Sample records for sample nucleic acid

  1. Method for isolating nucleic acids

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

    Hurt, Jr., Richard Ashley; Elias, Dwayne A.

    The current disclosure provides methods and kits for isolating nucleic acid from an environmental sample. The current methods and compositions further provide methods for isolating nucleic acids by reducing adsorption of nucleic acids by charged ions and particles within an environmental sample. The methods of the current disclosure provide methods for isolating nucleic acids by releasing adsorbed nucleic acids from charged particles during the nucleic acid isolation process. The current disclosure facilitates the isolation of nucleic acids of sufficient quality and quantity to enable one of ordinary skill in the art to utilize or analyze the isolated nucleic acids formore » a wide variety of applications including, sequencing or species population analysis.« less

  2. Universal nucleic acids sample preparation method for cells, spores and their mixture

    DOEpatents

    Bavykin, Sergei [Darien, IL

    2011-01-18

    The present invention relates to a method for extracting nucleic acids from biological samples. More specifically the invention relates to a universal method for extracting nucleic acids from unidentified biological samples. An advantage of the presently invented method is its ability to effectively and efficiently extract nucleic acids from a variety of different cell types including but not limited to prokaryotic or eukaryotic cells and/or recalcitrant organisms (i.e. spores). Unlike prior art methods which are focused on extracting nucleic acids from vegetative cell or spores, the present invention effectively extracts nucleic acids from spores, multiple cell types or mixtures thereof using a single method. Important that the invented method has demonstrated an ability to extract nucleic acids from spores and vegetative bacterial cells with similar levels effectiveness. The invented method employs a multi-step protocol which erodes the cell structure of the biological sample, isolates, labels, fragments nucleic acids and purifies labeled samples from the excess of dye.

  3. Method for isolating chromosomal DNA in preparation for hybridization in suspension

    DOEpatents

    Lucas, Joe N.

    2000-01-01

    A method is provided for detecting nucleic acid sequence aberrations using two immobilization steps. According to the method, a nucleic acid sequence aberration is detected by detecting nucleic acid sequences having both a first nucleic acid sequence type (e.g., from a first chromosome) and a second nucleic acid sequence type (e.g., from a second chromosome), the presence of the first and the second nucleic acid sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. In the method, immobilization of a first hybridization probe is used to isolate a first set of nucleic acids in the sample which contain the first nucleic acid sequence type. Immobilization of a second hybridization probe is then used to isolate a second set of nucleic acids from within the first set of nucleic acids which contain the second nucleic acid sequence type. The second set of nucleic acids are then detected, their presence indicating the presence of a nucleic acid sequence aberration. Chromosomal DNA in a sample containing cell debris is prepared for hybridization in suspension by treating the mixture with RNase. The treated DNA can also be fixed prior to hybridization.

  4. Multiplexed microfluidic approach for nucleic acid enrichment

    DOEpatents

    VanderNoot, Victoria A.; Langevin, Stanley Alan; Bent, Zachary; Renzi, Ronald F.; Ferko, Scott M.; Van De Vreugde, James L.; Lane, Todd; Patel, Kamlesh; Branda, Steven

    2016-04-26

    A system for enhancing a nucleic acid sample may include a one pump, a denaturing chamber; a microfluidic hydroxyapatite chromatography device configured for performing hydroxyapatite chromatography on the nucleic acid sample, a sample collector, and tubing connecting the pump with the denaturing chamber, the hydroxyapatite chromatography device and the sample collector such that the pump may be used to move the nucleic acid sample from the denaturing chamber to the hydroxyapatite chromatography device and then to the sample collector.

  5. Method for nucleic acid isolation using supercritical fluids

    DOEpatents

    Nivens, David E.; Applegate, Bruce M.

    1999-01-01

    A method for detecting the presence of a microorganism in an environmental sample involves contacting the sample with a supercritical fluid to isolate nucleic acid from the microorganism, then detecting the presence of a particular sequence within the isolated nucleic acid. The nucleic acid may optionally be subjected to further purification.

  6. Quantitative detection of pathogens in centrifugal microfluidic disks

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

    Koh, Chung-Yan; Schaff, Ulrich Y.; Sommer, Gregory Jon

    A system and methods for detection of a nucleic acid including forming a plurality of nucleic acid detection complexes are described, each of the complexes including a nucleic acid analyte, a detection agent and a functionalized probe. The method further including binding the nucleic acid detection complexes to a plurality of functionalized particles in a fluid sample and separating the functionalized particles having the nucleic acid detection complexes bound thereto from the fluid sample using a density media. The nucleic acid analyte is detected by detecting the detection agent.

  7. Method for nucleic acid isolation using supercritical fluids

    DOEpatents

    Nivens, D.E.; Applegate, B.M.

    1999-07-13

    A method is disclosed for detecting the presence of a microorganism in an environmental sample involves contacting the sample with a supercritical fluid to isolate nucleic acid from the microorganism, then detecting the presence of a particular sequence within the isolated nucleic acid. The nucleic acid may optionally be subjected to further purification. 4 figs.

  8. Nucleic acid arrays and methods of synthesis

    DOEpatents

    Sabanayagam, Chandran R.; Sano, Takeshi; Misasi, John; Hatch, Anson; Cantor, Charles

    2001-01-01

    The present invention generally relates to high density nucleic acid arrays and methods of synthesizing nucleic acid sequences on a solid surface. Specifically, the present invention contemplates the use of stabilized nucleic acid primer sequences immobilized on solid surfaces, and circular nucleic acid sequence templates combined with the use of isothermal rolling circle amplification to thereby increase nucleic acid sequence concentrations in a sample or on an array of nucleic acid sequences.

  9. Highly simplified lateral flow-based nucleic acid sample preparation and passive fluid flow control

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

    Cary, Robert E.

    2015-12-08

    Highly simplified lateral flow chromatographic nucleic acid sample preparation methods, devices, and integrated systems are provided for the efficient concentration of trace samples and the removal of nucleic acid amplification inhibitors. Methods for capturing and reducing inhibitors of nucleic acid amplification reactions, such as humic acid, using polyvinylpyrrolidone treated elements of the lateral flow device are also provided. Further provided are passive fluid control methods and systems for use in lateral flow assays.

  10. Highly simplified lateral flow-based nucleic acid sample preparation and passive fluid flow control

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

    Cary, Robert B.

    Highly simplified lateral flow chromatographic nucleic acid sample preparation methods, devices, and integrated systems are provided for the efficient concentration of trace samples and the removal of nucleic acid amplification inhibitors. Methods for capturing and reducing inhibitors of nucleic acid amplification reactions, such as humic acid, using polyvinylpyrrolidone treated elements of the lateral flow device are also provided. Further provided are passive fluid control methods and systems for use in lateral flow assays.

  11. Methods for making nucleotide probes for sequencing and synthesis

    DOEpatents

    Church, George M; Zhang, Kun; Chou, Joseph

    2014-07-08

    Compositions and methods for making a plurality of probes for analyzing a plurality of nucleic acid samples are provided. Compositions and methods for analyzing a plurality of nucleic acid samples to obtain sequence information in each nucleic acid sample are also provided.

  12. Polymerase chain reaction system using magnetic beads for analyzing a sample that includes nucleic acid

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

    Nasarabadi, Shanavaz

    2011-01-11

    A polymerase chain reaction system for analyzing a sample containing nucleic acid includes providing magnetic beads; providing a flow channel having a polymerase chain reaction chamber, a pre polymerase chain reaction magnet position adjacent the polymerase chain reaction chamber, and a post pre polymerase magnet position adjacent the polymerase chain reaction chamber. The nucleic acid is bound to the magnetic beads. The magnetic beads with the nucleic acid flow to the pre polymerase chain reaction magnet position in the flow channel. The magnetic beads and the nucleic acid are washed with ethanol. The nucleic acid in the polymerase chain reactionmore » chamber is amplified. The magnetic beads and the nucleic acid are separated into a waste stream containing the magnetic beads and a post polymerase chain reaction mix containing the nucleic acid. The reaction mix containing the nucleic acid flows to an analysis unit in the channel for analysis.« less

  13. Solid phase sequencing of double-stranded nucleic acids

    DOEpatents

    Fu, Dong-Jing; Cantor, Charles R.; Koster, Hubert; Smith, Cassandra L.

    2002-01-01

    This invention relates to methods for detecting and sequencing of target double-stranded nucleic acid sequences, to nucleic acid probes and arrays of probes useful in these methods, and to kits and systems which contain these probes. Useful methods involve hybridizing the nucleic acids or nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes. These probe comprise a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. The molecular weights of the hybridized nucleic acids of the set can be determined by mass spectroscopy, and the sequence of the target determined from the molecular weights of the fragments. Nucleic acids whose sequences can be determined include nucleic acids in biological samples such as patient biopsies and environmental samples. Probes may be fixed to a solid support such as a hybridization chip to facilitate automated determination of molecular weights and identification of the target sequence.

  14. Identification of random nucleic acid sequence aberrations using dual capture probes which hybridize to different chromosome regions

    DOEpatents

    Lucas, J.N.; Straume, T.; Bogen, K.T.

    1998-03-24

    A method is provided for detecting nucleic acid sequence aberrations using two immobilization steps. According to the method, a nucleic acid sequence aberration is detected by detecting nucleic acid sequences having both a first nucleic acid sequence type (e.g., from a first chromosome) and a second nucleic acid sequence type (e.g., from a second chromosome), the presence of the first and the second nucleic acid sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. In the method, immobilization of a first hybridization probe is used to isolate a first set of nucleic acids in the sample which contain the first nucleic acid sequence type. Immobilization of a second hybridization probe is then used to isolate a second set of nucleic acids from within the first set of nucleic acids which contain the second nucleic acid sequence type. The second set of nucleic acids are then detected, their presence indicating the presence of a nucleic acid sequence aberration. 14 figs.

  15. Identification of random nucleic acid sequence aberrations using dual capture probes which hybridize to different chromosome regions

    DOEpatents

    Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

    1998-01-01

    A method is provided for detecting nucleic acid sequence aberrations using two immobilization steps. According to the method, a nucleic acid sequence aberration is detected by detecting nucleic acid sequences having both a first nucleic acid sequence type (e.g., from a first chromosome) and a second nucleic acid sequence type (e.g., from a second chromosome), the presence of the first and the second nucleic acid sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. In the method, immobilization of a first hybridization probe is used to isolate a first set of nucleic acids in the sample which contain the first nucleic acid sequence type. Immobilization of a second hybridization probe is then used to isolate a second set of nucleic acids from within the first set of nucleic acids which contain the second nucleic acid sequence type. The second set of nucleic acids are then detected, their presence indicating the presence of a nucleic acid sequence aberration.

  16. Apparatus for point-of-care detection of nucleic acid in a sample

    DOEpatents

    Bearinger, Jane P.; Dugan, Lawrence C.

    2016-04-19

    Provided herein are methods and apparatus for detecting a target nucleic acid in a sample and related methods and apparatus for diagnosing a condition in an individual. The condition is associated with presence of nucleic acid produced by certain pathogens in the individual.

  17. Methods for point-of-care detection of nucleic acid in a sample

    DOEpatents

    Bearinger, Jane P.; Dugan, Lawrence C.

    2015-12-29

    Provided herein are methods and apparatus for detecting a target nucleic acid in a sample and related methods and apparatus for diagnosing a condition in an individual. The condition is associated with presence of nucleic acid produced by certain pathogens in the individual.

  18. Solid phase sequencing of biopolymers

    DOEpatents

    Cantor, Charles; Koster, Hubert

    2010-09-28

    This invention relates to methods for detecting and sequencing target nucleic acid sequences, to mass modified nucleic acid probes and arrays of probes useful in these methods, and to kits and systems which contain these probes. Useful methods involve hybridizing the nucleic acids or nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes. These probes comprise a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. The molecular weights of the hybridized nucleic acids of the set can be determined by mass spectroscopy, and the sequence of the target determined from the molecular weights of the fragments. Nucleic acids whose sequences can be determined include DNA or RNA in biological samples such as patient biopsies and environmental samples. Probes may be fixed to a solid support such as a hybridization chip to facilitate automated molecular weight analysis and identification of the target sequence.

  19. Integrated sample-to-detection chip for nucleic acid test assays.

    PubMed

    Prakash, R; Pabbaraju, K; Wong, S; Tellier, R; Kaler, K V I S

    2016-06-01

    Nucleic acid based diagnostic techniques are routinely used for the detection of infectious agents. Most of these assays rely on nucleic acid extraction platforms for the extraction and purification of nucleic acids and a separate real-time PCR platform for quantitative nucleic acid amplification tests (NATs). Several microfluidic lab on chip (LOC) technologies have been developed, where mechanical and chemical methods are used for the extraction and purification of nucleic acids. Microfluidic technologies have also been effectively utilized for chip based real-time PCR assays. However, there are few examples of microfluidic systems which have successfully integrated these two key processes. In this study, we have implemented an electro-actuation based LOC micro-device that leverages multi-frequency actuation of samples and reagents droplets for chip based nucleic acid extraction and real-time, reverse transcription (RT) PCR (qRT-PCR) amplification from clinical samples. Our prototype micro-device combines chemical lysis with electric field assisted isolation of nucleic acid in a four channel parallel processing scheme. Furthermore, a four channel parallel qRT-PCR amplification and detection assay is integrated to deliver the sample-to-detection NAT chip. The NAT chip combines dielectrophoresis and electrostatic/electrowetting actuation methods with resistive micro-heaters and temperature sensors to perform chip based integrated NATs. The two chip modules have been validated using different panels of clinical samples and their performance compared with standard platforms. This study has established that our integrated NAT chip system has a sensitivity and specificity comparable to that of the standard platforms while providing up to 10 fold reduction in sample/reagent volumes.

  20. Nucleic Acid Extraction from Synthetic Mars Analog Soils for in situ Life Detection.

    PubMed

    Mojarro, Angel; Ruvkun, Gary; Zuber, Maria T; Carr, Christopher E

    2017-08-01

    Biological informational polymers such as nucleic acids have the potential to provide unambiguous evidence of life beyond Earth. To this end, we are developing an automated in situ life-detection instrument that integrates nucleic acid extraction and nanopore sequencing: the Search for Extra-Terrestrial Genomes (SETG) instrument. Our goal is to isolate and determine the sequence of nucleic acids from extant or preserved life on Mars, if, for example, there is common ancestry to life on Mars and Earth. As is true of metagenomic analysis of terrestrial environmental samples, the SETG instrument must isolate nucleic acids from crude samples and then determine the DNA sequence of the unknown nucleic acids. Our initial DNA extraction experiments resulted in low to undetectable amounts of DNA due to soil chemistry-dependent soil-DNA interactions, namely adsorption to mineral surfaces, binding to divalent/trivalent cations, destruction by iron redox cycling, and acidic conditions. Subsequently, we developed soil-specific extraction protocols that increase DNA yields through a combination of desalting, utilization of competitive binders, and promotion of anaerobic conditions. Our results suggest that a combination of desalting and utilizing competitive binders may establish a "universal" nucleic acid extraction protocol suitable for analyzing samples from diverse soils on Mars. Key Words: Life-detection instruments-Nucleic acids-Mars-Panspermia. Astrobiology 17, 747-760.

  1. Method for nucleic acid hybridization using single-stranded DNA binding protein

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1996-01-01

    Method of nucleic acid hybridization for detecting the presence of a specific nucleic acid sequence in a population of different nucleic acid sequences using a nucleic acid probe. The nucleic acid probe hybridizes with the specific nucleic acid sequence but not with other nucleic acid sequences in the population. The method includes contacting a sample (potentially including the nucleic acid sequence) with the nucleic acid probe under hybridizing conditions in the presence of a single-stranded DNA binding protein provided in an amount which stimulates renaturation of a dilute solution (i.e., one in which the t.sub.1/2 of renaturation is longer than 3 weeks) of single-stranded DNA greater than 500 fold (i.e., to a t.sub.1/2 less than 60 min, preferably less than 5 min, and most preferably about 1 min.) in the absence of nucleotide triphosphates.

  2. Enabling environmental metagenomics and extremophile discovery through SCODA DNA purification

    NASA Astrophysics Data System (ADS)

    Lum, T.; Maydan, J.

    2016-12-01

    A major challenge in nucleic acid preparation from environmental samples is in the ability to separate DNA and RNA from contaminants that often co-purify with methods commonly used. This becomes even more challenging when nucleic acids are in low abundance or when enriching for high molecular weight fragments. Many column- and bead-based methods rely upon selective chemical affinity which is insufficient in dealing with similarly charged contaminants, and also often result in over fragmentation nucleic acids and substantial sample loss. Here we present a unique and alternative parameter for the separation nucleic acids based on the nonlinear response of long, charged polymers to electrophoretic fields. The synchronous coefficient of drag alteration (SCODA) technology is capable of purifying nucleic acids from highly contaminated sample matrices, with molecular weight ranges from 300 bp to over 1 Mbp, and from very low biomass origins. Using a combination of rotating dipole and quadrupole electric fields, SCODA technology concentrates ultrapure nucleic acids that enable PCR, NGS, and optical mapping applications on sample types that are otherwise difficult or impossible to analyze.

  3. Validation of internal controls for extraction and amplification of nucleic acids from enteric viruses in water samples.

    PubMed

    Hata, Akihiko; Katayama, Hiroyuki; Kitajima, Masaaki; Visvanathan, Chettiyappan; Nol, Chea; Furumai, Hiroaki

    2011-07-01

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

  4. Nucleic Acid Extraction from Synthetic Mars Analog Soils for in situ Life Detection

    PubMed Central

    Mojarro, Angel; Ruvkun, Gary; Zuber, Maria T.

