copy number at a set of 40 human loci, including detection of deletions causing Duchenne muscular dystrophy and Prader-Willi/Angelman syndromes. Assembly of other probe sets will allow novel, technically simple approaches to a wide variety of genetic analyses, including the potential for extension to high resolution genome-wide screens for deletions and amplifications.

MAMMALIAN DNA IN PCR REAGENTS

EPA Science Inventory

Ancient DNA analysis is becoming widespread. These studies use polymerase chain reaction (PCR) to amplify minute quantities of heavily damaged template. Unusual steps are taken to achieve the sensitivity necessary to detect ancient DNA, including high- cycle PCR amplification t...

Length and sequence heterogeneity in 5S rDNA of Populus deltoides.

PubMed

Negi, Madan S; Rajagopal, Jyothi; Chauhan, Neeti; Cronn, Richard; Lakshmikumaran, Malathi

2002-12-01

The 5S rRNA genes and their associated non-transcribed spacer (NTS) regions are present as repeat units arranged in tandem arrays in plant genomes. Length heterogeneity in 5S rDNA repeats was previously identified in Populus deltoides and was also observed in the present study. Primers were designed to amplify the 5S rDNA NTS variants from the P. deltoides genome. The PCR-amplified products from the two accessions of P. deltoides (G3 and G48) suggested the presence of length heterogeneity of 5S rDNA units within and among accessions, and the size of the spacers ranged from 385 to 434 bp. Sequence analysis of the non-transcribed spacer (NTS) revealed two distinct classes of 5S rDNA within both accessions: class 1, which contained GAA trinucleotide microsatellite repeats, and class 2, which lacked the repeats. The class 1 spacer shows length variation owing to the microsatellite, with two clones exhibiting 10 GAA repeat units and one clone exhibiting 16 such repeat units. However, distance analysis shows that class 1 spacer sequences are highly similar inter se, yielding nucleotide diversity (pi) estimates that are less than 0.15% of those obtained for class 2 spacers (pi = 0.0183 vs. 0.1433, respectively). The presence of microsatellite in the NTS region leading to variation in spacer length is reported and discussed for the first time in P. deltoides.

Replication and Transcription of Eukaryotic DNA in Esherichia coli

PubMed Central

Morrow, John F.; Cohen, Stanley N.; Chang, Annie C. Y.; Boyer, Herbert W.; Goodman, Howard M.; Helling, Robert B.

1974-01-01

Fragments of amplified Xenopus laevis DNA, coding for 18S and 28S ribosomal RNA and generated by EcoRI restriction endonuclease, have been linked in vitro to the bacterial plasmid pSC101; and the recombinant molecular species have been introduced into E. coli by transformation. These recombinant plasmids, containing both eukaryotic and prokaryotic DNA, replicate stably in E. coli. RNA isolated from E. coli minicells harboring the plasmids hybridizes to amplified X. laevis rDNA. Images PMID:4600264

Diversity, molecular phylogeny and fingerprint profiles of airborne Aspergillus species using random amplified polymorphic DNA.

PubMed

Kermani, Firoozeh; Shams-Ghahfarokhi, Masoomeh; Gholami-Shabani, Mohammadhassan; Razzaghi-Abyaneh, Mehdi

2016-06-01

In the present study, diversity and phylogenetic relationship of Aspergillus species isolated from Tehran air was studied using random amplified polymorphic DNA (RAPD)-polymerase chain reaction (RAPD-PCR). Thirty-eight Aspergillus isolates belonging to 12 species i.e. A. niger (28.94 %, 11 isolates), A. flavus (18.42 %, 7 isolates), A. tubingensis (13.15 %, 5 isolates), A. japonicus (10.52 %, 4 isolates), A. ochraceus (10.52 %, 4 isolates), and 2.63 %, 1 isolate from each A. nidulans, A. amstelodami, A. oryzae, A. terreus, A. versicolor, A. flavipes and A. fumigatus were obtained by settle plate method which they were distributed in 18 out of 22 sampling sites examined. Fungal DNA was extracted from cultured mycelia of all Aspergillus isolates on Sabouraud Dextrose Agar and used for amplification of gene fragments in RAPD-PCR using 11 primers. RAPD-PCR data was analyzed using UPGMA software. Resulting dendrogram of combined selected primers including PM1, OPW-04, OPW-05, P160, P54, P10 and OPA14 indicated the distribution of 12 Aspergillus species in 8 major clusters. The similarity coefficient of all 38 Aspergillus isolates ranged from 0.02 to 0.40 indicating a wide degree of similarities and differences within and between species. Taken together, our results showed that various Aspergillus species including some important human pathogenic ones exist in the outdoor air of Tehran by different extents in distribution and diversity and suggested inter- and intra-species genetic diversity among Aspergillus species by RAPD-PCR as a rapid, sensitive and reproducible method.

Successful isolation and PCR amplification of DNA from National Institute of Standards and Technology herbal dietary supplement standard reference material powders and extracts.

PubMed

Cimino, Matthew T

2010-03-01

Twenty-four herbal dietary supplement powder and extract reference standards provided by the National Institute of Standards and Technology (NIST) were investigated using three different commercially available DNA extraction kits to evaluate DNA availability for downstream nucleotide-based applications. The material included samples of Camellia, Citrus, Ephedra, Ginkgo, Hypericum, Serenoa, And Vaccinium. Protocols from Qiagen, MoBio, and Phytopure were used to isolate and purify DNA from the NIST standards. The resulting DNA concentration was quantified using SYBR Green fluorometry. Each of the 24 samples yielded DNA, though the concentration of DNA from each approach was notably different. The Phytopure method consistently yielded more DNA. The average yield ratio was 22 : 3 : 1 (ng/microL; Phytopure : Qiagen : MoBio). Amplification of the internal transcribed spacer II region using PCR was ultimately successful in 22 of the 24 samples. Direct sequencing chromatograms of the amplified material suggested that most of the samples were comprised of mixtures. However, the sequencing chromatograms of 12 of the 24 samples were sufficient to confirm the identity of the target material. The successful extraction, amplification, and sequencing of DNA from these herbal dietary supplement extracts and powders supports a continued effort to explore nucleotide sequence-based tools for the authentication and identification of plants in dietary supplements. (c) Georg Thieme Verlag KG Stuttgart . New York.

Exonuclease III-assisted graphene oxide amplified fluorescence anisotropy strategy for ricin detection.

PubMed

Xiao, Xue; Tao, Jing; Zhang, Hong Zhi; Huang, Cheng Zhi; Zhen, Shu Jun

2016-11-15

Graphene oxide (GO) is an excellent fluorescence anisotropy (FA) amplifier. However, in the conventional GO amplified FA strategy, one target can only induce the FA change of one fluorophore on probe, which limits the detection sensitivity. Herein, we developed an exonuclease III (Exo III) aided GO amplified FA strategy by using aptamer as an recognition element and ricin B-chain as a proof-of-concept target. The aptamer was hybridized with a blocker sequence and linked onto the surface of magnetic beads (MBs). Upon the addition of ricin B-chain, blocker was released from the surface of MBs and hybridized with the dye-modified probe DNA on the surface of GO through the toehold-mediated strand exchange reaction. The formed blocker-probe DNA duplex triggered the Exo III-assisted cyclic signal amplification by repeating the hybridization and digestion of probe DNA, liberating the fluorophore with several nucleotides (low FA value). Thus, ricin B-chain could be sensitively detected by the significantly decreased FA. The linear range was from 1.0μg/mL to 13.3μg/mL and the limit of detection (LOD) was 400ng/mL. This method improved the sensitivity of FA assay and it could be generalized to any kind of target detection based on the use of an appropriate aptamer. Copyright © 2016 Elsevier B.V. All rights reserved.

Assessing the Fidelity of Ancient DNA Sequences Amplified From Nuclear Genes

PubMed Central

Binladen, Jonas; Wiuf, Carsten; Gilbert, M. Thomas P.; Bunce, Michael; Barnett, Ross; Larson, Greger; Greenwood, Alex D.; Haile, James; Ho, Simon Y. W.; Hansen, Anders J.; Willerslev, Eske

2006-01-01

To date, the field of ancient DNA has relied almost exclusively on mitochondrial DNA (mtDNA) sequences. However, a number of recent studies have reported the successful recovery of ancient nuclear DNA (nuDNA) sequences, thereby allowing the characterization of genetic loci directly involved in phenotypic traits of extinct taxa. It is well documented that postmortem damage in ancient mtDNA can lead to the generation of artifactual sequences. However, as yet no one has thoroughly investigated the damage spectrum in ancient nuDNA. By comparing clone sequences from 23 fossil specimens, recovered from environments ranging from permafrost to desert, we demonstrate the presence of miscoding lesion damage in both the mtDNA and nuDNA, resulting in insertion of erroneous bases during amplification. Interestingly, no significant differences in the frequency of miscoding lesion damage are recorded between mtDNA and nuDNA despite great differences in cellular copy numbers. For both mtDNA and nuDNA, we find significant positive correlations between total sequence heterogeneity and the rates of type 1 transitions (adenine → guanine and thymine → cytosine) and type 2 transitions (cytosine → thymine and guanine → adenine), respectively. Type 2 transitions are by far the most dominant and increase relative to those of type 1 with damage load. The results suggest that the deamination of cytosine (and 5-methyl cytosine) to uracil (and thymine) is the main cause of miscoding lesions in both ancient mtDNA and nuDNA sequences. We argue that the problems presented by postmortem damage, as well as problems with contamination from exogenous sources of conserved nuclear genes, allelic variation, and the reliance on single nucleotide polymorphisms, call for great caution in studies relying on ancient nuDNA sequences. PMID:16299392

Common source cascode amplifiers for integrating IR-FPA applications

NASA Technical Reports Server (NTRS)

Woolaway, James T.; Young, Erick T.

1989-01-01

Space based astronomical infrared measurements present stringent performance requirements on the infrared detector arrays and their associated readout circuitry. To evaluate the usefulness of commercial CMOS technology for astronomical readout applications a theoretical and experimental evaluation was performed on source follower and common-source cascode integrating amplifiers. Theoretical analysis indicates that for conditions where the input amplifier integration capacitance is limited by the detectors capacitance the input referred rms noise electrons of each amplifier should be equivalent. For conditions of input gate limited capacitance the source follower should provide lower noise. Measurements of test circuits containing both source follower and common source cascode circuits showed substantially lower input referred noise for the common-source cascode input circuits. Noise measurements yielded 4.8 input referred rms noise electrons for an 8.5 minute integration. The signal and noise gain of the common-source cascode amplifier appears to offer substantial advantages in acheiving predicted noise levels.

Hemolysis, Toxicity, and Randomly Amplified Polymorphic DNA Analysis of Stachybotrys chartarum Strains

PubMed Central

Vesper, Stephen J.; Dearborn, Dorr G.; Yike, Iwona; Sorenson, W. G.; Haugland, Richard A.

1999-01-01

Stachybotrys chartarum is an indoor air, toxigenic fungus that has been associated with a number of human and veterinary health problems. Most notable among these has been a cluster of idiopathic pulmonary hemorrhage cases that were observed in the Cleveland, Ohio, area. In this study, 16 strains of S. chartarum isolated from case (n = 8) or control (n = 8) homes in Cleveland and 12 non-Cleveland strains from diverse geographic locations were analyzed for hemolytic activity, conidial toxicity, and randomly amplified polymorphic DNA banding patterns. In tests for hemolytic activity, strains were grown at 23°C on wet wallboard pieces for an 8-week test period. Conidia from these wallboard pieces were subcultured on sheep’s blood agar once a week over this period and examined for growth and clearing of the medium at 37 or 23°C. Five of the Cleveland strains (all from case homes) showed hemolytic activity at 37°C throughout the 8-week test compared to 3 of the non-Cleveland strains. Five of the Cleveland strains, compared to two of the non-Cleveland strains, produced highly toxic conidia (>90 μg of T2 toxin equivalents per g [wet weight] of conidia) after 10 and 30 days of growth on wet wallboard. Only 3 of the 28 strains examined both were consistently hemolytic and produced highly toxic conidia. Each of these strains was isolated from a house in Cleveland where an infant had idiopathic pulmonary hemorrhage. PMID:10388719

Hemolysis, toxicity, and randomly amplified polymorphic DNA analysis of Stachybotrys chartarum strains.

PubMed

Vesper, S J; Dearborn, D G; Yike, I; Sorenson, W G; Haugland, R A

1999-07-01

Stachybotrys chartarum is an indoor air, toxigenic fungus that has been associated with a number of human and veterinary health problems. Most notable among these has been a cluster of idiopathic pulmonary hemorrhage cases that were observed in the Cleveland, Ohio, area. In this study, 16 strains of S. chartarum isolated from case (n = 8) or control (n = 8) homes in Cleveland and 12 non-Cleveland strains from diverse geographic locations were analyzed for hemolytic activity, conidial toxicity, and randomly amplified polymorphic DNA banding patterns. In tests for hemolytic activity, strains were grown at 23 degrees C on wet wallboard pieces for an 8-week test period. Conidia from these wallboard pieces were subcultured on sheep's blood agar once a week over this period and examined for growth and clearing of the medium at 37 or 23 degrees C. Five of the Cleveland strains (all from case homes) showed hemolytic activity at 37 degrees C throughout the 8-week test compared to 3 of the non-Cleveland strains. Five of the Cleveland strains, compared to two of the non-Cleveland strains, produced highly toxic conidia (>90 microgram of T2 toxin equivalents per g [wet weight] of conidia) after 10 and 30 days of growth on wet wallboard. Only 3 of the 28 strains examined both were consistently hemolytic and produced highly toxic conidia. Each of these strains was isolated from a house in Cleveland where an infant had idiopathic pulmonary hemorrhage.

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.

The Potential of Cosmetic Applicators as a Source of DNA for Forensic Analysis.

PubMed

Adamowicz, Michael S; Labonte, Renáe D; Schienman, John E

2015-07-01

Personal products, such as toothbrushes, have been used as both known reference and evidentiary samples for forensic DNA analysis. This study examined the viability of a broad selection of cosmetic applicators for use as targets for human DNA extraction and short tandem repeat (STR) analysis using standard polymerase chain reaction (PCR) conditions. Applicator types included eyeliner smudgers, pencils and crayons, eye shadow sponges, mascara wands, concealer wands, face makeup sponges, pads and brushes, lipsticks and balms, and lip gloss wands. The quantity and quality of DNA extracted from each type of applicator were examined by assessing the number of loci successfully amplified and the peak balance of the heterozygous alleles in each full STR profile. While degraded DNA, stochastic amplification, and PCR inhibition were observed for some items, full STR profiles were developed for 14 of 76 applicators. The face makeup sponge applicators yielded the highest proportional number of full STR profiles (4/7). © 2015 American Academy of Forensic Sciences.

Self-amplifying mRNA vaccines.

PubMed

Brito, Luis A; Kommareddy, Sushma; Maione, Domenico; Uematsu, Yasushi; Giovani, Cinzia; Berlanda Scorza, Francesco; Otten, Gillis R; Yu, Dong; Mandl, Christian W; Mason, Peter W; Dormitzer, Philip R; Ulmer, Jeffrey B; Geall, Andrew J

2015-01-01

This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization. Copyright © 2015 Elsevier Inc. All rights reserved.

Amplified DNAs in laboratory stocks of Leishmania tarentolae: extrachromosomal circles structurally and functionally similar to the inverted-H-region amplification of methotrexate-resistant Leishmania major

DOE Office of Scientific and Technical Information (OSTI.GOV)

Petrillo-Peixoto, M.L.; Beverley, S.M.

1988-12-01

We describe the structure of amplified DNA that was discovered in two laboratory stocks of the protozoan parasite Leishmania tarentolae. Restriction mapping and molecular cloning revealed that a region of 42 kilobases was amplified 8- to 30-fold in these lines. Southern blot analyses of digested DNAs or chromosomes separated by pulsed-field electrophoresis showed that the amplified DNA corresponded to the H region, a locus defined originally by its amplification in methotrexate-resistant Leishmania major. Similarities between the amplified DNA of the two species included (i) extensive cross-hybridization; (ii) approximate conservation of sequence order; (iii) extrachromosomal localization; (iv) an overall inverted, head-to-headmore » configuration as a circular 140-kilobase tetrameric molecule; (v) two regions of DNA sequence rearrangement, each of which was closely associated with the two centers of the inverted repeats; (vi) association with methotrexate resistance; and (vii) phenotypically conservative amplification, in which the wild-type chromosomal arrangement was retained without apparent modification. Our data showed that amplified DNA mediating drug resistance arose in unselected L. tarentolae, although the pressures leading to apparently spontaneous amplification and maintenance of the H region are not known. The simple structure and limited extent of DNA amplified in these and other Leishmania lines suggests that the study of gene amplification in Leishmania spp. offers an attractive model system for the study of amplification in cultured mammalian cells and tumors. We also introduced a method for measuring the size of large circular DNAs, using gamma-irradiation to introduce limited double-strand breaks followed by sizing of the linear DNAs by pulsed-field electrophoresis.« less

Amplified amperometric aptasensor for selective detection of protein using catalase-functional DNA-PtNPs dendrimer as a synergetic signal amplification label.

PubMed

Zhang, Juan; Yuan, Yali; biXie, Shun; Chai, Yaqin; Yuan, Ruo

2014-10-15

In this work, we present a new strategy to construct an electrochemical aptasensor for sensitive detection of platelet-derived growth factor BB (PDGF-BB) based on the synergetic amplification of a three-dimensional (3D) nanoscale catalase (CAT) enzyme-functional DNA-platinum nanoparticles (PtNPs) dendrimer through autonomous layer-by-layer assembly. Firstly, polyamidoaminedendrimer (PAMAM) with a hyper-branched and three-dimensional structure was served as nanocarriers to coimmobilize a large number of PDGF-BB binding aptamer (PBA II) and ssDNA 1 (S1) to form PBA II-PAMAM-S1 bioconjugate. In the presence of PDGF-BB, the bioconjugate was self-assembled on the electrode by sandwich assay. Following that, the carried S1 propagated a chain reaction of hybridization events between CAT-PtNPs-S1 and CAT-PtNPs-ssDNA 2 (S2) to form a 3D nanoscale CAT-functional PtNPs-DNA dendrimer, which successfully immobilized substantial CAT enzyme and PtNPs with superior catalysis activity. In this process, the formed negatively charged double-helix DNA could cause the intercalation of hexaammineruthenium(III) chloride (RuHex) into the groove via electrostatic interactions. Thus, numerous RuHex redox probes and CAT were decorated inside/outside of the dendrimer. In the presence of H2O2 in electrolytic cell, the synergistic reaction of CAT and PtNPs towards electrocatalysis could further amplify electrochemical signal. Under optimal condition, the CAT-PtNPs-DNA dendrimer-based sensing system presented a linear dependence between the reduction peak currents and logarithm of PDGF-BB concentrations in the range of 0.00005-35 nM with a relatively low detection limit of 0.02 pM. Copyright © 2014 Elsevier B.V. All rights reserved.

SQUARE WAVE AMPLIFIER

DOEpatents

Leavitt, M.A.; Lutz, I.C.

1958-08-01

An amplifier circuit is described for amplifying sigmals having an alternating current component superimposed upon a direct current component, without loss of any segnnent of the alternating current component. The general circuit arrangement includes a vibrator, two square wave amplifiers, and recombination means. The amplifier input is connected to the vibrating element of the vibrator and is thereby alternately applied to the input of each square wave amplifier. The detailed circuitry of the recombination means constitutes the novelty of the annplifier and consists of a separate, dual triode amplifier coupled to the output of each square wave amplifier with a recombination connection from the plate of one amplifier section to a grid of one section of the other amplifier. The recombination circuit has provisions for correcting distortion caused by overlapping of the two square wave voltages from the square wave amplifiers.

Whole genome amplification of DNA extracted from FFPE tissues.

PubMed

Bosso, Mira; Al-Mulla, Fahd

2011-01-01

Whole genome amplification systems were developed to meet the increasing research demands on DNA resources and to avoid DNA shortage. The technology enables amplification of nanogram amounts of DNA into microgram quantities and is increasingly used in the amplification of DNA from multiple origins such as blood, fresh frozen tissue, formalin-fixed paraffin-embedded tissues, saliva, buccal swabs, bacteria, and plant and animal sources. This chapter focuses on the use of GenomePlex(®) tissue Whole Genome Amplification Kit, to amplify DNA directly from archived tissue. In addition, this chapter documents our unique experience with the utilization of GenomePlex(®) amplified DNA using several molecular techniques including metaphase Comparative Genomic Hybridization, array Comparative Genomic Hybridization, and real-time quantitative polymerase chain reaction assays. GenomePlex(®) is a registered trademark of Rubicon Genomics Incorporation.

[Establishment of multiple regression model for virulence factors of Saccharomyces albicans by random amplified polymorphic DNA bands].

PubMed

Liu, Qi; Wu, Youcong; Yuan, Youhua; Bai, Li; Niu, Kun

2011-12-01

To research the relationship between the virulence factors of Saccharomyces albicans (S. albicans) and the random amplified polymorphic DNA (RAPD) bands of them, and establish the regression model by multiple regression analysis. Extracellular phospholipase, secreted proteinase, ability to generate germ tubes and adhere to oral mucosal cells of 92 strains of S. albicans were measured in vitro; RAPD-polymerase chain reaction (RAPD-PCR) was used to get their bands. Multiple regression for virulence factors of S. albicans and RAPD-PCR bands was established. The extracellular phospholipase activity was associated with 4 RAPD bands: 350, 450, 650 and 1 300 bp (P < 0.05); secreted proteinase activity of S. albicans was associated with 2 bands: 350 and 1 200 bp (P < 0.05); the ability of germ tube produce was associated with 2 bands: 400 and 550 bp (P < 0.05). Some RAPD bands will reflect the virulence factors of S. albicans indirectly. These bands would contain some important messages for regulation of S. albicans virulence factors.

A cationic conjugated polymer and graphene oxide: Application to amplified fluorescence detection of sinapine.

PubMed

Zhang, Zhen; Xiang, Xia; Shi, Jianbin; Huang, Fenghong; Xia, Xiaoyang; Zheng, Mingming; Han, Ling; Tang, Hu

2018-10-05

An amplified fluorescence strategy is described for the detection of sinapine (SP) by using a cationic conjugated polymer (PFP) and graphene oxide (GO). It is observed that the fluorescein (FAM)-labeled single-stranded DNA (FAM-DNA) is absorbed on the surface of GO if SP is absent. This causes that fluorescence resonance energy transfer (FRET) from PFP to FAM is inefficient when adding PFP into FAM-DNA/GO complex. If SP is added to FAM-DNA/GO complex, FAM-DNA is desorbed from GO surface due to the competitive binding of SP and FAM-DNA toward GO. In this case, FAM-DNA is close to PFP in the presence of PFP through strong electrostatic interaction, leading to the occurrence of efficient FRET. Based on the above phenomenon, we demonstrate a method to amplify fluorescence signal of traditional GO-based SP assay by introducing PFP. In comparison to the use of single GO, the combination of PFP with GO-based strategy displays high turn-on ratio and enhanced sensitivity with a limit of detection as low as 7.3 ng mL -1 for SP detection. Satisfactory results in practical samples are also obtained by the recovery experiments, demonstrating the potential application of cationic conjugated polymer in plant-derived small molecule. Copyright © 2018 Elsevier B.V. All rights reserved.

Molecular characterization of a distinct begomovirus species from Vernonia cinerea and its associated DNA-beta using the bacteriophage Phi 29 DNA polymerase.

PubMed

Packialakshmi, R M; Srivastava, N; Girish, K R; Usha, R

2010-08-01

Vernonia cinerea plants with yellow vein symptoms were collected around crop fields in Madurai. A portion (550 bp) of the AV1 gene amplified using degenerate primers from the total DNA purified from diseased leaf sample was cloned and sequenced. Specific primers derived from the above sequence were used to amplify 2,745 nucleotides with the typical genome organization of begomoviral DNA A (EMBL Accession No. AM182232). Sequence comparison with other begomoviruses revealed the greatest identity (82.4%) with Emilia yellow vein virus (EmYVV-[Fz1]) from China and less than 80% with all other known begomoviruses. The International Committee on Taxonomy of Viruses (ICTV) has therefore recognized Vernonia yellow vein virus (VeYVV) as a distinct begomovirus species. Conventional PCR could not amplify the DNA B or DNA beta from the diseased tissue. However, the beta DNA (1364 bp) associated with the disease was obtained (Accession No. FN435836) by the rolling circle amplification-restriction fragment length polymorphism method (RCA-RFLP) using Phi 29 DNA polymerase. Sequence analysis shows that DNA beta of VeYVV has the highest identity (56.8%) with DNA beta of Sigesbeckia yellow vein Guangxi betasatellite (SibYVGxB-[CN: Gx111:05]) and 56-53% with DNA beta associated with other begomoviruses. This is the first report of the molecular characterization of VeYVV from V. cinerea in India. The complete molecular characterization, phylogenetic analysis, and putative recombination events in VeYVV are reported.

Extracellular DNA in single- and multiple-species unsaturated biofilms.

PubMed

Steinberger, R E; Holden, P A

2005-09-01

The extracellular polymeric substances (EPS) of bacterial biofilms form a hydrated barrier between cells and their external environment. Better characterization of EPS could be useful in understanding biofilm physiology. The EPS are chemically complex, changing with both bacterial strain and culture conditions. Previously, we reported that Pseudomonas aeruginosa unsaturated biofilm EPS contains large amounts of extracellular DNA (eDNA) (R. E. Steinberger, A. R. Allen, H. G. Hansma, and P. A. Holden, Microb. Ecol. 43:416-423, 2002). Here, we investigated the compositional similarity of eDNA to cellular DNA, the relative quantity of eDNA, and the terminal restriction fragment length polymorphism (TRFLP) community profile of eDNA in multiple-species biofilms. By randomly amplified polymorphic DNA analysis, cellular DNA and eDNA appear identical for P. aeruginosa biofilms. Significantly more eDNA was produced in P. aeruginosa and Pseudomonas putida biofilms than in Rhodococcus erythropolis or Variovorax paradoxus biofilms. While the amount of eDNA in dual-species biofilms was of the same order of magnitude as that of of single-species biofilms, the amounts were not predictable from single-strain measurements. By the Shannon diversity index and principle components analysis of TRFLP profiles generated from 16S rRNA genes, eDNA of four-species biofilms differed significantly from either cellular or total DNA of the same biofilm. However, total DNA- and cellular DNA-based TRFLP analyses of this biofilm community yielded identical results. We conclude that extracellular DNA production in unsaturated biofilms is species dependent and that the phylogenetic information contained in this DNA pool is quantifiable and distinct from either total or cellular DNA.

DNA and bone structure preservation in medieval human skeletons.

PubMed

Coulson-Thomas, Yvette M; Norton, Andrew L; Coulson-Thomas, Vivien J; Florencio-Silva, Rinaldo; Ali, Nadir; Elmrghni, Samir; Gil, Cristiane D; Sasso, Gisela R S; Dixon, Ronald A; Nader, Helena B

2015-06-01

Morphological and ultrastructural data from archaeological human bones are scarce, particularly data that have been correlated with information on the preservation of molecules such as DNA. Here we examine the bone structure of macroscopically well-preserved medieval human skeletons by transmission electron microscopy and immunohistochemistry, and the quantity and quality of DNA extracted from these skeletons. DNA technology has been increasingly used for analyzing physical evidence in archaeological forensics; however, the isolation of ancient DNA is difficult since it is highly degraded, extraction yields are low and the co-extraction of PCR inhibitors is a problem. We adapted and optimised a method that is frequently used for isolating DNA from modern samples, Chelex(®) 100 (Bio-Rad) extraction, for isolating DNA from archaeological human bones and teeth. The isolated DNA was analysed by real-time PCR using primers targeting the sex determining region on the Y chromosome (SRY) and STR typing using the AmpFlSTR(®) Identifiler PCR Amplification kit. Our results clearly show the preservation of bone matrix in medieval bones and the presence of intact osteocytes with well preserved encapsulated nuclei. In addition, we show how effective Chelex(®) 100 is for isolating ancient DNA from archaeological bones and teeth. This optimised method is suitable for STR typing using kits aimed specifically at degraded and difficult DNA templates since amplicons of up to 250bp were successfully amplified. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A Novel Terminator Primer and Enhancer Reagents for Direct Expression of PCR-Amplified Genes in Mammalian Cells.

PubMed

Nakamura, Mikiko; Suzuki, Ayako; Akada, Junko; Yarimizu, Tohru; Iwakiri, Ryo; Hoshida, Hisashi; Akada, Rinji

2015-08-01

Escherichia coli plasmids are commonly used for gene expression experiments in mammalian cells, while PCR-amplified DNAs are rarely used even though PCR is a much faster and easier method to construct recombinant DNAs. One difficulty may be the limited amount of DNA produced by PCR. For direct utilization of PCR-amplified DNA in transfection experiments, efficient transfection with a smaller amount of DNA should be attained. For this purpose, we investigated two enhancer reagents, polyethylene glycol and tRNA, for a chemical transfection method. The addition of the enhancers to a commercial transfection reagent individually and synergistically exhibited higher transfection efficiency applicable for several mammalian cell culture lines in a 96-well plate. By taking advantage of a simple transfection procedure using PCR-amplified DNA, SV40 and rabbit β-globin terminator lengths were minimized. The terminator length is short enough to design in oligonucleotides; thus, terminator primers can be used for the construction and analysis of numerous mutations, deletions, insertions, and tag-fusions at the 3'-terminus of any gene. The PCR-mediated gene manipulation with the terminator primers will transform gene expression by allowing for extremely simple and high-throughput experiments with small-scale, multi-well, and mammalian cell cultures.

Differentiation of Trichophyton rubrum clinical isolates from Japanese and Chinese patients by randomly amplified polymorphic DNA and DNA sequence analysis of the non-transcribed spacer region of the rRNA gene.

PubMed

Yang, Xiumin; Sugita, Takashi; Takashima, Masako; Hiruma, Masataro; Li, Ruoyu; Sudo, Hajime; Ogawa, Hideoki; Ikeda, Shigaku

2009-04-01

Trichophyton rubrum is the most common pathogen causing dermatophytosis worldwide. Recent genetic investigations showed that the microorganism originated in Africa and then spread to Europe and North America via Asia. We investigated the intraspecific diversity of T. rubrum isolated from two closely located Asian countries, Japan and China. A total of 150 clinical isolates of T. rubrum obtained from Japanese and Chinese patients were analyzed by randomly amplified polymorphic DNA (RAPD) and DNA sequence analysis of the non-transcribed spacer (NTS) region in the rRNA gene. RAPD analysis divided the 150 strains into two major clusters, A and B. Of the Japanese isolates, 30% belonged to cluster A and 70% belonged to cluster B, whereas 91% of the Chinese isolates were in cluster A. The NTS region of the rRNA gene was divided into four major groups (I-IV) based on DNA sequencing. The majority of Japanese isolates were type IV (51%), and the majority of Chinese isolates were type III (75%). These results suggest that although Japan and China are neighboring countries, the origins of T. rubrum isolates from these countries may not be identical. These findings provide information useful for tracing the global transmission routes of T. rubrum.

LOGARITHMIC AMPLIFIER

DOEpatents

De Shong, J.A. Jr.

1957-12-31

A logarithmic current amplifier circuit having a high sensitivity and fast response is described. The inventor discovered the time constant of the input circuit of a system utilizing a feedback amplifier, ionization chamber, and a diode, is inversely proportional to the input current, and that the amplifier becomes unstable in amplifying signals in the upper frequency range when the amplifier's forward gain time constant equals the input circuit time constant. The described device incorporates impedance networks having low frequency response characteristic at various points in the circuit to change the forward gain of the amplifler at a rate of 0.7 of the gain magnitude for every two times increased in frequency. As a result of this improvement, the time constant of the input circuit is greatly reduced at high frequencies, and the amplifier response is increased.

The Evolution of Ribosomal DNA: Divergent Paralogues and Phylogenetic Implications

PubMed Central

Buckler-IV, E. S.; Ippolito, A.; Holtsford, T. P.

1997-01-01

Although nuclear ribosomal DNA (rDNA) repeats evolve together through concerted evolution, some genomes contain a considerable diversity of paralogous rDNA. This diversity includes not only multiple functional loci but also putative pseudogenes and recombinants. We examined the occurrence of divergent paralogues and recombinants in Gossypium, Nicotiana, Tripsacum, Winteraceae, and Zea ribosomal internal transcribed spacer (ITS) sequences. Some of the divergent paralogues are probably rDNA pseudogenes, since they have low predicted secondary structure stability, high substitution rates, and many deamination-driven substitutions at methylation sites. Under standard PCR conditions, the low stability paralogues amplified well, while many high-stability paralogues amplified poorly. Under highly denaturing PCR conditions (i.e., with dimethylsulfoxide), both low- and high-stability paralogues amplified well. We also found recombination between divergent paralogues. For phylogenetics, divergent ribosomal paralogues can aid in reconstructing ancestral states and thus serve as good outgroups. Divergent paralogues can also provide companion rDNA phylogenies. However, phylogeneticists must discriminate among families of divergent paralogues and recombinants or suffer from muddled and inaccurate organismal phylogenies. PMID:9055091

Application of amplified ribosomal DNA restriction analysis in identification of Acinetobacter baumannii from a tertiary teaching hospital, Malaysia.

PubMed

Kong, B H; Hanifah, Y A; Yusof, M Y; Thong, K L

2011-12-01

Acinetobacter baumannii, genomic species 3 and 13TU are being increasingly reported as the most important Acinetobacter species that cause infections in hospitalized patients. These Acinetobacter species are grouped in the Acinetobacter calcoaceticus- Acinetobacter baumannii (Acb) complex. Differentiation of the species in the Acb-complex is limited by phenotypic methods. Therefore, in this study, amplified ribosomal DNA restriction analysis (ARDRA) was applied to confirm the identity A. baumannii strains as well as to differentiate between the subspecies. One hundred and eighty-five strains from Intensive Care Unit, Universiti Malaya Medical Center (UMMC) were successfully identified as A. baumannii by ARDRA. Acinetobacter genomic species 13TU and 15TU were identified in 3 and 1 strains, respectively. ARDRA provides an accurate, rapid and definitive approach towards the identification of the species level in the genus Acinetobacter. This paper reports the first application ARDRA in genospecies identification of Acinetobacter in Malaysia.

Competition for DNA binding sites using Promega DNA IQ™ paramagnetic beads.