    2017-01-01

    Abstract Biological informational polymers such as nucleic acids have the potential to provide unambiguous evidence of life beyond Earth. To this end, we are developing an automated in situ life-detection instrument that integrates nucleic acid extraction and nanopore sequencing: the Search for Extra-Terrestrial Genomes (SETG) instrument. Our goal is to isolate and determine the sequence of nucleic acids from extant or preserved life on Mars, if, for example, there is common ancestry to life on Mars and Earth. As is true of metagenomic analysis of terrestrial environmental samples, the SETG instrument must isolate nucleic acids from crude samples and then determine the DNA sequence of the unknown nucleic acids. Our initial DNA extraction experiments resulted in low to undetectable amounts of DNA due to soil chemistry–dependent soil-DNA interactions, namely adsorption to mineral surfaces, binding to divalent/trivalent cations, destruction by iron redox cycling, and acidic conditions. Subsequently, we developed soil-specific extraction protocols that increase DNA yields through a combination of desalting, utilization of competitive binders, and promotion of anaerobic conditions. Our results suggest that a combination of desalting and utilizing competitive binders may establish a “universal” nucleic acid extraction protocol suitable for analyzing samples from diverse soils on Mars. Key Words: Life-detection instruments—Nucleic acids—Mars—Panspermia. Astrobiology 17, 747–760. PMID:28704064

  5. Nucleic Acid Extraction from Synthetic Mars Analog Soils for in situ Life Detection

    NASA Astrophysics Data System (ADS)

    Mojarro, Angel; Ruvkun, Gary; Zuber, Maria T.; Carr, Christopher E.

    2017-08-01

    Biological informational polymers such as nucleic acids have the potential to provide unambiguous evidence of life beyond Earth. To this end, we are developing an automated in situ life-detection instrument that integrates nucleic acid extraction and nanopore sequencing: the Search for Extra-Terrestrial Genomes (SETG) instrument. Our goal is to isolate and determine the sequence of nucleic acids from extant or preserved life on Mars, if, for example, there is common ancestry to life on Mars and Earth. As is true of metagenomic analysis of terrestrial environmental samples, the SETG instrument must isolate nucleic acids from crude samples and then determine the DNA sequence of the unknown nucleic acids. Our initial DNA extraction experiments resulted in low to undetectable amounts of DNA due to soil chemistry-dependent soil-DNA interactions, namely adsorption to mineral surfaces, binding to divalent/trivalent cations, destruction by iron redox cycling, and acidic conditions. Subsequently, we developed soil-specific extraction protocols that increase DNA yields through a combination of desalting, utilization of competitive binders, and promotion of anaerobic conditions. Our results suggest that a combination of desalting and utilizing competitive binders may establish a "universal" nucleic acid extraction protocol suitable for analyzing samples from diverse soils on Mars.

  6. Nucleic acid purification from plants, animals and microbes in under 30 seconds

    PubMed Central

    Zou, Yiping; Wang, Yuling; Wee, Eugene; Turni, Conny; Blackall, Patrick J.; Trau, Matt; Botella, Jose Ramon

    2017-01-01

    Nucleic acid amplification is a powerful molecular biology tool, although its use outside the modern laboratory environment is limited due to the relatively cumbersome methods required to extract nucleic acids from biological samples. To address this issue, we investigated a variety of materials for their suitability for nucleic acid capture and purification. We report here that untreated cellulose-based paper can rapidly capture nucleic acids within seconds and retain them during a single washing step, while contaminants present in complex biological samples are quickly removed. Building on this knowledge, we have successfully created an equipment-free nucleic acid extraction dipstick methodology that can obtain amplification-ready DNA and RNA from plants, animals, and microbes from difficult biological samples such as blood and leaves from adult trees in less than 30 seconds. The simplicity and speed of this method as well as the low cost and availability of suitable materials (e.g., common paper towelling), means that nucleic acid extraction is now more accessible and affordable for researchers and the broader community. Furthermore, when combined with recent advancements in isothermal amplification and naked eye DNA visualization techniques, the dipstick extraction technology makes performing molecular diagnostic assays achievable in limited resource settings including university and high school classrooms, field-based environments, and developing countries. PMID:29161268

  7. Portable nucleic acid thermocyclers.

    PubMed

    Almassian, David R; Cockrell, Lisa M; Nelson, William M

    2013-11-21

    A nucleic acid thermal cycler is considered to be portable if it is under ten pounds, easily carried by one individual, and battery powered. Nucleic acid amplification includes both polymerase chain reaction (e.g. PCR, RT-PCR) and isothermal amplification (e.g. RPA, HDA, LAMP, NASBA, RCA, ICAN, SMART, SDA). There are valuable applications for portable nucleic acid thermocyclers in fields that include clinical diagnostics, biothreat detection, and veterinary testing. A system that is portable allows for the distributed detection of targets at the point of care and a reduction of the time from sample to answer. The designer of a portable nucleic acid thermocycler must carefully consider both thermal control and the detection of amplification. In addition to thermal control and detection, the designer may consider the integration of a sample preparation subsystem with the nucleic acid thermocycler. There are a variety of technologies that can achieve accurate thermal control and the detection of nucleic acid amplification. Important evaluation criteria for each technology include maturity, power requirements, cost, sensitivity, speed, and manufacturability. Ultimately the needs of a particular market will lead to user requirements that drive the decision between available technologies.

  8. Detection of nucleic acids by multiple sequential invasive cleavages

    DOEpatents

    Hall, Jeff G.; Lyamichev, Victor I.; Mast, Andrea L.; Brow, Mary Ann D.

    1999-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

  9. Nucleic acid detection kits

    DOEpatents

    Hall, Jeff G.; Lyamichev, Victor I.; Mast, Andrea L.; Brow, Mary Ann; Kwiatkowski, Robert W.; Vavra, Stephanie H.

    2005-03-29

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of nucleic acid from various viruses in a sample.

  10. Detection of nucleic acids by multiple sequential invasive cleavages 02

    DOEpatents

    Hall, Jeff G.; Lyamichev, Victor I.; Mast, Andrea L.; Brow, Mary Ann D.

    2002-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

  11. Detection of nucleic acids by multiple sequential invasive cleavages

    DOEpatents

    Hall, Jeff G; Lyamichev, Victor I; Mast, Andrea L; Brow, Mary Ann D

    2012-10-16

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

  12. [Detection of Avian Influenza Virus in Environmental Samples Collected from Live Poultry Markets in China during 2009-2013].

    PubMed

    Zhang, Ye; Li, Xiaodan; Zou, Shumei; Bo, Hong; Dong, Libo; Gao, Rongbao; Wang, Dayan; Shu, Yuelong

    2015-11-01

    Abstract: To investigate the distribution of avian influenza virus in environmental samples from live poultry markets (LPM) in China, samples were collected and tested by nucleic acid during 2009-2013 season. Each sample was tested by real-time RT PCR using flu A specific primers. If any real-time PCR was positive, the sample was inoculated into specific-pathogen-free (SPF) embryonated chicken eggs for viral isolation. The results indicated that the positive rate of nucleic acid in enviromental samples exhibited seasonality. The positive rate of nucleic acid was significantly higher in Winter and Spring. The positive rate of nucleic acid in LPM located in the south of China was higher than in northern China. Samples of Sewage for cleaning poultry and chopping board showed that higher positive rate of nucleic acid than other samples. The Subtype identification showed that H5 and H9 were main subtypes in the enviromental samples. Viral isolation indicated H5 subtypes was more than H9 subtypes between 2009 and 2013 while H9 subtypes increased in 2013. Our findings suggested the significance of public health based on LPM surveillance and provided the basis of prevention and early warning for avian flu infection human.

  13. Quantification of Parvovirus B19 DNA Using COBAS AmpliPrep Automated Sample Preparation and LightCycler Real-Time PCR

    PubMed Central

    Schorling, Stefan; Schalasta, Gunnar; Enders, Gisela; Zauke, Michael

    2004-01-01

    The COBAS AmpliPrep instrument (Roche Diagnostics GmbH, D-68305 Mannheim, Germany) automates the entire sample preparation process of nucleic acid isolation from serum or plasma for polymerase chain reaction analysis. We report the analytical performance of the LightCycler Parvovirus B19 Quantification Kit (Roche Diagnostics) using nucleic acids isolated with the COBAS AmpliPrep instrument. Nucleic acids were extracted using the Total Nucleic Acid Isolation Kit (Roche Diagnostics) and amplified with the LightCycler Parvovirus B19 Quantification Kit. The kit combination processes 72 samples per 8-hour shift. The lower detection limit is 234 IU/ml at a 95% hit-rate, linear range approximately 104-1010 IU/ml, and overall precision 16 to 40%. Relative sensitivity and specificity in routine samples from pregnant women are 100% and 93%, respectively. Identification of a persistent parvovirus B19-infected individual by the polymerase chain reaction among 51 anti-parvovirus B19 IgM-negative samples underlines the importance of additional nucleic acid testing in pregnancy and its superiority to serology in identifying the risk of parvovirus B19 transmission via blood or blood products. Combination of the Total Nucleic Acid Isolation Kit on the COBAS AmpliPrep instrument with the LightCycler Parvovirus B19 Quantification Kit provides a reliable and time-saving tool for sensitive and accurate detection of parvovirus B19 DNA. PMID:14736825

  14. Use of Dimethyl Pimelimidate with Microfluidic System for Nucleic Acids Extraction without Electricity.

    PubMed

    Jin, Choong Eun; Lee, Tae Yoon; Koo, Bonhan; Choi, Kyung-Chul; Chang, Suhwan; Park, Se Yoon; Kim, Ji Yeun; Kim, Sung-Han; Shin, Yong

    2017-07-18

    The isolation of nucleic acids in the lab on a chip is crucial to achieve the maximal effectiveness of point-of-care testing for detection in clinical applications. Here, we report on the use of a simple and versatile single-channel microfluidic platform that combines dimethyl pimelimidate (DMP) for nucleic acids (both RNA and DNA) extraction without electricity using a thin-film system. The system is based on the adaption of DMP into nonchaotropic-based nucleic acids and the capture of reagents into a low-cost thin-film platform for use as a microfluidic total analysis system, which can be utilized for sample processing in clinical diagnostics. Moreover, we assessed the use of the DMP system for the extraction of nucleic acids from various samples, including mammalian cells, bacterial cells, and viruses from human disease, and we also confirmed that the quality and quantity of the nucleic acids extracted were sufficient to allow for the robust detection of biomarkers and/or pathogens in downstream analysis. Furthermore, this DMP system does not require any instruments and electricity, and has improved time efficiency, portability, and affordability. Thus, we believe that the DMP system may change the paradigm of sample processing in clinical diagnostics.

  15. An instrument for automated purification of nucleic acids from contaminated forensic samples

    PubMed Central

    Broemeling, David J; Pel, Joel; Gunn, Dylan C; Mai, Laura; Thompson, Jason D; Poon, Hiron; Marziali, Andre

    2008-01-01

    Forensic crime scene sample analysis, by its nature, often deals with samples in which there are low amounts of nucleic acids, on substrates that often lead to inhibition of subsequent enzymatic reactions such as PCR amplification for STR profiling. Common substrates include denim from blue jeans, which yields indigo dye as a PCR inhibitor, and soil, which yields humic substances as inhibitors. These inhibitors frequently co-extract with nucleic acids in standard column or bead-based preps, leading to frequent failure of STR profiling. We present a novel instrument for DNA purification of forensic samples that is capable of highly effective concentration of nucleic acids from soil particulates, fabric, and other complex samples including solid components. The novel concentration process, known as SCODA, is inherently selective for long charged polymers such as DNA, and therefore is able to effectively reject known contaminants. We present an automated sample preparation instrument based on this process, and preliminary results based on mock forensic samples. PMID:18438455

  16. FTA Cards for Preservation of Nucleic Acids for Molecular Assays: A Review on the Use of Cytologic/Tissue Samples.

    PubMed

    da Cunha Santos, Gilda

    2018-03-01

    - Traditional methods for storing histologic and cytologic specimens for future use in molecular assays have consisted of either snap-freezing with cryopreservation or formalin-fixing, paraffin-embedding the samples. Although snap-freezing with cryopreservation is recommended for better preservation of nucleic acids, the infrastructure and space required for archiving impose challenges for high-volume pathology laboratories. Cost-effective, long-term storage at room temperature; relatively easy shipment; and standardized handling can be achieved with formalin-fixed, paraffin-embedded samples, but formalin fixation induces fragmentation and chemical modification of nucleic acids. Advances in next-generation sequencing platforms, coupled with an increase in diagnostic, prognostic, and predictive molecular biomarkers have created a demand for high-quality nucleic acids. To address issues of the quality of nucleic acid and logistics in sample acquisition, alternatives for specimen preservation and long-term storage have been described and include novel universal tissue fixatives, stabilizers, and technologies. - To collect, retrieve, and review information from studies describing the use of nucleic acids recovered from cytologic/tissue specimens stored on Flinders Technology Associates (FTA, GE Whatman, Maidstone, Kent, United Kingdom) cards for downstream molecular applications. - An electronic literature search in the PubMed (National Center for Biotechnology Information, Bethesda, Maryland) database allowed the selection of manuscripts addressing the use of FTA cards for storage of cytologic samples for molecular analysis. Only articles published in English were retrieved. - The use of FTA cards is a versatile method for fostering multicenter, international collaborations and clinical trials that require centralized testing, long-distance shipment, and high-quality nucleic acids for molecular techniques. Studies with controlled temperature are required to test the quality of recovered RNA after long-term storage.

  17. Extraction of nucleic acids from yeast cells and plant tissues using ethanol as medium for sample preservation and cell disruption.

    PubMed

    Linke, Bettina; Schröder, Kersten; Arter, Juliane; Gasperazzo, Tatiana; Woehlecke, Holger; Ehwald, Rudolf

    2010-09-01

    Here we report that dehydrated ethanol is an excellent medium for both in situ preservation of nucleic acids and cell disruption of plant and yeast cells. Cell disruption was strongly facilitated by prior dehydration of the ethanol using dehydrated zeolite. Following removal of ethanol, nucleic acids were extracted from the homogenate pellet using denaturing buffers. The method provided DNA and RNA of high yield and integrity. Whereas cell wall disruption was essential for extraction of DNA and large RNA molecules, smaller molecules such as tRNAs could be selectively extracted from undisrupted, ethanol-treated yeast cells. Our results demonstrate the utility of absolute ethanol for sample fixation, cell membrane and cell wall disruption, as well as preservation of nucleic acids during sample storage.

  18. Detection and isolation of nucleic acid sequences using a bifunctional hybridization probe

    DOEpatents

    Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

    2000-01-01

    A method for detecting and isolating a target sequence in a sample of nucleic acids is provided using a bifunctional hybridization probe capable of hybridizing to the target sequence that includes a detectable marker and a first complexing agent capable of forming a binding pair with a second complexing agent. A kit is also provided for detecting a target sequence in a sample of nucleic acids using a bifunctional hybridization probe according to this method.

  19. Concentration methods for high-resolution THz spectroscopy of nucleic-acid biomolecules and crystals

    NASA Astrophysics Data System (ADS)

    Brown, E. R.; Zhang, W.; Mendoza, E. A.; Kuznetsova, Y.; Brueck, S. R. J.; Rahman, M.; Norton, M. L.

    2012-03-01

    Biomolecules can exhibit low-lying vibrational modes in the THz region which are detectable in transmission given a strong molecular dipole moment and optical depth, and a spectrometer of adequate sensitivity. The nucleic acids are particularly interesting because of applications such as label-free gene assay, bio-agent detection, etc. However for nucleic acids, sample preparation and THz coupling are of paramount importance because of the strong absorption by liquid water and the small concentration of molecules present in physiological solutions. Concentration methods become necessary to make the THz vibrational modes detectable, either by concentrating the nucleic-acid sample itself in a small volume but large area, or by concentrating the THz radiation down to the volume of the sample. This paper summarizes one type of the first method: nanofluidic channel arrays for biological nucleic acids; and two types of the second method: (1) a circular-waveguide pinhole, and (2) a circular-waveguide, conical-horn coupling structure, both for DNA crystals. The first method has been demonstrated on a very short artificial nucleic acid [small-interfering (si) RNA (17-to-25 bp)] and a much longer, biological molecule [Lambda-phage DNA (48.5 kbp)]. The second method has been demonstrated on small (~100 micron) single crystals of DNA grown by the sitting-drop method.

  20. Does the high nucleic acid content of individual bacterial cells allow us to discriminate between active cells and inactive cells in aquatic systems?

    PubMed

    Lebaron, P; Servais, P; Agogué, H; Courties, C; Joux, F

    2001-04-01

    The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems.

  1. Does the High Nucleic Acid Content of Individual Bacterial Cells Allow Us To Discriminate between Active Cells and Inactive Cells in Aquatic Systems?

    PubMed Central

    Lebaron, Philippe; Servais, Pierre; Agogué, Helene; Courties, Claude; Joux, Fabien

    2001-01-01

    The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems. PMID:11282632

  2. Nucleic Acid analysis by fourier transform ion cyclotron resonance mass spectrometry at the beginning of the twenty-first century.

    PubMed

    Frahm, J L; Muddiman, D C

    2005-01-01

    Mass spectrometers measure an intrinsic property (i.e., mass) of a molecule, which makes it an ideal platform for nucleic acid analysis. Importantly, the unparalleled capabilities of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry further extend its usefulness for nucleic acid analysis. The beginning of the twenty-first century has been marked with notable advances in the field of FT-ICR mass spectrometry analysis of nucleic acids. Some of these accomplishments include fundamental studies of nucleic acid properties, improvements in sample clean up and preparation, better methods to obtain higher mass measurement accuracy, analysis of noncovalent complexes, tandem mass spectrometry, and characterization of peptide nucleic acids. This diverse range of studies will be presented herein.

  3. Integrated printed circuit board device for cell lysis and nucleic acid extraction.

    PubMed

    Marshall, Lewis A; Wu, Liang Li; Babikian, Sarkis; Bachman, Mark; Santiago, Juan G

    2012-11-06

    Preparation of raw, untreated biological samples remains a major challenge in microfluidics. We present a novel microfluidic device based on the integration of printed circuit boards and an isotachophoresis assay for sample preparation of nucleic acids from biological samples. The device has integrated resistive heaters and temperature sensors as well as a 70 μm × 300 μm × 3.7 cm microfluidic channel connecting two 15 μL reservoirs. We demonstrated this device by extracting pathogenic nucleic acids from 1 μL dispensed volume of whole blood spiked with Plasmodium falciparum. We dispensed whole blood directly onto an on-chip reservoir, and the system's integrated heaters simultaneously lysed and mixed the sample. We used isotachophoresis to extract the nucleic acids into a secondary buffer via isotachophoresis. We analyzed the convective mixing action with micro particle image velocimetry (micro-PIV) and verified the purity and amount of extracted nucleic acids using off-chip quantitative polymerase chain reaction (PCR). We achieved a clinically relevant limit of detection of 500 parasites per microliter. The system has no moving parts, and the process is potentially compatible with a wide range of on-chip hybridization or amplification assays.

  4. Automated Nucleic Acid Extraction Systems for Detecting Cytomegalovirus and Epstein-Barr Virus Using Real-Time PCR: A Comparison Study Between the QIAsymphony RGQ and QIAcube Systems.

    PubMed

    Kim, Hanah; Hur, Mina; Kim, Ji Young; Moon, Hee Won; Yun, Yeo Min; Cho, Hyun Chan

    2017-03-01

    Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) are increasingly important in immunocompromised patients. Nucleic acid extraction methods could affect the results of viral nucleic acid amplification tests. We compared two automated nucleic acid extraction systems for detecting CMV and EBV using real-time PCR assays. One hundred and fifty-three whole blood (WB) samples were tested for CMV detection, and 117 WB samples were tested for EBV detection. Viral nucleic acid was extracted in parallel by using QIAsymphony RGQ and QIAcube (Qiagen GmbH, Germany), and real-time PCR assays for CMV and EBV were performed with a Rotor-Gene Q real-time PCR cycler (Qiagen). Detection rates for CMV and EBV were compared, and agreements between the two systems were analyzed. The detection rate of CMV and EBV differed significantly between the QIAsymphony RGQ and QIAcube systems (CMV, 59.5% [91/153] vs 43.8% [67/153], P=0.0005; EBV, 59.0% [69/117] vs 42.7% [50/117], P=0.0008). The two systems showed moderate agreement for CMV and EBV detection (kappa=0.43 and 0.52, respectively). QIAsymphony RGQ showed a negligible correlation with QIAcube for quantitative EBV detection. QIAcube exhibited EBV PCR inhibition in 23.9% (28/117) of samples. Automated nucleic acid extraction systems have different performances and significantly affect the detection of viral pathogens. The QIAsymphony RGQ system appears to be superior to the QIAcube system for detecting CMV and EBV. A suitable sample preparation system should be considered for optimized nucleic acid amplification in clinical laboratories.

  5. Outbreak or illusion: consequences of 'improved' diagnostics for gonorrhoea.

    PubMed

    Bennett, Amy; Jeffery, Katie; O'Neill, Eunan; Sherrard, Jackie

    2017-06-01

    The sexual health service in Oxford introduced gonorrhoea nucleic amplification acid testing using the BD Viper XTR™ System. For practical reasons, a confirmatory nucleic amplification acid testing using a different platform was not used initially. Following the introduction of nucleic amplification acid testing, the rates of gonorrhoea increased threefold. Concerns were raised that this increase represented an outbreak. A retrospective review of cases over six months suggested that there may have been a number of false-positive results. A prospective study was then undertaken over six months, where all gonorrhoea positive samples were sent for confirmatory testing. This evaluation of all gonorrhoea cases in an English county found that the overall presumptive false-positive rates for gonorrhoea nucleic amplification acid testing using BD Viper XTR™ in our population are significant at 27% of female samples, 13.2% of heterosexual male samples, 3.5% of anogenital multiple site men who have sex with men samples and 62.8% of pharyngeal only men who have sex with men samples. The data demonstrate the need for confirmatory testing using a second nucleic acid target, as per BASHH/Public Health England guidelines, especially in low-prevalence settings and extragenital sites, due to cross-reactivity with commensal Neisseria species and low positive predictive values.

  6. Chemical amplification based on fluid partitioning

    DOEpatents

    Anderson, Brian L [Lodi, CA; Colston, Jr., Billy W.; Elkin, Chris [San Ramon, CA

    2006-05-09

    A system for nucleic acid amplification of a sample comprises partitioning the sample into partitioned sections and performing PCR on the partitioned sections of the sample. Another embodiment of the invention provides a system for nucleic acid amplification and detection of a sample comprising partitioning the sample into partitioned sections, performing PCR on the partitioned sections of the sample, and detecting and analyzing the partitioned sections of the sample.

  7. Detection and isolation of nucleic acid sequences using competitive hybridization probes

    DOEpatents

    Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

    1997-01-01

    A method for detecting a target nucleic acid sequence in a sample is provided using hybridization probes which competitively hybridize to a target nucleic acid. According to the method, a target nucleic acid sequence is hybridized to first and second hybridization probes which are complementary to overlapping portions of the target nucleic acid sequence, the first hybridization probe including a first complexing agent capable of forming a binding pair with a second complexing agent and the second hybridization probe including a detectable marker. The first complexing agent attached to the first hybridization probe is contacted with a second complexing agent, the second complexing agent being attached to a solid support such that when the first and second complexing agents are attached, target nucleic acid sequences hybridized to the first hybridization probe become immobilized on to the solid support. The immobilized target nucleic acids are then separated and detected by detecting the detectable marker attached to the second hybridization probe. A kit for performing the method is also provided.

  8. Detection and isolation of nucleic acid sequences using competitive hybridization probes

    DOEpatents

    Lucas, J.N.; Straume, T.; Bogen, K.T.

    1997-04-01

    A method for detecting a target nucleic acid sequence in a sample is provided using hybridization probes which competitively hybridize to a target nucleic acid. According to the method, a target nucleic acid sequence is hybridized to first and second hybridization probes which are complementary to overlapping portions of the target nucleic acid sequence, the first hybridization probe including a first complexing agent capable of forming a binding pair with a second complexing agent and the second hybridization probe including a detectable marker. The first complexing agent attached to the first hybridization probe is contacted with a second complexing agent, the second complexing agent being attached to a solid support such that when the first and second complexing agents are attached, target nucleic acid sequences hybridized to the first hybridization probe become immobilized on to the solid support. The immobilized target nucleic acids are then separated and detected by detecting the detectable marker attached to the second hybridization probe. A kit for performing the method is also provided. 7 figs.

  9. Laboratory procedures to generate viral metagenomes.

    PubMed

    Thurber, Rebecca V; Haynes, Matthew; Breitbart, Mya; Wegley, Linda; Rohwer, Forest

    2009-01-01

    This collection of laboratory protocols describes the steps to collect viruses from various samples with the specific aim of generating viral metagenome sequence libraries (viromes). Viral metagenomics, the study of uncultured viral nucleic acid sequences from different biomes, relies on several concentration, purification, extraction, sequencing and heuristic bioinformatic methods. No single technique can provide an all-inclusive approach, and therefore the protocols presented here will be discussed in terms of hypothetical projects. However, care must be taken to individualize each step depending on the source and type of viral-particles. This protocol is a description of the processes we have successfully used to: (i) concentrate viral particles from various types of samples, (ii) eliminate contaminating cells and free nucleic acids and (iii) extract, amplify and purify viral nucleic acids. Overall, a sample can be processed to isolate viral nucleic acids suitable for high-throughput sequencing in approximately 1 week.

  10. Optimized methods for total nucleic acid extraction and quantification of the bat white-nose syndrome fungus, Pseudogymnoascus destructans, from swab and environmental samples.

    PubMed

    Verant, Michelle L; Bohuski, Elizabeth A; Lorch, Jeffery M; Blehert, David S

    2016-03-01

    The continued spread of white-nose syndrome and its impacts on hibernating bat populations across North America has prompted nationwide surveillance efforts and the need for high-throughput, noninvasive diagnostic tools. Quantitative real-time polymerase chain reaction (qPCR) analysis has been increasingly used for detection of the causative fungus, Pseudogymnoascus destructans, in both bat- and environment-associated samples and provides a tool for quantification of fungal DNA useful for research and monitoring purposes. However, precise quantification of nucleic acid from P. destructans is dependent on effective and standardized methods for extracting nucleic acid from various relevant sample types. We describe optimized methodologies for extracting fungal nucleic acids from sediment, guano, and swab-based samples using commercial kits together with a combination of chemical, enzymatic, and mechanical modifications. Additionally, we define modifications to a previously published intergenic spacer-based qPCR test for P. destructans to refine quantification capabilities of this assay. © 2016 The Author(s).

  11. Optimized methods for total nucleic acid extraction and quantification of the bat white-nose syndrome fungus, Pseudogymnoascus destructans, from swab and environmental samples

    USGS Publications Warehouse

    Verant, Michelle; Bohuski, Elizabeth A.; Lorch, Jeffrey M.; Blehert, David

    2016-01-01

    The continued spread of white-nose syndrome and its impacts on hibernating bat populations across North America has prompted nationwide surveillance efforts and the need for high-throughput, noninvasive diagnostic tools. Quantitative real-time polymerase chain reaction (qPCR) analysis has been increasingly used for detection of the causative fungus, Pseudogymnoascus destructans, in both bat- and environment-associated samples and provides a tool for quantification of fungal DNA useful for research and monitoring purposes. However, precise quantification of nucleic acid fromP. destructans is dependent on effective and standardized methods for extracting nucleic acid from various relevant sample types. We describe optimized methodologies for extracting fungal nucleic acids from sediment, guano, and swab-based samples using commercial kits together with a combination of chemical, enzymatic, and mechanical modifications. Additionally, we define modifications to a previously published intergenic spacer–based qPCR test for P. destructans to refine quantification capabilities of this assay.

  12. A novel automated device for rapid nucleic acid extraction utilizing a zigzag motion of magnetic silica beads.

    PubMed

    Yamaguchi, Akemi; Matsuda, Kazuyuki; Uehara, Masayuki; Honda, Takayuki; Saito, Yasunori

    2016-02-04

    We report a novel automated device for nucleic acid extraction, which consists of a mechanical control system and a disposable cassette. The cassette is composed of a bottle, a capillary tube, and a chamber. After sample injection in the bottle, the sample is lysed, and nucleic acids are adsorbed on the surface of magnetic silica beads. These magnetic beads are transported and are vibrated through the washing reagents in the capillary tube under the control of the mechanical control system, and thus, the nucleic acid is purified without centrifugation. The purified nucleic acid is automatically extracted in 3 min for the polymerase chain reaction (PCR). The nucleic acid extraction is dependent on the transport speed and the vibration frequency of the magnetic beads, and optimizing these two parameters provided better PCR efficiency than the conventional manual procedure. There was no difference between the detection limits of our novel device and that of the conventional manual procedure. We have already developed the droplet-PCR machine, which can amplify and detect specific nucleic acids rapidly and automatically. Connecting the droplet-PCR machine to our novel automated extraction device enables PCR analysis within 15 min, and this system can be made available as a point-of-care testing in clinics as well as general hospitals. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Chemical amplification based on fluid partitioning in an immiscible liquid

    DOEpatents

    Anderson, Brian L.; Colston, Bill W.; Elkin, Christopher J.

    2010-09-28

    A system for nucleic acid amplification of a sample comprises partitioning the sample into partitioned sections and performing PCR on the partitioned sections of the sample. Another embodiment of the invention provides a system for nucleic acid amplification and detection of a sample comprising partitioning the sample into partitioned sections, performing PCR on the partitioned sections of the sample, and detecting and analyzing the partitioned sections of the sample.

  14. Development of Sorbents for Extraction and Stabilization of Nucleic Acids

    DTIC Science & Technology

    2016-09-13

    ensure safe food and water supplies and to maintain the health and readiness of deployed troops. Identification of molecular signatures (genomic...biological, environmental, forensics, and food safety, drive the need for preservation of nucleic acid integrity during sample collection, transportation... antimicrobial activity as well as the potential for multiple and complex cationic interactions with nucleic acids (Fig. 10). Two different approaches were used

  15. An integrated paper-based sample-to-answer biosensor for nucleic acid testing at the point of care.

    PubMed

    Choi, Jane Ru; Hu, Jie; Tang, Ruihua; Gong, Yan; Feng, Shangsheng; Ren, Hui; Wen, Ting; Li, XiuJun; Wan Abas, Wan Abu Bakar; Pingguan-Murphy, Belinda; Xu, Feng

    2016-02-07

    With advances in point-of-care testing (POCT), lateral flow assays (LFAs) have been explored for nucleic acid detection. However, biological samples generally contain complex compositions and low amounts of target nucleic acids, and currently require laborious off-chip nucleic acid extraction and amplification processes (e.g., tube-based extraction and polymerase chain reaction (PCR)) prior to detection. To the best of our knowledge, even though the integration of DNA extraction and amplification into a paper-based biosensor has been reported, a combination of LFA with the aforementioned steps for simple colorimetric readout has not yet been demonstrated. Here, we demonstrate for the first time an integrated paper-based biosensor incorporating nucleic acid extraction, amplification and visual detection or quantification using a smartphone. A handheld battery-powered heating device was specially developed for nucleic acid amplification in POC settings, which is coupled with this simple assay for rapid target detection. The biosensor can successfully detect Escherichia coli (as a model analyte) in spiked drinking water, milk, blood, and spinach with a detection limit of as low as 10-1000 CFU mL(-1), and Streptococcus pneumonia in clinical blood samples, highlighting its potential use in medical diagnostics, food safety analysis and environmental monitoring. As compared to the lengthy conventional assay, which requires more than 5 hours for the entire sample-to-answer process, it takes about 1 hour for our integrated biosensor. The integrated biosensor holds great potential for detection of various target analytes for wide applications in the near future.

  16. 75 FR 22814 - Guidance for Industry: Nucleic Acid Testing (NAT) for Human Immunodeficiency Virus Type 1 (HIV-1...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-30

    ... Immunodeficiency Virus Type 1 (HIV-1) and Hepatitis C Virus (HCV): Testing, Product Disposition, and Donor Deferral... Industry: Nucleic Acid Testing (NAT) for Human Immunodeficiency Virus Type 1 (HIV-1) and Hepatitis C Virus... Acid Test (NAT) and Hepatitis C Virus (HCV) NAT, on testing individual samples or pooled samples from...

  17. Collecting, archiving and processing DNA from wildlife samples using FTA® databasing paper

    PubMed Central

    Smith, LM; Burgoyne, LA

    2004-01-01

    Background Methods involving the analysis of nucleic acids have become widespread in the fields of traditional biology and ecology, however the storage and transport of samples collected in the field to the laboratory in such a manner to allow purification of intact nucleic acids can prove problematical. Results FTA® databasing paper is widely used in human forensic analysis for the storage of biological samples and for purification of nucleic acids. The possible uses of FTA® databasing paper in the purification of DNA from samples of wildlife origin were examined, with particular reference to problems expected due to the nature of samples of wildlife origin. The processing of blood and tissue samples, the possibility of excess DNA in blood samples due to nucleated erythrocytes, and the analysis of degraded samples were all examined, as was the question of long term storage of blood samples on FTA® paper. Examples of the end use of the purified DNA are given for all protocols and the rationale behind the processing procedures is also explained to allow the end user to adjust the protocols as required. Conclusions FTA® paper is eminently suitable for collection of, and purification of nucleic acids from, biological samples from a wide range of wildlife species. This technology makes the collection and storage of such samples much simpler. PMID:15072582

  18. Ultra high-throughput nucleic acid sequencing as a tool for virus discovery in the turkey gut.

    USDA-ARS?s Scientific Manuscript database

    Recently, the use of the next generation of nucleic acid sequencing technology (i.e., 454 pyrosequencing, as developed by Roche/454 Life Sciences) has allowed an in-depth look at the uncultivated microorganisms present in complex environmental samples, including samples with agricultural importance....

  19. Apparatus for chemical amplification based on fluid partitioning in an immiscible liquid

    DOEpatents

    Anderson, Brian L [Lodi, CA; Colston, Bill W [San Ramon, CA; Elkin, Christopher J [San Ramon, CA

    2012-05-08

    A system for nucleic acid amplification of a sample comprises partitioning the sample into partitioned sections and performing PCR on the partitioned sections of the sample. Another embodiment of the invention provides a system for nucleic acid amplification and detection of a sample comprising partitioning the sample into partitioned sections, performing PCR on the partitioned sections of the sample, and detecting and analyzing the partitioned sections of the sample.