PubMed

Frégeau, Chantal J; De Moors, Anick

2012-09-01

The Promega DNA IQ™ system is easily amenable to automation and has been an integral part of standard operating procedures for many forensic laboratories including those of the Royal Canadian Mounted Police (RCMP) since 2004. Due to some failure to extract DNA from samples that should have produced DNA using our validated automated DNA IQ™-based protocol, the competition for binding sites on the DNA IQ™ magnetic beads was more closely examined. Heme from heavily blooded samples interfered slightly with DNA binding. Increasing the concentration of Proteinase K during lysis of these samples did not enhance DNA recovery. However, diluting the sample lysate following lysis prior to DNA extraction overcame the reduction in DNA yield and preserved portions of the lysates for subsequent manual or automated extraction. Dye/chemicals from black denim lysates competed for binding sites on the DNA IQ™ beads and significantly reduced DNA recovery. Increasing the size or number of black denim cuttings during lysis had a direct adverse effect on DNA yield from various blood volumes. The dilution approach was successful on these samples and permitted the extraction of high DNA yields. Alternatively, shortening the incubation time for cell lysis to 30 min instead of the usual overnight at 56 °C prevented competition from black denim dye/chemicals and increased DNA yields. Crown Copyright © 2011. Published by Elsevier Ireland Ltd. All rights reserved.

Electronic amplifiers: A compilation

NASA Technical Reports Server (NTRS)

1971-01-01

Several types of amplifiers and amplifier systems are considered. These include preamplifiers, high power amplifiers, buffer and isolation amplifiers, amplifier circuits, and general purpose amplifiers.

Minimum envelope roughness pulse design for reduced amplifier distortion in parallel excitation.

PubMed

Grissom, William A; Kerr, Adam B; Stang, Pascal; Scott, Greig C; Pauly, John M

2010-11-01

Parallel excitation uses multiple transmit channels and coils, each driven by independent waveforms, to afford the pulse designer an additional spatial encoding mechanism that complements gradient encoding. In contrast to parallel reception, parallel excitation requires individual power amplifiers for each transmit channel, which can be cost prohibitive. Several groups have explored the use of low-cost power amplifiers for parallel excitation; however, such amplifiers commonly exhibit nonlinear memory effects that distort radio frequency pulses. This is especially true for pulses with rapidly varying envelopes, which are common in parallel excitation. To overcome this problem, we introduce a technique for parallel excitation pulse design that yields pulses with smoother envelopes. We demonstrate experimentally that pulses designed with the new technique suffer less amplifier distortion than unregularized pulses and pulses designed with conventional regularization.

Microbial Degradation of Forensic Samples of Biological Origin: Potential Threat to Human DNA Typing.

PubMed

Dash, Hirak Ranjan; Das, Surajit

2018-02-01

Forensic biology is a sub-discipline of biological science with an amalgam of other branches of science used in the criminal justice system. Any nucleated cell/tissue harbouring DNA, either live or dead, can be used as forensic exhibits, a source of investigation through DNA typing. These biological materials of human origin are rich source of proteins, carbohydrates, lipids, trace elements as well as water and, thus, provide a virtuous milieu for the growth of microbes. The obstinate microbial growth augments the degradation process and is amplified with the passage of time and improper storage of the biological materials. Degradation of these biological materials carriages a huge challenge in the downstream processes of forensic DNA typing technique, such as short tandem repeats (STR) DNA typing. Microbial degradation yields improper or no PCR amplification, heterozygous peak imbalance, DNA contamination from non-human sources, degradation of DNA by microbial by-products, etc. Consequently, the most precise STR DNA typing technique is nullified and definite opinion can be hardly given with degraded forensic exhibits. Thus, suitable precautionary measures should be taken for proper storage and processing of the biological exhibits to minimize their decaying process by micro-organisms.

[Analysis of methylation-sensitive amplified polymorphism in wheat genome under the wheat leaf rust stress].

PubMed

Fu, Sheng-Jie; Wang, Hui; Feng, Li-Na; Sun, Yi; Yang, Wen-Xiang; Liu, Da-Qun

2009-03-01

Intrinsic DNA methylation pattern is an integral component of the epigenetic network in many eukaryotes. DNA methylation plays an important role in regulating gene expression in eukaryotes. Biological stress in plant provides an inherent epigenetic driving force of evolution. Study of DNA methylation patterns arising from biological stress will help us fully understand the epigenetic regulation of gene expression and DNA methylation of biological functions. The wheat near-isogenic lines TcLr19 and TcLr41 were resistant to races THTT and TKTJ, respectively, and Thatcher is compatible in the interaction with Puccinia triticina THTT and TKTJ, respectively. By means of methylation-sensitive amplified polymorphism (MSAP) analysis, the patterns of cytosine methylation in TcLr19, TcLr41, and Thatcher inoculated with P. triticina THTT and TKTJ were compared with those of the untreated samples. All the DNA fragments, each representing a recognition site cleaved by each or both of isoschizomers, were amplified using 60 pairs of selective primers. The results indicated that there was no significant difference between the challenged and unchallenged plants at DNA methylation level. However, epigenetic difference between the near-isogenic line for wheat leaf rust resistance gene Lr41 and Thatcher was present.

Traceability of plant contribution in olive oil by amplified fragment length polymorphisms.

PubMed

Pafundo, Simona; Agrimonti, Caterina; Marmiroli, Nelson

2005-09-07

Application of DNA molecular markers to traceability of foods is thought to bring new benefit to consumer's protection. Even in a complex matrix such as olive oil, DNA could be traced with PCR markers such as the amplified fragment length polymorphisms (AFLPs). In this work, fluorescent AFLPs were optimized for the characterization of olive oil DNA, to obtain highly reproducible, high-quality fingerprints, testing different parameters: the concentrations of dNTPs and labeled primer, the kind of Taq DNA polymerase and thermal cycler, and the quantity of DNA employed. It was found that correspondence of fingerprinting by comparing results in oils and in plants was close to 70% and that the DNA extraction from olive oil was the limiting step for the reliability of AFLP profiles, due to the complex matrix analyzed.

Different DNA methylation patterns detected by the Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) technique among various cell types of bulls.

PubMed

Phutikanit, Nawapen; Suwimonteerabutr, Junpen; Harrison, Dion; D'Occhio, Michael; Carroll, Bernie; Techakumphu, Mongkol

2010-03-05

The purpose of this study was to apply an arbitrarily primed methylation sensitive polymerase chain reaction (PCR) assay called Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) to investigate the methylation profiles of somatic and germ cells obtained from Holstein bulls. Genomic DNA was extracted from sperm, leukocytes and fibroblasts obtained from three bulls and digested with a methylation sensitive endonuclease (HpaII). The native genomic and enzyme treated DNA samples were used as templates in an arbitrarily primed-PCR assay with 30 sets of single short oligonucleotide primer. The PCR products were separated on silver stained denaturing polyacrylamide gels. Three types of PCR markers; digestion resistant-, digestion sensitive-, and digestion dependent markers, were analyzed based on the presence/absence polymorphism of the markers between the two templates. Approximately 1,000 PCR markers per sample were produced from 27 sets of primer and most of them (>90%) were digestion resistant markers. The highest percentage of digestion resistant markers was found in leukocytic DNA (94.8%) and the lowest in fibroblastic DNA (92.3%, P < or = 0.05). Spermatozoa contained a higher number of digestion sensitive markers when compared with the others (3.6% vs. 2.2% and 2.6% in leukocytes and fibroblasts respectively, P < or = 0.05). The powerfulness of the AMP PCR assay was the generation of methylation-associated markers without any prior knowledge of the genomic sequence. The data obtained from different primers provided an overview of genome wide DNA methylation content in different cell types. By using this technique, we found that DNA methylation profile is tissue-specific. Male germ cells were hypomethylated at the HpaII locations when compared with somatic cells, while the chromatin of the well-characterized somatic cells was heavily methylated when compared with that of the versatile somatic cells.

Improving the recovery of qPCR-grade DNA from sludge and sediment.

PubMed

Bonot, Sébastien; Courtois, Sophie; Block, Jean-Claude; Merlin, Christophe

2010-08-01

DNA extraction is often considered as the limiting step of most molecular approaches in ecology and environmental microbiology. Ten existing DNA extraction protocols were compared for recovery of DNA from sludge and a modified version of the protocol described by Porteous et al. (Mol Ecol 6:787-791, 1997) was determined to be the best method for recovery of DNA suitable for PCR. In this respect, it appeared that the commonly used guanidine isothiocyanate could impair the quality of the extracted DNA unless its concentration is lowered. Second, conditioning the samples as liquors as opposed to pellets critically impacts the outcome of the extraction. The suitability of the modified Porteous protocol for quantitative PCR applications is demonstrated in a series of experiments showing the absence of interfering coextracted inhibitors and the linear correspondence between the concentrations of input target DNA and PCR product. Interestingly, it is also shown that the nature of the environmental matrices affects the recovery yield of both circular plasmids and chromosomal DNA, resulting in an apparent fluctuation of the plasmid copy number per cell. This means that quantitative data obtained by PCR remain comparable as long as they apply to an identical target sequence extracted from a similar environment and amplified under the same conditions.

DNA book.

PubMed

Kawai, Jun; Hayashizaki, Yoshihide

2003-06-01

We propose herein a new method of DNA distribution, whereby DNA clones or PCR products are printed directly onto the pages of books and delivered to users along with relevant scientific information. DNA sheets, comprising water-soluble paper onto which DNA is spotted, can be bound into books. Readers can easily extract the DNA fragments from DNA sheets and amplify them using PCR. We show that DNA sheets can withstand various conditions that may be experienced during bookbinding and delivery, such as high temperatures and humidity. Almost all genes (95%-100% of randomly selected RIKEN mouse cDNA clones) were recovered successfully by use of PCR. Readers can start their experiments after a 2-h PCR amplification without waiting for the delivery of DNA clones. The DNA Book thus provides a novel method for delivering DNA in a timely and cost-effective manner. A sample DNA sheet (carrying RIKEN mouse cDNA clones encoding genes of enzymes for the TCA cycle) is included in this issue for field-testing. We would greatly appreciate it if readers could attempt to extract DNA and report the results and whether the DNA sheet was shipped to readers in good condition.

DNA damage and genetic methylation changes caused by Cd in Arabidopsis thaliana seedlings.

PubMed

Li, Zhaoling; Liu, Zhihong; Chen, Ruijuan; Li, Xiaojun; Tai, Peidong; Gong, Zongqiang; Jia, Chunyun; Liu, Wan

2015-09-01

Amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MASP) techniques are sensitive to deoxyribonucleic acid (DNA) damage and genetic methylation, respectively. Using these 2 techniques, Arabidopsis thaliana cultured with 0 mg/L (control), 0.5 mg/L, 1.5 mg/L, and 5.0 mg/L Cd(2+) for 16 d was used to analyze the DNA damage and methylation changes as a result of cadmium (Cd). The DNA was amplified by 14 AFLP primer pairs and 13 MSAP primer combinations. In the AFLP experiment, 62 polymorphic sites were found in the patterns of 11 primer combinations and a total of 1116 fragments were obtained in these patterns. There were no polymorphic bands in the remaining 3 pairs. The proportions of polymorphic sites in the 0.5-mg/L Cd(2+) and 5.0-mg/L Cd(2+) treatments were significantly different. Seven polymorphic fragments were then separated and successfully sequenced, yielding 6 nucleobase substitutions and 1 nucleobase deletion. Similarly, in the MSAP experiment, the MSAP% and number of demethylated-type bands were unchanged after Cd treatment, but the number of methylated-type bands was increased significantly in the 5.0-mg/L Cd(2+) treatment group, a finding that may be associated with the AFLP results. The polymorphic bands were also sequenced and the functions of their homologous genes were determined. The DNA damage and methylation changes may be the primary cause of certain pathology changes as a result of Cd uptake in plants. © 2015 SETAC.

Kilohertz Cr:forsterite regenerative amplifier.

PubMed

Evans, J M; Petri Evi, V; Alfano, R R; Fu, Q

1998-11-01

We report on a tunable regenerative amplifier that is operational in the near-infrared spectral region from 1230 to 1280 nm based on the vibronic laser material Cr:forsterite. Utilizing the technique of chirped-pulse amplification, we generated pulses as short as 150 fs at 1255 nm at a repetition rate of 1 kHz. Pulse amplification of more than 5 x 10(5) times was observed, with recorded output pulse energies of 34 muJ . Implementation of a second-harmonic generator yielded 110-fs-duration pulses of 7-muJ energy at 625 nm.

Cross-differential amplifier

NASA Technical Reports Server (NTRS)

Hajimiri, Seyed-Ali (Inventor); Kee, Scott D. (Inventor); Aoki, Ichiro (Inventor)

2010-01-01

A cross-differential amplifier is provided. The cross-differential amplifier includes an inductor connected to a direct current power source at a first terminal. A first and second switch, such as transistors, are connected to the inductor at a second terminal. A first and second amplifier are connected at their supply terminals to the first and second switch. The first and second switches are operated to commutate the inductor between the amplifiers so as to provide an amplified signal while limiting the ripple voltage on the inductor and thus limiting the maximum voltage imposed across the amplifiers and switches.

Cross-differential amplifier

NASA Technical Reports Server (NTRS)

Hajimiri, Seyed-Ali (Inventor); Kee, Scott D. (Inventor); Aoki, Ichiro (Inventor)

2011-01-01

A cross-differential amplifier is provided. The cross-differential amplifier includes an inductor connected to a direct current power source at a first terminal. A first and second switch, such as transistors, are connected to the inductor at a second terminal. A first and second amplifier are connected at their supply terminals to the first and second switch. The first and second switches are operated to commutate the inductor between the amplifiers so as to provide an amplified signal while limiting the ripple voltage on the inductor and thus limiting the maximum voltage imposed across the amplifiers and switches.

Hybrid matrix amplifier

DOEpatents

Martens, J.S.; Hietala, V.M.; Plut, T.A.

1995-01-03

The present invention comprises a novel matrix amplifier. The matrix amplifier includes an active superconducting power divider (ASPD) having N output ports; N distributed amplifiers each operatively connected to one of the N output ports of the ASPD; and a power combiner having N input ports each operatively connected to one of the N distributed amplifiers. The distributed amplifier can included M stages of amplification by cascading superconducting active devices. The power combiner can include N active elements. The resulting (N[times]M) matrix amplifier can produce signals of high output power, large bandwidth, and low noise. 6 figures.

Cross-differential amplifier

NASA Technical Reports Server (NTRS)

Aoki, Ichiro (Inventor); Hajimiri, Seyed-Ali (Inventor); Kee, Scott D. (Inventor)

2013-01-01

A cross-differential amplifier is provided. The cross-differential amplifier includes an inductor connected to a direct current power source at a first terminal. A first and second switch, such as transistors, are connected to the inductor at a second terminal. A first and second amplifier are connected at their supply terminals to the first and second switch. The first and second switches are operated to commutate the inductor between the amplifiers so as to provide an amplified signal while limiting the ripple voltage on the inductor and thus limiting the maximum voltage imposed across the amplifiers and switches.

Hybrid matrix amplifier

DOEpatents

Martens, Jon S.; Hietala, Vincent M.; Plut, Thomas A.

1995-01-01

The present invention comprises a novel matrix amplifier. The matrix amplifier includes an active superconducting power divider (ASPD) having N output ports; N distributed amplifiers each operatively connected to one of the N output ports of the ASPD; and a power combiner having N input ports each operatively connected to one of the N distributed amplifiers. The distributed amplifier can included M stages of amplification by cascading superconducting active devices. The power combiner can include N active elements. The resulting (N.times.M) matrix amplifier can produce signals of high output power, large bandwidth, and low noise.

Cross-differential amplifier

NASA Technical Reports Server (NTRS)

Hajimiri, Seyed-Ali (Inventor); Kee, Scott D. (Inventor); Aoki, Ichiro (Inventor)

2008-01-01

A cross-differential amplifier is provided. The cross-differential amplifier includes an inductor connected to a direct current power source at a first terminal. A first and second switch, such as transistors, are connected to the inductor at a second terminal. A first and second amplifier are connected at their supply terminals to the first and second switch. The first and second switches are operated to commutate the inductor between the amplifiers so as to provide an amplified signal while limiting the ripple voltage on the inductor and thus limiting the maximum voltage imposed across the amplifiers and switches.

Significant variance in genetic diversity among populations of Schistosoma haematobium detected using microsatellite DNA loci from a genome-wide database.

PubMed

Glenn, Travis C; Lance, Stacey L; McKee, Anna M; Webster, Bonnie L; Emery, Aidan M; Zerlotini, Adhemar; Oliveira, Guilherme; Rollinson, David; Faircloth, Brant C

2013-10-17

Urogenital schistosomiasis caused by Schistosoma haematobium is widely distributed across Africa and is increasingly being targeted for control. Genome sequences and population genetic parameters can give insight into the potential for population- or species-level drug resistance. Microsatellite DNA loci are genetic markers in wide use by Schistosoma researchers, but there are few primers available for S. haematobium. We sequenced 1,058,114 random DNA fragments from clonal cercariae collected from a snail infected with a single Schistosoma haematobium miracidium. We assembled and aligned the S. haematobium sequences to the genomes of S. mansoni and S. japonicum, identifying microsatellite DNA loci across all three species and designing primers to amplify the loci in S. haematobium. To validate our primers, we screened 32 randomly selected primer pairs with population samples of S. haematobium. We designed >13,790 primer pairs to amplify unique microsatellite loci in S. haematobium, (available at http://www.cebio.org/projetos/schistosoma-haematobium-genome). The three Schistosoma genomes contained similar overall frequencies of microsatellites, but the frequency and length distributions of specific motifs differed among species. We identified 15 primer pairs that amplified consistently and were easily scored. We genotyped these 15 loci in S. haematobium individuals from six locations: Zanzibar had the highest levels of diversity; Malawi, Mauritius, Nigeria, and Senegal were nearly as diverse; but the sample from South Africa was much less diverse. About half of the primers in the database of Schistosoma haematobium microsatellite DNA loci should yield amplifiable and easily scored polymorphic markers, thus providing thousands of potential markers. Sequence conservation among S. haematobium, S. japonicum, and S. mansoni is relatively high, thus it should now be possible to identify markers that are universal among Schistosoma species (i.e., using DNA sequences

Ultrasensitive Electrochemical Detection of mRNA Using Branched DNA Amplifiers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mao, Xun; Liu, Guodong; Wang, Shengfu

2008-11-01

We describe here an ultrasensitive electrochemical detection of m RNA protocol without RNA purification and PCR amplification. The new m RNA electrical detection capability is coupled to the amplification feature of branched DNA (bDNA) technology and with the nagnetic beads based electrochemical bioassay.

Exposure of DNA bases induced by the interaction of DNA and calf thymus DNA helix-destabilizing protein.

PubMed Central

Kohwi-Shigematsu, T; Enomoto, T; Yamada, M A; Nakanishi, M; Tsuboi, M

1978-01-01

The reaction of chloroacetaldehyde with adenine bases in DNA to give a fluorescent product was used to study the availability to intermolecular reaction of positions 1 and 6 of adenine in DNA complexes with calf thymus DNA helix-destabilizing protein. No inhibition of this reaction was observed when heat-denatured DNA was complexed with the protein at a protein/DNA weight ratio of 10:1, compared to free DNA. On the contrary, the same reaction was inhibited markedly for denatured DNA in the presence of calf thymus histone HI at protein/DNA weight ratio of 2:1. Furthermore, the exchange rate for hydrogens of amino and imide groups of DNA bases in DNA strands with deuterium in the solvent was totally unaffected upon complexing of DNA with the DNA helix-destabilizing protein as examined by stopped-flow ultraviolet spectroscopy. These results indicate that the DNA helix-destabilizing protein forms a complex with single-stranded DNA, leaving DNA bases uncovered by the protein. The fluorescence intensity of DNA pretreated with chloroacetaldehyde was amplified by nearly 3-fold upon addition of the DNA helix-destabilizing protein. The possibility of "unstacking" of DNA bases induced by the protein is discussed. PMID:216994

Palindromic Sequence Artifacts Generated during Next Generation Sequencing Library Preparation from Historic and Ancient DNA

PubMed Central

Star, Bastiaan; Nederbragt, Alexander J.; Hansen, Marianne H. S.; Skage, Morten; Gilfillan, Gregor D.; Bradbury, Ian R.; Pampoulie, Christophe; Stenseth, Nils Chr; Jakobsen, Kjetill S.; Jentoft, Sissel

2014-01-01

Degradation-specific processes and variation in laboratory protocols can bias the DNA sequence composition from samples of ancient or historic origin. Here, we identify a novel artifact in sequences from historic samples of Atlantic cod (Gadus morhua), which forms interrupted palindromes consisting of reverse complementary sequence at the 5′ and 3′-ends of sequencing reads. The palindromic sequences themselves have specific properties – the bases at the 5′-end align well to the reference genome, whereas extensive misalignments exists among the bases at the terminal 3′-end. The terminal 3′ bases are artificial extensions likely caused by the occurrence of hairpin loops in single stranded DNA (ssDNA), which can be ligated and amplified in particular library creation protocols. We propose that such hairpin loops allow the inclusion of erroneous nucleotides, specifically at the 3′-end of DNA strands, with the 5′-end of the same strand providing the template. We also find these palindromes in previously published ancient DNA (aDNA) datasets, albeit at varying and substantially lower frequencies. This artifact can negatively affect the yield of endogenous DNA in these types of samples and introduces sequence bias. PMID:24608104

Ultrasensitive Biosensor for the Detection of Vibrio cholerae DNA with Polystyrene-co-acrylic Acid Composite Nanospheres

NASA Astrophysics Data System (ADS)

Rahman, Mahbubur; Heng, Lee Yook; Futra, Dedi; Ling, Tan Ling

2017-08-01

An ultrasensitive electrochemical biosensor for the determination of pathogenic Vibrio cholerae ( V. cholerae) DNA was developed based on polystyrene-co-acrylic acid (PSA) latex nanospheres-gold nanoparticles composite (PSA-AuNPs) DNA carrier matrix. Differential pulse voltammetry (DPV) using an electroactive anthraquninone oligonucleotide label was used for measuring the biosensor response. Loading of gold nanoparticles (AuNPs) on the DNA-latex particle electrode has significantly amplified the faradaic current of DNA hybridisation. Together with the use of a reported probe, the biosensor has demonstrated high sensitivity. The DNA biosensor yielded a reproducible and wide linear response range to target DNA from 1.0 × 10-21 to 1.0 × 10-8 M (relative standard deviation, RSD = 4.5%, n = 5) with a limit of detection (LOD) of 1.0 × 10-21 M ( R 2 = 0.99). The biosensor obtained satisfactory recovery values between 91 and 109% ( n = 3) for the detection of V. cholerae DNA in spiked samples and could be reused for six consecutive DNA assays with a repeatability RSD value of 5% ( n = 5). The electrochemical biosensor response was stable and maintainable at 95% of its original response up to 58 days of storage period.

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

Peptostreptococcus micros in primary endodontic infections as detected by 16S rDNA-based polymerase chain reaction.

PubMed

Siqueira, José F; Rôças, Isabela N; Andrade, Arnaldo F B; de Uzeda, Milton

2003-02-01

A 16S rDNA-based polymerase chain reaction (PCR) method was used to detect Peptostreptococcus micros in primary root canal infections. Samples were collected from 50 teeth having carious lesions, necrotic pulps, and different forms of periradicular diseases. DNA extracted from the samples was amplified using the PCR assay, which yielded a specific fragment of P. micros 16S rDNA. P. micros was detected in 6 of 22 root canals associated with asymptomatic chronic periradicular lesions (27.3%), 2 of 8 teeth with acute apical periodontitis (25%), and 6 of 20 cases of acute periradicular abscess (30%). In general, P. micros was found in 14 of 50 cases (28%). There was no correlation between the presence of P. micros and the occurrence of symptoms. Findings suggested that P. micros can be involved in the pathogenesis of different forms of periradicular lesions.

DR-78, a novel Drosophila melanogaster genomic DNA fragment highly homologous to the DNA-binding domain of thyroid hormone-retinoic acid-vitamin D receptor subfamily.

PubMed

Martín-Blanco, E; Kornberg, T B

1993-11-16

Degenerate oligodeoxyribonucleotides were designed for both ends of the DNA-binding domain of members of the nuclear receptor superfamily. PCR amplified Drosophila melanogaster DNA was purified and cloned (DR plasmids). Genomic lambda DASH clones were identified at high stringency with an amplified DR-78 plasmid DNA and isolated. The partial sequence shows a very probable open reading frame which would encode a peptide highly homologous to members of the thyroid hormone-retinoic acid-vitamin D receptor subfamily. The fragment corresponds to a single copy gene and was mapped at position 78D of chromosome three by in situ hybridization.

Persistence of dead-cell bacterial DNA in ex vivo root canals and influence of nucleases on DNA decay in vitro.

PubMed

Brundin, Malin; Figdor, David; Roth, Chrissie; Davies, John K; Sundqvist, Göran; Sjögren, Ulf

2010-12-01

The fate of DNA from bacteria that do not survive in the root canal is uncertain, yet DNA longevity may confound recovery of authentic etiologic participants in the disease process. This study assessed the recovery of PCR-detectable DNA in ex vivo human root canals and some environmental factors on the decay of microbial DNA. Heat-killed Enterococcus faecalis cells were inoculated into instrumented human root canals ex vivo, and samples were taken at intervals over 2 years and analyzed by polymerase chain reaction. In an in vitro assay, heat-killed E. faecalis cells and extracted E. faecalis DNA were inoculated into various media, DNase, and culture of a DNase-producing species, Prevotella intermedia. Recovery of DNA was assessed by gel electrophoresis. In ex vivo human teeth, amplifiable DNA was recovered after 1 and 2 years (in 14/15 and 21/25 teeth, respectively). In vitro experiments showed that extracted DNA incubated in different media (water, 10%-50% sera, and DNase) progressively decomposed to levels below the detection limit. In corresponding assays, cell-bound DNA was more resistant to decay. Amplifiable DNA is preserved after cell death, but the critical determinant is the form of DNA. Free DNA undergoes spontaneous and enzymatic decomposition, whereas cell-bound E. faecalis DNA persists for long periods. Copyright © 2010 Mosby, Inc. All rights reserved.

Isolation of PCR quality microbial community DNA from heavily contaminated environments.

PubMed

Gunawardana, Manjula; Chang, Simon; Jimenez, Abraham; Holland-Moritz, Daniel; Holland-Moritz, Hannah; La Val, Taylor P; Lund, Craig; Mullen, Madeline; Olsen, John; Sztain, Terra A; Yoo, Jennifer; Moss, John A; Baum, Marc M

2014-07-01

Asphalts, biochemically degraded oil, contain persistent, water-soluble compounds that pose a significant challenge to the isolation of PCR quality DNA. The adaptation of existing DNA purification protocols and commercial kits proved unsuccessful at overcoming this hurdle. Treatment of aqueous asphalt extracts with a polyamide resin afforded genomic microbial DNA templates that could readily be amplified by PCR. Physicochemically distinct asphalt samples from five natural oil seeps successfully generated the expected 291 bp amplicons targeting a region of the 16S rRNA gene, illustrating the robustness of the method. DNA recovery yields were in the 50-80% range depending on how the asphalt sample was seeded with exogenous DNA. The scope of the new method was expanded to include soil with high humic acid content. DNA from soil samples spiked with a range of humic acid concentrations was extracted with a commercial kit followed by treatment with the polyamide resin. The additional step significantly improved the purity of the DNA templates, especially at high humic acid concentrations, based on qPCR analysis of the bacterial 16S rRNA genes. The new method has the advantages of being inexpensive, simple, and rapid and should provide a valuable addition to protocols in the field of petroleum and soil microbiology. Copyright © 2014 Elsevier B.V. All rights reserved.

Multifrequency Raman amplifiers

NASA Astrophysics Data System (ADS)

Barth, Ido; Fisch, Nathaniel J.

2018-03-01

In its usual implementation, the Raman amplifier features only one pump carrier frequency. However, pulses with well-separated frequencies can also be Raman amplified while compressed in time. Amplification with frequency-separated pumps is shown to hold even in the highly nonlinear, pump-depletion regime, as derived through a fluid model, and demonstrated via particle-in-cell simulations. The resulting efficiency is similar to single-frequency amplifiers, but, due to the beat-wave waveform of both the pump lasers and the amplified seed pulses, these amplifiers feature higher seed intensities with a shorter spike duration. Advantageously, these amplifiers also suffer less noise backscattering, because the total fluence is split between the different spectral components.

PUMA amplifies necroptosis signaling by activating cytosolic DNA sensors

PubMed Central

Tong, Jingshan; Yang, Liheng; Wei, Liang; Stolz, Donna B.; Yu, Jian; Zhang, Jianke; Zhang, Lin

2018-01-01

Necroptosis, a form of regulated necrotic cell death, is governed by RIP1/RIP3-mediated activation of MLKL. However, the signaling process leading to necroptotic death remains to be elucidated. In this study, we found that PUMA, a proapoptotic BH3-only Bcl-2 family member, is transcriptionally activated in an RIP3/MLKL-dependent manner following induction of necroptosis. The induction of PUMA, which is mediated by autocrine TNF-α and enhanced NF-κB activity, contributes to necroptotic death in RIP3-expressing cells with caspases inhibited. On induction, PUMA promotes the cytosolic release of mitochondrial DNA and activation of the DNA sensors DAI/Zbp1 and STING, leading to enhanced RIP3 and MLKL phosphorylation in a positive feedback loop. Furthermore, deletion of PUMA partially rescues necroptosis-mediated developmental defects in FADD-deficient embryos. Collectively, our results reveal a signal amplification mechanism mediated by PUMA and cytosolic DNA sensors that is involved in TNF-driven necroptotic death in vitro and in vivo. PMID:29581256

Development of SCAR Markers for the DNA-Based Detection of the Asian Long-Horned Beetle; Anoplophora glabripennis (Motschulsky)

Treesearch

Damodar R. Kethidi; David B. Roden; Tim R. Ladd; Peter J. Krell; Arthur Ratnakaran; Qili Feng

2003-01-01

DNA markers were identified for the molecular detection of the Asian long-horned beetle (ALB), Anoplophora glabripennis (Mot.), based on sequence charaterized amplified regions (SCARS) derived from random amplified polymorphic DNA (RAPD) fragments. A 2,740-bp DNA fragment that was present only in ALB and not in other Cerambycids was identified after...

Modified salting-out method: high-yield, high-quality genomic DNA extraction from whole blood using laundry detergent.

PubMed

Nasiri, H; Forouzandeh, M; Rasaee, M J; Rahbarizadeh, F

2005-01-01

Different approaches have been used to extract DNA from whole blood. In most of these methods enzymes (such as proteinase K and RNAse A) or toxic organic solvents (such as phenol or guanidine isothiocyanate) are used. Since these enzymes are expensive, and most of the materials that are used routinely are toxic, it is desirable to apply an efficient DNA extraction procedure that does not require the use of such materials. In this study, genomic DNA was extracted by the salting-out method, but instead of using an analytical-grade enzyme and chemical detergents, as normally used for DNA isolation, a common laundry powder was used. Different concentrations of the powder were tested, and proteins were precipitated by NaCl-saturated distilled water. Finally, DNA precipitation was performed with the use of 96% ethanol. From the results, we conclude that the optimum concentration of laundry powder for the highest yield and purity of isolated DNA is 30 mg/mL. The procedure was optimized, and a final protocol is suggested. Following the same protocol, DNA was extracted from 100 blood samples, and their amounts were found to be >50 microg/mL of whole blood. The integrity of the DNA fragments was confirmed by agarose gel electrophoresis. Furthermore, the extracted DNA was used as a template for PCR reaction. The results obtained from PCR showed that the final solutions of extracted DNA did not contain any inhibitory material for the enzyme used in the PCR reaction, and indicated that the isolated DNA was of good quality. These results show that this method is simple, fast, safe, and cost-effective, and can be used in medical laboratories and research centers. Copyright 2005 Wiley-Liss, Inc.

Laser amplifier chain

DOEpatents

Hackel, R.P.

1992-10-20

A laser amplifier chain has a plurality of laser amplifiers arranged in a chain to sequentially amplify a low-power signal beam to produce a significantly higher-power output beam. Overall efficiency of such a chain is improved if high-gain, low efficiency amplifiers are placed on the upstream side of the chain where only a very small fraction of the total pumped power is received by the chain and low-gain, high-efficiency amplifiers are placed on the downstream side where a majority of pumping energy is received by the chain. 6 figs.

Laser amplifier chain

DOEpatents

Hackel, Richard P.

1992-01-01

A laser amplifier chain has a plurality of laser amplifiers arranged in a chain to sequentially amplify a low-power signal beam to produce a significantly higher-power output beam. Overall efficiency of such a chain is improved if high-gain, low efficiency amplifiers are placed on the upstream side of the chain where only a very small fraction of the total pumped power is received by the chain and low-gain, high-efficiency amplifiers are placed on the downstream side where a majority of pumping energy is received by the chain.

Directly transforming PCR-amplified DNA fragments into plant cells is a versatile system that facilitates the transient expression assay.

PubMed

Lu, Yuming; Chen, Xi; Wu, Yuxuan; Wang, Yanping; He, Yuqing; Wu, Yan

2013-01-01

A circular plasmid containing a gene coding sequence has been broadly used for studying gene regulation in cells. However, to accommodate a quick screen plasmid construction and preparation can be time consuming. Here we report a PCR amplified dsDNA fragments (PCR-fragments) based transient expression system (PCR-TES) for suiting in the study of gene regulation in plant cells. Instead of transforming plasmids into plant cells, transient expression of PCR-fragments can be applicable. The transformation efficiency and expression property of PCR-fragments are comparable to transformation using plasmids. We analyzed the transformation efficiency in PCR-TES at transcription and protein levels. Our results indicate that the PCR-TES is as versatile as the conventional transformation system using plasmid DNA. Through reconstituting PYR1-mediated ABA signaling pathway in Arabidopsis mesophyll protoplasts, we were not only validating the practicality of PCR-TES but also screening potential candidates of CDPK family members which might be involved in the ABA signaling. Moreover, we determined that phosphorylation of ABF2 by CPK4 could be mediated by ABA-induced PYR1 and ABI1, demonstrating a crucial role of CDPKs in the ABA signaling. In summary, PCR-TES can be applicable to facilitate analyzing gene regulation and for the screen of putative regulatory molecules at the high throughput level in plant cells.