  20. Method for chemical amplification based on fluid partitioning in an immiscible liquid

    DOEpatents

    Anderson, Brian L.; Colston, Bill W.; Elkin, Christopher J.

    2015-06-02

    A system for nucleic acid amplification of a sample comprises partitioning the sample into partitioned sections and performing PCR on the partitioned sections of the sample. Another embodiment of the invention provides a system for nucleic acid amplification and detection of a sample comprising partitioning the sample into partitioned sections, performing PCR on the partitioned sections of the sample, and detecting and analyzing the partitioned sections of the sample.

  1. Method for chemical amplification based on fluid partitioning in an immiscible liquid

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

    Anderson, Brian L.; Colston, Bill W.; Elkin, Christopher J.

    A system for nucleic acid amplification of a sample comprises partitioning the sample into partitioned sections and performing PCR on the partitioned sections of the sample. Another embodiment of the invention provides a system for nucleic acid amplification and detection of a sample comprising partitioning the sample into partitioned sections, performing PCR on the partitioned sections of the sample, and detecting and analyzing the partitioned sections of the sample.

  2. Lateral flow devices

    DOEpatents

    Mazumdar, Debapriya; Liu, Juewen; Lu, Yi

    2010-09-21

    An analytical test for an analyte comprises (a) a base, having a reaction area and a visualization area, (b) a capture species, on the base in the visualization area, comprising nucleic acid, and (c) analysis chemistry reagents, on the base in the reaction area. The analysis chemistry reagents comprise (i) a substrate comprising nucleic acid and a first label, and (ii) a reactor comprising nucleic acid. The analysis chemistry reagents can react with a sample comprising the analyte and water, to produce a visualization species comprising nucleic acid and the first label, and the capture species can bind the visualization species.

  3. Zinc complexes as fluorescent chemosensors for nucleic acids: new perspectives for a "boring" element.

    PubMed

    Terenzi, Alessio; Lauria, Antonino; Almerico, Anna Maria; Barone, Giampaolo

    2015-02-28

    Zinc(II) complexes are effective and selective nucleic acid-binders and strongly fluorescent molecules in the low energy range, from the visible to the near infrared. These two properties have often been exploited to quantitatively detect nucleic acids in biological samples, in both in vitro and in vivo models. In particular, the fluorescent emission of several zinc(II) complexes is drastically enhanced or quenched by the binding to nucleic acids and/or upon visible light exposure, in a different fashion in bulk solution and when bound to DNA. The twofold objective of this perspective is (1) to review recent utilisations of zinc(II) complexes as selective fluorescent probes for nucleic acids and (2) to highlight their novel potential applications as diagnostic tools based on their photophysical properties.

  4. Modular microfluidic system for biological sample preparation

    DOEpatents

    Rose, Klint A.; Mariella, Jr., Raymond P.; Bailey, Christopher G.; Ness, Kevin Dean

    2015-09-29

    A reconfigurable modular microfluidic system for preparation of a biological sample including a series of reconfigurable modules for automated sample preparation adapted to selectively include a) a microfluidic acoustic focusing filter module, b) a dielectrophoresis bacteria filter module, c) a dielectrophoresis virus filter module, d) an isotachophoresis nucleic acid filter module, e) a lyses module, and f) an isotachophoresis-based nucleic acid filter.

  5. An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids.

    PubMed

    Chen, Dafeng; Mauk, Michael; Qiu, Xianbo; Liu, Changchun; Kim, Jitae; Ramprasad, Sudhir; Ongagna, Serge; Abrams, William R; Malamud, Daniel; Corstjens, Paul L A M; Bau, Haim H

    2010-08-01

    A self-contained, integrated, disposable, sample-to-answer, polycarbonate microfluidic cassette for nucleic acid-based detection of pathogens at the point of care was designed, constructed, and tested. The cassette comprises on-chip sample lysis, nucleic acid isolation, enzymatic amplification (polymerase chain reaction and, when needed, reverse transcription), amplicon labeling, and detection. On-chip pouches and valves facilitate fluid flow control. All the liquids and dry reagents needed for the various reactions are pre-stored in the cassette. The liquid reagents are stored in flexible pouches formed on the chip surface. Dry (RT-)PCR reagents are pre-stored in the thermal cycling, reaction chamber. The process operations include sample introduction; lysis of cells and viruses; solid-phase extraction, concentration, and purification of nucleic acids from the lysate; elution of the nucleic acids into a thermal cycling chamber and mixing with pre-stored (RT-)PCR dry reagents; thermal cycling; and detection. The PCR amplicons are labeled with digoxigenin and biotin and transmitted onto a lateral flow strip, where the target analytes bind to a test line consisting of immobilized avidin-D. The immobilized nucleic acids are labeled with up-converting phosphor (UCP) reporter particles. The operation of the cassette is automatically controlled by an analyzer that provides pouch and valve actuation with electrical motors and heating for the thermal cycling. The functionality of the device is demonstrated by detecting the presence of bacterial B.Cereus, viral armored RNA HIV, and HIV I virus in saliva samples. The cassette and actuator described here can be used to detect other diseases as well as the presence of bacterial and viral pathogens in the water supply and other fluids.

  6. Molecular Dynamics Simulations of Nucleic Acids. From Tetranucleotides to the Ribosome.

    PubMed

    Šponer, Jiří; Banáš, Pavel; Jurečka, Petr; Zgarbová, Marie; Kührová, Petra; Havrila, Marek; Krepl, Miroslav; Stadlbauer, Petr; Otyepka, Michal

    2014-05-15

    We present a brief overview of explicit solvent molecular dynamics (MD) simulations of nucleic acids. We explain physical chemistry limitations of the simulations, namely, the molecular mechanics (MM) force field (FF) approximation and limited time scale. Further, we discuss relations and differences between simulations and experiments, compare standard and enhanced sampling simulations, discuss the role of starting structures, comment on different versions of nucleic acid FFs, and relate MM computations with contemporary quantum chemistry. Despite its limitations, we show that MD is a powerful technique for studying the structural dynamics of nucleic acids with a fast growing potential that substantially complements experimental results and aids their interpretation.

  7. A simple nucleic acid hybridization/latex agglutination assay for the rapid detection of polymerase chain reaction amplicons.

    PubMed

    Vollenhofer-Schrumpf, Sabine; Buresch, Ronald; Schinkinger, Manfred

    2007-03-01

    We have developed a new method for the detection of nucleic acid hybridization, based on a simple latex agglutination test that can be evaluated by the unaided eye. Nucleic acid, e.g., a polymerase chain reaction (PCR) product, is denatured and incubated with polystyrene beads carrying covalently bound complementary oligonucleotide sequences. Hybridization of the nucleic acids leads to aggregation of the latex particles, thereby verifying the presence of target sequence. The test is performed at room temperature, and results are available within 10 min. As a proof of principle, the hybridization/latex agglutination assay was applied to the detection of purified PCR fragments either specific for Salmonella spp. or a synthetic sequence, and to the detection of Salmonella enterica in artificially contaminated chicken samples. A few nanograms of purified PCR fragments were detectable. In artificially contaminated chicken samples, 3 colony-forming units (cfu)/25 g were detected in one of three replicates, and 30 cfu/25 g were detected in both of two replicates when samples for PCR were taken directly from primary enrichment, demonstrating the practical applicability of this test system. Even multiplex detection might be achievable. This novel kind of assay could be useful for a range of applications where hybridization of nucleic acids, e.g., PCR fragments, is to be detected.

  8. Computer-aided visualization and analysis system for sequence evaluation

    DOEpatents

    Chee, M.S.

    1998-08-18

    A computer system for analyzing nucleic acid sequences is provided. The computer system is used to perform multiple methods for determining unknown bases by analyzing the fluorescence intensities of hybridized nucleic acid probes. The results of individual experiments are improved by processing nucleic acid sequences together. Comparative analysis of multiple experiments is also provided by displaying reference sequences in one area and sample sequences in another area on a display device. 27 figs.

  9. Computer-aided visualization and analysis system for sequence evaluation

    DOEpatents

    Chee, Mark S.; Wang, Chunwei; Jevons, Luis C.; Bernhart, Derek H.; Lipshutz, Robert J.

    2004-05-11

    A computer system for analyzing nucleic acid sequences is provided. The computer system is used to perform multiple methods for determining unknown bases by analyzing the fluorescence intensities of hybridized nucleic acid probes. The results of individual experiments are improved by processing nucleic acid sequences together. Comparative analysis of multiple experiments is also provided by displaying reference sequences in one area and sample sequences in another area on a display device.

  10. Computer-aided visualization and analysis system for sequence evaluation

    DOEpatents

    Chee, Mark S.

    1998-08-18

    A computer system for analyzing nucleic acid sequences is provided. The computer system is used to perform multiple methods for determining unknown bases by analyzing the fluorescence intensities of hybridized nucleic acid probes. The results of individual experiments are improved by processing nucleic acid sequences together. Comparative analysis of multiple experiments is also provided by displaying reference sequences in one area and sample sequences in another area on a display device.

  11. Computer-aided visualization and analysis system for sequence evaluation

    DOEpatents

    Chee, Mark S.

    2003-08-19

    A computer system for analyzing nucleic acid sequences is provided. The computer system is used to perform multiple methods for determining unknown bases by analyzing the fluorescence intensities of hybridized nucleic acid probes. The results of individual experiments may be improved by processing nucleic acid sequences together. Comparative analysis of multiple experiments is also provided by displaying reference sequences in one area and sample sequences in another area on a display device.

  12. Computer-aided visualization and analysis system for sequence evaluation

    DOEpatents

    Chee, Mark S.

    1999-10-26

    A computer system (1) for analyzing nucleic acid sequences is provided. The computer system is used to perform multiple methods for determining unknown bases by analyzing the fluorescence intensities of hybridized nucleic acid probes. The results of individual experiments may be improved by processing nucleic acid sequences together. Comparative analysis of multiple experiments is also provided by displaying reference sequences in one area (814) and sample sequences in another area (816) on a display device (3).

  13. Computer-aided visualization and analysis system for sequence evaluation

    DOEpatents

    Chee, Mark S.

    2001-06-05

    A computer system (1) for analyzing nucleic acid sequences is provided. The computer system is used to perform multiple methods for determining unknown bases by analyzing the fluorescence intensities of hybridized nucleic acid probes. The results of individual experiments may be improved by processing nucleic acid sequences together. Comparative analysis of multiple experiments is also provided by displaying reference sequences in one area (814) and sample sequences in another area (816) on a display device (3).

  14. The relationship between odd- and branched-chain fatty acids and microbial nucleic acid bases in rumen

    PubMed Central

    Liu, Keyuan; Hao, Xiaoyan; Li, Yang; Luo, Guobin; Zhang, Yonggen; Xin, Hangshu

    2017-01-01

    Objective This study aims to identify the relationship between odd- and branched-chain fatty acids (OBCFAs) and microbial nucleic acid bases in the rumen, and to establish a model to accurately predict microbial protein flow by using OBCFA. Methods To develop the regression equations, data on the rumen contents of individual cows were obtained from 2 feeding experiments. In the first experiment, 3 rumen-fistulated dry dairy cows arranged in a 3×3 Latin square were fed diets of differing forage to concentration ratios (F:C). The second experiment consisted of 9 lactating Holstein dairy cows of similar body weights at the same stage of pregnancy. For each lactation stage, 3 cows with similar milk production were selected. The rumen contents were sampled at 4 time points of every two hours after morning feeding 6 h, and then to analyse the concentrations of OBCFA and microbial nucleic acid bases in the rumen samples. Results The ruminal bacteria nucleic acid bases were significantly influenced by feeding diets of differing forge to concentration ratios and lactation stages of dairy cows (p<0.05). The concentrations of OBCFAs, especially odd-chain fatty acids and C15:0 isomers, strongly correlated with the microbial nucleic acid bases in the rumen (p<0.05). The equations of ruminal microbial nucleic acid bases established by ruminal OBCFAs contents showed a good predictive capacity, as indicated by reasonably low standard errors and high R-squared values. Conclusion This finding suggests that the rumen OBCFA composition could be used as an internal marker of rumen microbial matter. PMID:28728386

  15. The relationship between odd- and branched-chain fatty acids and microbial nucleic acid bases in rumen.

    PubMed

    Liu, Keyuan; Hao, Xiaoyan; Li, Yang; Luo, Guobin; Zhang, Yonggen; Xin, Hangshu

    2017-11-01

    This study aims to identify the relationship between odd- and branched-chain fatty acids (OBCFAs) and microbial nucleic acid bases in the rumen, and to establish a model to accurately predict microbial protein flow by using OBCFA. To develop the regression equations, data on the rumen contents of individual cows were obtained from 2 feeding experiments. In the first experiment, 3 rumen-fistulated dry dairy cows arranged in a 3×3 Latin square were fed diets of differing forage to concentration ratios (F:C). The second experiment consisted of 9 lactating Holstein dairy cows of similar body weights at the same stage of pregnancy. For each lactation stage, 3 cows with similar milk production were selected. The rumen contents were sampled at 4 time points of every two hours after morning feeding 6 h, and then to analyse the concentrations of OBCFA and microbial nucleic acid bases in the rumen samples. The ruminal bacteria nucleic acid bases were significantly influenced by feeding diets of differing forge to concentration ratios and lactation stages of dairy cows (p<0.05). The concentrations of OBCFAs, especially odd-chain fatty acids and C15:0 isomers, strongly correlated with the microbial nucleic acid bases in the rumen (p<0.05). The equations of ruminal microbial nucleic acid bases established by ruminal OBCFAs contents showed a good predictive capacity, as indicated by reasonably low standard errors and high R-squared values. This finding suggests that the rumen OBCFA composition could be used as an internal marker of rumen microbial matter.

  16. Comparison of point-of-care-compatible lysis methods for bacteria and viruses.

    PubMed

    Heiniger, Erin K; Buser, Joshua R; Mireles, Lillian; Zhang, Xiaohong; Ladd, Paula D; Lutz, Barry R; Yager, Paul

    2016-09-01

    Nucleic acid sample preparation has been an especially challenging barrier to point-of-care nucleic acid amplification tests in low-resource settings. Here we provide a head-to-head comparison of methods for lysis of, and nucleic acid release from, several pathogenic bacteria and viruses-methods that are adaptable to point-of-care usage in low-resource settings. Digestion with achromopeptidase, a mixture of proteases and peptidoglycan-specific hydrolases, followed by thermal deactivation in a boiling water bath, effectively released amplifiable nucleic acid from Staphylococcus aureus, Bordetella pertussis, respiratory syncytial virus, and influenza virus. Achromopeptidase was functional after dehydration and reconstitution, even after eleven months of dry storage without refrigeration. Mechanical lysis methods proved to be effective against a hard-to-lyse Mycobacterium species, and a miniature bead-mill, the AudioLyse, is shown to be capable of releasing amplifiable DNA and RNA from this species. We conclude that point-of-care-compatible sample preparation methods for nucleic acid tests need not introduce amplification inhibitors, and can provide amplification-ready lysates from a wide range of bacterial and viral pathogens. Copyright © 2016. Published by Elsevier B.V.

  17. An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids

    PubMed Central

    Chen, Dafeng; Mauk, Michael; Qiu, Xianbo; Liu, Changchun; Kim, Jitae; Ramprasad, Sudhir; Ongagna, Serge; Abrams, William R.; Malamud, Daniel; Corstjens, Paul L. A. M.

    2010-01-01

    A self-contained, integrated, disposable, sample-to-answer, polycarbonate microfluidic cassette for nucleic acid—based detection of pathogens at the point of care was designed, constructed, and tested. The cassette comprises on-chip sample lysis, nucleic acid isolation, enzymatic amplification (polymerase chain reaction and, when needed, reverse transcription), amplicon labeling, and detection. On-chip pouches and valves facilitate fluid flow control. All the liquids and dry reagents needed for the various reactions are pre-stored in the cassette. The liquid reagents are stored in flexible pouches formed on the chip surface. Dry (RT-)PCR reagents are pre-stored in the thermal cycling, reaction chamber. The process operations include sample introduction; lysis of cells and viruses; solid-phase extraction, concentration, and purification of nucleic acids from the lysate; elution of the nucleic acids into a thermal cycling chamber and mixing with pre-stored (RT-)PCR dry reagents; thermal cycling; and detection. The PCR amplicons are labeled with digoxigenin and biotin and transmitted onto a lateral flow strip, where the target analytes bind to a test line consisting of immobilized avidin-D. The immobilized nucleic acids are labeled with up-converting phosphor (UCP) reporter particles. The operation of the cassette is automatically controlled by an analyzer that provides pouch and valve actuation with electrical motors and heating for the thermal cycling. The functionality of the device is demonstrated by detecting the presence of bacterial B.Cereus, viral armored RNA HIV, and HIV I virus in saliva samples. The cassette and actuator described here can be used to detect other diseases as well as the presence of bacterial and viral pathogens in the water supply and other fluids. PMID:20401537

  18. Self-powered integrated microfluidic point-of-care low-cost enabling (SIMPLE) chip

    PubMed Central

    Yeh, Erh-Chia; Fu, Chi-Cheng; Hu, Lucy; Thakur, Rohan; Feng, Jeffrey; Lee, Luke P.

    2017-01-01

    Portable, low-cost, and quantitative nucleic acid detection is desirable for point-of-care diagnostics; however, current polymerase chain reaction testing often requires time-consuming multiple steps and costly equipment. We report an integrated microfluidic diagnostic device capable of on-site quantitative nucleic acid detection directly from the blood without separate sample preparation steps. First, we prepatterned the amplification initiator [magnesium acetate (MgOAc)] on the chip to enable digital nucleic acid amplification. Second, a simplified sample preparation step is demonstrated, where the plasma is separated autonomously into 224 microwells (100 nl per well) without any hemolysis. Furthermore, self-powered microfluidic pumping without any external pumps, controllers, or power sources is accomplished by an integrated vacuum battery on the chip. This simple chip allows rapid quantitative digital nucleic acid detection directly from human blood samples (10 to 105 copies of methicillin-resistant Staphylococcus aureus DNA per microliter, ~30 min, via isothermal recombinase polymerase amplification). These autonomous, portable, lab-on-chip technologies provide promising foundations for future low-cost molecular diagnostic assays. PMID:28345028

  19. Rapid Nucleic Acid Extraction and Purification Using a Miniature Ultrasonic Technique

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

    Branch, Darren W.; Vreeland, Erika C.; McClain, Jamie L.

    Miniature ultrasonic lysis for biological sample preparation is a promising technique for efficient and rapid extraction of nucleic acids and proteins from a wide variety of biological sources. Acoustic methods achieve rapid, unbiased, and efficacious disruption of cellular membranes while avoiding the use of harsh chemicals and enzymes, which interfere with detection assays. In this work, a miniature acoustic nucleic acid extraction system is presented. Using a miniature bulk acoustic wave (BAW) transducer array based on 36° Y-cut lithium niobate, acoustic waves were coupled into disposable laminate-based microfluidic cartridges. To verify the lysing effectiveness, the amount of liberated ATP andmore » the cell viability were measured and compared to untreated samples. The relationship between input power, energy dose, flow-rate, and lysing efficiency were determined. DNA was purified on-chip using three approaches implemented in the cartridges: a silica-based sol-gel silica-bead filled microchannel, nucleic acid binding magnetic beads, and Nafion-coated electrodes. Using E. coli, the lysing dose defined as ATP released per joule was 2.2× greater, releasing 6.1× more ATP for the miniature BAW array compared to a bench-top acoustic lysis system. An electric field-based nucleic acid purification approach using Nafion films yielded an extraction efficiency of 69.2% in 10 min for 50 µL samples.« less

  20. Rapid Nucleic Acid Extraction and Purification Using a Miniature Ultrasonic Technique

    DOE PAGES

    Branch, Darren W.; Vreeland, Erika C.; McClain, Jamie L.; ...

    2017-07-21

    Miniature ultrasonic lysis for biological sample preparation is a promising technique for efficient and rapid extraction of nucleic acids and proteins from a wide variety of biological sources. Acoustic methods achieve rapid, unbiased, and efficacious disruption of cellular membranes while avoiding the use of harsh chemicals and enzymes, which interfere with detection assays. In this work, a miniature acoustic nucleic acid extraction system is presented. Using a miniature bulk acoustic wave (BAW) transducer array based on 36° Y-cut lithium niobate, acoustic waves were coupled into disposable laminate-based microfluidic cartridges. To verify the lysing effectiveness, the amount of liberated ATP andmore » the cell viability were measured and compared to untreated samples. The relationship between input power, energy dose, flow-rate, and lysing efficiency were determined. DNA was purified on-chip using three approaches implemented in the cartridges: a silica-based sol-gel silica-bead filled microchannel, nucleic acid binding magnetic beads, and Nafion-coated electrodes. Using E. coli, the lysing dose defined as ATP released per joule was 2.2× greater, releasing 6.1× more ATP for the miniature BAW array compared to a bench-top acoustic lysis system. An electric field-based nucleic acid purification approach using Nafion films yielded an extraction efficiency of 69.2% in 10 min for 50 µL samples.« less

  1. NanoDrop Microvolume Quantitation of Nucleic Acids

    PubMed Central

    Desjardins, Philippe; Conklin, Deborah

    2010-01-01

    Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation. The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results. The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 μL of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 μL or less are now possible using a dedicated microvolume fluorospectrometer. Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ μL to 15,000 ng/ μL with minimal consumption of sample. PMID:21189466

  2. Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time

    PubMed Central

    Gandelman, Olga A.; Church, Vicki L.; Moore, Cathy A.; Kiddle, Guy; Carne, Christopher A.; Parmar, Surendra; Jalal, Hamid; Tisi, Laurence C.; Murray, James A. H.

    2010-01-01

    Background The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. Principal Findings Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs) enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART) continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi) produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi) produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. Conclusions The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a simple light detector, the iNAAT-BART combination is ideal for molecular diagnostic assays in both laboratory and low resource settings. PMID:21152399

  3. Low-Cost 3D Printers Enable High-Quality and Automated Sample Preparation and Molecular Detection

    PubMed Central

    Chan, Kamfai; Coen, Mauricio; Hardick, Justin; Gaydos, Charlotte A.; Wong, Kah-Yat; Smith, Clayton; Wilson, Scott A.; Vayugundla, Siva Praneeth; Wong, Season

    2016-01-01

    Most molecular diagnostic assays require upfront sample preparation steps to isolate the target’s nucleic acids, followed by its amplification and detection using various nucleic acid amplification techniques. Because molecular diagnostic methods are generally rather difficult to perform manually without highly trained users, automated and integrated systems are highly desirable but too costly for use at point-of-care or low-resource settings. Here, we showcase the development of a low-cost and rapid nucleic acid isolation and amplification platform by modifying entry-level 3D printers that cost between $400 and $750. Our modifications consisted of replacing the extruder with a tip-comb attachment that houses magnets to conduct magnetic particle-based nucleic acid extraction. We then programmed the 3D printer to conduct motions that can perform high-quality extraction protocols. Up to 12 samples can be processed simultaneously in under 13 minutes and the efficiency of nucleic acid isolation matches well against gold-standard spin-column-based extraction technology. Additionally, we used the 3D printer’s heated bed to supply heat to perform water bath-based polymerase chain reactions (PCRs). Using another attachment to hold PCR tubes, the 3D printer was programmed to automate the process of shuttling PCR tubes between water baths. By eliminating the temperature ramping needed in most commercial thermal cyclers, the run time of a 35-cycle PCR protocol was shortened by 33%. This article demonstrates that for applications in resource-limited settings, expensive nucleic acid extraction devices and thermal cyclers that are used in many central laboratories can be potentially replaced by a device modified from inexpensive entry-level 3D printers. PMID:27362424

  4. 76 FR 72950 - Draft Guidance for Industry: Use of Nucleic Acid Tests on Pooled and Individual Samples From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-28

    ... Hepatitis B Virus AGENCY: Food and Drug Administration, HHS. ACTION: Notice. SUMMARY: The Food and Drug... Risk of Transmission of Hepatitis B Virus (HBV), and Requalification of Donors Who Test HBV NAT...-licensed nucleic acid tests (NAT) to screen blood donors for hepatitis B virus (HBV) deoxyribonucleic acid...

  5. Concentration determination of nucleic acids and proteins using the micro-volume BioSpec-nano-spectrophotometer.

    PubMed

    Sukumaran, Suja

    2011-02-17

    Nucleic acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning. In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is: Sample Concentration = DF · (OD260-OD320)· NACF (1) Where DF = sample dilution factor and NACF = nucleic acid concentration factor. The Nucleic Acid concentration factor is set in accordance with the analyte selected. Protein concentration results can be expressed as μg/mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation. In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

  6. Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

    PubMed Central

    Sukumaran, Suja

    2011-01-01

    Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning. In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is: Sample Concentration = DF · (OD260-OD320)· NACF (1) Where DF = sample dilution factor and NACF = nucleic acid concentration factor. The Nucleic Acid concentration factor is set in accordance with the analyte selected1. Protein concentration results can be expressed as μg/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation. In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells. PMID:21372788

  7. System for portable nucleic acid testing in low resource settings

    NASA Astrophysics Data System (ADS)

    Lu, Hsiang-Wei; Roskos, Kristina; Hickerson, Anna I.; Carey, Thomas; Niemz, Angelika

    2013-03-01

    Our overall goal is to enable timely diagnosis of infectious diseases through nucleic acid testing at the point-of-care and in low resource settings, via a compact system that integrates nucleic acid sample preparation, isothermal DNA amplification, and nucleic acid lateral flow (NALF) detection. We herein present an interim milestone, the design of the amplification and detection subsystem, and the characterization of thermal and fluidic control and assay execution within this system. Using an earlier prototype of the amplification and detection unit, comprised of a disposable cartridge containing flexible pouches, passive valves, and electrolysis-driven pumps, in conjunction with a small heater, we have demonstrated successful execution of an established and clinically validated isothermal loop-mediated amplification (LAMP) reaction targeting Mycobacterium tuberculosis (M.tb) DNA, coupled to NALF detection. The refined design presented herein incorporates miniaturized and integrated electrolytic pumps, novel passive valves, overall design changes to facilitate integration with an upstream sample preparation unit, and a refined instrument design that automates pumping, heating, and timing. Nucleic acid amplification occurs in a two-layer pouch that facilitates fluid handling and appropriate thermal control. The disposable cartridge is manufactured using low-cost and scalable techniques and forms a closed system to prevent workplace contamination by amplicons. In a parallel effort, we are developing a sample preparation unit based on similar design principles, which performs mechanical lysis of mycobacteria and DNA extraction from liquefied and disinfected sputum. Our next step is to combine sample preparation, amplification, and detection in a final integrated cartridge and device, to enable fully automated sample-in to answer-out diagnosis of active tuberculosis in primary care facilities of low-resource and high-burden countries.

  8. Devices, systems, and methods for detecting nucleic acids using sedimentation

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

    Koh, Chung-Yan; Schaff, Ulrich Y.; Sommer, Gregory J.

    Embodiments of the present invention are directed toward devices, systems, and method for conducting nucleic acid purification and quantification using sedimentation. In one example, a method includes generating complexes which bind to a plurality of beads in a fluid sample, individual ones of the complexes comprising a nucleic acid molecule such as DNA or RNA and a labeling agent. The plurality of beads including the complexes may be transported through a density media, wherein the density media has a density lower than a density of the beads and higher than a density of the fluid sample, and wherein the transportingmore » occurs, at least in part, by sedimentation. Signal may be detected from the labeling agents of the complexes.« less

  9. Tracking fetal development through molecular analysis of maternal biofluids☆

    PubMed Central

    Edlow, Andrea G.; Bianchi, Diana W.

    2015-01-01

    Current monitoring of fetal development includes fetal ultrasonography, chorionic villus sampling or amniocentesis for chromosome analysis, and maternal serum biochemical screening for analytes associated with aneuploidy and open neural tube defects. Over the last 15 years, significant advances in noninvasive prenatal diagnosis (NIPD) via cell-free fetal (cff) nucleic acids in maternal plasma have resulted in the ability to determine fetal sex, RhD genotype, and aneuploidy. Cff nucleic acids in the maternal circulation originate primarily from the placenta. This contrasts with cff nucleic acids in amniotic fluid, which derive from the fetus, and are present in significantly higher concentrations than in maternal blood. The fetal origin of cff nucleic acids in the amniotic fluid permits the acquisition of real-time information about fetal development and gene expression. This review seeks to provide a comprehensive summary of the molecular analysis of cff nucleic acids in maternal biofluids to elucidate mechanisms of fetal development, physiology, and pathology. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure. PMID:22542507

  10. Extraction of Total Nucleic Acids From Ticks for the Detection of Bacterial and Viral Pathogens

    PubMed Central

    Crowder, Chris D.; Rounds, Megan A.; Phillipson, Curtis A.; Picuri, John M.; Matthews, Heather E.; Halverson, Justina; Schutzer, Steven E.; Ecker, David J.; Eshoo, Mark W.

    2010-01-01

    Ticks harbor numerous bacterial, protozoal, and viral pathogens that can cause serious infections in humans and domestic animals. Active surveillance of the tick vector can provide insight into the frequency and distribution of important pathogens in the environment. Nucleic-acid based detection of tick-borne bacterial, protozoan, and viral pathogens requires the extraction of both DNA and RNA (total nucleic acids) from ticks. Traditional methods for nucleic acid extraction are limited to extraction of either DNA or the RNA from a sample. Here we present a simple bead-beating based protocol for extraction of DNA and RNA from a single tick and show detection of Borrelia burgdorferi and Powassan virus from individual, infected Ixodes scapularis ticks. We determined expected yields for total nucleic acids by this protocol for a variety of adult tick species. The method is applicable to a variety of arthropod vectors, including fleas and mosquitoes, and was partially automated on a liquid handling robot. PMID:20180313

  11. Methods And Devices For Characterizing Duplex Nucleic Acid Molecules

    DOEpatents

    Akeson, Mark; Vercoutere, Wenonah; Haussler, David; Winters-Hilt, Stephen

    2005-08-30

    Methods and devices are provided for characterizing a duplex nucleic acid, e.g., a duplex DNA molecule. In the subject methods, a fluid conducting medium that includes a duplex nucleic acid molecule is contacted with a nanopore under the influence of an applied electric field and the resulting changes in current through the nanopore caused by the duplex nucleic acid molecule are monitored. The observed changes in current through the nanopore are then employed as a set of data values to characterize the duplex nucleic acid, where the set of data values may be employed in raw form or manipulated, e.g., into a current blockade profile. Also provided are nanopore devices for practicing the subject methods, where the subject nanopore devices are characterized by the presence of an algorithm which directs a processing means to employ monitored changes in current through a nanopore to characterize a duplex nucleic acid molecule responsible for the current changes. The subject methods and devices find use in a variety of applications, including, among other applications, the identification of an analyte duplex DNA molecule in a sample, the specific base sequence at a single nulceotide polymorphism (SNP), and the sequencing of duplex DNA molecules.

  12. Virus inactivation by nucleic acid extraction reagents.

    PubMed

    Blow, Jamie A; Dohm, David J; Negley, Diane L; Mores, Christopher N

    2004-08-01

    Many assume that common methods to extract viral nucleic acids are able to render a sample non-infectious. It may be that inactivation of infectious virus is incomplete during viral nucleic acid extraction methods. Accordingly, two common viral nucleic acid extraction techniques were evaluated for the ability to inactivate high viral titer specimens. In particular, the potential for TRIzol LS Reagent (Invitrogen Corp., Carlsbad, CA) and AVL Buffer (Qiagen, Valencia, CA) were examined to render suspensions of alphaviruses, flaviviruses, filoviruses and a bunyavirus non-infectious to tissue culture assay. The dilution series for both extraction reagents consistently caused cell death through a 100-fold dilution. Except for the DEN subtype 4 positive control, all viruses had titers of at least 10(6)pfu/ml. No plaques were detected in any extraction reagent plus virus combination in this study, therefore, the extraction reagents appeared to inactivate completely each of the high-titer viruses used in this study. These results support the reliance upon either TRIzol LS Reagent or AVL Buffer to render clinical or environmental samples non-infectious, which has implications for the handling and processing of samples under austere field conditions and low level containment.

  13. Rapid Bedside Inactivation of Ebola Virus for Safe Nucleic Acid Tests.

    PubMed

    Rosenstierne, Maiken Worsøe; Karlberg, Helen; Bragstad, Karoline; Lindegren, Gunnel; Stoltz, Malin Lundahl; Salata, Cristiano; Kran, Anne-Marte Bakken; Dudman, Susanne Gjeruldsen; Mirazimi, Ali; Fomsgaard, Anders

    2016-10-01

    Rapid bedside inactivation of Ebola virus would be a solution for the safety of medical and technical staff, risk containment, sample transport, and high-throughput or rapid diagnostic testing during an outbreak. We show that the commercially available Magna Pure lysis/binding buffer used for nucleic acid extraction inactivates Ebola virus. A rapid bedside inactivation method for nucleic acid tests is obtained by simply adding Magna Pure lysis/binding buffer directly into vacuum blood collection EDTA tubes using a thin needle and syringe prior to sampling. The ready-to-use inactivation vacuum tubes are stable for more than 4 months, and Ebola virus RNA is preserved in the Magna Pure lysis/binding buffer for at least 5 weeks independent of the storage temperature. We also show that Ebola virus RNA can be manually extracted from Magna Pure lysis/binding buffer-inactivated samples using the QIAamp viral RNA minikit. We present an easy and convenient method for bedside inactivation using available blood collection vacuum tubes and reagents. We propose to use this simple method for fast, safe, and easy bedside inactivation of Ebola virus for safe transport and routine nucleic acid detection. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  14. Tumor suppressor molecules and methods of use

    DOEpatents

    Welch, Peter J.; Barber, Jack R.

    2004-09-07

    The invention provides substantially pure tumor suppressor nucleic acid molecules and tumor suppressor polypeptides. The invention also provides hairpin ribozymes and antibodies selective for these tumor suppressor molecules. Also provided are methods of detecting a neoplastic cell in a sample using detectable agents specific for the tumor suppressor nucleic acids and polypeptides.