Directly Transforming PCR-Amplified DNA Fragments into Plant Cells Is a Versatile System That Facilitates the Transient Expression Assay

PubMed Central

Lu, Yuming; Chen, Xi; Wu, Yuxuan; Wang, Yanping; He, Yuqing; Wu, Yan

2013-01-01

A circular plasmid containing a gene coding sequence has been broadly used for studying gene regulation in cells. However, to accommodate a quick screen plasmid construction and preparation can be time consuming. Here we report a PCR amplified dsDNA fragments (PCR-fragments) based transient expression system (PCR-TES) for suiting in the study of gene regulation in plant cells. Instead of transforming plasmids into plant cells, transient expression of PCR-fragments can be applicable. The transformation efficiency and expression property of PCR-fragments are comparable to transformation using plasmids. We analyzed the transformation efficiency in PCR-TES at transcription and protein levels. Our results indicate that the PCR-TES is as versatile as the conventional transformation system using plasmid DNA. Through reconstituting PYR1-mediated ABA signaling pathway in Arabidopsis mesophyll protoplasts, we were not only validating the practicality of PCR-TES but also screening potential candidates of CDPK family members which might be involved in the ABA signaling. Moreover, we determined that phosphorylation of ABF2 by CPK4 could be mediated by ABA-induced PYR1 and ABI1, demonstrating a crucial role of CDPKs in the ABA signaling. In summary, PCR-TES can be applicable to facilitate analyzing gene regulation and for the screen of putative regulatory molecules at the high throughput level in plant cells. PMID:23468926

DNA Book

PubMed Central

Kawai, Jun; Hayashizaki, Yoshihide

2003-01-01

We propose herein a new method of DNA distribution, whereby DNA clones or PCR products are printed directly onto the pages of books and delivered to users along with relevant scientific information. DNA sheets, comprising water-soluble paper onto which DNA is spotted, can be bound into books. Readers can easily extract the DNA fragments from DNA sheets and amplify them using PCR. We show that DNA sheets can withstand various conditions that may be experienced during bookbinding and delivery, such as high temperatures and humidity. Almost all genes (95%–100% of randomly selected RIKEN mouse cDNA clones) were recovered successfully by use of PCR. Readers can start their experiments after a 2-h PCR amplification without waiting for the delivery of DNA clones. The DNA Book thus provides a novel method for delivering DNA in a timely and cost-effective manner. A sample DNA sheet (carrying RIKEN mouse cDNA clones encoding genes of enzymes for the TCA cycle) is included in this issue for field-testing. We would greatly appreciate it if readers could attempt to extract DNA and report the results and whether the DNA sheet was shipped to readers in good condition. PMID:12819147

Checking of individuality by DNA profiling.

PubMed

Brdicka, R; Nürnberg, P

1993-08-25

A review of methods of DNA analysis used in forensic medicine for identification, paternity testing, etc. is provided. Among other techniques, DNA fingerprinting using different probes and polymerase chain reaction-based techniques such as amplified sequence polymorphisms and minisatellite variant repeat mapping are thoroughly described and both theoretical and practical aspects are discussed.

Multifrequency Raman amplifiers

DOE PAGES

Barth, Ido; Fisch, Nathaniel J.

2018-03-08

In its usual implementation, the Raman amplifier features only one pump carrier frequency. However, pulses with well-separated frequencies can also be Raman amplified while compressed in time. Amplification with frequency-separated pumps is shown to hold even in the highly nonlinear, pump-depletion regime, as derived through a fluid model, and demonstrated via particle-in-cell (PIC) simulations. The resulting efficiency is similar to single-frequency amplifiers, but, due to the beat-wave waveform of both the pump lasers and the amplified seed pulses, these amplifiers feature higher seed intensities with a shorter spike duration. Advantageously, these amplifiers also suffer less noise backscattering, because the totalmore » fluence is split between the different spectral components.« less

Multifrequency Raman amplifiers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barth, Ido; Fisch, Nathaniel J.

In its usual implementation, the Raman amplifier features only one pump carrier frequency. However, pulses with well-separated frequencies can also be Raman amplified while compressed in time. Amplification with frequency-separated pumps is shown to hold even in the highly nonlinear, pump-depletion regime, as derived through a fluid model, and demonstrated via particle-in-cell (PIC) simulations. The resulting efficiency is similar to single-frequency amplifiers, but, due to the beat-wave waveform of both the pump lasers and the amplified seed pulses, these amplifiers feature higher seed intensities with a shorter spike duration. Advantageously, these amplifiers also suffer less noise backscattering, because the totalmore » fluence is split between the different spectral components.« less

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.

Redox polymer and probe DNA tethered to gold electrodes for enzyme-amplified amperometric detection of DNA hybridization.

PubMed

Kavanagh, Paul; Leech, Dónal

2006-04-15

The detection of nucleic acids based upon recognition surfaces formed by co-immobilization of a redox polymer mediator and DNA probe sequences on gold electrodes is described. The recognition surface consists of a redox polymer, [Os(2,2'-bipyridine)2(polyvinylimidazole)(10)Cl](+/2+), and a model single DNA strand cross-linked and tethered to a gold electrode via an anchoring self-assembled monolayer (SAM) of cysteamine. Hybridization between the immobilized probe DNA of the recognition surface and a biotin-conjugated target DNA sequence (designed from the ssrA gene of Listeria monocytogenes), followed by addition of an enzyme (glucose oxidase)-avidin conjugate, results in electrical contact between the enzyme and the mediating redox polymer. In the presence of glucose, the current generated due to the catalytic oxidation of glucose to gluconolactone is measured, and a response is obtained that is binding-dependent. The tethering of the probe DNA and redox polymer to the SAM improves the stability of the surface to assay conditions of rigorous washing and high salt concentration (1 M). These conditions eliminate nonspecific interaction of both the target DNA and the enzyme-avidin conjugate with the recognition surfaces. The sensor response increases linearly with increasing concentration of target DNA in the range of 1 x 10(-9) to 2 x 10(-6) M. The detection limit is approximately 1.4 fmol, (corresponding to 0.2 nM of target DNA). Regeneration of the recognition surface is possible by treatment with 0.25 M NaOH solution. After rehybridization of the regenerated surface with the target DNA sequence, >95% of the current is recovered, indicating that the redox polymer and probe DNA are strongly bound to the surface. These results demonstrate the utility of the proposed approach.

Use of DNA markers in forest tree improvement research

Treesearch

D.B. Neale; M.E. Devey; K.D. Jermstad; M.R. Ahuja; M.C. Alosi; K.A. Marshall

1992-01-01

DNA markers are rapidly being developed for forest trees. The most important markers are restriction fragment length polymorphisms (RFLPs), polymerase chain reaction- (PCR) based markers such as random amplified polymorphic DNA (RAPD), and fingerprinting markers. DNA markers can supplement isozyme markers for monitoring tree improvement activities such as; estimating...

High preservation of DNA standards diluted in 50% glycerol.

PubMed

Schaudien, Dirk; Baumgärtner, Wolfgang; Herden, Christiane

2007-09-01

Standard curves are important tools in real-time quantitative polymerase chain reaction (PCR) to precisely analyze gene expression patterns under physiologic and pathologic conditions. Handling of DNA standards often implies multiple cycles of freezing and thawing that might affect DNA stability and integrity. This in turn might influence the reliability and reproducibility of quantitative measurements in real-time PCR assays. In this study, 3 DNA standards such as murine tumor necrosis factor (TNF) alpha, interferon (IFN) gamma, and kainat-1 receptor were diluted in 50% glycerol or water after 1, 4, and 16 cycles of freezing and thawing and amplified copy numbers after real-time PCR were compared. The standards diluted in water showed a reduction to 83%, 55%, and 50% after 4 cycles, to 24%, 5%, and 4% after 16 cycles for kainat-1 receptor, TNFalpha, and IFNgamma standards, respectively, when compared with a single cycle of freezing and thawing. Interestingly, all cDNA samples diluted in 50% glycerol were amplified in comparable copy numbers even after 16 cycles of freezing and thawing. The effect of the standards undergoing different cycles of freezing and thawing on sample values was demonstrated by amplifying cDNA obtained from Borna disease virus infected and noninfected TNF-transgenic mice brain. This revealed significant differences of measured cDNA copy numbers using water-diluted DNA standards. In contrast, sample values did not vary using glycerol-diluted standards that were frozen and thawed for 16 times. In conclusion, glycerol storage of DNA standards represents a suitable tool for the accurate and reproducible quantification of cDNA samples in real-time PCR analysis.

High-speed detection of DNA translocation in nanopipettes

NASA Astrophysics Data System (ADS)

Fraccari, Raquel L.; Ciccarella, Pietro; Bahrami, Azadeh; Carminati, Marco; Ferrari, Giorgio; Albrecht, Tim

2016-03-01

We present a high-speed electrical detection scheme based on a custom-designed CMOS amplifier which allows the analysis of DNA translocation in glass nanopipettes on a microsecond timescale. Translocation of different DNA lengths in KCl electrolyte provides a scaling factor of the DNA translocation time equal to p = 1.22, which is different from values observed previously with nanopipettes in LiCl electrolyte or with nanopores. Based on a theoretical model involving electrophoresis, hydrodynamics and surface friction, we show that the experimentally observed range of p-values may be the result of, or at least be affected by DNA adsorption and friction between the DNA and the substrate surface.We present a high-speed electrical detection scheme based on a custom-designed CMOS amplifier which allows the analysis of DNA translocation in glass nanopipettes on a microsecond timescale. Translocation of different DNA lengths in KCl electrolyte provides a scaling factor of the DNA translocation time equal to p = 1.22, which is different from values observed previously with nanopipettes in LiCl electrolyte or with nanopores. Based on a theoretical model involving electrophoresis, hydrodynamics and surface friction, we show that the experimentally observed range of p-values may be the result of, or at least be affected by DNA adsorption and friction between the DNA and the substrate surface. Electronic supplementary information (ESI) available: Gel electrophoresis confirming lengths and purity of DNA samples, comparison between Axopatch 200B and custom-built setup, comprehensive low-noise amplifier characterization, representative I-V curves of nanopipettes used, typical scatter plots of τ vs. peak amplitude for the four LDNA's used, table of most probable τ values, a comparison between different fitting models for the DNA translocation time distribution, further details on the stochastic numerical simulation of the scaling statistics and the derivation of the extended

Hybridization chain reaction-based instantaneous derivatization technology for chemiluminescence detection of specific DNA sequences.

PubMed

Wang, Xin; Lau, Choiwan; Kai, Masaaki; Lu, Jianzhong

2013-05-07

We propose here a new amplifying strategy that uses hybridization chain reaction (HCR) to detect specific sequences of DNA, where stable DNA monomers assemble on the magnetic beads only upon exposure to a target DNA. Briefly, in the HCR process, two complementary stable species of hairpins coexist in solution until the introduction of initiator reporter strands triggers a cascade of hybridization events that yield nicked double helices analogous to alternating copolymers. Moreover, a "sandwich-type" detection strategy is employed in our design. Magnetic beads, which are functionalized with capture DNA, are reacted with the target, and sandwiched with the above nicked double helices. Then, chemiluminescence (CL) detection proceeds via an instantaneous derivatization reaction between a specific CL reagent, 3,4,5-trimethoxylphenylglyoxal (TMPG), and the guanine nucleotides within the target DNA, reporter strands and DNA monomers for the generation of light. Our results clearly show that the amplification detection of specific sequences of DNA achieves a better performance (e.g. wide linear response range, low detection limit, and high specificity) as compared to the traditional sandwich type (capture/target/reporter) assays. Upon modification, the approach presented could be extended to detect other types of targets. We believe that this simple technique is promising for improving medical diagnosis and treatment.

Influence of heavy metal stress on antioxidant status and DNA damage in Urtica dioica.

PubMed

Gjorgieva, Darinka; Kadifkova Panovska, Tatjana; Ruskovska, Tatjana; Bačeva, Katerina; Stafilov, Trajče

2013-01-01

Heavy metals have the potential to interact and induce several stress responses in the plants; thus, effects of heavy metal stress on DNA damages and total antioxidants level in Urtica dioica leaves and stems were investigated. The samples are sampled from areas with different metal exposition. Metal content was analyzed by Inductively Coupled Plasma-Atomic Emission Spectrometer (ICP-AES), for total antioxidants level assessment the Ferric-Reducing Antioxidant Power (FRAP) assay was used, and genomic DNA isolation from frozen plant samples was performed to obtain DNA fingerprints of investigated plant. It was found that heavy metal contents in stems generally changed synchronously with those in leaves of the plant, and extraneous metals led to imbalance of mineral nutrient elements. DNA damages were investigated by Random Amplified Polymorphic DNA (RAPD) technique, and the results demonstrated that the samples exposed to metals yielded a large number of new fragments (total 12) in comparison with the control sample. This study showed that DNA stability is highly affected by metal pollution which was identified by RAPD markers. Results suggested that heavy metal stress influences antioxidant status and also induces DNA damages in U. dioica which may help to understand the mechanisms of metals genotoxicity.

Influence of Heavy Metal Stress on Antioxidant Status and DNA Damage in Urtica dioica

PubMed Central

Kadifkova Panovska, Tatjana; Bačeva, Katerina; Stafilov, Trajče

2013-01-01

Heavy metals have the potential to interact and induce several stress responses in the plants; thus, effects of heavy metal stress on DNA damages and total antioxidants level in Urtica dioica leaves and stems were investigated. The samples are sampled from areas with different metal exposition. Metal content was analyzed by Inductively Coupled Plasma-Atomic Emission Spectrometer (ICP-AES), for total antioxidants level assessment the Ferric-Reducing Antioxidant Power (FRAP) assay was used, and genomic DNA isolation from frozen plant samples was performed to obtain DNA fingerprints of investigated plant. It was found that heavy metal contents in stems generally changed synchronously with those in leaves of the plant, and extraneous metals led to imbalance of mineral nutrient elements. DNA damages were investigated by Random Amplified Polymorphic DNA (RAPD) technique, and the results demonstrated that the samples exposed to metals yielded a large number of new fragments (total 12) in comparison with the control sample. This study showed that DNA stability is highly affected by metal pollution which was identified by RAPD markers. Results suggested that heavy metal stress influences antioxidant status and also induces DNA damages in U. dioica which may help to understand the mechanisms of metals genotoxicity. PMID:23862140

DNA methylation in sugarcane somaclonal variants assessed through methylation-sensitive amplified polymorphism.

PubMed

Francischini, J H M B; Kemper, E L; Costa, J B; Manechini, J R V; Pinto, L R

2017-05-04

Micropropagation is an important tool for large-scale multiplication of plant superior genotypes. However, somaclonal variation is one of the drawbacks of this process. Changes in DNA methylation have been widely reported as one of the main causes of somaclonal variations in plants. In order to investigate the occurrence of changes in the methylation pattern of sugarcane somaclonal variants, the MSAP (methylation-sensitive amplified polymorphism) technique was applied to micro-propagated plantlets sampled at the third subculture phase. The mother plant, in vitro normal plantlets, and in vitro abnormal plantlets (somaclonal variants) of four sugarcane clones were screened against 16 MSAP selective primers for EcoRI/MspI and EcoRI/HpaII restriction enzymes. A total of 1005 and 1200 MSAP-derived markers with polymorphism percentages of 28.36 and 40.67 were obtained for EcoRI/HpaII and EcoRI/MspI restriction enzyme combinations, respectively. The genetic similarity between the mother plant and the somaclonal variants ranged from 0.877 to 0.911 (EcoRI/MspI) and from 0.928 to 0.955 (EcoRI/HpaII). Most of the MASPs among mother plant and micro-propagated plantlets were derived from EcoRI/MspI restriction enzymes suggesting alteration due to gain or loss of internal cytosine methylation. A higher rate of loss of methylation (hypomethylation) than gain of methylation (hypermethylation) was observed in the abnormal in vitro sugarcane plantlets. Although changes in the methylation pattern were also observed in the in vitro normal plantlets, they were lower than those observed for the in vitro abnormal plantlets. The MASP technique proved to be a promising tool to early assessment of genetic fidelity of micro-propagated sugarcane plants.

Primers for polymerase chain reaction to detect genomic DNA of Toxocara canis and T. cati.

PubMed

Wu, Z; Nagano, I; Xu, D; Takahashi, Y

1997-03-01

Primers for polymerase chain reaction to amplify genomic DNA of both Toxocara canis and T. cati were constructed by adapting cloning and sequencing random amplified polymorphic DNA. The primers are expected to detect eggs and/or larvae of T. canis and T. cati, both of which are known to cause toxocariasis in humans.

Auto-Zero Differential Amplifier

NASA Technical Reports Server (NTRS)

Quilligan, Gerard T. (Inventor); Aslam, Shahid (Inventor)

2017-01-01

An autozero amplifier may include a window comparator network to monitor an output offset of a differential amplifier. The autozero amplifier may also include an integrator to receive a signal from a latched window comparator network, and send an adjustment signal back to the differential amplifier to reduce an offset of the differential amplifier.

Genotyping by randomly amplified polymorphic DNA of bacteriocin producing Lactobacillus acidophilus strains from Nigeria.

PubMed

Alli, John Adeolu; Iwalokun, Bamidele A; Oluwadun, Afolabi; Okonko, Iheanyi Omezuruike

2015-01-01

Yogurt and starter culture producers are still searching strains of Lactobacillus acidophilus to produce healthier yogurt with a longer shelf life and better texture, taste, and quality. This study determined the genotyping of bacteriocin producing Lactobacillus acidophilus strains recovered from Nigerian yogurts. Yogurt samples were collected from four different states of South West regions of Nigeria. Isolates were obtained from MRS Medium and biochemically characterized. This was further confirmed by API50CH. The bacteriocin positivity and activity was determined. Genomic characterization of our Lactobacillus acidophilus strains was done with randomly amplified polymorphic DNA-PCR. All yogurt samples containing Lactobacillus acidophilus strains meet the probiotic requirement of ≥10(6) cfu/mL. The gel picture revealed 6 RAPD clonal types of Lactobacillus acidophilus strains with RAPD type C observed to be more common. Significant differences existed in the mean growth inhibition zone (t = -7.32, P < 0.05 for E. coli ATCC; t = -6.19, P < 0.05 for E. coli clinical isolates; t = -6.16, P < 0.05 for Enterobacter sp; t = -11.92, P < 0.05 for Salmonella typhi, t = -1.10, P > 0.05 Staphylococcus aureus). No correlation between the bacteriocin production, activity, and their RAPD clonal division (X(2) = 7.49, P = 0.1610, df = 5). In conclusion, L. acidophilus isolated in Nigeria samples met the probiotic requirements of ≥10(6) cfu/mL and produce bacteriocins with good spectrum of activity.

Development and characterization of 79 nuclear markers amplifying in viviparous and oviparous clades of the European common lizard.

PubMed

Horreo, J L; Peláez, M L; Suárez, T; Fitze, P S

2018-02-01

The European common lizard (Zootoca vivipara) is a widely distributed species across Europe and Asia exhibiting two reproductive modes (oviparity/viviparity), six major lineages and several sublineages. It has been used to tackle a large variety of research questions, nevertheless, few nuclear DNA sequence markers have been developed for this species. Here we developed 79 new nuclear DNA sequence markers using a clonation protocol. These markers were amplified in several oviparous and viviparous specimens including samples of all extant clades, to test the amplification success and their diversity. 49.4% of the markers were polymorphic and of those, 51.3% amplified in all and 94.9% amplified in 5-7 of the extant Z. vivipara clades. These new markers will be very useful for the study of the population structure, population dynamics, and micro/macro evolution of Z. vivipara. Cross-species amplification in four lizard species (Psammodromus edwardsianus, Podarcis muralis, Lacerta bilineata, and Takydromus sexlineatus) was positive in several of the markers, and six makers amplified in all five species. The large genetic distance between P. edwardsianus and Z. vivipara further suggests that these markers may as well be employed in many other species.

Optimized DNA extraction from neonatal dried blood spots: application in methylome profiling

PubMed Central

2014-01-01

Background Neonatal dried blood spots (DBS) represent an inexpensive method for long-term biobanking worldwide and are considered gold mines for research for several human diseases, including those of metabolic, infectious, genetic and epigenetic origin. However, the utility of DBS is restricted by the limited amount and quality of extractable biomolecules (including DNA), especially for genome wide profiling. Degradation of DNA in DBS often occurs during storage and extraction. Moreover, amplifying small quantities of DNA often leads to a bias in subsequent data, particularly in methylome profiles. Thus it is important to develop methodologies that maximize both the yield and quality of DNA from DBS for downstream analyses. Results Using combinations of in-house-derived and modified commercial extraction kits, we developed a robust and efficient protocol, compatible with methylome studies, many of which require stringent bisulfite conversion steps. Several parameters were tested in a step-wise manner, including blood extraction, cell lysis, protein digestion, and DNA precipitation, purification and elution. DNA quality was assessed based on spectrophotometric measurements, DNA detectability by PCR, and DNA integrity by gel electrophoresis and bioanalyzer analyses. Genome scale Infinium HumanMethylation450 and locus-specific pyrosequencing data generated using the refined DBS extraction protocol were of high quality, reproducible and consistent. Conclusions This study may prove useful to meet the increased demand for research on prenatal, particularly epigenetic, origins of human diseases and for newborn screening programs, all of which are often based on DNA extracted from DBS. PMID:24980254

Mitochondrial DNA mutations in single human blood cells.

PubMed

Yao, Yong-Gang; Kajigaya, Sachiko; Young, Neal S

2015-09-01

Determination mitochondrial DNA (mtDNA) sequences from extremely small amounts of DNA extracted from tissue of limited amounts and/or degraded samples is frequently employed in medical, forensic, and anthropologic studies. Polymerase chain reaction (PCR) amplification followed by DNA cloning is a routine method, especially to examine heteroplasmy of mtDNA mutations. In this review, we compare the mtDNA mutation patterns detected by three different sequencing strategies. Cloning and sequencing methods that are based on PCR amplification of DNA extracted from either single cells or pooled cells yield a high frequency of mutations, partly due to the artifacts introduced by PCR and/or the DNA cloning process. Direct sequencing of PCR product which has been amplified from DNA in individual cells is able to detect the low levels of mtDNA mutations present within a cell. We further summarize the findings in our recent studies that utilized this single cell method to assay mtDNA mutation patterns in different human blood cells. Our data show that many somatic mutations observed in the end-stage differentiated cells are found in hematopoietic stem cells (HSCs) and progenitors within the CD34(+) cell compartment. Accumulation of mtDNA variations in the individual CD34+ cells is affected by both aging and family genetic background. Granulocytes harbor higher numbers of mutations compared with the other cells, such as CD34(+) cells and lymphocytes. Serial assessment of mtDNA mutations in a population of single CD34(+) cells obtained from the same donor over time suggests stability of some somatic mutations. CD34(+) cell clones from a donor marked by specific mtDNA somatic mutations can be found in the recipient after transplantation. The significance of these findings is discussed in terms of the lineage tracing of HSCs, aging effect on accumulation of mtDNA mutations and the usage of mtDNA sequence in forensic identification. Copyright © 2015 Elsevier B.V. All rights

Successful amplification of DNA aboard the International Space Station.

PubMed

Boguraev, Anna-Sophia; Christensen, Holly C; Bonneau, Ashley R; Pezza, John A; Nichols, Nicole M; Giraldez, Antonio J; Gray, Michelle M; Wagner, Brandon M; Aken, Jordan T; Foley, Kevin D; Copeland, D Scott; Kraves, Sebastian; Alvarez Saavedra, Ezequiel

2017-01-01

As the range and duration of human ventures into space increase, it becomes imperative that we understand the effects of the cosmic environment on astronaut health. Molecular technologies now widely used in research and medicine will need to become available in space to ensure appropriate care of astronauts. The polymerase chain reaction (PCR) is the gold standard for DNA analysis, yet its potential for use on-orbit remains under-explored. We describe DNA amplification aboard the International Space Station (ISS) through the use of a miniaturized miniPCR system. Target sequences in plasmid, zebrafish genomic DNA, and bisulfite-treated DNA were successfully amplified under a variety of conditions. Methylation-specific primers differentially amplified bisulfite-treated samples as would be expected under standard laboratory conditions. Our findings establish proof of concept for targeted detection of DNA sequences during spaceflight and lay a foundation for future uses ranging from environmental monitoring to on-orbit diagnostics.

Low noise tuned amplifier

NASA Technical Reports Server (NTRS)

Kleinberg, L. L. (Inventor)

1984-01-01

A bandpass amplifier employing a field effect transistor amplifier first stage is described with a resistive load either a.c. or directly coupled to the non-inverting input of an operational amplifier second stage which is loaded in a Wien Bridge configuration. The bandpass amplifier may be operated with a signal injected into the gate terminal of the field effect transistor and the signal output taken from the output terminal of the operational amplifier. The operational amplifier stage appears as an inductive reactance, capacitive reactance and negative resistance at the non-inverting input of the operational amplifier, all of which appear in parallel with the resistive load of the field effect transistor.

Ancient Mitochondrial DNA Analyses of Ascaris Eggs Discovered in Coprolites from Joseon Tomb

PubMed Central

Oh, Chang Seok; Seo, Min; Hong, Jong Ha; Chai, Jong-Yil; Oh, Seung Whan; Park, Jun Bum; Shin, Dong Hoon

2015-01-01

Analysis of ancient DNA (aDNA) extracted from Ascaris is very important for understanding the phylogenetic lineage of the parasite species. When aDNAs obtained from a Joseon tomb (SN2-19-1) coprolite in which Ascaris eggs were identified were amplified with primers for cytochrome b (cyt b) and 18S small subunit ribosomal RNA (18S rRNA) gene, the outcome exhibited Ascaris specific amplicon bands. By cloning, sequencing, and analysis of the amplified DNA, we obtained information valuable for comprehending genetic lineage of Ascaris prevalent among pre-modern Joseon peoples. PMID:25925186

Digital droplet multiple displacement amplification (ddMDA) for whole genome sequencing of limited DNA samples

DOE PAGES

Rhee, Minsoung; Light, Yooli K.; Meagher, Robert J.; ...

2016-05-04

Here, multiple displacement amplification (MDA) is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples) before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA) technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently,more » the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet), ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli) compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology.« less

Digital droplet multiple displacement amplification (ddMDA) for whole genome sequencing of limited DNA samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rhee, Minsoung; Light, Yooli K.; Meagher, Robert J.

Here, multiple displacement amplification (MDA) is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples) before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA) technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently,more » the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet), ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli) compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology.« less

Portable musical instrument amplifier

DOEpatents

Christian, David E.

1990-07-24

The present invention relates to a musical instrument amplifier which is particularly useful for electric guitars. The amplifier has a rigid body for housing both the electronic system for amplifying and processing signals from the guitar and the system's power supply. An input plug connected to and projecting from the body is electrically coupled to the signal amplifying and processing system. When the plug is inserted into an output jack for an electric guitar, the body is rigidly carried by the guitar, and the guitar is operatively connected to the electrical amplifying and signal processing system without use of a loose interconnection cable. The amplifier is provided with an output jack, into which headphones are plugged to receive amplified signals from the guitar. By eliminating the conventional interconnection cable, the amplifier of the present invention can be used by musicians with increased flexibility and greater freedom of movement.

A new mutation in the CFTR gene, composed of two adjacent DNA alterations, is a common cause of cystic fibrosis among Georgian Jews

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shoshani, T.; Berkun, Y.; Yahav, Y.

Five Jewish cystic fibrosis (CF) patients from four unrelated families, all of whom emigrated from what was Soviet Georgia were studied. The parents in two of the families are first-degree relatives. The clinical phenotype of the patients seems to be associated with a severe disease, as reflected by early age of diagnosis (before the age of 1 year), high sweat chloride level (105-140 meq/liter), and pancreatic insufficiency. The pulmonary function and nutritional status of these patients are normal. These patients were tested for [Delta]F508 by analysis of heteroduplex DNA (4). None of the CF chromosomes was found to carry themore » [Delta]F508 mutation. Subsequently, PCR-amplified genomic DNA samples from two of these patients were subjected to direct sequencing (5) of regions containing exons 7, 9-12, an 19-21 of the CF gene using the oligonucleotides previously described (3, 6). In exon 7, two DNA alterations 3 bp apart were identified in both patients. The first alteration in a C [yields] A transversion at nucleotide position 1207, changing the glutamine codon to lysine (Q359K). The second DNA alteration is a C [yields] A transversion at nucleotide position 1211 changing the threonine codon to lysine (T360K). The two DNA alterations cause nonconservative amino acid substitutions, changing each of the two uncharged polar amino acids (glutamine and threonine) to a basic amino acid, lysine. The Q359K substitution destroys an Rsal recognition site and can be detected by PCR amplification of exon 7 using 7i-5 and 7i-3 oligonucleotides (6), followed by Rsal digestion and electrophoresis on 10% polyacrylamide gels. Two Rsal sites are found in a normal amplified DNA fragment, resulting in three restriction fragments of 292, 68, and 50 bp. Digestion of the PCR fragment of an individual homozygous for this substitution resulted in only two fragments of 342 and 68 bp. 6 refs., 3 figs.« less

Isolation of a sex-linked DNA sequence in cranes.

PubMed

Duan, W; Fuerst, P A

2001-01-01

A female-specific DNA fragment (CSL-W; crane sex-linked DNA on W chromosome) was cloned from female whooping cranes (Grus americana). From the nucleotide sequence of CSL-W, a set of polymerase chain reaction (PCR) primers was identified which amplify a 227-230 bp female-specific fragment from all existing crane species and some other noncrane species. A duplicated versions of the DNA segment, which is found to have a larger size (231-235 bp) than CSL-W in both sexes, was also identified, and was designated CSL-NW (crane sex-linked DNA on non-W chromosome). The nucleotide similarity between the sequences of CSL-W and CSL-NW from whooping cranes was 86.3%. The CSL primers do not amplify any sequence from mammalian DNA, limiting the potential for contamination from human sources. Using the CSL primers in combination with a quick DNA extraction method allows the noninvasive identification of crane gender in less than 10 h. A test of the methodology was carried out on fully developed body feathers from 18 captive cranes and resulted in 100% successful identification.

Detection of sequence variation in parasite ribosomal DNA by electrophoresis in agarose gels supplemented with a DNA-intercalating agent.

PubMed

Zhu, X Q; Chilton, N B; Gasser, R B

1998-05-01

This study evaluated the use of a commercially available DNA intercalating agent (Resolver Gold) in agarose gels for the direct detection of sequence variation in ribosomal DNA (rDNA). This agent binds preferentially to AT sequence motifs in DNA. Regions of nuclear rDNA, known to provide genetic markers for the identification of species of parasitic ascarid nematodes (order Ascaridida), were amplified by polymerase chain reaction (PCR) and subjected to electrophoresis in standard agarose gels versus gels supplemented with Resolver Gold. Individual taxa examined could not be distinguished reliably based on the size of their amplicons in standard agarose gels, whereas they could be readily delineated based on mobility using Resolver Gold-supplemented gels. The latter was achieved because of differences (approximately 0.1-8.2%) in the AT content of the fragments among different taxa, which were associated with significant interspecific differences (approximately 11-39%) in the rDNA sequences employed. There was a tendency for fragments with higher AT content to migrate slower in supplemented agarose gels compared with those of lower AT content. The results indicate the usefulness of this electrophoretic approach to rapidly screen for sequence variability within or among PCR-amplified rDNA fragments of similar sizes but differing AT contents. Although evaluated on rDNA of parasites, the approach has potential to be applied to a range of genes of different groups of infectious organisms.

DNA degrades during storage in formalin-fixed and paraffin-embedded tissue blocks.

PubMed

Guyard, Alice; Boyez, Alice; Pujals, Anaïs; Robe, Cyrielle; Tran Van Nhieu, Jeanne; Allory, Yves; Moroch, Julien; Georges, Odette; Fournet, Jean-Christophe; Zafrani, Elie-Serge; Leroy, Karen

2017-10-01

Formalin-fixed paraffin-embedded (FFPE) tissue blocks are widely used to identify clinically actionable molecular alterations or perform retrospective molecular studies. Our goal was to quantify degradation of DNA occurring during mid to long-term storage of samples in usual conditions. We selected 46 FFPE samples of surgically resected carcinomas of lung, colon, and urothelial tract, of which DNA had been previously extracted. We performed a second DNA extraction on the same blocks under identical conditions after a median period of storage of 5.5 years. Quantitation of DNA by fluorimetry showed a 53% decrease in DNA quantity after storage. Quantitative PCR (qPCR) targeting KRAS exon 2 showed delayed amplification of DNA extracted after storage in all samples but one. The qPCR/fluorimetry quantification ratio decreased from 56 to 15% after storage (p < 0.001). Overall, remaining proportion of DNA analyzable by qPCR represented only 11% of the amount obtained at first extraction. Maximal length of amplifiable DNA fragments assessed with a multiplex PCR was reduced in DNA extracted from stored tissue, indicating that DNA fragmentation had increased in the paraffin blocks during storage. Next-generation sequencing was performed on 12 samples and showed a mean 3.3-fold decrease in library yield and a mean 4.5-fold increase in the number of single-nucleotide variants detected after storage. In conclusion, we observed significant degradation of DNA extracted from the same FFPE block after 4 to 6 years of storage. Better preservation strategies should be considered for storage of FFPE biopsy specimens.

Modified midi- and mini-multiplex PCR systems for mitochondrial DNA control region sequence analysis in degraded samples.

PubMed

Kim, Na Young; Lee, Hwan Young; Park, Sun Joo; Yang, Woo Ick; Shin, Kyoung-Jin

2013-05-01

Two multiplex polymerase chain reaction (PCR) systems (Midiplex and Miniplex) were developed for the amplification of the mitochondrial DNA (mtDNA) control region, and the efficiencies of the multiplexes for amplifying degraded DNA were validated using old skeletal remains. The Midiplex system consisted of two multiplex PCRs to amplify six overlapping amplicons ranging in length from 227 to 267 bp. The Miniplex system consisted of three multiplex PCRs to amplify 10 overlapping short amplicons ranging in length from 142 to 185 bp. Most mtDNA control region sequences of several 60-year-old and 400-500-year-old skeletal remains were successfully obtained using both PCR systems and consistent with those previously obtained by monoplex amplification. The multiplex system consisting of smaller amplicons is effective for mtDNA sequence analyses of ancient and forensic degraded samples, saving time, cost, and the amount of DNA sample consumed during analysis. © 2013 American Academy of Forensic Sciences.

High-gain cryogenic amplifier assembly employing a commercial CMOS operational amplifier.

PubMed

Proctor, J E; Smith, A W; Jung, T M; Woods, S I

2015-07-01

We have developed a cryogenic amplifier for the measurement of small current signals (10 fA-100 nA) from cryogenic optical detectors. Typically operated with gain near 10(7) V/A, the amplifier performs well from DC to greater than 30 kHz and exhibits noise level near the Johnson limit. Care has been taken in the design and materials to control heat flow and temperatures throughout the entire detector-amplifier assembly. A simple one-board version of the amplifier assembly dissipates 8 mW to our detector cryostat cold stage, and a two-board version can dissipate as little as 17 μW to the detector cold stage. With current noise baseline of about 10 fA/(Hz)(1/2), the cryogenic amplifier is generally useful for cooled infrared detectors, and using blocked impurity band detectors operated at 10 K, the amplifier enables noise power levels of 2.5 fW/(Hz)(1/2) for detection of optical wavelengths near 10 μm.