  15. A new comprehensive method for detection of livestock-related pathogenic viruses using a target enrichment system.

    PubMed

    Oba, Mami; Tsuchiaka, Shinobu; Omatsu, Tsutomu; Katayama, Yukie; Otomaru, Konosuke; Hirata, Teppei; Aoki, Hiroshi; Murata, Yoshiteru; Makino, Shinji; Nagai, Makoto; Mizutani, Tetsuya

    2018-01-08

    We tested usefulness of a target enrichment system SureSelect, a comprehensive viral nucleic acid detection method, for rapid identification of viral pathogens in feces samples of cattle, pigs and goats. This system enriches nucleic acids of target viruses in clinical/field samples by using a library of biotinylated RNAs with sequences complementary to the target viruses. The enriched nucleic acids are amplified by PCR and subjected to next generation sequencing to identify the target viruses. In many samples, SureSelect target enrichment method increased efficiencies for detection of the viruses listed in the biotinylated RNA library. Furthermore, this method enabled us to determine nearly full-length genome sequence of porcine parainfluenza virus 1 and greatly increased Breadth, a value indicating the ratio of the mapping consensus length in the reference genome, in pig samples. Our data showed usefulness of SureSelect target enrichment system for comprehensive analysis of genomic information of various viruses in field samples. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Solar-thermal complex sample processing for nucleic acid based diagnostics in limited resource settings

    PubMed Central

    Gumus, Abdurrahman; Ahsan, Syed; Dogan, Belgin; Jiang, Li; Snodgrass, Ryan; Gardner, Andrea; Lu, Zhengda; Simpson, Kenneth; Erickson, David

    2016-01-01

    The use of point-of-care (POC) devices in limited resource settings where access to commonly used infrastructure, such as water and electricity, can be restricted represents simultaneously one of the best application fits for POC systems as well as one of the most challenging places to deploy them. Of the many challenges involved in these systems, the preparation and processing of complex samples like stool, vomit, and biopsies are particularly difficult due to the high number and varied nature of mechanical and chemical interferents present in the sample. Previously we have demonstrated the ability to use solar-thermal energy to perform PCR based nucleic acid amplifications. In this work demonstrate how the technique, using similar infrastructure, can also be used to perform solar-thermal based sample processing system for extracting and isolating Vibrio Cholerae nucleic acids from fecal samples. The use of opto-thermal energy enables the use of sunlight to drive thermal lysing reactions in large volumes without the need for external electrical power. Using the system demonstrate the ability to reach a 95°C threshold in less than 5 minutes and maintain a stable sample temperature of +/− 2°C following the ramp up. The system is demonstrated to provide linear results between 104 and 108 CFU/mL when the released nucleic acids were quantified via traditional means. Additionally, we couple the sample processing unit with our previously demonstrated solar-thermal PCR and tablet based detection system to demonstrate very low power sample-in-answer-out detection. PMID:27231636

  17. [Comparison of two nucleic acid extraction methods for norovirus in oysters].

    PubMed

    Yuan, Qiao; Li, Hui; Deng, Xiaoling; Mo, Yanling; Fang, Ling; Ke, Changwen

    2013-04-01

    To explore a convenient and effective method for norovirus nucleic acid extraction from oysters suitable for long-term viral surveillance. Two methods, namely method A (glycine washing and polyethylene glycol precipitation of the virus followed by silica gel centrifugal column) and method B (protease K digestion followed by application of paramagnetic silicon) were compared for their performance in norovirus nucleic acid extraction from oysters. Real-time RT-PCR was used to detect norovirus in naturally infected oysters and in oysters with induced infection. The two methods yielded comparable positive detection rates for the samples, but the recovery rate of the virus was higher with method B than with method A. Method B is a more convenient and rapid method for norovirus nucleic acid extraction from oysters and suitable for long-term surveillance of norovirus.

  18. Silicon Dioxide Thin Film Mediated Single Cell Nucleic Acid Isolation

    PubMed Central

    Bogdanov, Evgeny; Dominova, Irina; Shusharina, Natalia; Botman, Stepan; Kasymov, Vitaliy; Patrushev, Maksim

    2013-01-01

    A limited amount of DNA extracted from single cells, and the development of single cell diagnostics make it necessary to create a new highly effective method for the single cells nucleic acids isolation. In this paper, we propose the DNA isolation method from biomaterials with limited DNA quantity in sample, and from samples with degradable DNA based on the use of solid-phase adsorbent silicon dioxide nanofilm deposited on the inner surface of PCR tube. PMID:23874571

  19. Capillary electrophoresis, a method for the determination of nucleic acid ligands covalently attached to quantum dots representing a donor of Förster resonance energy transfer.

    PubMed

    Datinská, Vladimíra; Klepárník, Karel; Belšánová, Barbora; Minárik, Marek; Foret, František

    2018-05-09

    The synthesis and determination of the structure of a Förster resonance energy transfer probe intended for the detection of specific nucleic acid sequences are described here. The probe is based on the hybridization of oligonucleotide modified quantum dots with a fluorescently labeled nucleic acid sample resulting in changes of the fluorescence emission due to the energy transfer effect. The stoichiometry distribution of oligonucleotides conjugated to quantum dots was determined by capillary electrophoresis separation. The results indicate that one to four molecules of oligonucleotide are conjugated to the surface of a single nanoparticle. This conclusion is confirmed by the course of the dependence of Förster resonance energy transfer efficiency on the concentration of fluorescently labeled complementary single-stranded nucleic acid, showing saturation. While the energy transfer efficiency of the probe hybridized with complementary nucleic acid strands was 30%, negligible efficiency was observed with a non-complementary strands. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  20. Isothermal Amplification Methods for the Detection of Nucleic Acids in Microfluidic Devices

    PubMed Central

    Zanoli, Laura Maria; Spoto, Giuseppe

    2012-01-01

    Diagnostic tools for biomolecular detection need to fulfill specific requirements in terms of sensitivity, selectivity and high-throughput in order to widen their applicability and to minimize the cost of the assay. The nucleic acid amplification is a key step in DNA detection assays. It contributes to improving the assay sensitivity by enabling the detection of a limited number of target molecules. The use of microfluidic devices to miniaturize amplification protocols reduces the required sample volume and the analysis times and offers new possibilities for the process automation and integration in one single device. The vast majority of miniaturized systems for nucleic acid analysis exploit the polymerase chain reaction (PCR) amplification method, which requires repeated cycles of three or two temperature-dependent steps during the amplification of the nucleic acid target sequence. In contrast, low temperature isothermal amplification methods have no need for thermal cycling thus requiring simplified microfluidic device features. Here, the use of miniaturized analysis systems using isothermal amplification reactions for the nucleic acid amplification will be discussed. PMID:25587397

  1. Detection of nucleic acid-protein interactions in plant leaves using fluorescence lifetime imaging microscopy.

    PubMed

    Camborde, Laurent; Jauneau, Alain; Brière, Christian; Deslandes, Laurent; Dumas, Bernard; Gaulin, Elodie

    2017-09-01

    DNA-binding proteins (DNA-BPs) and RNA-binding proteins (RNA-BPs) have critical roles in living cells in all kingdoms of life. Various experimental approaches exist for the study of nucleic acid-protein interactions in vitro and in vivo, but the detection of such interactions at the subcellular level remains challenging. Here we describe how to detect nucleic acid-protein interactions in plant leaves by using a fluorescence resonance energy transfer (FRET) approach coupled to fluorescence lifetime imaging microscopy (FLIM). Proteins of interest (POI) are tagged with a GFP and transiently expressed in plant cells to serve as donor fluorophore. After sample fixation and cell wall permeabilization, leaves are treated with Sytox Orange, a nucleic acid dye that can function as a FRET acceptor. Upon close association of the GFP-tagged POI with Sytox-Orange-stained nucleic acids, a substantial decrease of the GFP lifetime due to FRET between the donor and the acceptor can be monitored. Treatment with RNase before FRET-FLIM measurements allows determination of whether the POI associates with DNA and/or RNA. A step-by-step protocol is provided for sample preparation, data acquisition and analysis. We describe how to calibrate the equipment and include a tutorial explaining the use of the FLIM software. To illustrate our approach, we provide experimental procedures to detect the interaction between plant DNA and two proteins (the AeCRN13 effector from the oomycete Aphanomyces euteiches and the AtWRKY22 defensive transcription factor from Arabidopsis). This protocol allows the detection of protein-nucleic acid interactions in plant cells and can be completed in <2 d.

  2. Development of a nucleic Acid extraction procedure for simultaneous recovery of DNA and RNA from diverse microbes in water.

    PubMed

    Hill, Vincent R; Narayanan, Jothikumar; Gallen, Rachel R; Ferdinand, Karen L; Cromeans, Theresa; Vinjé, Jan

    2015-05-26

    Drinking and environmental water samples contain a diverse array of constituents that can interfere with molecular testing techniques, especially when large volumes of water are concentrated to the small volumes needed for effective molecular analysis. In this study, a suite of enteric viruses, bacteria, and protozoan parasites were seeded into concentrated source water and finished drinking water samples, in order to investigate the relative performance of nucleic acid extraction techniques for molecular testing. Real-time PCR and reverse transcription-PCR crossing threshold (CT) values were used as the metrics for evaluating relative performance. Experimental results were used to develop a guanidinium isothiocyanate-based lysis buffer (UNEX buffer) that enabled effective simultaneous extraction and recovery of DNA and RNA from the suite of study microbes. Procedures for bead beating, nucleic acid purification, and PCR facilitation were also developed and integrated in the protocol. The final lysis buffer and sample preparation procedure was found to be effective for a panel of drinking water and source water concentrates when compared to commercial nucleic acid extraction kits. The UNEX buffer-based extraction protocol enabled PCR detection of six study microbes, in 100 L finished water samples from four drinking water treatment facilities, within three CT values (i.e., within 90% difference) of the reagent-grade water control. The results from this study indicate that this newly formulated lysis buffer and sample preparation procedure can be useful for standardized molecular testing of drinking and environmental waters.

  3. Development of a Nucleic Acid Extraction Procedure for Simultaneous Recovery of DNA and RNA from Diverse Microbes in Water

    PubMed Central

    Hill, Vincent R.; Narayanan, Jothikumar; Gallen, Rachel R.; Ferdinand, Karen L.; Cromeans, Theresa; Vinjé, Jan

    2015-01-01

    Drinking and environmental water samples contain a diverse array of constituents that can interfere with molecular testing techniques, especially when large volumes of water are concentrated to the small volumes needed for effective molecular analysis. In this study, a suite of enteric viruses, bacteria, and protozoan parasites were seeded into concentrated source water and finished drinking water samples, in order to investigate the relative performance of nucleic acid extraction techniques for molecular testing. Real-time PCR and reverse transcription-PCR crossing threshold (CT) values were used as the metrics for evaluating relative performance. Experimental results were used to develop a guanidinium isothiocyanate-based lysis buffer (UNEX buffer) that enabled effective simultaneous extraction and recovery of DNA and RNA from the suite of study microbes. Procedures for bead beating, nucleic acid purification, and PCR facilitation were also developed and integrated in the protocol. The final lysis buffer and sample preparation procedure was found to be effective for a panel of drinking water and source water concentrates when compared to commercial nucleic acid extraction kits. The UNEX buffer-based extraction protocol enabled PCR detection of six study microbes, in 100 L finished water samples from four drinking water treatment facilities, within three CT values (i.e., within 90% difference) of the reagent-grade water control. The results from this study indicate that this newly formulated lysis buffer and sample preparation procedure can be useful for standardized molecular testing of drinking and environmental waters. PMID:26016775

  4. Extraction of total nucleic acid based on silica-coated magnetic particles for RT-qPCR detection of plant RNA virus/viroid.

    PubMed

    Sun, Ning; Deng, Congliang; Zhao, Xiaoli; Zhou, Qi; Ge, Guanglu; Liu, Yi; Yan, Wenlong; Xia, Qiang

    2014-02-01

    In this study, a nucleic acid extraction method based on silica-coated magnetic particles (SMPs) and RT-qPCR assay was developed to detect Arabis mosaic virus (ArMV), Lily symptomless virus (LSV), Hop stunt viroid (HSVd) and grape yellow speckle viroid 1 (GYSVd-1). The amplification sequences of RT-qPCR were reversely transcribed in vitro as RNA standard templates. The standard curves covered six or seven orders of magnitude with a detection limit of 100 copies per each assay. Extraction efficiency of the SMPs method was evaluated by recovering spiked ssRNAs from plant samples and compared to two commercial kits (TRIzol and RNeasy Plant mini kit). Results showed that the recovery rate of SMPs method was comparable to the commercial kits when spiked ssRNAs were extracted from lily leaves, whereas it was two or three times higher than commercial kits when spiked ssRNAs were extracted from grapevine leaves. SMPs method was also used to extract viral nucleic acid from15 ArMV-positive lily leaf samples and 15 LSV-positive lily leaf samples. SMPs method did not show statistically significant difference from other methods on detecting ArMV, but LSV. The SMPs method has the same level of virus load as the TRIzol, and its mean virus load of was 0.5log10 lower than the RNeasy Plant mini kit. Nucleic acid was extracted from 19 grapevine-leaf samples with SMPs and the two commercial kits and subsequently screened for HSVd and GYSVd-1 by RT-qPCR. Regardless of HSVd or GYSVd-1, SMPs method outperforms other methods on both positive rate and the viroid load. In conclusion, SMPs method was able to efficiently extract the nucleic acid of RNA viruses or viroids, especially grapevine viroids, from lily-leaf or grapevine-leaf samples for RT-qPCR detection. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Identification of multiple mRNA and DNA sequences from small tissue samples isolated by laser-assisted microdissection.

    PubMed

    Bernsen, M R; Dijkman, H B; de Vries, E; Figdor, C G; Ruiter, D J; Adema, G J; van Muijen, G N

    1998-10-01

    Molecular analysis of small tissue samples has become increasingly important in biomedical studies. Using a laser dissection microscope and modified nucleic acid isolation protocols, we demonstrate that multiple mRNA as well as DNA sequences can be identified from a single-cell sample. In addition, we show that the specificity of procurement of tissue samples is not compromised by smear contamination resulting from scraping of the microtome knife during sectioning of lesions. The procedures described herein thus allow for efficient RT-PCR or PCR analysis of multiple nucleic acid sequences from small tissue samples obtained by laser-assisted microdissection.

  6. Preservation of Biospecimens at Ambient Temperature: Special Focus on Nucleic Acids and Opportunities for the Biobanking Community.

    PubMed

    Muller, Rolf; Betsou, Fay; Barnes, Michael G; Harding, Keith; Bonnet, Jacques; Kofanova, Olga; Crowe, John H

    2016-04-01

    Several approaches to the preservation of biological materials at ambient temperature and the relative impact on sample stability and degradation are reviewed, with a focus on nucleic acids. This appraisal is undertaken within the framework of biobank risk, quality management systems, and accreditation, with a view to assessing how best to apply ambient temperature sample storage to ensure stability, reduce costs, improve handling logistics, and increase the efficiency of biobank procedures.

  7. High-throughput, automated extraction of DNA and RNA from clinical samples using TruTip technology on common liquid handling robots.

    PubMed

    Holmberg, Rebecca C; Gindlesperger, Alissa; Stokes, Tinsley; Brady, Dane; Thakore, Nitu; Belgrader, Philip; Cooney, Christopher G; Chandler, Darrell P

    2013-06-11

    TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. The geometry of the monolith can be adapted for specific pipette tips ranging in volume from 1.0 to 5.0 ml. The large porosity of the monolith enables viscous or complex samples to readily pass through it with minimal fluidic backpressure. Bi-directional flow maximizes residence time between the monolith and sample, and enables large sample volumes to be processed within a single TruTip. The fundamental steps, irrespective of sample volume or TruTip geometry, include cell lysis, nucleic acid binding to the inner pores of the TruTip monolith, washing away unbound sample components and lysis buffers, and eluting purified and concentrated nucleic acids into an appropriate buffer. The attributes and adaptability of TruTip are demonstrated in three automated clinical sample processing protocols using an Eppendorf epMotion 5070, Hamilton STAR and STARplus liquid handling robots, including RNA isolation from nasopharyngeal aspirate, genomic DNA isolation from whole blood, and fetal DNA extraction and enrichment from large volumes of maternal plasma (respectively).

  8. Evaluation of three automated nucleic acid extraction systems for identification of respiratory viruses in clinical specimens by multiplex real-time PCR.

    PubMed

    Kim, Yoonjung; Han, Mi-Soon; Kim, Juwon; Kwon, Aerin; Lee, Kyung-A

    2014-01-01

    A total of 84 nasopharyngeal swab specimens were collected from 84 patients. Viral nucleic acid was extracted by three automated extraction systems: QIAcube (Qiagen, Germany), EZ1 Advanced XL (Qiagen), and MICROLAB Nimbus IVD (Hamilton, USA). Fourteen RNA viruses and two DNA viruses were detected using the Anyplex II RV16 Detection kit (Seegene, Republic of Korea). The EZ1 Advanced XL system demonstrated the best analytical sensitivity for all the three viral strains. The nucleic acids extracted by EZ1 Advanced XL showed higher positive rates for virus detection than the others. Meanwhile, the MICROLAB Nimbus IVD system was comprised of fully automated steps from nucleic extraction to PCR setup function that could reduce human errors. For the nucleic acids recovered from nasopharyngeal swab specimens, the QIAcube system showed the fewest false negative results and the best concordance rate, and it may be more suitable for detecting various viruses including RNA and DNA virus strains. Each system showed different sensitivity and specificity for detection of certain viral pathogens and demonstrated different characteristics such as turnaround time and sample capacity. Therefore, these factors should be considered when new nucleic acid extraction systems are introduced to the laboratory.

  9. Variables Influencing Extraction of Nucleic Acids from Microbial Plankton (Viruses, Bacteria, and Protists) Collected on Nanoporous Aluminum Oxide Filters

    PubMed Central

    Mueller, Jaclyn A.; Culley, Alexander I.

    2014-01-01

    Anodic aluminum oxide (AAO) filters have high porosity and can be manufactured with a pore size that is small enough to quantitatively capture viruses. These properties make the filters potentially useful for harvesting total microbial communities from water samples for molecular analyses, but their performance for nucleic acid extraction has not been systematically or quantitatively evaluated. In this study, we characterized the flux of water through commercially produced nanoporous (0.02 μm) AAO filters (Anotop; Whatman) and used isolates (a virus, a bacterium, and a protist) and natural seawater samples to test variables that we expected would influence the efficiency with which nucleic acids are recovered from the filters. Extraction chemistry had a significant effect on DNA yield, and back flushing the filters during extraction was found to improve yields of high-molecular-weight DNA. Using the back-flush protocol, the mass of DNA recovered from microorganisms collected on AAO filters was ≥100% of that extracted from pellets of cells and viruses and 94% ± 9% of that obtained by direct extraction of a liquid bacterial culture. The latter is a minimum estimate of the relative recovery of microbial DNA, since liquid cultures include dissolved nucleic acids that are retained inefficiently by the filter. In conclusion, we demonstrate that nucleic acids can be extracted from microorganisms on AAO filters with an efficiency similar to that achievable by direct extraction of microbes in suspension or in pellets. These filters are therefore a convenient means by which to harvest total microbial communities from multiple aqueous samples in parallel for subsequent molecular analyses. PMID:24747903

  10. [Comparison of manual and automated (MagNA Pure) nucleic acid isolation methods in molecular diagnosis of HIV infections].

    PubMed

    Alp, Alpaslan; Us, Dürdal; Hasçelik, Gülşen

    2004-01-01

    Rapid quantitative molecular methods are very important for the diagnosis of human immunodeficiency virus (HIV) infections, assessment of prognosis and follow up. The purpose of this study was to compare and evaluate the performances of conventional manual extraction method and automated MagNA Pure system, for the nucleic acid isolation step which is the first and most important step in molecular diagnosis of HIV infections. Plasma samples of 35 patients in which anti-HIV antibodies were found as positive by microparticule enzyme immunoassay and confirmed by immunoblotting method, were included in the study. The nucleic acids obtained simultaneously by manual isolation kit (Cobas Amplicor, HIV-1 Monitor Test, version 1.5, Roche Diagnostics) and automated system (MagNA Pure LC Total Nucleic Acid Isolation Kit, Roche Diagnostics), were amplified and detected in Cobas Amplicor (Roche Diagnostics) instrument. Twenty three of 35 samples (65.7%) were found to be positive, and 9 (25.7%) were negative by both of the methods. The agreement between the methods were detected as 91.4%, for qualitative results. Viral RNA copies detected by manual and MagNA Pure isolation methods were found between 76.0-7.590.000 (mean: 487.143) and 113.0-20.300.0000 (mean: 2.174.097) copies/ml, respectively. When both of the overall and individual results were evaluated, the number of RNA copies obtained with automatized system, were found higher than the manual method (p<0.05). Three samples which had low numbers of nucleic acids (113, 773, 857, respectively) with MagNA Pure, yielded negative results with manual method. In conclusion, the automatized MagNA Pure system was found to be a reliable, rapid and practical method for the isolation of HIV-RNA.

  11. New nucleic acid testing devices to diagnose infectious diseases in resource-limited settings.

    PubMed

    Maffert, P; Reverchon, S; Nasser, W; Rozand, C; Abaibou, H

    2017-10-01

    Point-of-care diagnosis based on nucleic acid testing aims to incorporate all the analytical steps, from sample preparation to nucleic acid amplification and detection, in a single device. This device needs to provide a low-cost, robust, sensitive, specific, and easily readable analysis. Microfluidics has great potential for handling small volumes of fluids on a single platform. Microfluidic technology has recently been applied to paper, which is already used in low-cost lateral flow tests. Nucleic acid extraction from a biological specimen usually requires cell filtration and lysis on specific membranes, while affinity matrices, such as chitosan or polydiacetylene, are well suited to concentrating nucleic acids for subsequent amplification. Access to electricity is often difficult in resource-limited areas, so the amplification step needs to be equipment-free. Consequently, the reaction has to be isothermal to alleviate the need for a thermocycler. LAMP, NASBA, HDA, and RPA are examples of the technologies available. Nucleic acid detection techniques are currently based on fluorescence, colorimetry, or chemiluminescence. For point-of-care diagnostics, the results should be readable with the naked eye. Nowadays, interpretation and communication of results to health professionals could rely on a smartphone, used as a telemedicine device. The major challenge of creating an "all-in-one" diagnostic test involves the design of an optimal solution and a sequence for each analytical step, as well as combining the execution of all these steps on a single device. This review provides an overview of available materials and technologies which seem to be adapted to point-of-care nucleic acid-based diagnosis, in low-resource areas.

  12. Electricity-Free, Sequential Nucleic Acid and Protein Isolation

    PubMed Central

    Pawlowski, David R.; Karalus, Richard J.

    2012-01-01

    Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis. Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4. By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden. PMID:22635135

  13. Electricity-free, sequential nucleic acid and protein isolation.

    PubMed

    Pawlowski, David R; Karalus, Richard J

    2012-05-15

    Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable (1). The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment (2). The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis. Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters (3). CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation(4). By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

  14. Nucleic acid probes as a diagnostic method for tick-borne hemoparasites of veterinary importance.

    PubMed

    Figueroa, J V; Buening, G M

    1995-03-01

    An increased number of articles on the use of nucleic acid-based hybridization techniques for diagnostic purposes have been recently published. This article reviews nucleic acid-based hybridization as an assay to detect hemoparasite infections of economic relevance in veterinary medicine. By using recombinant DNA techniques, selected clones containing inserts of Anaplasma, Babesia, Cowdria or Theileria genomic DNA sequences have been obtained, and they are now available to be utilized as specific, highly sensitive DNA or RNA probes to detect the presence of the hemoparasite DNA in an infected animal. Either in an isotopic or non-isotopic detection system, probes have allowed scientists to test for--originally in samples collected from experimentally infected animals and later in samples collected in the field--the presence of hemoparasites during the prepatent, patent, convalescent, and chronic periods of the infection in the host. Nucleic acid probes have given researchers the opportunity to carry out genomic analysis of parasite DNA to differentiate hemoparasite species and to identify genetically distinct populations among and within isolates, strains and clonal populations. Prevalence of parasite infection in the tick vector can now be accomplished more specifically with the nucleic acid probes. Lately, with the advent of the polymerase chain reaction technique, small numbers of hemoparasites can be positively identified in the vertebrate host and tick vector. These techniques can be used to assess the veterinary epidemiological situation in a particular geographical region for the planning of control measures.

  15. Towards a “Sample-In, Answer-Out” Point-of-Care Platform for Nucleic Acid Extraction and Amplification: Using an HPV E6/E7 mRNA Model System

    PubMed Central

    Gulliksen, Anja; Keegan, Helen; Martin, Cara; O'Leary, John; Solli, Lars A.; Falang, Inger Marie; Grønn, Petter; Karlgård, Aina; Mielnik, Michal M.; Johansen, Ib-Rune; Tofteberg, Terje R.; Baier, Tobias; Gransee, Rainer; Drese, Klaus; Hansen-Hagge, Thomas; Riegger, Lutz; Koltay, Peter; Zengerle, Roland; Karlsen, Frank; Ausen, Dag; Furuberg, Liv

    2012-01-01

    The paper presents the development of a “proof-of-principle” hands-free and self-contained diagnostic platform for detection of human papillomavirus (HPV) E6/E7 mRNA in clinical specimens. The automated platform performs chip-based sample preconcentration, nucleic acid extraction, amplification, and real-time fluorescent detection with minimal user interfacing. It consists of two modular prototypes, one for sample preparation and one for amplification and detection; however, a common interface is available to facilitate later integration into one single module. Nucleic acid extracts (n = 28) from cervical cytology specimens extracted on the sample preparation chip were tested using the PreTect HPV-Proofer and achieved an overall detection rate for HPV across all dilutions of 50%–85.7%. A subset of 6 clinical samples extracted on the sample preparation chip module was chosen for complete validation on the NASBA chip module. For 4 of the samples, a 100% amplification for HPV 16 or 33 was obtained at the 1 : 10 dilution for microfluidic channels that filled correctly. The modules of a “sample-in, answer-out” diagnostic platform have been demonstrated from clinical sample input through sample preparation, amplification and final detection. PMID:22235204

  16. Comparison of MagNA Pure 96, Chemagic MSM1, and QIAamp MinElute for hepatitis B virus nucleic acid extraction.

    PubMed

    Kang, Seong-Ho; Lee, Eun Hee; Park, Geon; Jang, Sook Jin; Moon, Dae Soo

    2012-01-01

    This study was designed to compare two automated systems and one manual system for hepatitis B virus (HBV) nucleic acid extraction. The two automated systems were the MagNA Pure 96 system (Roche Applied Science, Manheim, Germany) and the Chemagic system (Chemagen, Baesweiler, Germany), and the manual system was the QIAamp system (Qiagen, Hilden, Germany). Sixty-eight samples that were within the detection range of the Cobas Ampliprep/Cobas TaqMan (CAP/CTM) platform (Roche Molecular Systems, Manheim, Germany) were selected. Extracted viral nucleic acids from the three systems were quantified using an AccuPower HBV Quantitative PCR kit (Bioneer, Daejon, Korea). The MagNA Pure 96 system and QIAamp system did not detect viral loads in one sample. The Chemagic system did not detect low viral loads in nine samples (range, 26-290 IU/mL by the CAP/CTM platform). Comparisons of the viral loads of the samples from the MagNA Pure 96 system, the Chemagic system, and the QIAamp system with those from the CAP/CTM platform yielded correlation coefficients of 0.977, 0.914, and 0.967, respectively. Comparisons of the MagNA Pure 96 system and the Chemagic system with the QIAamp system yielded correlation coefficients of 0.987 and 0.939, respectively. The MagNA Pure 96 system demonstrated better performance than the Chemagic system for HBV nucleic acid extraction. The MagNA Pure 96 system demonstrated comparable performance with the QIAamp system.

  17. Topography-Assisted Electromagnetic Platform for Blood-to-PCR in a Droplet

    PubMed Central

    Chiou, Chi-Han; Shin, Dong Jin; Zhang, Yi; Wang, Tza-Huei

    2013-01-01

    This paper presents an electromagnetically actuated platform for automated sample preparation and detection of nucleic acids. The proposed platform integrates nucleic acid extraction using silica-coated magnetic particles with real-time polymerase chain reaction (PCR) on a single cartridge. Extraction of genomic material was automated by manipulating magnetic particles in droplets using a series of planar coil electromagnets assisted by topographical features, enabling efficient fluidic processing over a variety of buffers and reagents. The functionality of the platform was demonstrated by performing nucleic acid extraction from whole blood, followed by real-time PCR detection of KRAS oncogene. Automated sample processing from whole blood to PCR-ready droplet was performed in 15 minutes. We took a modular approach of decoupling the modules of magnetic manipulation and optical detection from the device itself, enabling a low-complexity cartridge that operates in tandem with simple external instruments. PMID:23835223

  18. Nucleic acids in mummified plant seeds: screening of twelve specimens by gel-electrophoresis, molecular hybridization and DNA cloning.

    PubMed

    Rollo, F; La Marca, A; Amici, A

    1987-02-01

    Twelve seed specimens of varying ages and from different archaeological sites were analyzed for the presence of polymerized DNA and RNA. Amongst the samples tested, one of Vitis vinifera from an archaeological site in Iran (2,000-3,000 B.C.) was found to be completely devoid of nucleic acids. Zea mais seeds of Precolumbial age from Peru (about 800 A.D.) contained depolymerized DNA and RNA. Samples of Vitis vinifera and Rubus sp. from a Lombard archaeological site (800 A.D.) as well as radiocarbon dated seeds from the site of the "Spring Sanctuary" near Metaponto (I-IV century B.C.) were found to contain polymerized DNA and rRNA bands. However the electrophoretic properties of the rRNAs in one case and hybridization experiments performed with cloned seed DNA in the other, clearly demonstrated that the polymerized nucleic acids were not of plant origin.

  19. Broad-range real-time PCR assay for the rapid identification of cell-line contaminants and clinically important mollicute species.

    PubMed

    Störmer, Melanie; Vollmer, Tanja; Henrich, Birgit; Kleesiek, Knut; Dreier, Jens

    2009-04-01

    Polymerase chain reaction assays have become widely used methods of confirming the presence of Mollicutes species in clinical samples and cell cultures. We have developed a broad-range real-time PCR assay using the locked nucleic acid technology to detect mollicute species causing human infection and cell line contamination. Primers and probes specifically for the conserved regions of the mycoplasmal tuf gene (encoding elongation factor Tu) were designed. Cell culture supernatants, clinical specimens (vaginal swabs, sputum, cryopreserved heart valve tissues), and reference strains were tested for mollicute contamination as well as to exclude cross-reaction to human nucleic acids and other bacterial species. Nucleic acids were extracted using magnetic separation technology. The coamplification of the human beta2-microglobulin DNA served as an internal control. The PCR assay was highly specific and obtained an analytical sensitivity of one copy per microl sample. The 95% detection limit was calculated to 10 copies per microl sample for Mycoplasma pneumoniae and M. orale. No false-positive results were observed due to cross-reaction of walled bacterial, fungal, and human nucleic acids. To evaluate the PCR, we compared the results to two commercialized test systems. Moreover, in combination with a previously developed broad-range RT-PCR assay for the detection of bacteria in blood products, both mollicute and walled bacterial contamination can be detected simultaneously using multiplex real-time RT-PCR.

  20. Composition for nucleic acid sequencing

    DOEpatents

    Korlach, Jonas [Ithaca, NY; Webb, Watt W [Ithaca, NY; Levene, Michael [Ithaca, NY; Turner, Stephen [Ithaca, NY; Craighead, Harold G [Ithaca, NY; Foquet, Mathieu [Ithaca, NY

    2008-08-26

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  1. Method for sequencing nucleic acid molecules

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2006-06-06

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  2. Method for sequencing nucleic acid molecules

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2006-05-30

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  3. Method for identifying and quantifying nucleic acid sequence aberrations

    DOEpatents

    Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

    1998-01-01

    A method for detecting nucleic acid sequence aberrations by detecting nucleic acid sequences having both a first and a second nucleic acid sequence type, the presence of the first and second sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. The method uses a first hybridization probe which includes a nucleic acid sequence that is complementary to a first sequence type and a first complexing agent capable of attaching to a second complexing agent and a second hybridization probe which includes a nucleic acid sequence that selectively hybridizes to the second nucleic acid sequence type over the first sequence type and includes a detectable marker for detecting the second hybridization probe.

  4. Method for identifying and quantifying nucleic acid sequence aberrations

    DOEpatents

    Lucas, J.N.; Straume, T.; Bogen, K.T.

    1998-07-21

    A method is disclosed for detecting nucleic acid sequence aberrations by detecting nucleic acid sequences having both a first and a second nucleic acid sequence type, the presence of the first and second sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. The method uses a first hybridization probe which includes a nucleic acid sequence that is complementary to a first sequence type and a first complexing agent capable of attaching to a second complexing agent and a second hybridization probe which includes a nucleic acid sequence that selectively hybridizes to the second nucleic acid sequence type over the first sequence type and includes a detectable marker for detecting the second hybridization probe. 11 figs.

  5. Replica amplification of nucleic acid arrays

    DOEpatents

    Church, George M.

    2002-01-01

    A method of producing a plurality of a nucleic acid array, comprising, in order, the steps of amplifying in situ nucleic acid molecules of a first randomly-patterned, immobilized nucleic acid array comprising a heterogeneous pool of nucleic acid molecules affixed to a support, transferring at least a subset of the nucleic acid molecules produced by such amplifying to a second support, and affixing the subset so transferred to the second support to form a second randomly-patterned, immobilized nucleic acid array, wherein the nucleic acid molecules of the second array occupy positions that correspond to those of the nucleic acid molecules from which they were amplified on the first array, so that the first array serves as a template to produce a plurality, is disclosed.

  6. Nucleic acid analysis using terminal-phosphate-labeled nucleotides

    DOEpatents

    Korlach, Jonas [Ithaca, NY; Webb, Watt W [Ithaca, NY; Levene, Michael [Ithaca, NY; Turner, Stephen [Ithaca, NY; Craighead, Harold G [Ithaca, NY; Foquet, Mathieu [Ithaca, NY

    2008-04-22

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  7. Kit for detecting nucleic acid sequences using competitive hybridization probes

    DOEpatents

    Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

    2001-01-01

    A kit is provided for detecting a target nucleic acid sequence in a sample, the kit comprising: a first hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a first portion of the target sequence, the first hybridization probe including a first complexing agent for forming a binding pair with a second complexing agent; and a second hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a second portion of the target sequence to which the first hybridization probe does not selectively hybridize, the second hybridization probe including a detectable marker; a third hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a first portion of the target sequence, the third hybridization probe including the same detectable marker as the second hybridization probe; and a fourth hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a second portion of the target sequence to which the third hybridization probe does not selectively hybridize, the fourth hybridization probe including the first complexing agent for forming a binding pair with the second complexing agent; wherein the first and second hybridization probes are capable of simultaneously hybridizing to the target sequence and the third and fourth hybridization probes are capable of simultaneously hybridizing to the target sequence, the detectable marker is not present on the first or fourth hybridization probes and the first, second, third, and fourth hybridization probes each include a competitive nucleic acid sequence which is sufficiently complementary to a third portion of the target sequence that the competitive sequences of the first, second, third, and fourth hybridization probes compete with each other to hybridize to the third portion of the target sequence.