Sequence-related amplified polymorphism (SRAP) markers: A potential resource for studies in plant molecular biology(1.).

PubMed

Robarts, Daniel W H; Wolfe, Andrea D

2014-07-01

In the past few decades, many investigations in the field of plant biology have employed selectively neutral, multilocus, dominant markers such as inter-simple sequence repeat (ISSR), random-amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP) to address hypotheses at lower taxonomic levels. More recently, sequence-related amplified polymorphism (SRAP) markers have been developed, which are used to amplify coding regions of DNA with primers targeting open reading frames. These markers have proven to be robust and highly variable, on par with AFLP, and are attained through a significantly less technically demanding process. SRAP markers have been used primarily for agronomic and horticultural purposes, developing quantitative trait loci in advanced hybrids and assessing genetic diversity of large germplasm collections. Here, we suggest that SRAP markers should be employed for research addressing hypotheses in plant systematics, biogeography, conservation, ecology, and beyond. We provide an overview of the SRAP literature to date, review descriptive statistics of SRAP markers in a subset of 171 publications, and present relevant case studies to demonstrate the applicability of SRAP markers to the diverse field of plant biology. Results of these selected works indicate that SRAP markers have the potential to enhance the current suite of molecular tools in a diversity of fields by providing an easy-to-use, highly variable marker with inherent biological significance.

Sequence-related amplified polymorphism (SRAP) markers: A potential resource for studies in plant molecular biology1

PubMed Central

Robarts, Daniel W. H.; Wolfe, Andrea D.

2014-01-01

In the past few decades, many investigations in the field of plant biology have employed selectively neutral, multilocus, dominant markers such as inter-simple sequence repeat (ISSR), random-amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP) to address hypotheses at lower taxonomic levels. More recently, sequence-related amplified polymorphism (SRAP) markers have been developed, which are used to amplify coding regions of DNA with primers targeting open reading frames. These markers have proven to be robust and highly variable, on par with AFLP, and are attained through a significantly less technically demanding process. SRAP markers have been used primarily for agronomic and horticultural purposes, developing quantitative trait loci in advanced hybrids and assessing genetic diversity of large germplasm collections. Here, we suggest that SRAP markers should be employed for research addressing hypotheses in plant systematics, biogeography, conservation, ecology, and beyond. We provide an overview of the SRAP literature to date, review descriptive statistics of SRAP markers in a subset of 171 publications, and present relevant case studies to demonstrate the applicability of SRAP markers to the diverse field of plant biology. Results of these selected works indicate that SRAP markers have the potential to enhance the current suite of molecular tools in a diversity of fields by providing an easy-to-use, highly variable marker with inherent biological significance. PMID:25202637

K-sam, an amplified gene in stomach cancer, is a member of the heparin-binding growth factor receptor genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hattori, Yutaka; Odagiri, Hiroki; Nakatani, Hiroshi

1990-08-01

DNA fragments amplified in a stomach cancer-derived cell line, KATO-III, were previously identified by the in-gel DNA renaturation method, and a 0.2-kilobase-pair fragment of the amplified sequence was subsequently cloned. By genomic walking, a portion of the exon of the gene flanking this 0.2-kilobase-pair fragment was cloned, and the gene was designated as K-sam ({und K}ATO-III cell-derived {und s}tomach cancer {und am}plified gene). The K-sam cDNAs, corresponding to the 3.5-kilobase K-sam mRNA, were cloned from the KATO-III cells. Sequence analysis revealed that this gene coded for 682 amino acid residues that satisfied the characteristics of the receptor tyrosine kinase. Themore » K-sam gene had significant homologies with bek, FLG, and chicken basic fibroblast growth factor receptor gene. The K-sam gene was amplified in KATO-III cells with the major transcript of 3.5-kilobases in size. This gene was also expressed in some other stomach cancer cells, a small cell lung cancer, and germ cell tumors.« less

Multiplex Amplification Coupled with COLD-PCR and High Resolution Melting Enables Identification of Low-Abundance Mutations in Cancer Samples with Low DNA Content

PubMed Central

Milbury, Coren A.; Chen, Clark C.; Mamon, Harvey; Liu, Pingfang; Santagata, Sandro; Makrigiorgos, G. Mike

2011-01-01

Thorough screening of cancer-specific biomarkers, such as DNA mutations, can require large amounts of genomic material; however, the amount of genomic material obtained from some specimens (such as biopsies, fine-needle aspirations, circulating-DNA or tumor cells, and histological slides) may limit the analyses that can be performed. Furthermore, mutant alleles may be at low-abundance relative to wild-type DNA, reducing detection ability. We present a multiplex-PCR approach tailored to amplify targets of interest from small amounts of precious specimens, for extensive downstream detection of low-abundance alleles. Using 3 ng of DNA (1000 genome-equivalents), we amplified the 1 coding exons (2-11) of TP53 via multiplex-PCR. Following multiplex-PCR, we performed COLD-PCR (co-amplification of major and minor alleles at lower denaturation temperature) to enrich low-abundance variants and high resolution melting (HRM) to screen for aberrant melting profiles. Mutation-positive samples were sequenced. Evaluation of mutation-containing dilutions revealed improved sensitivities after COLD-PCR over conventional-PCR. COLD-PCR improved HRM sensitivity by approximately threefold to sixfold. Similarly, COLD-PCR improved mutation identification in sequence-chromatograms over conventional PCR. In clinical specimens, eight mutations were detected via conventional-PCR-HRM, whereas 12 were detected by COLD-PCR-HRM, yielding a 33% improvement in mutation detection. In summary, we demonstrate an efficient approach to increase screening capabilities from limited DNA material via multiplex-PCR and improve mutation detection sensitivity via COLD-PCR amplification. PMID:21354058

High stability amplifier

NASA Technical Reports Server (NTRS)

Adams, W. A.; Reinhardt, V. S. (Inventor)

1983-01-01

An electrical RF signal amplifier for providing high temperature stability and RF isolation and comprised of an integrated circuit voltage regulator, a single transistor, and an integrated circuit operational amplifier mounted on a circuit board such that passive circuit elements are located on side of the circuit board while the active circuit elements are located on the other side is described. The active circuit elements are embedded in a common heat sink so that a common temperature reference is provided for changes in ambient temperature. The single transistor and operational amplifier are connected together to form a feedback amplifier powered from the voltage regulator with transistor implementing primarily the desired signal gain while the operational amplifier implements signal isolation. Further RF isolation is provided by the voltage regulator which inhibits cross-talk from other like amplifiers powered from a common power supply. Input and output terminals consisting of coaxial connectors are located on the sides of a housing in which all the circuit components and heat sink are located.

Dendritic Cell-Based Immunotherapy of Breast Cancer: Modulation by CpG DNA

DTIC Science & Technology

2005-09-01

tumor-associated antigens and bacterial DNA oligodeoxynucleotides containing unmethylated CpG sequences (CpG DNA) further augment the immune priming...associated antigens by cytotoxic T lymphocytes, and bacterial DNA oligodeoxy- nucleotides containing unmethylated CpG sequences (CpG DNA) can further...further amplify their immunostimulatory capacity and bacterial DNA oligodeoxynucleotides (ODN) containing unmethylated CpG sequences (CpG DNA) provide such

Increased yield of PCR products by addition of T4 gene 32 protein to the SMART PCR cDNA synthesis system.

PubMed

Villalva, C; Touriol, C; Seurat, P; Trempat, P; Delsol, G; Brousset, P

2001-07-01

Under certain conditions, T4 gene 32 protein is known to increase the efficiency of different enzymes, such as Taq DNA polymerase, reverse transcriptase, and telomerase. In this study, we compared the efficiency of the SMART PCR cDNA synthesis kit with and without the T4 gene 32 protein. The use of this cDNA synthesis procedure, in combination with T4 gene 32 protein, increases the yield of RT-PCR products from approximately 90% to 150%. This effect is even observed for long mRNA templates and low concentrations of total RNA (25 ng). Therefore, we suggest the addition of T4 gene 32 protein in the RT-PCR mixture to increase the efficiency of cDNA synthesis, particularly in cases when low amounts of tissue are used.

Histological analysis and ancient DNA amplification of human bone remains found in caius iulius polybius house in pompeii.

PubMed

Cipollaro, M; Di Bernado, G; Forte, A; Galano, G; De Masi, L; Galderisi, U; Guarino, F M; Angelini, F; Cascino, A

1999-09-01

Thirteen skeletons found in the Caius Iulius Polybius house, which has been the object of intensive study since its discovery in Pompeii 250 years ago, have provided an opportunity to study either bone diagenesis by histological investigation or ancient DNA by polymerase chain reaction analysis. DNA analysis was done by amplifying both X- and Y-chromosomes amelogenin loci and Y-specific alphoid repeat locus. The von Willebrand factor (vWF) microsatellite locus on chromosome 12 was also analyzed for personal identification in two individuals showing alleles with 10/11 and 12/12 TCTA repeats, respectively. Technical problems were the scarcity of DNA content from osteocytes, DNA molecule fragmentation, microbial contamination which change bone structure, contaminating human DNA which results from mishandling, and frequent presence of Taq DNA polymerase inhibiting molecules like polyphenols and heavy metals. The results suggest that the remains contain endogenous human DNA that can be amplified and analyzed. The amplifiability of DNA corresponds to the bone preservation and dynamics of the burial conditions subsequent to the 79 A.D. eruption.

Protein Detection via Direct Enzymatic Amplification of Short DNA Aptamers

PubMed Central

Fischer, Nicholas O.; Tarasow, Theodore M.; Tok, Jeffrey B.-H.

2008-01-01

Aptamers are single-stranded nucleic acids that fold into defined tertiary structures to bind target molecules with high specificities and affinities. DNA aptamers have garnered much interest as recognition elements for biodetection and diagnostic applications due to their small size, ease of discovery and synthesis, and chemical and thermal stability. Herein, we describe the design and application of a short DNA molecule capable of both protein target binding and amplifiable bioreadout processes. As both recognition and readout capabilities are incorporated into a single DNA molecule, tedious conjugation procedures required for protein-DNA hybrids can be omitted. The DNA aptamer is designed to be amplified directly by either the polymerase chain reaction (PCR) or rolling circle amplification (RCA) processes, taking advantage of real-time amplification monitoring techniques for target detection. A combination of both RCA and PCR provides a wide protein target dynamic range (1 μM to 10 pM). PMID:17980857

2000 Year-old ancient equids: an ancient-DNA lesson from pompeii remains.

PubMed

Di Bernardo, Giovanni; Del Gaudio, Stefania; Galderisi, Umberto; Cipollaro, Marilena

2004-11-15

Ancient DNA extracted from 2000 year-old equine bones was examined in order to amplify mitochondrial and nuclear DNA fragments. A specific equine satellite-type sequence representing 3.7%-11% of the entire equine genome, proved to be a suitable target to address the question of the presence of aDNA in ancient bones. The PCR strategy designed to investigate this specific target also allowed us to calculate the molecular weight of amplifiable DNA fragments. Sequencing of a 370 bp DNA fragment of mitochondrial control region allowed the comparison of ancient DNA sequences with those of modern horses to assess their genetic relationship. The 16S rRNA mitochondrial gene was also examined to unravel the post-mortem base modification feature and to test the status of Pompeian equids taxon on the basis of a Mae III restriction site polymorphism. Copyright 2004 Wiley-Liss, Inc.

Human genomic DNA quantitation system, H-Quant: development and validation for use in forensic casework.

PubMed

Shewale, Jaiprakash G; Schneida, Elaine; Wilson, Jonathan; Walker, Jerilyn A; Batzer, Mark A; Sinha, Sudhir K

2007-03-01

The human DNA quantification (H-Quant) system, developed for use in human identification, enables quantitation of human genomic DNA in biological samples. The assay is based on real-time amplification of AluYb8 insertions in hominoid primates. The relatively high copy number of subfamily-specific Alu repeats in the human genome enables quantification of very small amounts of human DNA. The oligonucleotide primers present in H-Quant are specific for human DNA and closely related great apes. During the real-time PCR, the SYBR Green I dye binds to the DNA that is synthesized by the human-specific AluYb8 oligonucleotide primers. The fluorescence of the bound SYBR Green I dye is measured at the end of each PCR cycle. The cycle at which the fluorescence crosses the chosen threshold correlates to the quantity of amplifiable DNA in that sample. The minimal sensitivity of the H-Quant system is 7.6 pg/microL of human DNA. The amplicon generated in the H-Quant assay is 216 bp, which is within the same range of the common amplifiable short tandem repeat (STR) amplicons. This size amplicon enables quantitation of amplifiable DNA as opposed to a quantitation of degraded or nonamplifiable DNA of smaller sizes. Development and validation studies were performed on the 7500 real-time PCR system following the Quality Assurance Standards for Forensic DNA Testing Laboratories.

Operational Amplifiers.

ERIC Educational Resources Information Center

Foxcroft, G. E.

1986-01-01

Addresses the introduction of low cost equipment into high school and college physical science classes. Examines the properties of an "ideal" operational amplifier and discusses how it might be used under saturated and non-saturated conditions. Notes the action of a "real" operational amplifier. (TW)

The amplification effect of functionalized gold nanoparticles on the binding of anticancer drug dacarbazine to DNA and DNA bases

NASA Astrophysics Data System (ADS)

Shen, Qin; Wang, Xuemei; Fu, Degang

2008-11-01

The promising application of functionalized gold nanoparticles to amplify the performance of biosensors and relevant biomolecular recognition processes has been explored in this paper. Our observations illustrate the apparent enhancement effect of the gold nanoparticles on the electrochemical response of the anticancer drug dacarbazine (DTIC) binding to DNA and DNA bases, indicating that these functionalized gold nanoparticles could readily facilitate the specific interactions between DTIC and DNA/DNA bases. This raises the potential valuable applications of these biocompatible nanoparticles in the promising biosensors and biomedical engineering.

Detection of sesame seed DNA in foods using real-time PCR.

PubMed

Brzezinski, Jennifer L

2007-04-01

The detection of potentially allergenic foods, such as sesame seeds, in food products is a major concern for the food-processing industry. A real-time PCR method was designed to determine if sesame seed DNA is present in food products. The PCR reaction amplifies a 66-bp fragment of the sesame seed 2S albumin gene, which is detected with a sesame-specific, dual-labeled TaqMan probe. This reaction will not amplify DNA derived from other seeds present in baked goods, such as pumpkin, poppy, and sunflower seeds. Additionally, this assay will not cross-react with DNA from several tree nut species, such as almond, Brazil nut, cashew, hazelnut, and walnut, as well as four varieties of peanut. This assay is sensitive enough to detect 5 pg of purified sesame seed DNA, as well as sesame seed DNA in a spiked wheat cracker sample.

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.

Probing DNA Translocations with Inplane Current Signals in a Graphene Nanoribbon with a Nanopore

PubMed Central

2018-01-01

Many theoretical studies predict that DNA sequencing should be feasible by monitoring the transverse current through a graphene nanoribbon while a DNA molecule translocates through a nanopore in that ribbon. Such a readout would benefit from the special transport properties of graphene, provide ultimate spatial resolution because of the single-atom layer thickness of graphene, and facilitate high-bandwidth measurements. Previous experimental attempts to measure such transverse inplane signals were however dominated by a trivial capacitive response. Here, we explore the feasibility of the approach using a custom-made differential current amplifier that discriminates between the capacitive current signal and the resistive response in the graphene. We fabricate well-defined short and narrow (30 nm × 30 nm) nanoribbons with a 5 nm nanopore in graphene with a high-temperature scanning transmission electron microscope to retain the crystallinity and sensitivity of the graphene. We show that, indeed, resistive modulations can be observed in the graphene current due to DNA translocation through the nanopore, thus demonstrating that DNA sensing with inplane currents in graphene nanostructures is possible. The approach is however exceedingly challenging due to low yields in device fabrication connected to the complex multistep device layout. PMID:29474060

Amplified spontaneous emission of Rhodamine 6G embedded in pure deoxyribonucleic acid

NASA Astrophysics Data System (ADS)

Rau, Ileana; Szukalski, Adam; Sznitko, Lech; Miniewicz, Andrzej; Bartkiewicz, Stanislaw; Kajzar, Francois; Sahraoui, Bouchta; Mysliwiec, Jaroslaw

2012-10-01

Deoxyribonucleic acid (DNA) is commonly viewed as a genetic information carrier. However, now it is recognized as a nanomaterial, rather than as a biological material, in the research field of nanotechnology. Here, we show that using pure DNA, doped with rhodamine 6G, we are able to observe amplified spontaneous emission (ASE) phenomenon. Moderate ASE threshold, photodegradation, and reasonable gain coefficient observed in this natural host gives some perspectives for practical applications of this system in biophotonics. Obtained results open the way and will be leading to construction of truly bio-lasers using nature made luminophores, such as anthocyanins.

Association Between Chloroplast DNA and Mitochondrial DNA Haplotypes in Prunus spinosa L. (Rosaceae) Populations across Europe

PubMed Central

MOHANTY, APARAJITA; MARTÍN, JUAN PEDRO; GONZÁLEZ, LUIS MIGUEL; AGUINAGALDE, ITZIAR

2003-01-01

Chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) were studied in 24 populations of Prunus spinosa sampled across Europe. The cpDNA and mtDNA fragments were amplified using universal primers and subsequently digested with restriction enzymes to obtain the polymorphisms. Combinations of all the polymorphisms resulted in 33 cpDNA haplotypes and two mtDNA haplotypes. Strict association between the cpDNA haplotypes and the mtDNA haplotypes was detected in most cases, indicating conjoint inheritance of the two genomes. The most frequent and abundant cpDNA haplotype (C20; frequency, 51 %) is always associated with the more frequent and abundant mtDNA haplotype (M1; frequency, 84 %). All but two of the cpDNA haplotypes associated with the less frequent mtDNA haplotype (M2) are private haplotypes. These private haplotypes are phylogenetically related but geographically unrelated. They form a separate cluster on the minimum‐length spanning tree. PMID:14534199

Characterizing DNA preservation in degraded specimens of Amara alpina (Carabidae: Coleoptera).

PubMed

Heintzman, Peter D; Elias, Scott A; Moore, Karen; Paszkiewicz, Konrad; Barnes, Ian

2014-05-01

DNA preserved in degraded beetle (Coleoptera) specimens, including those derived from dry-stored museum and ancient permafrost-preserved environments, could provide a valuable resource for researchers interested in species and population histories over timescales from decades to millenia. However, the potential of these samples as genetic resources is currently unassessed. Here, using Sanger and Illumina shotgun sequence data, we explored DNA preservation in specimens of the ground beetle Amara alpina, from both museum and ancient environments. Nearly all museum specimens had amplifiable DNA, with the maximum amplifiable fragment length decreasing with age. Amplification of DNA was only possible in 45% of ancient specimens. Preserved mitochondrial DNA fragments were significantly longer than those of nuclear DNA in both museum and ancient specimens. Metagenomic characterization of extracted DNA demonstrated that parasite-derived sequences, including Wolbachia and Spiroplasma, are recoverable from museum beetle specimens. Ancient DNA extracts contained beetle DNA in amounts comparable to museum specimens. Overall, our data demonstrate that there is great potential for both museum and ancient specimens of beetles in future genetic studies, and we see no reason why this would not be the case for other orders of insect. © 2013 John Wiley & Sons Ltd.

Identification of hallucinogenic fungi from the genera Psilocybe and Panaeolus by amplified fragment length polymorphism.

PubMed

Lee, J C; Cole, M; Linacre, A

2000-05-01

Unambiguous identification of the hallucinogenic fungi of the genera Psilocybe and Panaeolus is required by national and international drug control legislation. We report on a DNA-based test using the technique of amplified fragment length polymorphism (AFLP). AFLP can differentiate species of the two genera Psilocybe and Panaeolus by using different primer sets. The identification of hallucinogenic fungi using a DNA-based test, which can be used in conjunction with morphological features, will assist in forensic investigations.

Evaluation of a manual DNA extraction protocol and an isothermal amplification assay for detecting HIV-1 DNA from dried blood spots for use in resource-limited settings.

PubMed

Jordan, Jeanne A; Ibe, Christine O; Moore, Miranda S; Host, Christel; Simon, Gary L

2012-05-01

In resource-limited settings (RLS) dried blood spots (DBS) are collected on infants and transported through provincial laboratories to a central facility where HIV-1 DNA PCR testing is performed using specialized equipment. Implementing a simpler approach not requiring such equipment or skilled personnel could allow the more numerous provincial laboratories to offer testing, improving turn-around-time to identify and treat infected infants sooner. Assess performances of a manual DNA extraction method and helicase-dependent amplification (HDA) assay for detecting HIV-1 DNA from DBS. 60 HIV-1 infected adults were enrolled, blood samples taken and DBS made. DBS extracts were assessed for DNA concentration and beta globin amplification using PCR and melt-curve analysis. These same extracts were then tested for HIV-1 DNA using HDA and compared to results generated by PCR and pyrosequencing. Finally, HDA limit of detection (LOD) studies were performed using DBS extracts prepared with known numbers of 8E5 cells. The manual extraction protocol consistently yielded high concentrations of amplifiable DNA from DBS. LOD assessment demonstrated HDA detected ∼470 copies/ml of HIV-1 DNA extracts in 4/4 replicates. No statistical difference was found using the McNemar's test when comparing HDA to PCR for detecting HIV-1 DNA from DBS. Using just a magnet, heat block and pipettes, the manual extraction protocol and HDA assay detected HIV-1 DNA from DBS at levels that would be useful for early infant diagnosis. Next steps will include assessing HDA for non-B HIV-1 subtypes recognition and comparison to Roche HIV-1 DNA v1.5 PCR assay. Copyright © 2012 Elsevier B.V. All rights reserved.

Improved Yield of High Molecular Weight DNA Coincides with Increased Microbial Diversity Access from Iron Oxide Cemented Sub-Surface Clay Environments

PubMed Central

Hurt, Richard A.; Robeson, Michael S.; Shakya, Migun; Moberly, James G.; Vishnivetskaya, Tatiana A.; Gu, Baohua; Elias, Dwayne A.

2014-01-01

Despite over three decades of progress, extraction of high molecular weight (HMW) DNA from high clay soils or iron oxide cemented clay has remained challenging. HMW DNA is desirable for next generation sequencing as it yields the most comprehensive coverage. Several DNA extraction procedures were compared from samples that exhibit strong nucleic acid adsorption. pH manipulation or use of alternative ion solutions offered no improvement in nucleic acid recovery. Lysis by liquid N2 grinding in concentrated guanidine followed by concentrated sodium phosphate extraction supported HMW DNA recovery from clays high in iron oxides. DNA recovered using 1 M sodium phosphate buffer (PB) as a competitive desorptive wash was 15.22±2.33 µg DNA/g clay, with most DNA consisting of >20 Kb fragments, compared to 2.46±0.25 µg DNA/g clay with the Powerlyzer system (MoBio). Increasing PB concentration in the lysis reagent coincided with increasing DNA fragment length during initial extraction. Rarefaction plots of 16S rRNA (V1–V3 region) pyrosequencing from A-horizon and clay soils showed an ∼80% and ∼400% larger accessed diversity compared to the Powerlyzer soil DNA system, respectively. The observed diversity from the Firmicutes showed the strongest increase with >3-fold more operational taxonomic units (OTU) recovered. PMID:25033199

A CMOS-MEMS clamped–clamped beam displacement amplifier for resonant switch applications

NASA Astrophysics Data System (ADS)

Liu, Jia-Ren; Lu, Shih-Chuan; Tsai, Chun-Pu; Li, Wei-Chang

2018-06-01

This paper presents a micromechanical clamped–clamped beam (CC-beam) displacement amplifier based on a CMOS-MEMS fabrication process platform. In particular, a 2.0 MHz resonant displacement amplifier composed of two identical CC-beams coupled by a mechanical beam at locations where the two beams have mismatched velocities exhibits a larger displacement, up to 9.96×, on one beam than that of the other. The displacement amplification prevents unwanted input impacting—the structure switches only to the output but not the input—required by resonant switch-based mechanical circuits (Kim et al 2009 22nd IEEE Int. Conf. on Micro Electro Mechanical Systems; Lin et al 2009 15th Int. Conf. on Solid-State Sensors, Actuators, & Microsystems (TRANSDUCERS’09) Li et al 2013 17th Int. Conf. on Solid-State Sensors, Actuators, & Microsystems (TRANSDUCERS’13)). Compared to a single CC-beam displacement amplifier, theory predicts that the displacement amplifying CC-beam array yields a larger overall output displacement for displacement gain beyond 1.13 thanks to the preserved input driving force. A complete analytical model predicts the resultant stiffness and displacement gain of the coupled CC-beam displacement amplifier that match well with finite element analysis (FEA) prediction and measured results.

Mechanisms of DNA Packaging by Large Double-Stranded DNA Viruses

PubMed Central

Rao, Venigalla B.; Feiss, Michael

2016-01-01

Translocation of viral double-stranded DNA (dsDNA) into the icosahedral prohead shell is catalyzed by TerL, a motor protein that has ATPase, endonuclease, and translocase activities. TerL, following endonucleolytic cleavage of immature viral DNA concatemer recognized by TerS, assembles into a pentameric ring motor on the prohead’s portal vertex and uses ATP hydrolysis energy for DNA translocation. TerL’s N-terminal ATPase is connected by a hinge to the C-terminal endonuclease. Inchworm models propose that modest domain motions accompanying ATP hydrolysis are amplified, through changes in electrostatic interactions, into larger movements of the C-terminal domain bound to DNA. In phage φ29, four of the five TerL subunits sequentially hydrolyze ATP, each powering translocation of 2.5 bp. After one viral genome is encapsidated, the internal pressure signals termination of packaging and ejection of the motor. Current focus is on the structures of packaging complexes and the dynamics of TerL during DNA packaging, endonuclease regulation, and motor mechanics. PMID:26958920

DNA Extraction from Museum Specimens of Parasitic Hymenoptera

PubMed Central

Andersen, Jeremy C.; Mills, Nicholas J.

2012-01-01

At the same time that molecular researchers are improving techniques to extract DNA from museum specimens, this increased demand for access to museum specimens has created tension between the need to preserve specimens for maintaining collections and morphological research and the desire to conduct molecular analyses. To address these concerns, we examined the suitability of non-invasive DNA extraction techniques on three species of parasitic Hymenoptera (Braconidae), and test the effects of body size (parasitoid species), age (time since collection), and DNA concentration from each extract on the probability of amplifying meaningful fragments of two commonly used genetic loci. We found that age was a significant factor for determining the probability of success for sequencing both 28S and COI fragments. While the size of the braconid parasitoids significantly affected the total amount of extracted DNA, neither size nor DNA concentration were significant factors for the amplification of either gene region. We also tested several primer combinations of various lengths, but were unable to amplify fragments longer than ∼150 base pairs. These short fragments of 28S and COI were however sufficient for species identification, and for the discovery of within species genetic variation. PMID:23077493

Development and evaluation of specific PCR primers targeting the ribosomal DNA-internal transcribed spacer (ITS) region of peritrich ciliates in environmental samples

NASA Astrophysics Data System (ADS)

Su, Lei; Zhang, Qianqian; Gong, Jun

2017-07-01

Peritrich ciliates are highly diverse and can be important bacterial grazers in aquatic ecosystems. Morphological identifications of peritrich species and assemblages in the environment are time-consuming and expertise-demanding. In this study, two peritrich-specific PCR primers were newly designed to amplify a fragment including the internal transcribed spacer (ITS) region of ribosomal rDNA from environmental samples. The primers showed high specificity in silico, and in tests with peritrich isolates and environmental DNA. Application of these primers in clone library construction and sequencing yielded exclusively sequences of peritrichs for water and sediment samples. We also found the ITS1, ITS2, ITS, D1 region of 28S rDNA, and ITS+D1 region co-varied with, and generally more variable than, the V9 region of 18S rDNA in peritrichs. The newly designed specific primers thus provide additional tools to study the molecular diversity, community composition, and phylogeography of these ecologically important protists in different systems.

Development of Specific Sequence-Characterized Amplified Region Markers for Detecting Histoplasma capsulatum in Clinical and Environmental Samples

PubMed Central

Frías De León, María Guadalupe; Arenas López, Gabina; Taylor, Maria Lucia; Acosta Altamirano, Gustavo

2012-01-01

Sequence-characterized amplified region (SCAR) markers, generated by randomly amplified polymorphic DNA (RAPD)-PCR, were developed to detect Histoplasma capsulatum selectively in clinical and environmental samples. A 1,200-bp RAPD-PCR-specific band produced with the 1281-1283 primers was cloned, sequenced, and used to design two SCAR markers, 1281-1283220 and 1281-1283230. The specificity of these markers was confirmed by Southern hybridization. To evaluate the relevance of the SCAR markers for the diagnosis of histoplasmosis, another molecular marker (M antigen probe) was used for comparison. To validate 1281-1283220 and 1281-1283230 as new tools for the identification of H. capsulatum, the specificity and sensitivity of these markers were assessed for the detection of the pathogen in 36 clinical (17 humans, as well as 9 experimentally and 10 naturally infected nonhuman mammals) and 20 environmental (10 contaminated soil and 10 guano) samples. Although the two SCAR markers and the M antigen probe identified H. capsulatum isolates from different geographic origins in America, the 1281-1283220 SCAR marker was the most specific and detected the pathogen in all samples tested. In contrast, the 1281-1283230 SCAR marker and the M antigen probe also amplified DNA from Aspergillus niger and Cryptococcus neoformans, respectively. Both SCAR markers detected as little as 0.001 ng of H. capsulatum DNA, while the M antigen probe detected 0.5 ng of fungal DNA. The SCAR markers revealed the fungal presence better than the M antigen probe in contaminated soil and guano samples. Based on our results, the 1281-1283220 marker can be used to detect and identify H. capsulatum in samples from different sources. PMID:22189121

Environmental DNA (eDNA) metabarcoding assays to detect invasive invertebrate species in the Great Lakes.

PubMed

Klymus, Katy E; Marshall, Nathaniel T; Stepien, Carol A

2017-01-01

Describing and monitoring biodiversity comprise integral parts of ecosystem management. Recent research coupling metabarcoding and environmental DNA (eDNA) demonstrate that these methods can serve as important tools for surveying biodiversity, while significantly decreasing the time, expense and resources spent on traditional survey methods. The literature emphasizes the importance of genetic marker development, as the markers dictate the applicability, sensitivity and resolution ability of an eDNA assay. The present study developed two metabarcoding eDNA assays using the mtDNA 16S RNA gene with Illumina MiSeq platform to detect invertebrate fauna in the Laurentian Great Lakes and surrounding waterways, with a focus for use on invasive bivalve and gastropod species monitoring. We employed careful primer design and in vitro testing with mock communities to assess ability of the markers to amplify and sequence targeted species DNA, while retaining rank abundance information. In our mock communities, read abundances reflected the initial input abundance, with regressions having significant slopes (p<0.05) and high coefficients of determination (R2) for all comparisons. Tests on field environmental samples revealed similar ability of our markers to measure relative abundance. Due to the limited reference sequence data available for these invertebrate species, care must be taken when analyzing results and identifying sequence reads to species level. These markers extend eDNA metabarcoding research for molluscs and appear relevant to other invertebrate taxa, such as rotifers and bryozoans. Furthermore, the sphaeriid mussel assay is group-specific, exclusively amplifying bivalves in the Sphaeridae family and providing species-level identification. Our assays provide useful tools for managers and conservation scientists, facilitating early detection of invasive species as well as improving resolution of mollusc diversity.

IC Ku-band Impatt Amplifier. [solid state spacecraft transmitter

NASA Technical Reports Server (NTRS)

Sokolov, V.; Namordi, M. R.; Doerbeck, F. H.; Wisseman, W. R.

1979-01-01

High efficiency GaAs low-high-low IMPATTs were investigated. Theoretical analyses were employed to establish a design window for the material parameters to maximize microwave performance. Single mesa devices yielded typically 2 to 3 W with 16 to 23% efficiency in waveguide oscillator test circuits. IMPATTs with high reliability Pt/TiW/Pt/Au metallizations were subjected to temperature stress, non-rf bias-temperature stress, and rf bias-temperature stress. Assuming that temperature is the driving force behind the dominant failure mechanism, a mean-time-to-failure considerably greater than 500,000 hours is indicated by the stress tests. A 15 GHz, 4W, 56 dB gain microstrip amplifier was realized using GaAs FETs and IMPATTs. Power combining using a 3 db Lange coupler is employed in the power output stage having an intrinsic power-added efficiency of 15.7%. Overall dc-to-rf efficiency of the amplifier is 10.8%. The amplifier has greater than a 250 MHz, 1 db bandwidth; operates over the 0 deg to 50 C (base plate) temperature range with less than 0.5 db change in the power output; weighs 444 grams; and has a volume of 220 cu cm.

LOGARITHMIC AMPLIFIER

DOEpatents

Wade, E.J.; Stone, R.S.

1959-03-10

Electronic,amplifier circuits, especially a logai-ithmic amplifier characterizxed by its greatly improved strability are discussed. According to the in ention, means are provided to feed bach the output valtagee to a diode in the amplifier input circuit, the diode being utilized to produce the logarithmic characteristics. The diode is tics, The diode isition therewith and having its filament operated from thc same source s the filament of the logarithmic diode. A bias current of relatively large value compareii with the signal current is continuously passed through the compiting dioie to render the diode insensitivy to variations in the signal current. by this odes kdu to variaelled, so that the stability of the amlifier will be unimpaired.

The DNA of ciliated protozoa.

PubMed Central

Prescott, D M

1994-01-01

Ciliates contain two types of nuclei: a micronucleus and a macronucleus. The micronucleus serves as the germ line nucleus but does not express its genes. The macronucleus provides the nuclear RNA for vegetative growth. Mating cells exchange haploid micronuclei, and a new macronucleus develops from a new diploid micronucleus. The old macronucleus is destroyed. This conversion consists of amplification, elimination, fragmentation, and splicing of DNA sequences on a massive scale. Fragmentation produces subchromosomal molecules in Tetrahymena and Paramecium cells and much smaller, gene-sized molecules in hypotrichous ciliates to which telomere sequences are added. These molecules are then amplified, some to higher copy numbers than others. rDNA is differentially amplified to thousands of copies per macronucleus. Eliminated sequences include transposonlike elements and sequences called internal eliminated sequences that interrupt gene coding regions in the micronuclear genome. Some, perhaps all, of these are excised as circular molecules and destroyed. In at least some hypotrichs, segments of some micronuclear genes are scrambled in a nonfunctional order and are recorded during macronuclear development. Vegetatively growing ciliates appear to possess a mechanism for adjusting copy numbers of individual genes, which corrects gene imbalances resulting from random distribution of DNA molecules during amitosis of the macronucleus. Other distinctive features of ciliate DNA include an altered use of the conventional stop codons. Images PMID:8078435

ULTRA-STABILIZED D. C. AMPLIFIER

DOEpatents

Hartwig, E.C.; Kuenning, R.W.; Acker, R.C.

1959-02-17

An improved circuit is described for stabilizing the drift and minimizing the noise and hum level of d-c amplifiers so that the output voltage will be zero when the input is zero. In its detailed aspects, the disclosed circuit incorporates a d-c amplifier having a signal input, a second input, and an output circuit coupled back to the first input of the amplifier through inverse feedback means. An electronically driven chopper having a pair of fixed contacts and a moveable contact alternately connects the two inputs of a difference amplifier to the signal input. The A. E. error signal produced in the difference amplifier is amplified, rectified, and applied to the second input of the amplifier as the d-c stabilizing voltage.

Enhanced sequencing coverage with digital droplet multiple displacement amplification

PubMed Central

Sidore, Angus M.; Lan, Freeman; Lim, Shaun W.; Abate, Adam R.

2016-01-01

Sequencing small quantities of DNA is important for applications ranging from the assembly of uncultivable microbial genomes to the identification of cancer-associated mutations. To obtain sufficient quantities of DNA for sequencing, the small amount of starting material must be amplified significantly. However, existing methods often yield errors or non-uniform coverage, reducing sequencing data quality. Here, we describe digital droplet multiple displacement amplification, a method that enables massive amplification of low-input material while maintaining sequence accuracy and uniformity. The low-input material is compartmentalized as single molecules in millions of picoliter droplets. Because the molecules are isolated in compartments, they amplify to saturation without competing for resources; this yields uniform representation of all sequences in the final product and, in turn, enhances the quality of the sequence data. We demonstrate the ability to uniformly amplify the genomes of single Escherichia coli cells, comprising just 4.7 fg of starting DNA, and obtain sequencing coverage distributions that rival that of unamplified material. Digital droplet multiple displacement amplification provides a simple and effective method for amplifying minute amounts of DNA for accurate and uniform sequencing. PMID:26704978

Buccal DNA collection: comparison of buccal swabs with FTA cards.

PubMed

Milne, Elizabeth; van Bockxmeer, Frank M; Robertson, Laila; Brisbane, Joanna M; Ashton, Lesley J; Scott, Rodney J; Armstrong, Bruce K

2006-04-01

Collection and analysis of DNA, most commonly from blood or buccal cells, is becoming more common in epidemiologic studies. Buccal samples, which are painless to take and relatively easily collected, are often the preferred source. There are several buccal cell collection methods: swabs, brushes, mouthwash, and treated cards, such as FTA or IsoCode cards. Few studies have systematically compared methods of buccal cell collection with respect to DNA yield and amplification success under similar conditions. We compared buccal DNA collection and amplification using buccal swabs and FTA cards in 122 control subjects from our Australian case-control study of childhood acute lymphoblastic leukaemia. Buccal DNA was quantified using a real-time PCR for beta-actin and genotyped at the loci of three polymorphisms (MTHFR 677C>T, ACE I/D, and XPD 1012G>A). PCR was successful with DNA from buccal swabs for 62% to 89% of subjects and from FTA cards for 83% to 100% of subjects, depending on the locus. The matched pair odds ratios (95% confidence interval) comparing success of FTA cards with buccal swabs are as follows: MTHFR 677C>T using PCR-RFLP, 12.5 (11.6-13.5) and using real-time PCR, 130.0 (113.1-152.8); ACE I/D using PCR-amplified fragment length polymorphism, 3.36 (3.2-3.5); XPD 1012G>A using real-time PCR, 150.0 (132.7-172.3). FTA cards are a robust DNA collection method and generally produce DNA suitable for PCR more reliably than buccal swabs. There are, however, technical challenges in handling discs punched from FTA cards that intending users should be aware of.

An analysis on DNA fingerprints of thirty papaya cultivars (Carica papaya L.), grown in Thailand with the use of amplified fragment length polymorphisms technique.

PubMed

Ratchadaporn, Janthasri; Sureeporn, Katengam; Khumcha, U

2007-09-15

The experiment was carried out at the Department of Horticulture, Ubon Ratchathani University, Ubon Ratchathani province, Northeast Thailand during June 2002 to May 2003 aims to identify DNA fingerprints of thirty papaya cultivars with the use of Amplified Fragment Length Polymorphisms (AFLP) technique. Papaya cultivars were collected from six different research centers in Thailand. Papaya plants of each cultivar were grown under field conditions up to four months then leaf numbers 2 and 3 of each cultivar (counted from top) were chosen for DNA extraction and the samples were used for AFLP analysis. Out of 64 random primers being used, 55 pairs gave an increase in DNA bands but only 12 pairs of random primers were randomly chosen for the final analysis of the experiment. The results showed that AFLP markers gave Polymorphic Information Contents (PIC) of three ranges i.e., AFLP markers of 235 lied on a PIC range of 0.003-0.05, 47 for a PIC range of 0.15-0.20 and 12 for a PIC range of 0.35-0.40. The results on dendrogram cluster analysis revealed that the thirty papaya cultivars were classified into six groups i.e., (1) Kaeg Dum and Malador (2) Kaeg Nuan (3) Pakchong and Solo (4) Taiwan (5) Co Coa Hai Nan and (6) Sitong. Nevertheless, in spite of the six papaya groups all papaya cultivars were genetically related to each other where diversity among the cultivars was not significantly found.

[Effect of BSA on random amplified polymorphic DNA (RAPD) in plants].

PubMed

Bian, Cai-Miao; Li, Jun-Min; Jin, Ze-Xin; Ge, Ming-Ju

2002-05-01

Using Metasequoia glyptostroboides and Heptacodium miconioides DNA as templates,the effect of bovine serum albumin (BSA) on RAPD in plants was studied. The results showed that suitable concentrations of BSA used in Metasequoia glyptostroboides and Heptacodium miconioides RAPD were different, which were 0.6 microg/microl and 1 microg/microl, respectively. The inhibition of acetylated BSA on the amplification of plant RAPD could be relieved by BSA. BSA could reduce the dosage of Taq DNA polymerase.

DNA methylation profiling of genomic DNA isolated from urine in diabetic chronic kidney disease: A pilot study

PubMed Central

Sexton-Oates, Alexandra; Carmody, Jake; Ekinci, Elif I.; Dwyer, Karen M.; Saffery, Richard

2018-01-01

Aim To characterise the genomic DNA (gDNA) yield from urine and quality of derived methylation data generated from the widely used Illuminia Infinium MethylationEPIC (HM850K) platform and compare this with buffy coat samples. Background DNA methylation is the most widely studied epigenetic mark and variations in DNA methylation profile have been implicated in diabetes which affects approximately 415 million people worldwide. Methods QIAamp Viral RNA Mini Kit and QIAamp DNA micro kit were used to extract DNA from frozen and fresh urine samples as well as increasing volumes of fresh urine. Matched buffy coats to the frozen urine were also obtained and DNA was extracted from the buffy coats using the QIAamp DNA Mini Kit. Genomic DNA of greater concentration than 20μg/ml were used for methylation analysis using the HM850K array. Results Irrespective of extraction technique or the use of fresh versus frozen urine samples, limited genomic DNA was obtained using a starting sample volume of 5ml (0–0.86μg/mL). In order to optimize the yield, we increased starting volumes to 50ml fresh urine, which yielded only 0–9.66μg/mL A different kit, QIAamp DNA Micro Kit, was trialled in six fresh urine samples and ten frozen urine samples with inadequate DNA yields from 0–17.7μg/mL and 0–1.6μg/mL respectively. Sufficient genomic DNA was obtained from only 4 of the initial 41 frozen urine samples (10%) for DNA methylation profiling. In comparison, all four buffy coat samples (100%) provided sufficient genomic DNA. Conclusion High quality data can be obtained provided a sufficient yield of genomic DNA is isolated. Despite optimizing various extraction methodologies, the modest amount of genomic DNA derived from urine, may limit the generalisability of this approach for the identification of DNA methylation biomarkers of chronic diabetic kidney disease. PMID:29462136

DNA methylation profiling of genomic DNA isolated from urine in diabetic chronic kidney disease: A pilot study.

PubMed

Lecamwasam, Ashani; Sexton-Oates, Alexandra; Carmody, Jake; Ekinci, Elif I; Dwyer, Karen M; Saffery, Richard

2018-01-01

To characterise the genomic DNA (gDNA) yield from urine and quality of derived methylation data generated from the widely used Illuminia Infinium MethylationEPIC (HM850K) platform and compare this with buffy coat samples. DNA methylation is the most widely studied epigenetic mark and variations in DNA methylation profile have been implicated in diabetes which affects approximately 415 million people worldwide. QIAamp Viral RNA Mini Kit and QIAamp DNA micro kit were used to extract DNA from frozen and fresh urine samples as well as increasing volumes of fresh urine. Matched buffy coats to the frozen urine were also obtained and DNA was extracted from the buffy coats using the QIAamp DNA Mini Kit. Genomic DNA of greater concentration than 20μg/ml were used for methylation analysis using the HM850K array. Irrespective of extraction technique or the use of fresh versus frozen urine samples, limited genomic DNA was obtained using a starting sample volume of 5ml (0-0.86μg/mL). In order to optimize the yield, we increased starting volumes to 50ml fresh urine, which yielded only 0-9.66μg/mL A different kit, QIAamp DNA Micro Kit, was trialled in six fresh urine samples and ten frozen urine samples with inadequate DNA yields from 0-17.7μg/mL and 0-1.6μg/mL respectively. Sufficient genomic DNA was obtained from only 4 of the initial 41 frozen urine samples (10%) for DNA methylation profiling. In comparison, all four buffy coat samples (100%) provided sufficient genomic DNA. High quality data can be obtained provided a sufficient yield of genomic DNA is isolated. Despite optimizing various extraction methodologies, the modest amount of genomic DNA derived from urine, may limit the generalisability of this approach for the identification of DNA methylation biomarkers of chronic diabetic kidney disease.

DISTRIBUTED AMPLIFIER INCORPORATING FEEDBACK

DOEpatents

Bell, P.R. Jr.

1958-10-21

An improved distributed amplifier system employing feedback for stabilization is presented. In accordance with the disclosed invention, a signal to be amplified is applled to one end of a suitable terminated grid transmission line. At intervals along the transmission line, the signal is fed to stable, resistance-capacitance coupled amplifiers incorporating feedback loops therein. The output current from each amplifier is passed through an additional tube to minimize the electrostatic capacitance between the tube elements of the last stage of the amplifier, and fed to appropriate points on an output transmission line, similar to the grid line, but terminated at the opposite (input) end. The output taken from the unterminated end of the plate transmission line is proportional to the input voltage impressed upon the grid line.

Polymerization amplified SPR-DNA assay on noncovalently functionalized graphene.

PubMed

Yuan, Pei-Xin; Deng, Sheng-Yuan; Yao, Chuan-Guang; Wan, Ying; Cosnier, Serge; Shan, Dan

2017-03-15

A highly efficient surface plasmon resonance (SPR)-based DNA assay was developed, by employing noncovalently functionalized graphene nanosheets as a substrate, and enzymatic catalysis-induced polymerization as mass relay. The objective of this strategy was manifold: first of all, to sensitize the overall SPR output by in situ optimized electrogeneration of graphene thin-film, which was characterized by atomic force microscopic topography; secondly, to regulate the self-assembly and orientation of biotinylated capture probes on nickel-chelated nitrilotriacetic acid (NTA) scaffolds, that anchored onto graphene-supported pyrenyl derivatives; and lastly, to synergize the signal amplification via real-time conversion of the additive aniline into polyaniline precipitation by horseradish peroxidase-tagged reporters. With this setup, a precise and replicable DNA sensing platform for specific targets was achieved with a detection limit down to femtomolar, thus demonstrating a beneficial exploration and exploitation of two-dimensional nanomaterials as unique SPR infrastructure. The possibility of such ″bottom-up″ architecture mounted with ″top-down″ weight reactor would be most likely extensible and adaptable to protein determinations. Copyright © 2016 Elsevier B.V. All rights reserved.

Dose rate, mitotic cycle duration, and sensitivity of cell transitions from G1 $Yields$ S and G2 $Yields$ M to protracted gamma radiation in root meristems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Evans, L.S.; Hof, J.V.

1975-11-01

Experiments were designed to determine the relative radiosensitivity of the cell transition points of G1 $Yields$ S and G2 $Yields$ M in root meristems of several plant species. Label and mitotic indices and microspectrophotometry were used to measure the proportions of cells in each mitotic cycle stage in root meristems after protracted gamma radiation. The criterion of radiosensitivity was the dose rate needed to produce a tissue with less than 1 percent cells in S and none in M after 3 days of continuous exposure. The results show that DNA is the primary radiation target in proliferative root meristems andmore » that the cycle duration stipulates the time interval of vulnerability. In each species, nonrandom reproducible cell proportions were established with 2C:4C:8C amounts of nuclear DNA after 3 days of exposure. Roots of Helianthus annuus, Crepis capillaris, and Tradescantia clone 02 had 80 percent cells with a 2C amount of DNA and 20 percent had a 4C amount of DNA. In these species the transition point of G1 $Yields$ S was more radiosensitive than G2 $Yields$ M. Roots of Pisum sativum and Triticum aestivum had cell proportions at 2C:4C:8C amounts of DNA in frequencies of 0.10 to 0.20:0.40 to 0.60:0.30 to 0.40. In these two species 0.30 to 0.40 cells underwent radiation-induced endoreduplication that resulted from a rapid inhibition of cell transit from G2 $Yields$ M and a slower impairment of G1 $Yields$ S. Cells increased from 2C to 4C and from 4C to 8C amounts of DNA during irradiation. The proportions of nuclei with 2C:4C:8C amounts of DNA were dependent in part upon the relative radiosensitivity of the G1 $Yields$ S and G2 $Yields$ M control points. The data show the relative radiosensitivity of the transition points from G1 $Yields$ S and from G2 $Yields$ M was species specific and unrelated to the cycle duration and mean nuclear DNA content of the plant species. (auth)« less

Estimation of the Relative Abundance of Different Bacteroides and Prevotella Ribotypes in Gut Samples by Restriction Enzyme Profiling of PCR-Amplified 16S rRNA Gene Sequences

PubMed Central

Wood, Jacqueline; Scott, Karen P.; Avguštin, Gorazd; Newbold, C. James; Flint, Harry J.

1998-01-01

We describe an approach for determining the genetic composition of Bacteroides and Prevotella populations in gut contents based on selective amplification of 16S rRNA gene sequences (rDNA) followed by cleavage of the amplified material with restriction enzymes. The relative contributions of different ribotypes to total Bacteroides and Prevotella 16S rDNA are estimated after end labelling of one of the PCR primers, and the contribution of Bacteroides and Prevotella sequences to total eubacterial 16S rDNA is estimated by measuring the binding of oligonucleotide probes to amplified DNA. Bacteroides and Prevotella 16S rDNA accounted for between 12 and 62% of total eubacterial 16S rDNA in samples of ruminal contents from six sheep and a cow. Ribotypes 4, 5, 6, and 7, which include most cultivated rumen Prevotella strains, together accounted for between 20 and 86% of the total amplified Bacteroides and Prevotella rDNA in these samples. The most abundant Bacteroides or Prevotella ribotype in four animals, however, was ribotype 8, for which there is only one known cultured isolate, while ribotypes 1 and 2, which include many colonic Bacteroides spp., were the most abundant in two animals. This indicates that some abundant Bacteroides and Prevotella groups in the rumen are underrepresented among cultured rumen Prevotella isolates. The approach described here provides a rapid, convenient, and widely applicable method for comparing the genotypic composition of bacterial populations in gut samples. PMID:9758785

Genetic Relatedness among Environmental, Clinical, and Diseased-Eel Vibrio vulnificus Isolates from Different Geographic Regions by Ribotyping and Randomly Amplified Polymorphic DNA PCR

PubMed Central

Arias, Covadonga R.; Pujalte, María Jesús; Garay, Esperanza; Aznar, Rosa

1998-01-01

Genetic relationships among 132 strains of Vibrio vulnificus (clinical, environmental, and diseased-eel isolates from different geographic origins, as well as seawater and shellfish isolates from the western Mediterranean coast, including reference strains) were analyzed by random amplified polymorphic DNA (RAPD) PCR. Results were validated by ribotyping. For ribotyping, DNAs were digested with KpnI and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene. Random amplification of DNA was performed with M13 and T3 universal primers. The comparison between ribotyping and RAPD PCR revealed an overall agreement regarding the high level of homogeneity of diseased-eel isolates in contrast to the genetic heterogeneity of Mediterranean isolates. The latter suggests the existence of autochthonous clones present in Mediterranean coastal waters. Both techniques have revealed a genetic proximity among Spanish fish farm isolates and a close relationship between four Spanish eel farm isolates and some Mediterranean isolates. Whereas the differentiation within diseased-eel isolates was only possible by ribotyping, RAPD PCR was able to differentiate phenotypically atypical isolates of V. vulnificus. On the basis of our results, RAPD PCR is proposed as a better technique than ribotyping for rapid typing in the routine analysis of new V. vulnificus isolates. PMID:9726889

A third-order class-D amplifier with and without ripple compensation

NASA Astrophysics Data System (ADS)

Cox, Stephen M.; du Toit Mouton, H.

2018-06-01

We analyse the nonlinear behaviour of a third-order class-D amplifier, and demonstrate the remarkable effectiveness of the recently introduced ripple compensation (RC) technique in reducing the audio distortion of the device. The amplifier converts an input audio signal to a high-frequency train of rectangular pulses, whose widths are modulated according to the input signal (pulse-width modulation) and employs negative feedback. After determining the steady-state operating point for constant input and calculating its stability, we derive a small-signal model (SSM), which yields in closed form the transfer function relating (infinitesimal) input and output disturbances. This SSM shows how the RC technique is able to linearise the small-signal response of the device. We extend this SSM through a fully nonlinear perturbation calculation of the dynamics of the amplifier, based on the disparity in time scales between the pulse train and the audio signal. We obtain the nonlinear response of the amplifier to a general audio signal, avoiding the linearisation inherent in the SSM; we thereby more precisely quantify the reduction in distortion achieved through RC. Finally, simulations corroborate our theoretical predictions and illustrate the dramatic deterioration in performance that occurs when the amplifier is operated in an unstable regime. The perturbation calculation is rather general, and may be adapted to quantify the way in which other nonlinear negative-feedback pulse-modulated devices track a time-varying input signal that slowly modulates the system parameters.

Low-Noise Band-Pass Amplifier

NASA Technical Reports Server (NTRS)

Kleinberg, L.

1982-01-01

Circuit uses standard components to overcome common limitation of JFET amplifiers. Low-noise band-pass amplifier employs JFET and operational amplifier. High gain and band-pass characteristics are achieved with suitable choice of resistances and capacitances. Circuit should find use as low-noise amplifier, for example as first stage instrumentation systems.

Compact laser amplifier system

DOEpatents

Carr, R.B.

1974-02-26

A compact laser amplifier system is described in which a plurality of face-pumped annular disks, aligned along a common axis, independently radially amplify a stimulating light pulse. Partially reflective or lasing means, coaxially positioned at the center of each annualar disk, radially deflects a stimulating light directed down the common axis uniformly into each disk for amplification, such that the light is amplified by the disks in a parallel manner. Circumferential reflecting means coaxially disposed around each disk directs amplified light emission, either toward a common point or in a common direction. (Official Gazette)

Stable photosensor amplifiers

NASA Technical Reports Server (NTRS)

Fujimoto, H.

1972-01-01

Minimization of common mode effects in differential amplifier arrangement which processes signals from two high impedance photosensors is achieved by connecting one photosensor in feedback loop of amplifier and using field effect transistors in the input circuit.

Environmental DNA (eDNA) metabarcoding assays to detect invasive invertebrate species in the Great Lakes

PubMed Central

Klymus, Katy E.; Marshall, Nathaniel T.

2017-01-01

Describing and monitoring biodiversity comprise integral parts of ecosystem management. Recent research coupling metabarcoding and environmental DNA (eDNA) demonstrate that these methods can serve as important tools for surveying biodiversity, while significantly decreasing the time, expense and resources spent on traditional survey methods. The literature emphasizes the importance of genetic marker development, as the markers dictate the applicability, sensitivity and resolution ability of an eDNA assay. The present study developed two metabarcoding eDNA assays using the mtDNA 16S RNA gene with Illumina MiSeq platform to detect invertebrate fauna in the Laurentian Great Lakes and surrounding waterways, with a focus for use on invasive bivalve and gastropod species monitoring. We employed careful primer design and in vitro testing with mock communities to assess ability of the markers to amplify and sequence targeted species DNA, while retaining rank abundance information. In our mock communities, read abundances reflected the initial input abundance, with regressions having significant slopes (p<0.05) and high coefficients of determination (R2) for all comparisons. Tests on field environmental samples revealed similar ability of our markers to measure relative abundance. Due to the limited reference sequence data available for these invertebrate species, care must be taken when analyzing results and identifying sequence reads to species level. These markers extend eDNA metabarcoding research for molluscs and appear relevant to other invertebrate taxa, such as rotifers and bryozoans. Furthermore, the sphaeriid mussel assay is group-specific, exclusively amplifying bivalves in the Sphaeridae family and providing species-level identification. Our assays provide useful tools for managers and conservation scientists, facilitating early detection of invasive species as well as improving resolution of mollusc diversity. PMID:28542313

Identification of druggable cancer driver genes amplified across TCGA datasets.

PubMed

Chen, Ying; McGee, Jeremy; Chen, Xianming; Doman, Thompson N; Gong, Xueqian; Zhang, Youyan; Hamm, Nicole; Ma, Xiwen; Higgs, Richard E; Bhagwat, Shripad V; Buchanan, Sean; Peng, Sheng-Bin; Staschke, Kirk A; Yadav, Vipin; Yue, Yong; Kouros-Mehr, Hosein

2014-01-01

The Cancer Genome Atlas (TCGA) projects have advanced our understanding of the driver mutations, genetic backgrounds, and key pathways activated across cancer types. Analysis of TCGA datasets have mostly focused on somatic mutations and translocations, with less emphasis placed on gene amplifications. Here we describe a bioinformatics screening strategy to identify putative cancer driver genes amplified across TCGA datasets. We carried out GISTIC2 analysis of TCGA datasets spanning 16 cancer subtypes and identified 486 genes that were amplified in two or more datasets. The list was narrowed to 75 cancer-associated genes with potential "druggable" properties. The majority of the genes were localized to 14 amplicons spread across the genome. To identify potential cancer driver genes, we analyzed gene copy number and mRNA expression data from individual patient samples and identified 42 putative cancer driver genes linked to diverse oncogenic processes. Oncogenic activity was further validated by siRNA/shRNA knockdown and by referencing the Project Achilles datasets. The amplified genes represented a number of gene families, including epigenetic regulators, cell cycle-associated genes, DNA damage response/repair genes, metabolic regulators, and genes linked to the Wnt, Notch, Hedgehog, JAK/STAT, NF-KB and MAPK signaling pathways. Among the 42 putative driver genes were known driver genes, such as EGFR, ERBB2 and PIK3CA. Wild-type KRAS was amplified in several cancer types, and KRAS-amplified cancer cell lines were most sensitive to KRAS shRNA, suggesting that KRAS amplification was an independent oncogenic event. A number of MAP kinase adapters were co-amplified with their receptor tyrosine kinases, such as the FGFR adapter FRS2 and the EGFR family adapters GRB2 and GRB7. The ubiquitin-like ligase DCUN1D1 and the histone methyltransferase NSD3 were also identified as novel putative cancer driver genes. We discuss the patient tailoring implications for existing cancer

Identification of Druggable Cancer Driver Genes Amplified across TCGA Datasets

PubMed Central

Chen, Ying; McGee, Jeremy; Chen, Xianming; Doman, Thompson N.; Gong, Xueqian; Zhang, Youyan; Hamm, Nicole; Ma, Xiwen; Higgs, Richard E.; Bhagwat, Shripad V.; Buchanan, Sean; Peng, Sheng-Bin; Staschke, Kirk A.; Yadav, Vipin; Yue, Yong; Kouros-Mehr, Hosein

2014-01-01

The Cancer Genome Atlas (TCGA) projects have advanced our understanding of the driver mutations, genetic backgrounds, and key pathways activated across cancer types. Analysis of TCGA datasets have mostly focused on somatic mutations and translocations, with less emphasis placed on gene amplifications. Here we describe a bioinformatics screening strategy to identify putative cancer driver genes amplified across TCGA datasets. We carried out GISTIC2 analysis of TCGA datasets spanning 14 cancer subtypes and identified 461 genes that were amplified in two or more datasets. The list was narrowed to 73 cancer-associated genes with potential “druggable” properties. The majority of the genes were localized to 14 amplicons spread across the genome. To identify potential cancer driver genes, we analyzed gene copy number and mRNA expression data from individual patient samples and identified 40 putative cancer driver genes linked to diverse oncogenic processes. Oncogenic activity was further validated by siRNA/shRNA knockdown and by referencing the Project Achilles datasets. The amplified genes represented a number of gene families, including epigenetic regulators, cell cycle-associated genes, DNA damage response/repair genes, metabolic regulators, and genes linked to the Wnt, Notch, Hedgehog, JAK/STAT, NF-KB and MAPK signaling pathways. Among the 40 putative driver genes were known driver genes, such as EGFR, ERBB2 and PIK3CA. Wild-type KRAS was amplified in several cancer types, and KRAS-amplified cancer cell lines were most sensitive to KRAS shRNA, suggesting that KRAS amplification was an independent oncogenic event. A number of MAP kinase adapters were co-amplified with their receptor tyrosine kinases, such as the FGFR adapter FRS2 and the EGFR family adapter GRB7. The ubiquitin-like ligase DCUN1D1 and the histone methyltransferase NSD3 were also identified as novel putative cancer driver genes. We discuss the patient tailoring implications for existing cancer drug

Optimization and evaluation of T7 based RNA linear amplification protocols for cDNA microarray analysis

PubMed Central

Zhao, Hongjuan; Hastie, Trevor; Whitfield, Michael L; Børresen-Dale, Anne-Lise; Jeffrey, Stefanie S

2002-01-01

Background T7 based linear amplification of RNA is used to obtain sufficient antisense RNA for microarray expression profiling. We optimized and systematically evaluated the fidelity and reproducibility of different amplification protocols using total RNA obtained from primary human breast carcinomas and high-density cDNA microarrays. Results Using an optimized protocol, the average correlation coefficient of gene expression of 11,123 cDNA clones between amplified and unamplified samples is 0.82 (0.85 when a virtual array was created using repeatedly amplified samples to minimize experimental variation). Less than 4% of genes show changes in expression level by 2-fold or greater after amplification compared to unamplified samples. Most changes due to amplification are not systematic both within one tumor sample and between different tumors. Amplification appears to dampen the variation of gene expression for some genes when compared to unamplified poly(A)+ RNA. The reproducibility between repeatedly amplified samples is 0.97 when performed on the same day, but drops to 0.90 when performed weeks apart. The fidelity and reproducibility of amplification is not affected by decreasing the amount of input total RNA in the 0.3–3 micrograms range. Adding template-switching primer, DNA ligase, or column purification of double-stranded cDNA does not improve the fidelity of amplification. The correlation coefficient between amplified and unamplified samples is higher when total RNA is used as template for both experimental and reference RNA amplification. Conclusion T7 based linear amplification reproducibly generates amplified RNA that closely approximates original sample for gene expression profiling using cDNA microarrays. PMID:12445333

Invasive reaction assisted strand-displacement signal amplification for sensitive DNA detection.

PubMed

Zou, Bingjie; Song, Qinxin; Wang, Jianping; Liu, Yunlong; Zhou, Guohua

2014-11-18

A novel DNA detection assay was proposed by invasive reaction coupled with molecular beacon assisted strand-displacement signal amplification (IRASA). Target DNAs are firstly hybridized to two probes to initiate invasive reaction to produce amplified flaps. Then these flaps are further amplified by strand-displacement signal amplification. The detection limit was around 0.2 pM.

Comparing different post-mortem human samples as DNA sources for downstream genotyping and identification.

PubMed

Calacal, Gayvelline C; Apaga, Dame Loveliness T; Salvador, Jazelyn M; Jimenez, Joseph Andrew D; Lagat, Ludivino J; Villacorta, Renato Pio F; Lim, Maria Cecilia F; Fortun, Raquel D R; Datar, Francisco A; De Ungria, Maria Corazon A

2015-11-01

The capability of DNA laboratories to perform genotyping procedures from post-mortem remains, including those that had undergone putrefaction, continues to be a challenge in the Philippines, a country characterized by very humid and warm conditions all year round. These environmental conditions accelerate the decomposition of human remains that were recovered after a disaster and those that were left abandoned after a crime. When considerable tissue decomposition of human remains has taken place, there is no other option but to extract DNA from bone and/or teeth samples. Routinely, femur shafts are obtained from recovered bodies for human identification because the calcium matrix protects the DNA contained in the osteocytes. In the Philippines, there is difficulty in collecting femur samples after natural disasters or even human-made disasters, because these events are usually characterized by a large number of fatalities. Identification of casualties is further delayed by limitation in human and material resources. Hence, it is imperative to test other types of biological samples that are easier to collect, transport, process and store. We analyzed DNA that were obtained from body fluid, bone marrow, muscle tissue, clavicle, femur, metatarsal, patella, rib and vertebral samples from five recently deceased untreated male cadavers and seven male human remains that were embalmed, buried for ∼ 1 month and then exhumed. The bodies had undergone different environmental conditions and were in various stages of putrefaction. A DNA extraction method utilizing a detergent-washing step followed by an organic procedure was used. The utility of bone marrow and vitreous fluid including bone marrow and vitreous fluid that was transferred on FTA(®) cards and subjected to autosomal STR and Y-STR DNA typing were also evaluated. DNA yield was measured and the presence or absence of PCR inhibitors in DNA extracts was assessed using Plexor(®)HY. All samples were amplified using

DIRECT COUPLED AMPLIFIER

DOEpatents

Dandl, R.A.

1961-09-19

A transistor amplifier is designed for vyery small currents below 10/sup -8/ amperes. The filrst and second amplifier stages use unusual selected transistors in which the current amplification increases markedly for values of base current below 10/sup -6/ amperes.

Pit-1 gene polymorphism, milk yield, and conformation traits for Italian Holstein-Friesian bulls.

PubMed

Renaville, R; Gengler, N; Vrech, E; Prandi, A; Massart, S; Corradini, C; Bertozzi, C; Mortiaux, F; Burny, A; Portetelle, D

1997-12-01

The growth hormone factor-1/pituitary-specific transcription factor Pit-1 is responsible for the expression of growth hormone in mammals. Mutations in Pit-1 have been found in growth hormone disorders of mice and humans. We studied the eventual association between Pit-1 polymorphism using the HinfI enzyme and the milk yield and conformation traits of 89 Italian Holstein-Friesian bulls. A strategy employing polymerase chain reaction was used to amplify a 451-bp fragment from semen DNA. Digestion of polymerase chain reaction products with HinfI revealed two alleles: allele A was not digested (451-bp fragment), and allele B was cut at one restriction site, generating two fragments of 244 and 207 bp. Three patterns were observed; frequencies were 2.2, 31.5, and 66.3% for AA, AB, and BB, respectively. Fixed and mixed linear models were fitted on daughter yield deviations for milk yields and on deregressed proofs for conformation traits. Predictions were weighted using the inverse of the estimated variance of records. The models used contained mean and gene substitution effects for Pit-1 A allele as fixed effects and random sire effect for the mixed model. The A allele was found to be superior for milk and protein yields, inferior for fat percentage, and superior for body depth, angularity, and rear leg set, which is difficult to explain. A canonical transformation revealed that Pit-1 had three actions, one linked to milk yield traits and angularity, a second linked to body depth and rear leg set, and a third linked to lower fat yields and to higher angularity.

Fast and simple DNA extraction from saliva and sperm cells obtained from the skin or isolated from swabs.

PubMed

von Wurmb-Schwark, Nicole; Mályusz, Victoria; Fremdt, Heike; Koch, Christine; Simeoni, Eva; Schwark, Thorsten

2006-05-01

The forensic scientist often has to cope with problematic samples from the crime scene due to their minute size and thus the low amount of extractable DNA. The retrieval of DNA from swabs taken from the surface of the skin, for example, in cases of strangulation, can be especially difficult. We systematically investigated swabs taken from the skin (to obtain a genetic profile from the victim and also from a possible offender) and from sperm cell containing swabs using two extraction kits: the Invisorb forensic and the Invisorb spin swab kit (both Invitek, Germany). DNA quality and quantity were tested on ethidium bromide containing agarose gels and in a highly sensitive duplex-PCR, which amplifies fragments specific for mitochondrial and nuclear DNA. Absolute quantification was done using real time PCR. Samples, which were positive in the duplex-PCR, were also employed to genetic fingerprinting using the Powerplex ES and the AmpFlSTRIdentifiler(TM) kits. Our study shows that the easy-to-use Invisorb spin swab kit is very suitable for DNA isolation from swabs taken from the skin and also from sperm cells. Retrieval of cells from the skin with swabs moistened in extraction buffer, not in distilled water, led to a significant higher DNA yield.

Development of internal amplification controls for DNA profiling with the AmpFℓSTR(®) SGM Plus(®) kit.

PubMed

Zahra, Nathalie; Hadi, Sibte; Smith, Judith A; Iyengar, Arati; Goodwin, William

2011-06-01

DNA extracted from forensic samples can be degraded and also contain co-extracted contaminants that inhibit PCR. The effects of DNA degradation and PCR inhibition are often indistinguishable when examining a DNA profile. Two internal amplification controls (IACs) were developed to improve quality control of PCR using the AmpFℓSTR® SGM Plus® kit. The co-amplification of these controls with DNA samples was used to monitor amplification efficiency and detect PCR inhibitors. IAC fragments of 90 and 410 bp (IAC₉₀ and IAC₄₁₀) were generated from the plasmid pBR322 using tailed primers and then amplified with ROX-labelled primers. Co-amplification of IAC₉₀ and IAC₄₁₀ was performed with varying amounts of template DNA, degraded DNA and DNA contaminated with humic acid, heme and indigo dye. Both IAC₉₀ and IAC₄₁₀ were successfully amplified with human DNA without significantly affecting the quality of the DNA profile, even with DNA amounts lower than 0.5 ng. In the presence of inhibitors, the IAC₉₀ signal was still present after all human DNA loci fail to amplify; in contrast, the IAC₄₁₀ signal was reduced or absent at low levels of inhibition. Amplification of the two IACs provided an internal PCR control and allowed partial profiles caused by inhibition to be distinguished from degraded DNA profiles. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

K-Band Power Enbedded Transmission Line (ETL) MMIC Amplifiers for Satellite Communication Applications

NASA Technical Reports Server (NTRS)

Tserng, Hua-Quen; Ketterson, Andrew; Saunier, Paul; McCarty, Larry; Davis, Steve

1998-01-01

The design, fabrication, and performance of K-band high-efficiency, linear power pHEMT amplifiers implemented in Embedded Transmission Line (ETL) MMIC configuration with unthinned GaAs substrate and topside grounding are reported. A three-stage amplifier achieved a power-added efficiency of 40.5% with 264 mW output at 20.2 GHz. The linear gain is 28.5 dB with 1-dB gain compression output power of 200 mW and 31% power-added efficiency. The carrier-to-third-order intermodulation ratio is approx. 20 dBc at the 1-dB compression point. A RF functional yield of more than 90% has been achieved.

Digital automatic gain amplifier

NASA Technical Reports Server (NTRS)

Holley, L. D.; Ward, J. O. (Inventor)

1978-01-01

A circuit is described for adjusting the amplitude of a reference signal to a predetermined level so as to permit subsequent data signals to be interpreted correctly. The circuit includes an operational amplifier having a feedback circuit connected between an output terminal and an input terminal; a bank of relays operably connected to a plurality of resistors; and a comparator comparing an output voltage of the amplifier with a reference voltage and generating a compared signal responsive thereto. Means is provided for selectively energizing the relays according to the compared signal from the comparator until the output signal from the amplifier equals to the reference signal. A second comparator is provided for comparing the output of the amplifier with a second voltage source so as to illuminate a lamp when the output signal from the amplifier exceeds the second voltage.

Nanoparticle sensor for label free detection of swine DNA in mixed biological samples

NASA Astrophysics Data System (ADS)

Ali, M. E.; Hashim, U.; Mustafa, S.; Che Man, Y. B.; Yusop, M. H. M.; Bari, M. F.; Islam, Kh N.; Hasan, M. F.

2011-05-01

We used 40 ± 5 nm gold nanoparticles (GNPs) as colorimetric sensor to visually detect swine-specific conserved sequence and nucleotide mismatch in PCR-amplified and non-amplified mitochondrial DNA mixtures to authenticate species. Colloidal GNPs changed color from pinkish-red to gray-purple in 2 mM PBS. Visually observed results were clearly reflected by the dramatic reduction of surface plasmon resonance peak at 530 nm and the appearance of new features in the 620-800 nm regions in their absorption spectra. The particles were stabilized against salt-induced aggregation upon the adsorption of single-stranded DNA. The PCR products, without any additional processing, were hybridized with a 17-base probe prior to exposure to GNPs. At a critical annealing temperature (55 °C) that differentiated matched and mismatched base pairing, the probe was hybridized to pig PCR product and dehybridized from the deer product. The dehybridized probe stuck to GNPs to prevent them from salt-induced aggregation and retained their characteristic red color. Hybridization of a 27-nucleotide probe to swine mitochondrial DNA identified them in pork-venison, pork-shad and venison-shad binary admixtures, eliminating the need of PCR amplification. Thus the assay was applied to authenticate species both in PCR-amplified and non-amplified heterogeneous biological samples. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The LOD (for genomic DNA) of the assay was 6 µg ml - 1 swine DNA in mixed meat samples. We believe the assay can be applied for species assignment in food analysis, mismatch detection in genetic screening and homology studies between closely related species.

Amplifier improvement circuit

NASA Technical Reports Server (NTRS)

Sturman, J.

1968-01-01

Stable input stage was designed for the use with a integrated circuit operational amplifier to provide improved performance as an instrumentation-type amplifier. The circuit provides high input impedance, stable gain, good common mode rejection, very low drift, and low output impedance.

Simple two-electrode biosignal amplifier.

PubMed

Dobrev, D; Neycheva, T; Mudrov, N

2005-11-01

A simple, cost effective circuit for a two-electrode non-differential biopotential amplifier is proposed. It uses a 'virtual ground' transimpedance amplifier and a parallel RC network for input common mode current equalisation, while the signal input impedance preserves its high value. With this innovative interface circuit, a simple non-inverting amplifier fully emulates high CMRR differential. The amplifier equivalent CMRR (typical range from 70-100 dB) is equal to the open loop gain of the operational amplifier used in the transimpedance interface stage. The circuit has very simple structure and utilises a small number of popular components. The amplifier is intended for use in various two-electrode applications, such as Holter-type monitors, defibrillators, ECG monitors, biotelemetry devices etc.

Spatial distribution and yield of DNA double-strand breaks induced by 3-7 MeV helium ions in human fibroblasts

NASA Technical Reports Server (NTRS)

Rydberg, Bjorn; Heilbronn, Lawrence; Holley, William R.; Lobrich, Markus; Zeitlin, Cary; Chatterjee, Aloke; Cooper, Priscilla K.

2002-01-01

Accelerated helium ions with mean energies at the target location of 3-7 MeV were used to simulate alpha-particle radiation from radon daughters. The experimental setup and calibration procedure allowed determination of the helium-ion energy distribution and dose in the nuclei of irradiated cells. Using this system, the induction of DNA double-strand breaks and their spatial distributions along DNA were studied in irradiated human fibroblasts. It was found that the apparent number of double-strand breaks as measured by a standard pulsed-field gel assay (FAR assay) decreased with increasing LET in the range 67-120 keV/microm (corresponding to the energy of 7-3 MeV). On the other hand, the generation of small and intermediate-size DNA fragments (0.1-100 kbp) increased with LET, indicating an increased intratrack long-range clustering of breaks. The fragment size distribution was measured in several size classes down to the smallest class of 0.1-2 kbp. When the clustering was taken into account, the actual number of DNA double-strand breaks (separated by at least 0.1 kbp) could be calculated and was found to be in the range 0.010-0.012 breaks/Mbp Gy(-1). This is two- to threefold higher than the apparent yield obtained by the FAR assay. The measured yield of double-strand breaks as a function of LET is compared with theoretical Monte Carlo calculations that simulate the track structure of energy depositions from helium ions as they interact with the 30-nm chromatin fiber. When the calculation is performed to include fragments larger than 0.1 kbp (to correspond to the experimental measurements), there is good agreement between experiment and theory.

Improved-Bandwidth Transimpedance Amplifier

NASA Technical Reports Server (NTRS)

Chapsky, Jacob

2009-01-01

The widest available operational amplifier, with the best voltage and current noise characteristics, is considered for transimpedance amplifier (TIA) applications where wide bandwidth is required to handle fast rising input signals (as for time-of-flight measurement cases). The added amplifier inside the TIA feedback loop can be configured to have slightly lower voltage gain than the bandwidth reduction factor.

Evidence of transmission of Mycobacterium tuberculosis by random amplified polymorphic DNA (RAPD) fingerprinting in Taipei City, Taiwan.

PubMed Central

Harn, H J; Shen, K L; Ho, L I; Yu, K W; Liu, G C; Yueh, K C; Lee, J H

1997-01-01

AIMS: To determine, by strain identification of Mycobacterium tuberculosis, whether transmission has occurred between individuals or whether new strains are present. METHODS: A rapid protocol for random amplified polymorphic DNA (RAPD) analysis was developed. This protocol was applied to 64 strains of M tuberculosis that had been confirmed by culture and microbiological methods. RESULTS: There are five groups of M tuberculosis prevalent in Taipei city, Taiwan. The major types are groups I and III. Groups I and II had been prevalent until the end of last year when, according to our group analysis, they had been eradicated. However, group III was continuously present from the middle of 1995 to the middle of 1996, and group IV was present at the end of both years, which indicated that both groups were transmitted continuously. These clustered strains had demographic characteristics consistent with a finding of transmission tuberculosis. Also, there were 13 of 64 strains with unique RAPD fingerprints that were inferred to be due primarily to the reactivation of infection. In the drug resistance analysis, the major type represented included group III and part of group IV. CONCLUSIONS: Our preliminary data imply, not only that the prevalence of M tuberculosis in Taipei city is due to transmission rather than reactivation, but that drug resistance also may play a role in tuberculosis transmission. Images PMID:9378819

Efficiency, error and yield in light-directed maskless synthesis of DNA microarrays

PubMed Central

2011-01-01

Background Light-directed in situ synthesis of DNA microarrays using computer-controlled projection from a digital micromirror device--maskless array synthesis (MAS)--has proved to be successful at both commercial and laboratory scales. The chemical synthetic cycle in MAS is quite similar to that of conventional solid-phase synthesis of oligonucleotides, but the complexity of microarrays and unique synthesis kinetics on the glass substrate require a careful tuning of parameters and unique modifications to the synthesis cycle to obtain optimal deprotection and phosphoramidite coupling. In addition, unintended deprotection due to scattering and diffraction introduce insertion errors that contribute significantly to the overall error rate. Results Stepwise phosphoramidite coupling yields have been greatly improved and are now comparable to those obtained in solid phase synthesis of oligonucleotides. Extended chemical exposure in the synthesis of complex, long oligonucleotide arrays result in lower--but still high--final average yields which approach 99%. The new synthesis chemistry includes elimination of the standard oxidation until the final step, and improved coupling and light deprotection. Coupling Insertions due to stray light are the limiting factor in sequence quality for oligonucleotide synthesis for gene assembly. Diffraction and local flare are by far the largest contributors to loss of optical contrast. Conclusions Maskless array synthesis is an efficient and versatile method for synthesizing high density arrays of long oligonucleotides for hybridization- and other molecular binding-based experiments. For applications requiring high sequence purity, such as gene assembly, diffraction and flare remain significant obstacles, but can be significantly reduced with straightforward experimental strategies. PMID:22152062

High-speed detection of DNA translocation in nanopipettes.

PubMed

Fraccari, Raquel L; Ciccarella, Pietro; Bahrami, Azadeh; Carminati, Marco; Ferrari, Giorgio; Albrecht, Tim

2016-04-14

We present a high-speed electrical detection scheme based on a custom-designed CMOS amplifier which allows the analysis of DNA translocation in glass nanopipettes on a microsecond timescale. Translocation of different DNA lengths in KCl electrolyte provides a scaling factor of the DNA translocation time equal to p = 1.22, which is different from values observed previously with nanopipettes in LiCl electrolyte or with nanopores. Based on a theoretical model involving electrophoresis, hydrodynamics and surface friction, we show that the experimentally observed range of p-values may be the result of, or at least be affected by DNA adsorption and friction between the DNA and the substrate surface.

DNA polymerase preference determines PCR priming efficiency.

PubMed

Pan, Wenjing; Byrne-Steele, Miranda; Wang, Chunlin; Lu, Stanley; Clemmons, Scott; Zahorchak, Robert J; Han, Jian

2014-01-30

Polymerase chain reaction (PCR) is one of the most important developments in modern biotechnology. However, PCR is known to introduce biases, especially during multiplex reactions. Recent studies have implicated the DNA polymerase as the primary source of bias, particularly initiation of polymerization on the template strand. In our study, amplification from a synthetic library containing a 12 nucleotide random portion was used to provide an in-depth characterization of DNA polymerase priming bias. The synthetic library was amplified with three commercially available DNA polymerases using an anchored primer with a random 3' hexamer end. After normalization, the next generation sequencing (NGS) results of the amplified libraries were directly compared to the unamplified synthetic library. Here, high throughput sequencing was used to systematically demonstrate and characterize DNA polymerase priming bias. We demonstrate that certain sequence motifs are preferred over others as primers where the six nucleotide sequences at the 3' end of the primer, as well as the sequences four base pairs downstream of the priming site, may influence priming efficiencies. DNA polymerases in the same family from two different commercial vendors prefer similar motifs, while another commercially available enzyme from a different DNA polymerase family prefers different motifs. Furthermore, the preferred priming motifs are GC-rich. The DNA polymerase preference for certain sequence motifs was verified by amplification from single-primer templates. We incorporated the observed DNA polymerase preference into a primer-design program that guides the placement of the primer to an optimal location on the template. DNA polymerase priming bias was characterized using a synthetic library amplification system and NGS. The characterization of DNA polymerase priming bias was then utilized to guide the primer-design process and demonstrate varying amplification efficiencies among three commercially

A DNA logic gate based on strand displacement reaction and rolling circle amplification, responding to multiple low-abundance DNA fragment input signals, and its application in detecting miRNAs.

PubMed

Chen, Yuqi; Song, Yanyan; Wu, Fan; Liu, Wenting; Fu, Boshi; Feng, Bingkun; Zhou, Xiang

2015-04-25

A conveniently amplified DNA AND logic gate platform was designed for the highly sensitive detection of low-abundance DNA fragment inputs based on strand displacement reaction and rolling circle amplification strategy. Compared with others, this system can detect miRNAs in biological samples. The success of this strategy demonstrates the potential of DNA logic gates in disease diagnosis.

A novel input-parasitic compensation technique for a nanopore-based CMOS DNA detection sensor

NASA Astrophysics Data System (ADS)

Kim, Jungsuk

2016-12-01

This paper presents a novel input-parasitic compensation (IPC) technique for a nanopore-based complementary metal-oxide-semiconductor (CMOS) DNA detection sensor. A resistive-feedback transimpedance amplifier is typically adopted as the headstage of a DNA detection sensor to amplify the minute ionic currents generated from a nanopore and convert them to a readable voltage range for digitization. But, parasitic capacitances arising from the headstage input and the nanopore often cause headstage saturation during nanopore sensing, thereby resulting in significant DNA data loss. To compensate for the unwanted saturation, in this work, we propose an area-efficient and automated IPC technique, customized for a low-noise DNA detection sensor, fabricated using a 0.35- μm CMOS process; we demonstrated this prototype in a benchtop test using an α-hemolysin ( α-HL) protein nanopore.

A Method for Preparing DNA Sequencing Templates Using a DNA-Binding Microplate

PubMed Central

Yang, Yu; Hebron, Haroun R.; Hang, Jun

2009-01-01

A DNA-binding matrix was immobilized on the surface of a 96-well microplate and used for plasmid DNA preparation for DNA sequencing. The same DNA-binding plate was used for bacterial growth, cell lysis, DNA purification, and storage. In a single step using one buffer, bacterial cells were lysed by enzymes, and released DNA was captured on the plate simultaneously. After two wash steps, DNA was eluted and stored in the same plate. Inclusion of phosphates in the culture medium was found to enhance the yield of plasmid significantly. Purified DNA samples were used successfully in DNA sequencing with high consistency and reproducibility. Eleven vectors and nine libraries were tested using this method. In 10 μl sequencing reactions using 3 μl sample and 0.25 μl BigDye Terminator v3.1, the results from a 3730xl sequencer gave a success rate of 90–95% and read-lengths of 700 bases or more. The method is fully automatable and convenient for manual operation as well. It enables reproducible, high-throughput, rapid production of DNA with purity and yields sufficient for high-quality DNA sequencing at a substantially reduced cost. PMID:19568455

DNA analysis of molluscs from a museum wet collection: a comparison of different extraction methods.

PubMed

Jaksch, Katharina; Eschner, Anita; Rintelen, Thomas V; Haring, Elisabeth

2016-07-18

DNA isolation and PCR amplification from molluscan taxa is considered as problematic because polysaccharides in tissue and mucus presumably co-precipitate with the DNA and inhibit the activity of DNA polymerase. In the present study we tested two common extraction methods on specimens from the mollusc collection of the Natural History Museum Vienna (NHMW). We analysed a broad variety of taxa covering a large temporal span (acquisition years 1877 to 1999), which distinguishes our study from previous ones where mostly fresh material was used. We also took other factors into account: effects of sample age, effects of formaldehyde treatment and taxon-specific problems. We used several primer combinations to amplify amplicons of different lengths of two mitochondrial genes: cytochrome c oxidase subunit 1 (COI) and 16S rRNA gene (16S). Overall PCR success was 43 % in the 576 extractions (including all primer combinations). The smallest amplicon (~240 bp) showed the best results (49 % positive reactions), followed by the 400 bp amplicon (40.5 %). Both short sections yielded significantly better results than the 700 bp long amplicon (27 %). Comparatively, the Gen-ial-First, All-tissue DNA-Kit-extraction method performed significantly better than Promega-Tissue and Hair Extraction Kit. Generally, PCR success is age-dependent. Nonetheless, we were able to obtain the longest amplicon even from 137-year-old material. Importantly, formaldehyde traces did not totally inhibit amplification success, although very high concentrations did. Museum material has gained importance for DNA analysis in recent years, especially for DNA barcoding projects. In some cases, however, the amplification of the standard barcoding region (partial sequence of the COI) is problematic with old material. Our study clearly shows that the COI barcoding region could be amplified in up to 49 % of PCRs (varying with amplicon length), which is, for museum samples, quite a high percentage. The

Improved Yield of High Molecular Weight DNA Coincides with Increased Microbial Diversity Access from Iron Oxide Cemented Sub-Surface Clay Environments

DOE PAGES

Hurt, Jr., Richard A.; Robeson II, Michael S.; Shakya, Migun; ...

2014-07-14

Despite more than three decades of progress, efficient nucleic acid extraction from microbial communities has remained difficult, particularly from clay environments. Lysis with concentrated guanidine followed by concentrated sodium phosphate extraction supported DNA and RNA recovery from high iron, low humus content clay. Alterating the extraction pH or using other ionic solutions (Na 2SO 4 and NH 4H 2PO 4) yielded no detectable nucleic acid. DNA recovered using a lysis solution with 500 mM phosphate buffer (PB) followed by a 1 M PB wash was 15.22±2.33 g DNA/g clay, with most DNA consisting of >20 Kb fragments, compared to 2.46±0.25more » g DNA/g clay with the Powerlyzer soil DNA system (MoBio). Increasing [PB] in the lysis reagent coincided with increasing DNA fragment length. Rarefaction plots based on16S rRNA (V1/V3 region) pyrosequencing libraries from A-horizon and clay soils showed an ~80% and ~400% larger accessed diversity compared to a previous grinding protocol or the Powerlyzer soil DNA system, respectively. The observed diversity from the Firmicutes showed the strongest increase with >3-fold more bacterial species recovered using this system. Additionally, some OTU's having more than 100 sequences in these libraries were absent in samples extracted using the PowerLyzer reagents or the previous lysis method.« less

Improved Yield of High Molecular Weight DNA Coincides with Increased Microbial Diversity Access from Iron Oxide Cemented Sub-Surface Clay Environments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hurt, Jr., Richard A.; Robeson II, Michael S.; Shakya, Migun

Despite more than three decades of progress, efficient nucleic acid extraction from microbial communities has remained difficult, particularly from clay environments. Lysis with concentrated guanidine followed by concentrated sodium phosphate extraction supported DNA and RNA recovery from high iron, low humus content clay. Alterating the extraction pH or using other ionic solutions (Na 2SO 4 and NH 4H 2PO 4) yielded no detectable nucleic acid. DNA recovered using a lysis solution with 500 mM phosphate buffer (PB) followed by a 1 M PB wash was 15.22±2.33 g DNA/g clay, with most DNA consisting of >20 Kb fragments, compared to 2.46±0.25more » g DNA/g clay with the Powerlyzer soil DNA system (MoBio). Increasing [PB] in the lysis reagent coincided with increasing DNA fragment length. Rarefaction plots based on16S rRNA (V1/V3 region) pyrosequencing libraries from A-horizon and clay soils showed an ~80% and ~400% larger accessed diversity compared to a previous grinding protocol or the Powerlyzer soil DNA system, respectively. The observed diversity from the Firmicutes showed the strongest increase with >3-fold more bacterial species recovered using this system. Additionally, some OTU's having more than 100 sequences in these libraries were absent in samples extracted using the PowerLyzer reagents or the previous lysis method.« less

Immobilization of microalgae cells in alginate facilitates isolation of DNA and RNA.

PubMed

Lopez, Blanca R; Hernandez, Juan-Pablo; Bashan, Yoav; de-Bashan, Luz E

2017-04-01

Isolation of nucleic acids from Chlorella is difficult, given the chemically complex nature of their cell walls and variable production of metabolites. Immobilization of microalgae in polymers adds additional difficulty. Here, we modified, amended, and standardized methods for isolation of nucleic acids and compared the yield of DNA and RNA from free-living and encapsulated microalgae C. sorokiniana. Isolation of nucleic acids from immobilized cells required two steps in dissolving the alginate matrix, releasing the cells, and mechanical disruption with glass beads. For DNA extraction, we used modified versions of a commercial kit along with the hexadecyltrimethylammonium bromide (CTAB) method. For RNA extraction, we used the commercial TRI reagent procedure and the CTAB-dithiotreitol method. Quantity and quality of nucleic acids in extracts varied with growth conditions, isolation procedures, and time of incubation of the original culture. There were consistently higher amounts of DNA and RNA in extracts from immobilized cells. Quantitatively, the modified procedure with the commercial Promega kit was the most reliable procedure for isolating DNA and a modified commercial TRI reagent procedure was the choice for isolating RNA. All four procedures eliminated proteins efficiently and had low levels of contamination from residual polysaccharides from the matrices and/or metabolites naturally produced by the microalgae. All DNA extracts under both growth conditions, time of incubation, and two isolation methods successfully amplified the 18S ribosomal RNA by PCR and quantitative reverse transcription (RT-qPCR). Copyright © 2017 Elsevier B.V. All rights reserved.

FLUIDIC AC AMPLIFIERS.

DTIC Science & Technology

Several fluidic tuned AC Amplifiers were designed and tested. Interstage tuning and feedback designs are considered. Good results were obtained...corresponding Q’s as high as 12. Element designs and test results of one, two, and three stage amplifiers are presented. AC Modulated Carrier Systems

A new approach for cloning hLIF cDNA from genomic DNA isolated from the oral mucous membrane.

PubMed

Cui, Y H; Zhu, G Q; Chen, Q J; Wang, Y F; Yang, M M; Song, Y X; Wang, J G; Cao, B Y

2011-11-25

Complementary DNA (cDNA) is valuable for investigating protein structure and function in the study of life science, but it is difficult to obtain by traditional reverse transcription. We employed a novel strategy to clone human leukemia inhibitory factor (hLIF) gene cDNA from genomic DNA, which was directly isolated from the mucous membrane of mouth. The hLIF sequence, which is 609 bp long and is composed of three exons, can be acquired within a few hours by amplifying each exon and splicing all of them using overlap-PCR. This new approach developed is simple, time- and cost-effective, without RNA preparation or cDNA synthesis, and is not limited to the specific tissues for a particular gene and the expression level of the gene.

Genetic analysis of tumorigenesis: XXXII. Localization of constitutionally amplified KRAS sequences to Chinese hamster chromosomes X and Y by in situ hybridization.

PubMed

Stenman, G; Anisowicz, A; Sager, R

1988-11-01

The KRAS gene is constitutionally amplified in the Chinese hamster. We have mapped the amplified sequences by in situ hybridization to two major sites on the X and Y chromosomes, Xq4 and Yp2. No autosomal site was detected despite a search under relaxed hybridization conditions. KRAS DNA is amplified about 50-fold compared to a human cell line known to have a diploid number of KRAS sequences, whereas mRNA expression is 5- to 10-fold lower than in normal human cells. While mRNA expression levels do not necessarily parallel gene copy number, the low expression level strongly suggests that the amplified sequences are transcriptionally silent. It is suggested that the amplified sequences arose from the original KRAS gene on chromosome 8 and that the KRAS sequences on the Y chromosome arose by X-Y recombination.

Right-handed double-helix ultrashort DNA yields chiral nematic phases with both right- and left-handed director twist

PubMed Central

Zanchetta, Giuliano; Giavazzi, Fabio; Nakata, Michi; Buscaglia, Marco; Cerbino, Roberto; Clark, Noel A.; Bellini, Tommaso

2010-01-01

Concentrated solutions of duplex-forming DNA oligomers organize into various mesophases among which is the nematic (N∗), which exhibits a macroscopic chiral helical precession of molecular orientation because of the chirality of the DNA molecule. Using a quantitative analysis of the transmission spectra in polarized optical microscopy, we have determined the handedness and pitch of this chiral nematic helix for a large number of sequences ranging from 8 to 20 bases. The B-DNA molecule exhibits a right-handed molecular double-helix structure that, for long molecules, always yields N∗ phases with left-handed pitch in the μm range. We report here that ultrashort oligomeric duplexes show an extremely diverse behavior, with both left- and right-handed N∗ helices and pitches ranging from macroscopic down to 0.3 μm. The behavior depends on the length and the sequence of the oligomers, and on the nature of the end-to-end interactions between helices. In particular, the N∗ handedness strongly correlates with the oligomer length and concentration. Right-handed phases are found only for oligomers shorter than 14 base pairs, and for the sequences having the transition to the N∗ phase at concentration larger than 620 mg/mL. Our findings indicate that in short DNA, the intermolecular double-helical interactions switch the preferred liquid crystal handedness when the columns of stacked duplexes are forced at high concentrations to separations comparable to the DNA double-helix pitch, a regime still to be theoretically described. PMID:20876125

Ce(III, IV)-MOF electrocatalyst as signal-amplifying tag for sensitive electrochemical aptasensing.

PubMed

Yu, Hua; Han, Jing; An, Shangjie; Xie, Gang; Chen, Sanping

2018-06-30

Metal-organic frameworks (MOFs) as a new class of porous materials have attracted increasing attention in the field of biomimetic catalysis. This study firstly reports a mixed valence state Ce-MOF possessing intrinsic catalytic activity towards thionine (Thi), and its application in constructing an amplified electrochemical aptasensor for thrombin detection. As noticed, the novel catalytic process combines the advantages of 3D infinite extension of the Ce(III, IV)-MOF skeleton containing large amounts of catalytic sites and spontaneous recycling of the Ce(III)/Ce(IV) for electrochemical reduction of Thi, thereby presenting amplified electrochemical signals. To further improve the aptasensor performance, the high selectivity of proximity binding-induced DNA strand displacement and high efficiency of exonuclease III-assisted recycling amplification were incorporated into the assay. The aptasensor was employed to detect thrombin in complex serum samples, which shows high sensitivity, specificity, stability and reproducibility. This work offers an opportunity to develop MOF-based electrocatalyst as signal-amplifying tag for versatile bioassays and catalytic applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Characterization of mutagen-activated cellular oncogenes that confer anchorage independence to human fibroblasts and tumorigenicity to NIH 3T3 cells: Sequence analysis of an enzymatically amplified mutant HRAS allele

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stevens, C.W.; Manoharan, T.H.; Fahl, W.E.

1988-06-01

Treatment of diploid human fibroblasts with an alkylating mutagen has been shown to induce stable, anchorage-independent cell populations at frequencies consistent with an activating mutation. After treatment of human foreskin fibroblasts with the mutagen benzo({alpha})pyrene ({plus minus})anti-7,8-dihydrodiol 9,10-epoxide and selection in soft agar, 17 anchorage-independent clones were isolated and expanded, and their cellular DNA was used to cotransfect NIH 3T3 cells along with pSV2neo. DNA from 11 of the 17 clones induced multiple NIH 3T3 cell tumors in recipient nude mice. Southern blot analyses showed the presence of human Alu repetitive sequences in all of the NIH 3T3 tumor cellmore » DNAs. Intact, human HRAS sequences were observed in 2 of the 11 tumor groups, whereas no hybridization was detected when human KRAS or NRAS probes were used. Slow-migrating ras p21 proteins, consistent with codon 12 mutations, were observed in the same two NIH 3T3 tumor cell groups that contained the human HRAS bands. Genomic DNA from one of these two human anchorage-independent cell populations (clone 21A) was used to enzymatically amplify a portion of exon 1 of the HRAS gene. The results demonstrate that exposure of normal human cells to a common environmental mutagen yields HRAS GC {yields} TA codon 12 transversions that have been commonly observed in human tumors.« less

Amplification of Mitochondrial DNA for detection of Plasmodiumvivax in Balochistan.

PubMed

Shahwani, Muhammad Naeem; Nisar, Samia; Aleem, Abdul; Panezai, Marina; Afridi, Sarwat; Malik, Shaukat Iqbal

2017-05-01

To access a new step using PCR to amplify the targeted mtDNA sequence for detecting specifically Plasmodium vivax and its co-infections, false positive and false negative results with Plasmodium falciparum. In this study we have standardized a new technical approach in which the target mitochondrial DNA sequence (mtDNA) was amplified by using a PCR technique as a tool to detect Plasmodium spp. Species specific primers were designed to hybridize with cytochrome c oxidase gene of P. vivax (cox I) and P. falciparum (cox III). Two hundred blood samples were collected on the basis of clinical symptoms which were initially examined through microscopic analysis after preparing Giemsa stained thick and thin blood smears. Afterwards genomic DNA was extracted from all samples and was then subjected to PCR amplification by using species specific primers and amplified segments were sequenced for confirmation of results. One-hundred and thirty-two blood samples were detected as positive for malaria by PCR, out of which 64 were found to be positive by PCR and 53 by both microscopy and PCR for P.vivax infection. Nine samples were found to be false negative, one P.vivax mono infection was declared as co infection by PCR and 3 samples identified as having P.falciparum gametes were confirmed as P.vivax by PCR amplification. Sensitivity and specificity were found to be 85% and 92% respectively. Results obtained through PCR method were comparatively better and reliable than microscopy.

Amplified spontaneous emission in phenylethylammonium methylammonium lead iodide quasi-2D perovskites.

PubMed

Leyden, Matthew R; Matsushima, Toshinori; Qin, Chuanjiang; Ruan, Shibin; Ye, Hao; Adachi, Chihaya

2018-06-06

Organo-metal-halide perovskites are a promising set of materials for optoelectronic applications such as solar cells, light emitting diodes and lasers. Perovskite thin films have demonstrated amplified spontaneous emission thresholds as low as 1.6 μJ cm-2 and lasing thresholds as low as 0.2 μJ cm-2. Recently the performance of perovskite light emitting diodes has rapidly risen due to the formation of quasi 2D films using bulky ligands such as phenylethylammonium. Despite the high photoluminescent yield and external quantum efficiency of quasi 2D perovskites, few reports exist on amplified spontaneous emission. We show within this report that the threshold for amplified spontaneous emission of quasi 2D perovskite films increases with the concentration of phenylethylammonium. We attribute this increasing threshold to a charge transfer state at the PEA interface that competes for excitons with the ASE process. Additionally, the comparatively slow inter-grain charge transfer process cannot significantly contribute to the fast radiative recombination in amplified spontaneous emission. These results suggest that relatively low order PEA based perovskite films that are suitable for LED applications are not well suited for lasing applications. However high order films were able to maintain their low threshold values and may still benefit from improved stability.

Forensic Analysis of Canine DNA Samples in the Undergraduate Biochemistry Laboratory

ERIC Educational Resources Information Center

Carson, Tobin M.; Bradley, Sharonda Q.; Fekete, Brenda L.; Millard, Julie T.; LaRiviere, Frederick J.

2009-01-01

Recent advances in canine genomics have allowed the development of highly distinguishing methods of analysis for both nuclear and mitochondrial DNA. We describe a laboratory exercise suitable for an undergraduate biochemistry course in which the polymerase chain reaction is used to amplify hypervariable regions of DNA from dog hair and saliva…

Use PCR and a Single Hair To Produce a "DNA Fingerprint."

ERIC Educational Resources Information Center

Campbell, A. Malcolm; And Others

1997-01-01

Presents a laboratory procedure that involves students extracting their own DNA from a single hair follicle, using the polymerase chain reaction (PCR) to amplify a polymorphic locus, performing electrophoresis on the PCR products on an agarose gel, and visualizing the alleles to generate a "DNA fingerprint." Discusses theoretical background,…

Aspergillus section Versicolores: nine new species and multilocus DNA sequence based phylogeny

USDA-ARS?s Scientific Manuscript database

ß-tubulin, calmodulin, internal transcribed spacer and partial lsu-rDNA, RNA polymerase, DNA replication licensing factor Mcm7, and pre-rRNA processing protein Tsr1 were amplified and sequenced from 62 A. versicolor clade isolates and analyzed phylogenetically using the concordance model to establis...

Aspergillus section Versicolores, nine new species and multilocus DNA sequence based phylogeny

USDA-ARS?s Scientific Manuscript database

ß-tubulin, calmodulin, internal transcribed spacer and partial lsu-rDNA, RNA polymerase, DNA replication licensing factor Mcm7, and pre-rRNA processing protein Tsr1 were amplified and sequenced from 62 A. versicolor clade isolates and analyzed phylogenetically using the concordance model to establis...

PCR performance of a thermostable heterodimeric archaeal DNA polymerase

PubMed Central

Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

2014-01-01

DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications. PMID:24847315

CMOS analogue amplifier circuits optimisation using hybrid backtracking search algorithm with differential evolution

NASA Astrophysics Data System (ADS)

Mallick, S.; Kar, R.; Mandal, D.; Ghoshal, S. P.

2016-07-01

This paper proposes a novel hybrid optimisation algorithm which combines the recently proposed evolutionary algorithm Backtracking Search Algorithm (BSA) with another widely accepted evolutionary algorithm, namely, Differential Evolution (DE). The proposed algorithm called BSA-DE is employed for the optimal designs of two commonly used analogue circuits, namely Complementary Metal Oxide Semiconductor (CMOS) differential amplifier circuit with current mirror load and CMOS two-stage operational amplifier (op-amp) circuit. BSA has a simple structure that is effective, fast and capable of solving multimodal problems. DE is a stochastic, population-based heuristic approach, having the capability to solve global optimisation problems. In this paper, the transistors' sizes are optimised using the proposed BSA-DE to minimise the areas occupied by the circuits and to improve the performances of the circuits. The simulation results justify the superiority of BSA-DE in global convergence properties and fine tuning ability, and prove it to be a promising candidate for the optimal design of the analogue CMOS amplifier circuits. The simulation results obtained for both the amplifier circuits prove the effectiveness of the proposed BSA-DE-based approach over DE, harmony search (HS), artificial bee colony (ABC) and PSO in terms of convergence speed, design specifications and design parameters of the optimal design of the analogue CMOS amplifier circuits. It is shown that BSA-DE-based design technique for each amplifier circuit yields the least MOS transistor area, and each designed circuit is shown to have the best performance parameters such as gain, power dissipation, etc., as compared with those of other recently reported literature.

Bio-isolated DC operational amplifier

NASA Technical Reports Server (NTRS)

Lee, R. D.

1974-01-01

Possibility of shocks from leakage currents can be reduced by use of isolated preamplifiers. Amplifier consists of battery-powered operational amplifier coupled by means of light-emitting diodes to another amplifier which may be grounded and operated from ac power mains or separate battery supply.

Isolation of genomic DNA from defatted oil seed residue of rapeseed (Brassica napus).

PubMed

Sadia, M; Rabbani, M A; Hameed, S; Pearce, S R; Malik, S A

2011-02-08

A simple protocol for obtaining pure, restrictable and amplifiable megabase genomic DNA from oil-free seed residue of Brassica napus, an important oil seed plant, has been developed. Oil from the dry seeds was completely recovered in an organic solvent and quantified gravimetrically followed by processing of the residual biomass (defatted seed residue) for genomic DNA isolation. The isolated DNA can be cut by a range of restriction enzymes. The method enables simultaneous isolation and recovery of lipids and genomic DNA from the same test sample, thus allowing two independent analyses from a single sample. Multiple micro-scale oil extraction from the commercial seeds gave approximately 39% oil, which is close to the usual oil recovery from standard oil seed. Most of the amplified fragments were scored in the range of 2.5 to 0.5 kb, best suited for scoring as molecular diagnostics.

Spiking of contemporary human template DNA with ancient DNA extracts induces mutations under PCR and generates nonauthentic mitochondrial sequences.

PubMed

Pusch, Carsten M; Bachmann, Lutz

2004-05-01

Proof of authenticity is the greatest challenge in palaeogenetic research, and many safeguards have become standard routine in laboratories specialized on ancient DNA research. Here we describe an as-yet unknown source of artifacts that will require special attention in the future. We show that ancient DNA extracts on their own can have an inhibitory and mutagenic effect under PCR. We have spiked PCR reactions including known human test DNA with 14 selected ancient DNA extracts from human and nonhuman sources. We find that the ancient DNA extracts inhibit the amplification of large fragments to different degrees, suggesting that the usual control against contaminations, i.e., the absence of long amplifiable fragments, is not sufficient. But even more important, we find that the extracts induce mutations in a nonrandom fashion. We have amplified a 148-bp stretch of the mitochondrial HVRI from contemporary human template DNA in spiked PCR reactions. Subsequent analysis of 547 sequences from cloned amplicons revealed that the vast majority (76.97%) differed from the correct sequence by single nucleotide substitutions and/or indels. In total, 34 positions of a 103-bp alignment are affected, and most mutations occur repeatedly in independent PCR amplifications. Several of the induced mutations occur at positions that have previously been detected in studies of ancient hominid sequences, including the Neandertal sequences. Our data imply that PCR-induced mutations are likely to be an intrinsic and general problem of PCR amplifications of ancient templates. Therefore, ancient DNA sequences should be considered with caution, at least as long as the molecular basis for the extract-induced mutations is not understood.

Capacities of quantum amplifier channels

NASA Astrophysics Data System (ADS)

Qi, Haoyu; Wilde, Mark M.

2017-01-01

Quantum amplifier channels are at the core of several physical processes. Not only do they model the optical process of spontaneous parametric down-conversion, but the transformation corresponding to an amplifier channel also describes the physics of the dynamical Casimir effect in superconducting circuits, the Unruh effect, and Hawking radiation. Here we study the communication capabilities of quantum amplifier channels. Invoking a recently established minimum output-entropy theorem for single-mode phase-insensitive Gaussian channels, we determine capacities of quantum-limited amplifier channels in three different scenarios. First, we establish the capacities of quantum-limited amplifier channels for one of the most general communication tasks, characterized by the trade-off between classical communication, quantum communication, and entanglement generation or consumption. Second, we establish capacities of quantum-limited amplifier channels for the trade-off between public classical communication, private classical communication, and secret key generation. Third, we determine the capacity region for a broadcast channel induced by the quantum-limited amplifier channel, and we also show that a fully quantum strategy outperforms those achieved by classical coherent-detection strategies. In all three scenarios, we find that the capacities significantly outperform communication rates achieved with a naive time-sharing strategy.

Optical Amplifier for Space Applications

NASA Technical Reports Server (NTRS)

Fork, Richard L.; Cole, Spencer T.; Gamble, Lisa J.; Diffey, William M.; Keys, Andrew S.

1999-01-01

We describe an optical amplifier designed to amplify a spatially sampled component of an optical wavefront to kilowatt average power. The goal is means for implementing a strategy of spatially segmenting a large aperture wavefront, amplifying the individual segments, maintaining the phase coherence of the segments by active means, and imaging the resultant amplified coherent field. Applications of interest are the transmission of space solar power over multi-megameter distances, as to distant spacecraft, or to remote sites with no preexisting power grid.

Improved Grid-Array Millimeter-Wave Amplifier

NASA Technical Reports Server (NTRS)

Rosenberg, James J.; Rutledge, David B.; Smith, R. Peter; Weikle, Robert

1993-01-01

Improved grid-array amplifiers operating at millimeter and submillimeter wavelengths developed for use in communications and radar. Feedback suppressed by making input polarizations orthogonal to output polarizations. Amplifier made to oscillate by introducing some feedback. Several grid-array amplifiers concatenated to form high-gain beam-amplifying unit.

NASA developments in solid state power amplifiers

NASA Technical Reports Server (NTRS)

Leonard, Regis F.

1990-01-01

Over the last ten years, NASA has undertaken an extensive program aimed at development of solid state power amplifiers for space applications. Historically, the program may be divided into three phases. The first efforts were carried out in support of the advanced communications technology satellite (ACTS) program, which is developing an experimental version of a Ka-band commercial communications system. These first amplifiers attempted to use hybrid technology. The second phase was still targeted at ACTS frequencies, but concentrated on monolithic implementations, while the current, third phase, is a monolithic effort that focusses on frequencies appropriate for other NASA programs and stresses amplifier efficiency. The topics covered include: (1) 20 GHz hybrid amplifiers; (2) 20 GHz monolithic MESFET power amplifiers; (3) Texas Instruments' (TI) 20 GHz variable power amplifier; (4) TI 20 GHz high power amplifier; (5) high efficiency monolithic power amplifiers; (6) GHz high efficiency variable power amplifier; (7) TI 32 GHz monolithic power amplifier performance; (8) design goals for Hughes' 32 GHz variable power amplifier; and (9) performance goals for Hughes' pseudomorphic 60 GHz power amplifier.

High input impedance amplifier

NASA Technical Reports Server (NTRS)

Kleinberg, Leonard L.

1995-01-01

High input impedance amplifiers are provided which reduce the input impedance solely to a capacitive reactance, or, in a somewhat more complex design, provide an extremely high essentially infinite, capacitive reactance. In one embodiment, where the input impedance is reduced in essence, to solely a capacitive reactance, an operational amplifier in a follower configuration is driven at its non-inverting input and a resistor with a predetermined magnitude is connected between the inverting and non-inverting inputs. A second embodiment eliminates the capacitance from the input by adding a second stage to the first embodiment. The second stage is a second operational amplifier in a non-inverting gain-stage configuration where the output of the first follower stage drives the non-inverting input of the second stage and the output of the second stage is fed back to the non-inverting input of the first stage through a capacitor of a predetermined magnitude. These amplifiers, while generally useful, are very useful as sensor buffer amplifiers that may eliminate significant sources of error.

Electrospun amplified fiber optics.

PubMed

Morello, Giovanni; Camposeo, Andrea; Moffa, Maria; Pisignano, Dario

2015-03-11

All-optical signal processing is the focus of much research aiming to obtain effective alternatives to existing data transmission platforms. Amplification of light in fiber optics, such as in Erbium-doped fiber amplifiers, is especially important for efficient signal transmission. However, the complex fabrication methods involving high-temperature processes performed in a highly pure environment slow the fabrication process and make amplified components expensive with respect to an ideal, high-throughput, room temperature production. Here, we report on near-infrared polymer fiber amplifiers working over a band of ∼20 nm. The fibers are cheap, spun with a process entirely carried out at room temperature, and shown to have amplified spontaneous emission with good gain coefficients and low levels of optical losses (a few cm(-1)). The amplification process is favored by high fiber quality and low self-absorption. The found performance metrics appear to be suitable for short-distance operations, and the large variety of commercially available doping dyes might allow for effective multiwavelength operations by electrospun amplified fiber optics.

Os11Gsk gene from a wild rice, Oryza rufipogon improves yield in rice.

PubMed

Thalapati, Sudhakar; Batchu, Anil K; Neelamraju, Sarla; Ramanan, Rajeshwari

2012-06-01

Chromosomal segments from wild rice species Oryza rufipogon, introgressed into an elite indica rice restorer line (KMR3) using molecular markers, resulted in significant increase in yield. Here we report the transcriptome analysis of flag leaves and fully emerged young panicles of one of the high yielding introgression lines IL50-7 in comparison to KMR3. A 66-fold upregulated gene Os11Gsk, which showed no transcript in KMR3 was highly expressed in O. rufipogon and IL50-7. A 5-kb genomic region including Os11Gsk and its flanking regions could be PCR amplified only from IL50-7, O. rufipogon, japonica varieties of rice-Nipponbare and Kitaake but not from the indica varieties, KMR3 and Taichung Native-1. Three sister lines of IL50-7 yielding higher than KMR3 showed presence of Os11Gsk, whereas the gene was absent in three other ILs from the same cross having lower yield than KMR3, indicating an association of the presence of Os11Gsk with high yield. Southern analysis showed additional bands in the genomic DNA of O. rufipogon and IL50-7 with Os11Gsk probe. Genomic sequence analysis of ten highly co-expressed differentially regulated genes revealed that two upregulated genes in IL50-7 were derived from O. rufipogon and most of the downregulated genes were either from KMR3 or common to KMR3, IL50-7, and O. rufipogon. Thus, we show that Os11Gsk is a wild rice-derived gene introduced in KMR3 background and increases yield either by regulating expression of functional genes sharing homology with it or by causing epigenetic modifications in the introgression line.

DNA-based species detection capabilities using laser transmission spectroscopy

PubMed Central

Mahon, A. R.; Barnes, M. A.; Li, F.; Egan, S. P.; Tanner, C. E.; Ruggiero, S. T.; Feder, J. L.; Lodge, D. M.

2013-01-01

Early detection of invasive species is critical for effective biocontrol to mitigate potential ecological and economic damage. Laser transmission spectroscopy (LTS) is a powerful solution offering real-time, DNA-based species detection in the field. LTS can measure the size, shape and number of nanoparticles in a solution and was used here to detect size shifts resulting from hybridization of the polymerase chain reaction product to nanoparticles functionalized with species-specific oligonucleotide probes or with the species-specific oligonucleotide probes alone. We carried out a series of DNA detection experiments using the invasive freshwater quagga mussel (Dreissena bugensis) to evaluate the capability of the LTS platform for invasive species detection. Specifically, we tested LTS sensitivity to (i) DNA concentrations of a single target species, (ii) the presence of a target species within a mixed sample of other closely related species, (iii) species-specific functionalized nanoparticles versus species-specific oligonucleotide probes alone, and (iv) amplified DNA fragments versus unamplified genomic DNA. We demonstrate that LTS is a highly sensitive technique for rapid target species detection, with detection limits in the picomolar range, capable of successful identification in multispecies samples containing target and non-target species DNA. These results indicate that the LTS DNA detection platform will be useful for field application of target species. Additionally, we find that LTS detection is effective with species-specific oligonucleotide tags alone or when they are attached to polystyrene nanobeads and with both amplified and unamplified DNA, indicating that the technique may also have versatility for broader applications. PMID:23015524

Simultaneous Recovery of Extracellular and Intracellular DNA Suitable for Molecular Studies from Marine Sediments

PubMed Central

Corinaldesi, Cinzia; Danovaro, Roberto; Dell'Anno, Antonio

2005-01-01

The occurrence of high extracellular DNA concentrations in aquatic sediments (concentrations that are 3 to 4 orders of magnitude greater than those in the water column) might play an important role in biogeochemical cycling, as well as in horizontal gene transfer through natural transformation. Since isolation of extracellular DNA from sediments is a difficult and unsolved task, in this study we developed an efficient procedure to recover simultaneously DNA associated with microbial cells and extracellular DNA from the same sediment sample. This procedure is specifically suitable for studying extracellular DNA because it avoids any contamination with DNA released by cell lysis during handling and extraction. Applying this procedure to different sediment types, we obtained extracellular DNA concentrations that were about 10 to 70 times higher than the intracellular DNA concentrations. Using specific targeted prokaryotic primers, we obtained evidence that extracellular DNA recovered from different sediments did not contain amplifiable 16S rRNA genes. By contrast, using DNA extracted from microbial cells as the template, we always amplified 16S rRNA genes. Although 16S rRNA genes were not detected in extracellular DNA, analyses of the sizes of extracellular DNA indicated the presence of high-molecular-weight fragments that might have contained other gene sequences. This protocol allows investigation of extracellular DNA and its possible participation in natural transformation processes. PMID:15640168

Evaluation and comparison of FTA card and CTAB DNA extraction methods for non-agricultural taxa.

PubMed

Siegel, Chloe S; Stevenson, Florence O; Zimmer, Elizabeth A

2017-02-01

An efficient, effective DNA extraction method is necessary for comprehensive analysis of plant genomes. This study analyzed the quality of DNA obtained using paper FTA cards prepared directly in the field when compared to the more traditional cetyltrimethylammonium bromide (CTAB)-based extraction methods from silica-dried samples. DNA was extracted using FTA cards according to the manufacturer's protocol. In parallel, CTAB-based extractions were done using the automated AutoGen DNA isolation system. DNA quality for both methods was determined for 15 non-agricultural species collected in situ, by gel separation, spectrophotometry, fluorometry, and successful amplification and sequencing of nuclear and chloroplast gene markers. The FTA card extraction method yielded less concentrated, but also less fragmented samples than the CTAB-based technique. The card-extracted samples provided DNA that could be successfully amplified and sequenced. The FTA cards are also useful because the collected samples do not require refrigeration, extensive laboratory expertise, or as many hazardous chemicals as extractions using the CTAB-based technique. The relative success of the FTA card method in our study suggested that this method could be a valuable tool for studies in plant population genetics and conservation biology that may involve screening of hundreds of individual plants. The FTA cards, like the silica gel samples, do not contain plant material capable of propagation, and therefore do not require permits from the U.S. Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS) for transportation.

Autoclave method for rapid preparation of bacterial PCR-template DNA.

PubMed

Simmon, Keith E; Steadman, Dewey D; Durkin, Sarah; Baldwin, Amy; Jeffrey, Wade H; Sheridan, Peter; Horton, Rene; Shields, Malcolm S

2004-02-01

An autoclave method for preparing bacterial DNA for PCR template is presented, it eliminates the use of detergents, organic solvents, and mechanical cellular disruption approaches, thereby significantly reducing processing time and costs while increasing reproducibility. Bacteria are lysed by rapid heating and depressurization in an autoclave. The lysate, cleared by microcentrifugation, was either used directly in the PCR reaction, or concentrated by ultrafiltration. This approach was compared with seven established methods of DNA template preparation from four bacterial sources which included boiling Triton X-100 and SDS, bead beating, lysozyme/proteinase K, and CTAB lysis method components. Bacteria examined were Enterococcus and Escherichia coli, a natural marine bacterial community and an Antarctic cyanobacterial-mat. DNAs were tested for their suitability as PCR templates by repetitive element random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE) analysis. The autoclave method produced PCR amplifiable template comparable or superior to the other methods, with greater reproducibility, much shorter processing time, and at a significantly lower cost.

Colony-PCR Is a Rapid Method for DNA Amplification of Hyphomycetes

PubMed Central

Walch, Georg; Knapp, Maria; Rainer, Georg; Peintner, Ursula

2016-01-01

Fungal pure cultures identified with both classical morphological methods and through barcoding sequences are a basic requirement for reliable reference sequences in public databases. Improved techniques for an accelerated DNA barcode reference library construction will result in considerably improved sequence databases covering a wider taxonomic range. Fast, cheap, and reliable methods for obtaining DNA sequences from fungal isolates are, therefore, a valuable tool for the scientific community. Direct colony PCR was already successfully established for yeasts, but has not been evaluated for a wide range of anamorphic soil fungi up to now, and a direct amplification protocol for hyphomycetes without tissue pre-treatment has not been published so far. Here, we present a colony PCR technique directly from fungal hyphae without previous DNA extraction or other prior manipulation. Seven hundred eighty-eight fungal strains from 48 genera were tested with a success rate of 86%. PCR success varied considerably: DNA of fungi belonging to the genera Cladosporium, Geomyces, Fusarium, and Mortierella could be amplified with high success. DNA of soil-borne yeasts was always successfully amplified. Absidia, Mucor, Trichoderma, and Penicillium isolates had noticeably lower PCR success. PMID:29376929

Detection of cashew nut DNA in spiked baked goods using a real-time polymerase chain reaction method.

PubMed

Brzezinski, Jennifer L

2006-01-01

The detection of potentially allergenic foods, such as tree nuts, in food products is a major concern for the food processing industry. A real-time polymerase chain reaction (PCR) method was designed to determine the presence of cashew DNA in food products. The PCR amplifies a 67 bp fragment of the cashew 2S albumin gene, which is detected with a cashew-specific, dual-labeled TaqMan probe. This reaction will not amplify DNA derived from other tree nut species, such as almond, Brazil nut, hazelnut, and walnut, as well as 4 varieties of peanut. This assay was sensitive enough to detect 5 pg purified cashew DNA as well as cashew DNA in a spiked chocolate cookie sample containing 0.01% (100 mg/kg) cashew.

Bidirectional amplifier

DOEpatents

Wright, J.T.

1984-02-02

A bilateral circuit is operable for transmitting signals in two directions without generation of ringing due to feedback caused by the insertion of the circuit. The circuit may include gain for each of the signals to provide a bidirectional amplifier. The signals are passed through two separate paths, with a unidirectional amplifier in each path. A controlled sampling device is provided in each path for sampling the two signals. Any feedback loop between the two signals is disrupted by providing a phase displacement between the control signals for the two sampling devices.

Bidirectional amplifier

DOEpatents

Wright, James T.

1986-01-01

A bilateral circuit is operable for transmitting signals in two directions without generation of ringing due to feedback caused by the insertion of the circuit. The circuit may include gain for each of the signals to provide a bidirectional amplifier. The signals are passed through two separate paths, with a unidirectional amplifier in each path. A controlled sampling device is provided in each path for sampling the two signals. Any feedback loop between the two signals is disrupted by providing a phase displacement between the control signals for the two sampling devices.

Ping-pong auto-zero amplifier with glitch reduction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Larson, Mark R

A ping-pong amplifier with reduced glitching is described. The ping-pong amplifier includes a nulling amplifier coupled to a switching network. The switching network is used to auto-zero a ping amplifier within a ping-pong amplifier. The nulling amplifier drives the output of a ping amplifier to a proper output voltage level during auto-zeroing of the ping amplifier. By being at a proper output voltage level, glitches associated with transitioning between a ping amplifier and a pong amplifier are reduced or eliminated.

DNA Origami-Graphene Hybrid Nanopore for DNA Detection.

PubMed

Barati Farimani, Amir; Dibaeinia, Payam; Aluru, Narayana R

2017-01-11

DNA origami nanostructures can be used to functionalize solid-state nanopores for single molecule studies. In this study, we characterized a nanopore in a DNA origami-graphene heterostructure for DNA detection. The DNA origami nanopore is functionalized with a specific nucleotide type at the edge of the pore. Using extensive molecular dynamics (MD) simulations, we computed and analyzed the ionic conductivity of nanopores in heterostructures carpeted with one or two layers of DNA origami on graphene. We demonstrate that a nanopore in DNA origami-graphene gives rise to distinguishable dwell times for the four DNA base types, whereas for a nanopore in bare graphene, the dwell time is almost the same for all types of bases. The specific interactions (hydrogen bonds) between DNA origami and the translocating DNA strand yield different residence times and ionic currents. We also conclude that the speed of DNA translocation decreases due to the friction between the dangling bases at the pore mouth and the sequencing DNA strands.

Characterization of North American Armillaria species: Genetic relationships determined by ribosomal DNA sequences and AFLP markers

Treesearch

M. -S. Kim; N. B. Klopfenstein; J. W. Hanna; G. I. McDonald

2006-01-01

Phylogenetic and genetic relationships among 10 North American Armillaria species were analysed using sequence data from ribosomal DNA (rDNA), including intergenic spacer (IGS-1), internal transcribed spacers with associated 5.8S (ITS + 5.8S), and nuclear large subunit rDNA (nLSU), and amplified fragment length polymorphism (AFLP) markers. Based on rDNA sequence data,...

FUNGAL-SPECIFIC PCR PRIMERS DEVELOPED FOR ANALYSIS OF THE ITS REGION OF ENVIRONMENTAL DNA EXTRACTS

EPA Science Inventory

Background The Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) are highly variable sequences of great importance in distinguishing fungal species by PCR analysis. Previously published PCR primers available for amplifying these sequences from environmenta...

High power RF solid state power amplifier system

NASA Technical Reports Server (NTRS)

Sims, III, William Herbert (Inventor); Chavers, Donald Gregory (Inventor); Richeson, James J. (Inventor)

2011-01-01

A high power, high frequency, solid state power amplifier system includes a plurality of input multiple port splitters for receiving a high-frequency input and for dividing the input into a plurality of outputs and a plurality of solid state amplifier units. Each amplifier unit includes a plurality of amplifiers, and each amplifier is individually connected to one of the outputs of multiport splitters and produces a corresponding amplified output. A plurality of multiport combiners combine the amplified outputs of the amplifiers of each of the amplifier units to a combined output. Automatic level control protection circuitry protects the amplifiers and maintains a substantial constant amplifier power output.

Direct solar-pumped iodine laser amplifier

NASA Technical Reports Server (NTRS)

Han, K. S.

1985-01-01

This semiannual progress report covers the period from April 1, 1985 to Sept. 30, 1985 under NASA grant NAS1-441 entitled direct solar pumped iodine laser amplifier. During this period the parametric studies of the iodine laser oscillator pumped by a Vortek simulator was carried out before the amplifier studies. The amplifier studies are postponed to the extended period following completion of the parametric studies. In addition, the kinetic modeling of a solar pumped iodine laser amplifier, and the experimental work for a solar pumped dye laser amplifier are in progress. This report contains three parts: (1) the radiation characteristics of solar simulator and the parametric characteristics of photodissociation iodine laser continuously pumped by a Vortek solar simulator; (2) kinetic modeling of a solar pumped iodine laser amplifier; and (3) the study of the dye laser amplifier pumped by a Tamarack solar simulator.

[Experimental study on TCRbeta idiotypic antigenic determinants DNA vaccine to induce anti-lymphoma antibodies].

PubMed

Zhang, Yeping; Zhu, Ping; Shi, Yongjin; Liu, Jihua; Pu, Dingfang; Cao, Xianghong; Zhu, Qiang; Wang, Yijia; Ma, Mingxin; Yu, Jiren

2002-02-01

To investigate the anti-human CEM lymphoma cell activities induced by TCR idiotypic DNA vaccine containing different antigen determinants in BALB/c mice. The specific rearranged gene fragment encoding TCRVbeta region of CEM cell line was obtained by RT-PCR technique. The PCR product was cloned into eukaryocytic expression vector pcDNA3, which was used as DNA vaccine and template for PCR amplifying different antigen determinant. Gene fragments encoding different antigen determinant were amplified and cloned into pcDNA3, separately. The experimental mice were immunized by intramuscular injection of the DNA vaccines. The specific anti-idiotype antibodies were detected by indirect immunofluorescence assay. TCRbetaV of CEM cell line contains five antigen determinants. Specific anti-idiotype antibody was detected in all of the six mice immunized with DNA vaccine containing all the five determinants (the highest titer was 1:480). Although the antibody could also be detected in four of the six mice immunized with DNA vaccine containing four of the five antigen determinants, the antibody titer was lower (the highest titer was 1:80). DNA vaccine containing two of the five determinants could not induce the specific antibody. The idiotypic DNA vaccine containing the whole TCRbetaV five antigen determinants could induce the specific anti-lymphoma idiotypic antibody in BALB/c mice.

Log amplifier with pole-zero compensation

DOEpatents

Brookshier, William

1987-01-01

A logarithmic amplifier circuit provides pole-zero compensation for improved stability and response time over 6-8 decades of input signal frequency. The amplifier circuit includes a first operational amplifier with a first feedback loop which includes a second, inverting operational amplifier in a second feedback loop. The compensated output signal is provided by the second operational amplifier with the log elements, i.e., resistors, and the compensating capacitors in each of the feedback loops having equal values so that each break point or pole is offset by a compensating break point or zero.

Detection of genetically modified organisms (GMOs) using isothermal amplification of target DNA sequences.

PubMed

Lee, David; La Mura, Maurizio; Allnutt, Theo R; Powell, Wayne

2009-02-02

The most common method of GMO detection is based upon the amplification of GMO-specific DNA amplicons using the polymerase chain reaction (PCR). Here we have applied the loop-mediated isothermal amplification (LAMP) method to amplify GMO-related DNA sequences, 'internal' commonly-used motifs for controlling transgene expression and event-specific (plant-transgene) junctions. We have tested the specificity and sensitivity of the technique for use in GMO studies. Results show that detection of 0.01% GMO in equivalent background DNA was possible and dilutions of template suggest that detection from single copies of the template may be possible using LAMP. This work shows that GMO detection can be carried out using LAMP for routine screening as well as for specific events detection. Moreover, the sensitivity and ability to amplify targets, even with a high background of DNA, here demonstrated, highlights the advantages of this isothermal amplification when applied for GMO detection.

Laser amplifier and method

DOEpatents

Backus, S.; Kapteyn, H.C.; Murnane, M.M.

1997-07-01

Laser amplifiers and methods for amplifying a laser beam are disclosed. A representative embodiment of the amplifier comprises first and second curved mirrors, a gain medium, a third mirror, and a mask. The gain medium is situated between the first and second curved mirrors at the focal point of each curved mirror. The first curved mirror directs and focuses a laser beam to pass through the gain medium to the second curved mirror which reflects and recollimates the laser beam. The gain medium amplifies and shapes the laser beam as the laser beam passes therethrough. The third mirror reflects the laser beam, reflected from the second curved mirror, so that the laser beam bypasses the gain medium and return to the first curved mirror, thereby completing a cycle of a ring traversed by the laser beam. The mask defines at least one beam-clipping aperture through which the laser beam passes during a cycle. The gain medium is pumped, preferably using a suitable pumping laser. The laser amplifier can be used to increase the energy of continuous-wave or, especially, pulsed laser beams including pulses of femtosecond duration and relatively high pulse rate. 7 figs.

Laser amplifier and method

DOEpatents

Backus, Sterling; Kapteyn, Henry C.; Murnane, Margaret M.

1997-01-01

Laser amplifiers and methods for amplifying a laser beam are disclosed. A representative embodiment of the amplifier comprises first and second curved mirrors, a gain medium, a third mirror, and a mask. The gain medium is situated between the first and second curved mirrors at the focal point of each curved mirror. The first curved mirror directs and focuses a laser beam to pass through the gain medium to the second curved mirror which reflects and recollimates the laser beam. The gain medium amplifies and shapes the laser beam as the laser beam passes therethough. The third mirror reflects the laser beam, reflected from the second curved mirror, so that the laser beam bypasses the gain medium and return to the first curved mirror, thereby completing a cycle of a ring traversed by the laser beam. The mask defines at least one beam-clipping aperture through which the laser beam passes during a cycle. The gain medium is pumped, preferably using a suitable pumping laser. The laser amplifier can be used to increase the energy of continuous-wave or, especially, pulsed laser beams including pulses of femtosecond duration and relatively high pulse rate.

Non-homologous end joining-mediated functional marker selection for DNA cloning in the yeast Kluyveromyces marxianus.

PubMed

Hoshida, Hisashi; Murakami, Nobutada; Suzuki, Ayako; Tamura, Ryoko; Asakawa, Jun; Abdel-Banat, Babiker M A; Nonklang, Sanom; Nakamura, Mikiko; Akada, Rinji

2014-01-01

The cloning of DNA fragments into vectors or host genomes has traditionally been performed using Escherichia coli with restriction enzymes and DNA ligase or homologous recombination-based reactions. We report here a novel DNA cloning method that does not require DNA end processing or homologous recombination, but that ensures highly accurate cloning. The method exploits the efficient non-homologous end-joining (NHEJ) activity of the yeast Kluyveromyces marxianus and consists of a novel functional marker selection system. First, to demonstrate the applicability of NHEJ to DNA cloning, a C-terminal-truncated non-functional ura3 selection marker and the truncated region were PCR-amplified separately, mixed and directly used for the transformation. URA3(+) transformants appeared on the selection plates, indicating that the two DNA fragments were correctly joined by NHEJ to generate a functional URA3 gene that had inserted into the yeast chromosome. To develop the cloning system, the shortest URA3 C-terminal encoding sequence that could restore the function of a truncated non-functional ura3 was determined by deletion analysis, and was included in the primers to amplify target DNAs for cloning. Transformation with PCR-amplified target DNAs and C-terminal truncated ura3 produced numerous transformant colonies, in which a functional URA3 gene was generated and was integrated into the chromosome with the target DNAs. Several K. marxianus circular plasmids with different selection markers were also developed for NHEJ-based cloning and recombinant DNA construction. The one-step DNA cloning method developed here is a relatively simple and reliable procedure among the DNA cloning systems developed to date. Copyright © 2013 John Wiley & Sons, Ltd.

A low-voltage sense amplifier with two-stage operational amplifier clamping for flash memory

NASA Astrophysics Data System (ADS)

Guo, Jiarong

2017-04-01

A low-voltage sense amplifier with reference current generator utilizing two-stage operational amplifier clamp structure for flash memory is presented in this paper, capable of operating with minimum supply voltage at 1 V. A new reference current generation circuit composed of a reference cell and a two-stage operational amplifier clamping the drain pole of the reference cell is used to generate the reference current, which avoids the threshold limitation caused by current mirror transistor in the traditional sense amplifier. A novel reference voltage generation circuit using dummy bit-line structure without pull-down current is also adopted, which not only improves the sense window enhancing read precision but also saves power consumption. The sense amplifier was implemented in a flash realized in 90 nm flash technology. Experimental results show the access time is 14.7 ns with power supply of 1.2 V and slow corner at 125 °C. Project supported by the National Natural Science Fundation of China (No. 61376028).

Real Time Calibration Method for Signal Conditioning Amplifiers

NASA Technical Reports Server (NTRS)

Medelius, Pedro J. (Inventor); Mata, Carlos T. (Inventor); Eckhoff, Anthony (Inventor); Perotti, Jose (Inventor); Lucena, Angel (Inventor)

2004-01-01

A signal conditioning amplifier receives an input signal from an input such as a transducer. The signal is amplified and processed through an analog to digital converter and sent to a processor. The processor estimates the input signal provided by the transducer to the amplifier via a multiplexer. The estimated input signal is provided as a calibration voltage to the amplifier immediately following the receipt of the amplified input signal. The calibration voltage is amplified by the amplifier and provided to the processor as an amplified calibration voltage. The amplified calibration voltage is compared to the amplified input signal, and if a significant error exists, the gain and/or offset of the amplifier may be adjusted as necessary.

High-efficiency solid state power amplifier

NASA Technical Reports Server (NTRS)

Wallis, Robert E. (Inventor); Cheng, Sheng (Inventor)

2005-01-01

A high-efficiency solid state power amplifier (SSPA) for specific use in a spacecraft is provided. The SSPA has a mass of less than 850 g and includes two different X-band power amplifier sections, i.e., a lumped power amplifier with a single 11-W output and a distributed power amplifier with eight 2.75-W outputs. These two amplifier sections provide output power that is scalable from 11 to 15 watts without major design changes. Five different hybrid microcircuits, including high-efficiency Heterostructure Field Effect Transistor (HFET) amplifiers and Monolithic Microwave Integrated Circuit (MMIC) phase shifters have been developed for use within the SSPA. A highly efficient packaging approach enables the integration of a large number of hybrid circuits into the SSPA.

Pentopyranosyl Oligonucleotide Systems. Part 11: Systems with Shortened Backbones: D)-beta-Ribopyranosyl-(4 yields 3 )- and (L)-alpha - Lyxopyranosyl-(4 yields 3 )-oligonucleotides

NASA Technical Reports Server (NTRS)

Wippo, Harald; Reck, Folkert; Kudick, Rene; Ramaseshan, Mahesh; Ceulemans, Griet; Bolli, Martin; Krishnamurthy, Ramanarayanan; Eschenmoser, Albert

2001-01-01

The (L)-a-lyxopyranosyl-(4'yields 3')-oligonucleotide system-a member of a pentopyranosyl oligonucleotide family containing a shortened backbone-is capable of cooperative base-pairing and of cross-pairing with DNA and RNA. In contrast, corresponding (D)-beta-ribopyransoyl-(4' yields 3')-oligonucleotides do not show base-pairing under similar conditions. We conclude that oligonucleotide systems can violate the six-bonds-per-backbone-unit rule by having five bonds instead, if their vicinally bound phosphodiester bridges can assume an antiperiplanar conformation. An additional structural feature that seems relevant to the cross-pairing capability of the (L)-a-lyxopyranosyl-(4' yields 3')-oligonucleotide system is its (small) backbone/basepair axes inclination. An inclination which is similar to that in B-DNA seems to be a prerequisite for an oligonucleotide system s capability to cross-pair with DNA.

[Construction and characterization of a cDNA library from human liver tissue of cirrhosis].

PubMed

Chen, Xiao-hong; Chen, Zhi; Chen, Feng; Zhu, Hai-hong; Zhou, Hong-juan; Yao, Hang-ping

2005-03-01

To construct a cDNA library from human liver tissue of cirrhosis. The total RNA from human liver tissue of cirrhosis was extracted using Trizol method, and the mRNA was purified using mRNA purification kit. SMART technique and CDSIII/3' primer were used for first-strand cDNA synthesis. Long distance PCR was then used to synthesize the double-strand cDNA that was then digested by proteinase K and Sfi I, and was fractionated by CHOMA SPIN-400 column. The cDNA fragments longer than 0.4 kb were collected and ligated to lambdaTripl Ex2 vector. Then lambda-phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries was strictly checked by conventional titer determination. Eleven plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. The titers of unamplifed and amplified libraries were 1.03 x 10(6) pfu/ml and 1.36 x 10(9) pfu/ml respectively. The percentages of recombinants from both libraries were 97.24 % in unamplified library and 99.02 % in amplified library. The lengths of the inserts were 1.02 kb in average (36.36 % 1 approximately equals 2 kb and 63.64 % 0.5 approximately equals 1.0 kb). A high quality cDNA library from human liver tissue of cirrhosis was constructed successfully, which can be used for screening and cloning new special genes associated with the occurrence of cirrhosis.

Amplified impedimetric aptasensor based on gold nanoparticles covalently bound graphene sheet for the picomolar detection of ochratoxin A.

PubMed

Jiang, Ling; Qian, Jing; Yang, Xingwang; Yan, Yuting; Liu, Qian; Wang, Kan; Wang, Kun

2014-01-02

An amplified electrochemical impedimetric aptasensor for ochratoxin A (OTA) was developed with picomolar sensitivity. A facile route to fabricate gold nanoparticles covalently bound reduced graphene oxide (AuNPs-rGO) resulted in a large number of well-dispersed AuNPs on graphene sheets with tremendous binding sites for DNA, since the single rGO sheet and each AuNP can be loaded with hundreds of DNA strands. An aptasensor with sandwich model was fabricated which involved thiolated capture DNA immobilized on a gold electrode to capture the aptamer, then the sensing interface was incubated with OTA at a desired concentration, followed by AuNPs-rGO functionalized reporter DNA hybridized with the residual aptamers. By exploiting the AuNPs-rGO as an excellent signal amplified platform, a single hybridization event between aptamer and reporter DNA was translated into more than 10(7) redox events, leading to a substantial increase in charge-transfer resistance (Rct) by 7~ orders of magnitude compared with that of the free aptamer modified electrode. Such designed aptasensor showed a decreased response of Rct to the increase of OTA concentrations over a wide range of 1 pg mL(-1)-50 ng mL(-1) and could detect extremely low OTA concentration, namely, 0.3 pg mL(-1) or 0.74 pM, which was much lower than that of most other existed impedimetric aptasensors. The signal amplification platform presented here would provide a promising model for the aptamer-based detection with a direct impedimetric method. Copyright © 2013 Elsevier B.V. All rights reserved.

Sequencing of the large dsDNA genome of Oryctes rhinoceros nudivirus using multiple displacement amplification of nanogram amounts of virus DNA.

PubMed

Wang, Yongjie; Kleespies, Regina G; Ramle, Moslim B; Jehle, Johannes A

2008-09-01

The genomic sequence analysis of many large dsDNA viruses is hampered by the lack of enough sample materials. Here, we report a whole genome amplification of the Oryctes rhinoceros nudivirus (OrNV) isolate Ma07 starting from as few as about 10 ng of purified viral DNA by application of phi29 DNA polymerase- and exonuclease-resistant random hexamer-based multiple displacement amplification (MDA) method. About 60 microg of high molecular weight DNA with fragment sizes of up to 25 kbp was amplified. A genomic DNA clone library was generated using the product DNA. After 8-fold sequencing coverage, the 127,615 bp of OrNV whole genome was sequenced successfully. The results demonstrate that the MDA-based whole genome amplification enables rapid access to genomic information from exiguous virus samples.

Identification and reproducibility of diagnostic DNA markers for tuber starch and yield optimization in a novel association mapping population of potato (Solanum tuberosum L.).

PubMed

Schönhals, E M; Ortega, F; Barandalla, L; Aragones, A; Ruiz de Galarreta, J I; Liao, J-C; Sanetomo, R; Walkemeier, B; Tacke, E; Ritter, E; Gebhardt, C

2016-04-01

SNPs in candidate genes Pain - 1, InvCD141 (invertases), SSIV (starch synthase), StCDF1 (transcription factor), LapN (leucine aminopeptidase), and cytoplasm type are associated with potato tuber yield, starch content and/or starch yield. Tuber yield (TY), starch content (TSC), and starch yield (TSY) are complex characters of high importance for the potato crop in general and for industrial starch production in particular. DNA markers associated with superior alleles of genes that control the natural variation of TY, TSC, and TSY could increase precision and speed of breeding new cultivars optimized for potato starch production. Diagnostic DNA markers are identified by association mapping in populations of tetraploid potato varieties and advanced breeding clones. A novel association mapping population of 282 genotypes including varieties, breeding clones and Andean landraces was assembled and field evaluated in Northern Spain for TY, TSC, TSY, tuber number (TN) and tuber weight (TW). The landraces had lower mean values of TY, TW, TN, and TSY. The population was genotyped for 183 microsatellite alleles, 221 single nucleotide polymorphisms (SNPs) in fourteen candidate genes and eight known diagnostic markers for TSC and TSY. Association test statistics including kinship and population structure reproduced five known marker-trait associations of candidate genes and discovered new ones, particularly for tuber yield and starch yield. The inclusion of landraces increased the number of detected marker-trait associations. Integration of the present association mapping results with previous QTL linkage mapping studies for TY, TSC, TSY, TW, TN, and tuberization revealed some hot spots of QTL for these traits in the potato genome. The genomic positions of markers linked or associated with QTL for complex tuber traits suggest high multiplicity and genome wide distribution of the underlying genes.

STABILIZED FEEDBACK AMPLIFIER

DOEpatents

Fishbine, H.L.; Sewell, C. Jr.

1957-08-01

Negative feedback amplifiers, and particularly a negative feedback circuit which is economical on amode power consumption, are described. Basically, the disclosed circuit comprises two tetrode tubes where the output of the first tube is capacitamce coupled to the grid of the second tube, which in turn has its plate coupled to the cathode of the first tube to form a degenerative feedback circuit. Operating potential for screen of the second tube is supplied by connecting the cathode resistor of the first tube to the screen, while the screen is by-passed to the cathode of its tube for the amplified frequencies. Also, the amplifier incorporates a circuit to stabilize the transconductance of the tubes by making the grid potential of each tube interdependent on anode currents of both lubes by voltage divider circuitry.

Multiple pass laser amplifier system

DOEpatents

Brueckner, Keith A.; Jorna, Siebe; Moncur, N. Kent

1977-01-01

A laser amplification method for increasing the energy extraction efficiency from laser amplifiers while reducing the energy flux that passes through a flux limited system which includes apparatus for decomposing a linearly polarized light beam into multiple components, passing the components through an amplifier in delayed time sequence and recombining the amplified components into an in phase linearly polarized beam.

Engineering a Cell-surface Aptamer Circuit for Targeted and Amplified Photodynamic Cancer Therapy

PubMed Central

Han, Da; Zhu, Guizhi; Wu, Cuichen; Zhu, Zhi; Chen, Tao; Zhang, Xiaobing

2013-01-01

Photodynamic therapy (PDT) is one of the most promising and noninvasive methods for clinical treatment of different malignant diseases. Here, we present a novel strategy of designing an aptamer-based DNA nanocircuit capable of the selective recognition of cancer cells, controllable activation of photosensitizer and amplification of photodynamic therapeutic effect. The aptamers can selectively recognize target cancer cells and bind to the specific proteins on cell membranes. Then the overhanging catalyst sequence on aptamer can trigger a toehold-mediated catalytic strand displacement to activate photosensitizer and achieve amplified therapeutic effect. The specific binding-induced activation allows the DNA circuit to distinguish diseased cells from healthy cells, reducing damage to nearby healthy cells. Moreover, the catalytic amplification reaction will only take place close to the target cancer cells, resulting in a high local concentration of singlet oxygen to selectively kill the target cells. The principle employed in this study demonstrated the feasibility of assembling a DNA circuit on cell membranes and could further broaden the utility of DNA circuits for applications in biology, biotechnology, and biomedicine. PMID:23397942

Homogeneous Entropy-Driven Amplified Detection of Biomolecular Interactions.

PubMed

Kim, Donghyuk; Garner, Omai B; Ozcan, Aydogan; Di Carlo, Dino

2016-08-23

While a range of artificial biochemical circuits is likely to play a significant role in biological engineering, one of the challenges in the field is the design of circuits that can transduce between biomolecule classes (e.g., moving beyond nucleic acid only circuits). Herein, we design a transduction mechanism whereby a protein signal is transduced into an amplified nucleic acid output using DNA nanotechnology. In this system, a protein is recognized by nucleic acid bound recognition elements to form a catalytic complex that drives a hybridization/displacement reaction on a multicomponent nucleic acid substrate, releasing multiple target single-stranded oligonucleotides in an amplified fashion. Amplification power and simple one-pot reaction conditions lead us to apply the scheme in an assay format, achieving homogeneous and rapid (∼10 min) analyte detection that is also robust (operable in whole blood and plasma). In addition, we demonstrate the assay in a microfluidic digital assay format leading to improved quantification and sensitivity approaching single-molecule levels. The present scheme we believe will have a significant impact on a range of applications from fundamental molecular interaction studies to design of artificial circuits in vivo to high-throughput, multiplexed assays for screening or point-of-care diagnostics.

A nonenzymatic DNA nanomachine for biomolecular detection by target recycling of hairpin DNA cascade amplification.

PubMed

Zheng, Jiao; Li, Ningxing; Li, Chunrong; Wang, Xinxin; Liu, Yucheng; Mao, Guobin; Ji, Xinghu; He, Zhike

2018-06-01

Synthetic enzyme-free DNA nanomachine performs quasi-mechanical movements in response to external intervention, suggesting the promise of constructing sensitive and specific biosensors. Herein, a smart DNA nanomachine biosensor for biomolecule (such as nucleic acid, thrombin and adenosine) detection is developed by target-assisted enzyme-free hairpin DNA cascade amplifier. The whole DNA nanomachine system is constructed on gold nanoparticle which decorated with hundreds of locked hairpin substrate strands serving as DNA tracks, and the DNA nanomachine could be activated by target molecule toehold-mediated exchange on gold nanoparticle surface, resulted in the fluorescence recovery of fluorophore. The process is repeated so that each copy of the target can open multiplex fluorophore-labeled hairpin substrate strands, resulted in amplification of the fluorescence signal. Compared with the conventional biosensors of catalytic hairpin assembly (CHA) without substrate in solution, the DNA nanomachine could generate 2-3 orders of magnitude higher fluorescence signal. Furthermore, the DNA nanomachine could be used for nucleic acid, thrombin and adenosine highly sensitive specific detection based on isothermal, and homogeneous hairpin DNA cascade signal amplification in both buffer and a complicated biomatrix, and this kind of DNA nanomachine could be efficiently applied in the field of biomedical analysis. Copyright © 2018 Elsevier B.V. All rights reserved.

Charging YOYO-1 on Capillary Wall for Online DNA Intercalation and Integrating This Approach with Multiplex PCR and Bare Narrow Capillary–Hydrodynamic Chromatography for Online DNA Analysis

PubMed Central

2016-01-01

Multiplex polymerase chain reaction (PCR) has been widely utilized for high-throughput pathogen identification. Often, a dye is used to intercalate the amplified DNA fragments, and identifications of the pathogens are carried out by DNA melting curve analysis or gel electrophoresis. Integrating DNA amplification and identification is a logic path toward maximizing the benefit of multiplex PCR. Although PCR and gel electrophoresis have been integrated, replenishing the gels after each run is tedious and time-consuming. In this technical note, we develop an approach to address this issue. We perform multiplex PCR inside a capillary, transfer the amplified fragments to a bare narrow capillary, and measure their lengths online using bare narrow capillary–hydrodynamic chromatography (BaNC-HDC), a new technique recently developed in our laboratory for free-solution DNA separation. To intercalate the DNA with YOYO-1 (a fluorescent dye) for BaNC-HDC, we flush the capillary column with a YOYO-1 solution; positively charged YOYO-1 is adsorbed (or charged) onto the negatively charged capillary wall. As DNA molecules are driven down the column for separation, they react with the YOYO-1 stored on the capillary wall and are online-intercalated with the dye. With a single YOYO-1 charging, the column can be used for more than 40 runs, although the fluorescence signal intensities of the DNA peaks decrease gradually. Although the dye-DNA intercalation occurs during the separation, it does not affect the retention times, separation efficiencies, or resolutions. PMID:25555111

Visualization of DNA in highly processed botanical materials.

PubMed

Lu, Zhengfei; Rubinsky, Maria; Babajanian, Silva; Zhang, Yanjun; Chang, Peter; Swanson, Gary

2018-04-15

DNA-based methods have been gaining recognition as a tool for botanical authentication in herbal medicine; however, their application in processed botanical materials is challenging due to the low quality and quantity of DNA left after extensive manufacturing processes. The low amount of DNA recovered from processed materials, especially extracts, is "invisible" by current technology, which has casted doubt on the presence of amplifiable botanical DNA. A method using adapter-ligation and PCR amplification was successfully applied to visualize the "invisible" DNA in botanical extracts. The size of the "invisible" DNA fragments in botanical extracts was around 20-220 bp compared to fragments of around 600 bp for the more easily visualized DNA in botanical powders. This technique is the first to allow characterization and visualization of small fragments of DNA in processed botanical materials and will provide key information to guide the development of appropriate DNA-based botanical authentication methods in the future. Copyright © 2017 Elsevier Ltd. All rights reserved.

Electrochemical DNA sensor for Neisseria meningitidis detection.

PubMed

Patel, Manoj K; Solanki, Pratima R; Kumar, Ashok; Khare, Shashi; Gupta, Sunil; Malhotra, Bansi D

2010-08-15

Meningitis sensor based on nucleic acid probe of Neisseria meningitidis has been fabricated by immobilization of 5'-thiol end labeled single stranded deoxyribonucleic acid probe (ssDNA-SH) onto gold (Au) coated glass electrode. This ssDNA-SH/Au electrode hybridized with the genomic DNA (G-dsDNA/Au) and amplified DNA (PCR-dsDNA/Au) has been characterized using atomic force microscopy (AFM), Fourier transforms infrared spectroscopy (FT-IR) and electrochemical techniques. The ssDNA-SH/Au electrode can specifically detect upto 10-60 ng/microl of G-dsDNA-SH/Au and PCR-dsDNA-SH/Au of meningitis within 60s of hybridization time at 25 degrees C by cyclic voltammetry (CV) using methylene blue (MB) as electro-active DNA hybridization indicator. The values of sensitivities of the G-dsDNA-SH/Au and PCR-dsDNA-SH/Au electrodes have been determined as 0.0115 microA/ng cm(-2) and 0.0056 microA/ng cm(-2), respectively with regression coefficient (R) as 0.999. This DNA bioelectrode is stable for about 4 months when stored at 4 degrees C. Copyright 2010 Elsevier B.V. All rights reserved.

Amplifier Module for 260-GHz Band Using Quartz Waveguide Transitions

NASA Technical Reports Server (NTRS)

Padmanabhan, Sharmila; Fung, King Man; Kangaslahti, Pekka P.; Peralta, Alejandro; Soria, Mary M.; Pukala, David M.; Sin, Seth; Samoska, Lorene A.; Sarkozy, Stephen; Lai, Richard

2012-01-01

Packaging of MMIC LNA (monolithic microwave integrated circuit low-noise amplifier) chips at frequencies over 200 GHz has always been problematic due to the high loss in the transition between the MMIC chip and the waveguide medium in which the chip will typically be used. In addition, above 200 GHz, wire-bond inductance between the LNA and the waveguide can severely limit the RF matching and bandwidth of the final waveguide amplifier module. This work resulted in the development of a low-loss quartz waveguide transition that includes a capacitive transmission line between the MMIC and the waveguide probe element. This capacitive transmission line tunes out the wirebond inductance (where the wire-bond is required to bond between the MMIC and the probe element). This inductance can severely limit the RF matching and bandwidth of the final waveguide amplifier module. The amplifier module consists of a quartz E-plane waveguide probe transition, a short capacitive tuning element, a short wire-bond to the MMIC, and the MMIC LNA. The output structure is similar, with a short wire-bond at the output of the MMIC, a quartz E-plane waveguide probe transition, and the output waveguide. The quartz probe element is made of 3-mil quartz, which is the thinnest commercially available material. The waveguide band used is WR4, from 170 to 260 GHz. This new transition and block design is an improvement over prior art because it provides for better RF matching, and will likely yield lower loss and better noise figure. The development of high-performance, low-noise amplifiers in the 180-to- 700-GHz range has applications for future earth science and planetary instruments with low power and volume, and astrophysics array instruments for molecular spectroscopy. This frequency band, while suitable for homeland security and commercial applications (such as millimeter-wave imaging, hidden weapons detection, crowd scanning, airport security, and communications), also has applications to

DNA Barcoding of the Endangered Aquilaria (Thymelaeaceae) and Its Application in Species Authentication of Agarwood Products Traded in the Market

PubMed Central

Lee, Shiou Yih; Ng, Wei Lun; Mahat, Mohd Noor; Nazre, Mohd; Mohamed, Rozi

2016-01-01

The identification of Aquilaria species from their resinous non-wood product, the agarwood, is challenging as conventional techniques alone are unable to ascertain the species origin. Aquilaria is a highly protected species due to the excessive exploitation of its precious agarwood. Here, we applied the DNA barcoding technique to generate barcode sequences for Aquilaria species and later applied the barcodes to identify the source species of agarwood found in the market. We developed a reference DNA barcode library using eight candidate barcode loci (matK, rbcL, rpoB, rpoC1, psbA-trnH, trnL-trnF, ITS, and ITS2) amplified from 24 leaf accessions of seven Aquilaria species obtained from living trees. Our results indicated that all single barcodes can be easily amplified and sequenced with the selected primers. The combination of trnL-trnF+ITS and trnL-trnF+ITS2 yielded the greatest species resolution using the least number of loci combination, while matK+trnL-trnF+ITS showed potential in detecting the geographical origins of Aquilaria species. We propose trnL-trnF+ITS2 as the best candidate barcode for Aquilaria as ITS2 has a shorter sequence length compared to ITS, which eases PCR amplification especially when using degraded DNA samples such as those extracted from processed agarwood products. A blind test conducted on eight agarwood samples in different forms using the proposed barcode combination proved successful in their identification up to the species level. Such potential of DNA barcoding in identifying the source species of agarwood will contribute to the international timber trade control, by providing an effective method for species identification and product authentication. PMID:27128309

Biomonitoring of marine vertebrates in Monterey Bay using eDNA metabarcoding.

PubMed

Andruszkiewicz, Elizabeth A; Starks, Hilary A; Chavez, Francisco P; Sassoubre, Lauren M; Block, Barbara A; Boehm, Alexandria B

2017-01-01

Molecular analysis of environmental DNA (eDNA) can be used to assess vertebrate biodiversity in aquatic systems, but limited work has applied eDNA technologies to marine waters. Further, there is limited understanding of the spatial distribution of vertebrate eDNA in marine waters. Here, we use an eDNA metabarcoding approach to target and amplify a hypervariable region of the mitochondrial 12S rRNA gene to characterize vertebrate communities at 10 oceanographic stations spanning 45 km within the Monterey Bay National Marine Sanctuary (MBNMS). In this study, we collected three biological replicates of small volume water samples (1 L) at 2 depths at each of the 10 stations. We amplified fish mitochondrial DNA using a universal primer set. We obtained 5,644,299 high quality Illumina sequence reads from the environmental samples. The sequence reads were annotated to the lowest taxonomic assignment using a bioinformatics pipeline. The eDNA survey identified, to the lowest taxonomic rank, 7 families, 3 subfamilies, 10 genera, and 72 species of vertebrates at the study sites. These 92 distinct taxa come from 33 unique marine vertebrate families. We observed significantly different vertebrate community composition between sampling depths (0 m and 20/40 m deep) across all stations and significantly different communities at stations located on the continental shelf (<200 m bottom depth) versus in the deeper waters of the canyons of Monterey Bay (>200 m bottom depth). All but 1 family identified using eDNA metabarcoding is known to occur in MBNMS. The study informs the implementation of eDNA metabarcoding for vertebrate biomonitoring.

Hybrid thin-film amplifier

NASA Technical Reports Server (NTRS)

Cleveland, G.

1977-01-01

Miniature amplifier for bioelectronic instrumentation consumes only about 100 mW and has frequency response flat to within 0.5 dB from 0.14 to 450 Hz. Device consists of five thin film substrates, which contain eight operational amplifiers and seven field-effect transistor dice.

The effect of common imaging and hot water maceration on DNA recovery from skeletal remains.

PubMed

Frank, Emilie M; Mundorff, Amy Z; Davoren, Jon M

2015-12-01

Identifying human remains often begins with cleaning and imaging the material. Hot water maceration is used to remove adherent soft tissue from bone and radiographs are taken to better visualize osseous details. Heat and radiation are known to have harmful effects on DNA, but their ability to degrade DNA when used for cleaning and imaging has not been well studied. To better understand their individual and combined effects on the recoverability of DNA from bone, skeletal samples were subjected to (1) hot water maceration (62 °C for 45 min); (2) CT scanning (0.6mm slices, 120 kV, 10.4s); (3) X-ray (50 kVp, 150 mA, 0.03 s, 40 in); and (4) all 3 treatments combined. Forty-eight DNA samples were extracted, quantified and amplified with the AmpFLSTR(®) Identifiler(®) system. Nearly all of the processed samples had reduced RFU values relative to the unprocessed samples, indicating some amount of genetic loss. This loss did not always translate into loss of profile completeness, since only a few samples had a reduction in the number of loci detected after processing. DNA yields were not significantly reduced by any one of the processing methods, however the results indicate that the damaging effects are additive. It is possible that processing may reduce a bone's DNA reservoir and as more procedures are preformed, the pool of available genetic information might be diminished. Many intrinsic and extrinsic factors can affect the recoverability of DNA from bone. Collecting a DNA sample prior to processing avoids the negative effects from hot water maceration and radiological imaging. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Fourier plane image amplifier

DOEpatents

Hackel, Lloyd A.; Hermann, Mark R.; Dane, C. Brent; Tiszauer, Detlev H.

1995-01-01

A solid state laser is frequency tripled to 0.3 .mu.m. A small portion of the laser is split off and generates a Stokes seed in a low power oscillator. The low power output passes through a mask with the appropriate hole pattern. Meanwhile, the bulk of the laser output is focused into a larger stimulated Brillouin scattering (SBS) amplifier. The low power beam is directed through the same cell in the opposite direction. The majority of the amplification takes place at the focus which is the fourier transform plane of the mask image. The small holes occupy large area at the focus and thus are preferentially amplified. The amplified output is now imaged onto the multichip module where the holes are drilled. Because of the fourier plane amplifier, only .about.1/10th the power of a competitive system is needed. This concept allows less expensive masks to be used in the process and requires much less laser power.

Fourier plane image amplifier

DOEpatents

Hackel, L.A.; Hermann, M.R.; Dane, C.B.; Tiszauer, D.H.

1995-12-12

A solid state laser is frequency tripled to 0.3 {micro}m. A small portion of the laser is split off and generates a Stokes seed in a low power oscillator. The low power output passes through a mask with the appropriate hole pattern. Meanwhile, the bulk of the laser output is focused into a larger stimulated Brillouin scattering (SBS) amplifier. The low power beam is directed through the same cell in the opposite direction. The majority of the amplification takes place at the focus which is the fourier transform plane of the mask image. The small holes occupy large area at the focus and thus are preferentially amplified. The amplified output is now imaged onto the multichip module where the holes are drilled. Because of the fourier plane amplifier, only about 1/10th the power of a competitive system is needed. This concept allows less expensive masks to be used in the process and requires much less laser power. 1 fig.

Reflex ring laser amplifier system

DOEpatents

Summers, M.A.

1983-08-31

The invention is a method and apparatus for providing a reflex ring laser system for amplifying an input laser pulse. The invention is particularly useful in laser fusion experiments where efficient production of high-energy and high power laser pulses is required. The invention comprises a large aperture laser amplifier in an unstable ring resonator which includes a combination spatial filter and beam expander having a magnification greater than unity. An input pulse is injected into the resonator, e.g., through an aperture in an input mirror. The injected pulse passes through the amplifier and spatial filter/expander components on each pass around the ring. The unstable resonator is designed to permit only a predetermined number of passes before the amplified pulse exits the resonator. On the first pass through the amplifier, the beam fills only a small central region of the gain medium. On each successive pass, the beam has been expanded to fill the next concentric non-overlapping region of the gain medium.

Enhanced performance CCD output amplifier

DOEpatents

Dunham, Mark E.; Morley, David W.

1996-01-01

A low-noise FET amplifier is connected to amplify output charge from a che coupled device (CCD). The FET has its gate connected to the CCD in common source configuration for receiving the output charge signal from the CCD and output an intermediate signal at a drain of the FET. An intermediate amplifier is connected to the drain of the FET for receiving the intermediate signal and outputting a low-noise signal functionally related to the output charge signal from the CCD. The amplifier is preferably connected as a virtual ground to the FET drain. The inherent shunt capacitance of the FET is selected to be at least equal to the sum of the remaining capacitances.

Limit circuit prevents overdriving of operational amplifier

NASA Technical Reports Server (NTRS)

Openshaw, F. L.

1967-01-01

Cutoff-type high gain amplifier coupled by a diode prevents overdriving of operational amplifier. An amplified feedback signal offsets the excess input signal that tends to cause the amplifier to exceed its preset limit. The output is, therfore, held to the set clamp level.

21 CFR 882.1835 - Physiological signal amplifier.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Physiological signal amplifier. 882.1835 Section... signal amplifier. (a) Identification. A physiological signal amplifier is a general purpose device used to electrically amplify signals derived from various physiological sources (e.g., the...

21 CFR 882.1835 - Physiological signal amplifier.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Physiological signal amplifier. 882.1835 Section... signal amplifier. (a) Identification. A physiological signal amplifier is a general purpose device used to electrically amplify signals derived from various physiological sources (e.g., the...

Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Kai; Roberts, Gareth A.; Stephanou, Augoustinos S.

2010-07-23

Research highlights: {yields} Successful fusion of GFP to M.EcoKI DNA methyltransferase. {yields} GFP located at C-terminal of sequence specificity subunit does not later enzyme activity. {yields} FRET confirms structural model of M.EcoKI bound to DNA. -- Abstract: We describe the fusion of enhanced green fluorescent protein to the C-terminus of the HsdS DNA sequence-specificity subunit of the Type I DNA modification methyltransferase M.EcoKI. The fusion expresses well in vivo and assembles with the two HsdM modification subunits. The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease. The purified enzyme shows Foerstermore » resonance energy transfer to fluorescently-labelled DNA duplexes containing the target sequence and to fluorescently-labelled ocr protein, a DNA mimic that binds to the M.EcoKI enzyme. Distances determined from the energy transfer experiments corroborate the structural model of M.EcoKI.« less

Post pulse shutter for laser amplifier

DOEpatents

Bradley, Laird P. [Livermore, CA; Carder, Bruce M. [Antioch, CA; Gagnon, William L. [Berkeley, CA

1981-03-17

Apparatus and method for quickly closing off the return path for an amplified laser pulse at the output of an amplifier so as to prevent damage to amplifiers and other optical components appearing earlier in the chain by the return of an amplified pulse. The apparatus consists of a fast retropulse or post pulse shutter to suppress target reflection and/or beam return. This is accomplished by either quickly placing a solid across the light transmitting aperture of a component in the chain, such as a spatial filter pinhole, or generating and directing a plasma with sufficiently high density across the aperture, so as to, in effect, close the aperture to the returning amplified energy pulse.

Direct solar-pumped iodine laser amplifier

NASA Technical Reports Server (NTRS)

Han, K. S.

1986-01-01

During this period the parametric studies of the iodine laser oscillator pumped by a Vortek simulator were carried out before amplifier studies. The amplifier studies are postponed to the extended period after completing the parametric studies. In addition, the kinetic modeling of a solar-pumped iodine laser amplifier, and the experimental work for a solar pumped dye laser amplifier are in progress. This report contains three parts: (1) a 10 W CW iodine laser pumped by a Vortek solar simulator; (2) kinetic modeling to predict the time to lasing threshold, lasing time, and energy output of solar-pumped iodine laser; and (3) the study of the dye laser amplifier pumped by a Tamarack solar simulator.

Carbon Nanotube Nanoelectrode Array for Ultrasensitive DNA Detection

NASA Technical Reports Server (NTRS)

Li, Jun; Koehne, Jessica; Chen, Hua; Cassell, Alan; Ng, Hou Tee; Fan, Wendy; Ye, Qi; Han, Jie; Meyyappan, M.

2003-01-01

A reliable nanoelectrode array based on vertically aligned multi-walled carbon nanotubes (MWNTs) embedded in SiO2 is used for ultrasensitive DNA detection. Characteristic nanoelectrode behavior is observed using low-density MWNT arrays for measuring both bulk and surface immobilized redox species such as K4Fe(CN)6. The open-end of MWNTs present similar properties as graphite edge-plane electrodes with wide potential window, flexible chemical functionalities, and good biocompatibility. Oligonucleotide probes are selectively functionalized at the open ends cf the nanotube array and specifically hybridized with oligonucleotide targets. The guanine groups are employed as the signal moieties in the electrochemical measurements. Ru(bpy)3(2+) mediator is used to further amplify the guanine oxidation signal. The hybridization of subattomoles of PCR amplified DNA targets is detected electrochemically by combining the MWNT nanoelectrode array with the Ru(bpy)32' amplification mechanism. This system provides a general platform of molecular diagnostics for applications requiring ultrahigh sensitivity, high-degree of miniaturization, and simple sample preparations.

Characterization and application of a quantitative DNA marker that discriminates sex in chinook salmon (Oncorhynchus tshawytscha)

USGS Publications Warehouse

Clifton, D.R.; Rodriguez, R.J.

1997-01-01

A qualitative male-specific DNA marker (OT-24) was amplified by spPCR (single-primer polymerase chain reaction) from chinook salmon (Oncorhynchus tshawytscha) DNA along with several non-sex-linked products. The termini of the male-specific product were sequenced, and a pair of PeR primers were constructed for marker-specific PCR amplification. Dual primer PCR (dpPCR), with the marker-specific primers, amplified a product from both nudes and females. The amount of dpPCR product amplified from males was at least 100-fold greater than that from females. The quantitative difference between males and females was consistent among geographically distinct populations from western U.S. rivers. In addition, DNA sequence analysis indicated that OT-24 was highly conserved among geographically distinct salmon populations. The qualitative spPCR product segregated through several genetic crosses indicating equal sex ratios among progeny. Identification of the male and female juveniles by dpPCR was consistent with the spPCR analysis. There was no tissue specificity observed by spPCR or dpPCR analysis of this marker. A rapid DNA extraction method and dpPCR analysis were used to nonlethally determine sex ratios in wild spring chinook salmon adults, withheld for genetic and behavioral studies, prior to their development of gross sexual differences in their external morphology.

Characterization and application of a quantitative DNA marker that discriminates sex in Chinook salmon (Oncorhynchus tshawytscha)

USGS Publications Warehouse

Clifton, D.R.; Rodriguez, R.J.

1997-01-01

A qualitative male-specific DNA marker (OT-24) was amplified by spPCR (single-primer polymerase chain reaction) from chinook salmon (Oncorhynchus tshawytscha) DNA along with several non-sex-linked products. The termini of the male-specific product were sequenced, and a pair of PeR primers were constructed for marker-specific PCR amplification. Dual primer PCR (dpPCR), with the marker-specific primers, amplified a product from both nudes and females. The amount of dpPCR product amplified from males was at least 100-fold greater than that from females. The quantitative difference between males and females was consistent among geographically distinct populations from western U.S. rivers. In addition, DNA sequence analysis indicated that OT-24 was highly conserved among geographically distinct salmon populations. The qualitative spPCR product segregated through several genetic crosses indicating equal sex ratios among progeny. Identification of the male and female juveniles by dpPCR was consistent with the spPCR analysis. There was no tissue specificity observed by spPCR or dpPCR analysis of this marker. A rapid DNA extraction method and dpPCR analysis were used to nonlethally determine sex ratios in wild spring chinook salmon adults, withheld for genetic and behavioral studies, prior to their development of gross sexual differences in their external morphology.

Meter circuit for tuning RF amplifiers

NASA Technical Reports Server (NTRS)

Longthorne, J. E.

1973-01-01

Circuit computes and indicates efficiency of RF amplifier as inputs and other parameters are varied. Voltage drop across internal resistance of ammeter is amplified by operational amplifier and applied to one multiplier input. Other input is obtained through two resistors from positive terminal of power supply.

SQUID amplifiers for axion search experiments

NASA Astrophysics Data System (ADS)

Matlashov, Andrei; Schmelz, Matthias; Zakosarenko, Vyacheslav; Stolz, Ronny; Semertzidis, Yannis K.

2018-04-01

In the experiments for dark-matter QCD-axion searches, very weak microwave signals from a low-temperature High-Q resonant cavity should be detected using the highest sensitivity. The best commercial low-noise cryogenic semiconductor amplifiers based on high electron mobility transistors have a lowest noise temperature above 1.0 K, even if they are cooled well below 1 K. Superconducting quantum interference devices can work as microwave amplifiers with temperature noise close to the standard quantum limit. Previous SQUID-based RF amplifiers designed for axion search experiments have a microstrip resonant input coil and are thus called micro-strip SQUID amplifiers or MSAs. Due to the resonant input coupling they usually have narrow bandwidth. In this paper we report on a SQUID-based wideband microwave amplifier fabricated using sub-micron size Josephson junctions with very low capacitance. A single amplifier can be used in a frequency range of approximately 1-5 GHz.

Competitor internal standards for quantitative detection of mycoplasma DNA.

PubMed

Sidhu, M K; Rashidbaigi, A; Testa, D; Liao, M J

1995-05-01

Homologous internal controls were used as competitor DNA in the polymerase chain reaction for the quantitative detection of mycoplasma DNA. PCR primer sets were designed on the basis of the most conserved nucleotide sequences of the 16S rRNA gene of mycoplasma species. Amplification of this gene was examined in five different mycoplasma species: Mycoplasma orale, M. hyorhinus, M. synoviae, M. gallisepticum and M. pneumoniae. To evaluate the primers, a number of different cell lines were assayed for the detection of mycoplasma infections. All positive cell lines showed a distinct product on agarose gels while uninfected cells showed no DNA amplification. Neither bacterial nor eukaryotic DNA produced any cross-reaction with the primers used, thus confirming their specificity. Internal control DNA to be used for quantitation was constructed by modifying the sizes of the wild-type amplified products and cloning them in plasmid vectors. These controls used the same primer binding sites as the wild-type and the amplified products were differentiated by a size difference. The detection limits for all the mycoplasma species by competitive quantitative PCR were estimated to range from 4 to 60 genome copies per assay as determined by ethidium bromide-stained agarose gels. These internal standards also serve as positive controls in PCR-based detection of mycoplasma DNA, and therefore accidental contamination of test samples with wild-type positive controls can be eliminated. The quantitative PCR method developed will be useful in monitoring the progression and significance of mycoplasma in the disease process.

Rapid detection of microbial DNA by a novel isothermal genome exponential amplification reaction (GEAR) assay.