  8. A fully disposable and integrated paper-based device for nucleic acid extraction, amplification and detection.

    PubMed

    Tang, Ruihua; Yang, Hui; Gong, Yan; You, MinLi; Liu, Zhi; Choi, Jane Ru; Wen, Ting; Qu, Zhiguo; Mei, Qibing; Xu, Feng

    2017-03-29

    Nucleic acid testing (NAT) has been widely used for disease diagnosis, food safety control and environmental monitoring. At present, NAT mainly involves nucleic acid extraction, amplification and detection steps that heavily rely on large equipment and skilled workers, making the test expensive, time-consuming, and thus less suitable for point-of-care (POC) applications. With advances in paper-based microfluidic technologies, various integrated paper-based devices have recently been developed for NAT, which however require off-chip reagent storage, complex operation steps and equipment-dependent nucleic acid amplification, restricting their use for POC testing. To overcome these challenges, we demonstrate a fully disposable and integrated paper-based sample-in-answer-out device for NAT by integrating nucleic acid extraction, helicase-dependent isothermal amplification and lateral flow assay detection into one paper device. This simple device allows on-chip dried reagent storage and equipment-free nucleic acid amplification with simple operation steps, which could be performed by untrained users in remote settings. The proposed device consists of a sponge-based reservoir and a paper-based valve for nucleic acid extraction, an integrated battery, a PTC ultrathin heater, temperature control switch and on-chip dried enzyme mix storage for isothermal amplification, and a lateral flow test strip for naked-eye detection. It can sensitively detect Salmonella typhimurium, as a model target, with a detection limit of as low as 10 2 CFU ml -1 in wastewater and egg, and 10 3 CFU ml -1 in milk and juice in about an hour. This fully disposable and integrated paper-based device has great potential for future POC applications in resource-limited settings.

  9. Labeled nucleotide phosphate (NP) probes

    DOEpatents

    Korlach, Jonas [Ithaca, NY; Webb, Watt W [Ithaca, NY; Levene, Michael [Ithaca, NY; Turner, Stephen [Ithaca, NY; Craighead, Harold G [Ithaca, NY; Foquet, Mathieu [Ithaca, NY

    2009-02-03

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  10. Study of nucleic acid-gold nanorod interactions and detecting nucleic acid hybridization using gold nanorod solutions in the presence of sodium citrate.

    PubMed

    Kanjanawarut, Roejarek; Su, Xiaodi

    2010-09-01

    In this study, the authors report that sodium citrate can aggregate hexadecyl-trimethyl-ammonium ion(+)-coated gold nanorods (AuNRs), and nucleic acids of different charge and structure properties, i.e., single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded peptide nucleic acid (PNA), and PNA-DNA complex, can bind to the AuNRs and therefore retard the sodium citrate-induced aggregation to different extents. The discovery that hybridized dsDNA (and the PNA-DNA complex) has a more pronounced protection effect than ssDNA (and PNA) allows the authors to develop a homogeneous phase AuNRs-based UV-visible (UV-vis) spectral assay for detecting specific sequences of oligonucleotides (20 mer) with a single-base-mismatch selectivity and a limit of detection of 5 nM. This assay involves no tedious bioconjugation and on-particle hybridization. The simple "set and test" format allows for a highly efficient hybridization in a homogeneous phase and a rapid display of the results in less than a minute. By measuring the degree of reduction in AuNR aggregation in the presence of different nucleic acid samples, one can assess how different nucleic acids interact with the AuNRs to complement the knowledge of spherical gold nanoparticles. Besides UV-vis characterization, transmission electron microscopy and zeta potential measurements were conduced to provide visual evidence of the particle aggregation and to support the discussion of the assay principle.

  11. Nucleic acid tool enzymes-aided signal amplification strategy for biochemical analysis: status and challenges.

    PubMed

    Qing, Taiping; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Xu, Fengzhou; Wen, Li; Shangguan, Jingfang; Mao, Zhengui; Lei, Yanli

    2016-04-01

    Owing to their highly efficient catalytic effects and substrate specificity, the nucleic acid tool enzymes are applied as 'nano-tools' for manipulating different nucleic acid substrates both in the test-tube and in living organisms. In addition to the function as molecular scissors and molecular glue in genetic engineering, the application of nucleic acid tool enzymes in biochemical analysis has also been extensively developed in the past few decades. Used as amplifying labels for biorecognition events, the nucleic acid tool enzymes are mainly applied in nucleic acids amplification sensing, as well as the amplification sensing of biorelated variations of nucleic acids. With the introduction of aptamers, which can bind different target molecules, the nucleic acid tool enzymes-aided signal amplification strategies can also be used to sense non-nucleic targets (e.g., ions, small molecules, proteins, and cells). This review describes and discusses the amplification strategies of nucleic acid tool enzymes-aided biosensors for biochemical analysis applications. Various analytes, including nucleic acids, ions, small molecules, proteins, and cells, are reviewed briefly. This work also addresses the future trends and outlooks for signal amplification in nucleic acid tool enzymes-aided biosensors.

  12. Colorimetric Nucleic Acid Detection on Paper Microchip Using Loop Mediated Isothermal Amplification and Crystal Violet Dye.

    PubMed

    Roy, Sharmili; Mohd-Naim, Noor Faizah; Safavieh, Mohammadali; Ahmed, Minhaz Uddin

    2017-11-22

    Nucleic acid detection is of paramount importance in monitoring of microbial pathogens in food safety and infectious disease diagnostic applications. To address these challenges, a rapid, cost-effective label-free technique for nucleic acid detection with minimal instrumentations is highly desired. Here, we present paper microchip to detect and quantify nucleic acid using colorimetric sensing modality. The extracted DNA from food samples of meat as well as microbial pathogens was amplified utilizing loop-mediated isothermal amplification (LAMP). LAMP amplicon was then detected and quantified on a paper microchip fabricated in a cellulose paper and a small wax chamber utilizing crystal violet dye. The affinity of crystal violet dye toward dsDNA and positive signal were identified by changing the color from colorless to purple. Using this method, detection of Sus scrofa (porcine) and Bacillus subtilis (bacteria) DNA was possible at concentrations as low as 1 pg/μL (3.43 × 10 -1 copies/μL) and 10 pg/μL (2.2 × 10 3 copies/μL), respectively. This strategy can be adapted for detection of other DNA samples, with potential for development of a new breed of simple and inexpensive paper microchip at the point-of-need.

  13. Method of Identifying a Base in a Nucleic Acid

    DOEpatents

    Fodor, Stephen P. A.; Lipshutz, Robert J.; Huang, Xiaohua

    1999-01-01

    Devices and techniques for hybridization of nucleic acids and for determining the sequence of nucleic acids. Arrays of nucleic acids are formed by techniques, preferably high resolution, light-directed techniques. Positions of hybridization of a target nucleic acid are determined by, e.g., epifluorescence microscopy. Devices and techniques are proposed to determine the sequence of a target nucleic acid more efficiently and more quickly through such synthesis and detection techniques.

  14. Identifying a base in a nucleic acid

    DOEpatents

    Fodor, Stephen P. A.; Lipshutz, Robert J.; Huang, Xiaohua

    2005-02-08

    Devices and techniques for hybridization of nucleic acids and for determining the sequence of nucleic acids. Arrays of nucleic acids are formed by techniques, preferably high resolution, light-directed techniques. Positions of hybridization of a target nucleic acid are determined by, e.g., epifluorescence microscopy. Devices and techniques are proposed to determine the sequence of a target nucleic acid more efficiently and more quickly through such synthesis and detection techniques.

  15. NMR studies of protein-nucleic acid interactions.

    PubMed

    Varani, Gabriele; Chen, Yu; Leeper, Thomas C

    2004-01-01

    Protein-DNA and protein-RNA complexes play key functional roles in every living organism. Therefore, the elucidation of their structure and dynamics is an important goal of structural and molecular biology. Nuclear magnetic resonance (NMR) studies of protein and nucleic acid complexes have common features with studies of protein-protein complexes: the interaction surfaces between the molecules must be carefully delineated, the relative orientation of the two species needs to be accurately and precisely determined, and close intermolecular contacts defined by nuclear Overhauser effects (NOEs) must be obtained. However, differences in NMR properties (e.g., chemical shifts) and biosynthetic pathways for sample productions generate important differences. Chemical shift differences between the protein and nucleic acid resonances can aid the NMR structure determination process; however, the relatively limited dispersion of the RNA ribose resonances makes the process of assigning intermolecular NOEs more difficult. The analysis of the resulting structures requires computational tools unique to nucleic acid interactions. This chapter summarizes the most important elements of the structure determination by NMR of protein-nucleic acid complexes and their analysis. The main emphasis is on recent developments (e.g., residual dipolar couplings and new Web-based analysis tools) that have facilitated NMR studies of these complexes and expanded the type of biological problems to which NMR techniques of structural elucidation can now be applied.

  16. Molecular Modeling of Nucleic Acid Structure: Electrostatics and Solvation

    PubMed Central

    Bergonzo, Christina; Galindo-Murillo, Rodrigo; Cheatham, Thomas E.

    2014-01-01

    This unit presents an overview of computer simulation techniques as applied to nucleic acid systems, ranging from simple in vacuo molecular modeling techniques to more complete all-atom molecular dynamics treatments that include an explicit representation of the environment. The third in a series of four units, this unit focuses on critical issues in solvation and the treatment of electrostatics. UNITS 7.5 & 7.8 introduced the modeling of nucleic acid structure at the molecular level. This included a discussion of how to generate an initial model, how to evaluate the utility or reliability of a given model, and ultimately how to manipulate this model to better understand the structure, dynamics, and interactions. Subject to an appropriate representation of the energy, such as a specifically parameterized empirical force field, the techniques of minimization and Monte Carlo simulation, as well as molecular dynamics (MD) methods, were introduced as means to sample conformational space for a better understanding of the relevance of a given model. From this discussion, the major limitations with modeling, in general, were highlighted. These are the difficult issues in sampling conformational space effectively—the multiple minima or conformational sampling problems—and accurately representing the underlying energy of interaction. In order to provide a realistic model of the underlying energetics for nucleic acids in their native environments, it is crucial to include some representation of solvation (by water) and also to properly treat the electrostatic interactions. These are discussed in detail in this unit. PMID:18428877

  17. Molecular modeling of nucleic Acid structure: electrostatics and solvation.

    PubMed

    Bergonzo, Christina; Galindo-Murillo, Rodrigo; Cheatham, Thomas E

    2014-12-19

    This unit presents an overview of computer simulation techniques as applied to nucleic acid systems, ranging from simple in vacuo molecular modeling techniques to more complete all-atom molecular dynamics treatments that include an explicit representation of the environment. The third in a series of four units, this unit focuses on critical issues in solvation and the treatment of electrostatics. UNITS 7.5 & 7.8 introduced the modeling of nucleic acid structure at the molecular level. This included a discussion of how to generate an initial model, how to evaluate the utility or reliability of a given model, and ultimately how to manipulate this model to better understand its structure, dynamics, and interactions. Subject to an appropriate representation of the energy, such as a specifically parameterized empirical force field, the techniques of minimization and Monte Carlo simulation, as well as molecular dynamics (MD) methods, were introduced as a way of sampling conformational space for a better understanding of the relevance of a given model. This discussion highlighted the major limitations with modeling in general. When sampling conformational space effectively, difficult issues are encountered, such as multiple minima or conformational sampling problems, and accurately representing the underlying energy of interaction. In order to provide a realistic model of the underlying energetics for nucleic acids in their native environments, it is crucial to include some representation of solvation (by water) and also to properly treat the electrostatic interactions. These subjects are discussed in detail in this unit. Copyright © 2014 John Wiley & Sons, Inc.

  18. Hybridization and sequencing of nucleic acids using base pair mismatches

    DOEpatents

    Fodor, Stephen P. A.; Lipshutz, Robert J.; Huang, Xiaohua

    2001-01-01

    Devices and techniques for hybridization of nucleic acids and for determining the sequence of nucleic acids. Arrays of nucleic acids are formed by techniques, preferably high resolution, light-directed techniques. Positions of hybridization of a target nucleic acid are determined by, e.g., epifluorescence microscopy. Devices and techniques are proposed to determine the sequence of a target nucleic acid more efficiently and more quickly through such synthesis and detection techniques.

  19. Probe kit for identifying a base in a nucleic acid

    DOEpatents

    Fodor, Stephen P. A.; Lipshutz, Robert J.; Huang, Xiaohua

    2001-01-01

    Devices and techniques for hybridization of nucleic acids and for determining the sequence of nucleic acids. Arrays of nucleic acids are formed by techniques, preferably high resolution, light-directed techniques. Positions of hybridization of a target nucleic acid are determined by, e.g., epifluorescence microscopy. Devices and techniques are proposed to determine the sequence of a target nucleic acid more efficiently and more quickly through such synthesis and detection techniques.

  20. Methods for Identifying Ligands that Target Nucleic Acid Molecules and Nucleic Acid Structural Motifs

    NASA Technical Reports Server (NTRS)

    Childs-Disney, Jessica L. (Inventor); Disney, Matthew D. (Inventor)

    2017-01-01

    Disclosed are methods for identifying a nucleic acid (e.g., RNA, DNA, etc.) motif which interacts with a ligand. The method includes providing a plurality of ligands immobilized on a support, wherein each particular ligand is immobilized at a discrete location on the support; contacting the plurality of immobilized ligands with a nucleic acid motif library under conditions effective for one or more members of the nucleic acid motif library to bind with the immobilized ligands; and identifying members of the nucleic acid motif library that are bound to a particular immobilized ligand. Also disclosed are methods for selecting, from a plurality of candidate ligands, one or more ligands that have increased likelihood of binding to a nucleic acid molecule comprising a particular nucleic acid motif, as well as methods for identifying a nucleic acid which interacts with a ligand.

  1. Screening for Trichomonas vaginalis in a Large High-Risk Population: Prevalence Among Men and Women Determined by Nucleic Acid Amplification Testing.

    PubMed

    Schwebke, Jane; Merriweather, Anthony; Massingale, Sharon; Scisney, Mary; Hill, Craig; Getman, Damon

    2018-05-01

    Men and women attending family planning and sexually transmitted disease clinics for sexually transmitted infection screening in 2012 to 2013 were tested for Trichomonas vaginalis (TV) using a sensitive nucleic acid amplification test. T. vaginalis prevalence in urogenital samples was 11.3% in 77,740 women and 6.1% in 12,604 men, and increased with age in both sexes.

  2. Quantitative and discriminative analysis of nucleic acid samples using luminometric nonspecific nanoparticle methods

    NASA Astrophysics Data System (ADS)

    Pihlasalo, S.; Mariani, L.; Härmä, H.

    2016-03-01

    Homogeneous simple assays utilizing luminescence quenching and time-resolved luminescence resonance energy transfer (TR-LRET) were developed for the quantification of nucleic acids without sequence information. Nucleic acids prevent the adsorption of a protein to europium nanoparticles which is detected as a luminescence quenching of europium nanoparticles with a soluble quencher or as a decrease of TR-LRET from europium nanoparticles to the acceptor dye. Contrary to the existing methods based on fluorescent dye binding to nucleic acids, equal sensitivities for both single- (ssDNA) and double-stranded DNA (dsDNA) were measured and a detection limit of 60 pg was calculated for the quenching assay. The average coefficient of variation was 5% for the quenching assay and 8% for the TR-LRET assay. The TR-LRET assay was also combined with a nucleic acid dye selective to dsDNA in a single tube assay to measure the total concentration of DNA and the ratio of ssDNA and dsDNA in the mixture. To our knowledge, such a multiplexed assay is not accomplished with commercially available assays.Homogeneous simple assays utilizing luminescence quenching and time-resolved luminescence resonance energy transfer (TR-LRET) were developed for the quantification of nucleic acids without sequence information. Nucleic acids prevent the adsorption of a protein to europium nanoparticles which is detected as a luminescence quenching of europium nanoparticles with a soluble quencher or as a decrease of TR-LRET from europium nanoparticles to the acceptor dye. Contrary to the existing methods based on fluorescent dye binding to nucleic acids, equal sensitivities for both single- (ssDNA) and double-stranded DNA (dsDNA) were measured and a detection limit of 60 pg was calculated for the quenching assay. The average coefficient of variation was 5% for the quenching assay and 8% for the TR-LRET assay. The TR-LRET assay was also combined with a nucleic acid dye selective to dsDNA in a single tube assay to measure the total concentration of DNA and the ratio of ssDNA and dsDNA in the mixture. To our knowledge, such a multiplexed assay is not accomplished with commercially available assays. Electronic supplementary information (ESI) available: The labeling of amino modified polystyrene nanoparticles with Eu3+ chelate and the experimental details and results for the optimization of nucleic acid binding protein and for the ratiometric measurement of DNA and RNA with quenching assay. See DOI: 10.1039/c5nr09252c

  3. Performance of nucleic acid amplification following extraction of 5 milliliters of whole blood for diagnosis of Mycobacterium tuberculosis bacteremia.

    PubMed

    Crump, John A; Tuohy, Marion J; Morrissey, Anne B; Ramadhani, Habib O; Njau, Boniface N; Maro, Venance P; Reller, L Barth; Procop, Gary W

    2012-01-01

    To investigate the performance of a nucleic acid amplification test (NAAT) for the diagnosis of Mycobacterium tuberculosis bacteremia, 5-ml aliquots of blood were inoculated into bioMérieux mycobacterial (MB) bottles and incubated, and 5-ml aliquots of blood were extracted and tested by real-time PCR. Of 25 samples from patients with M. tuberculosis bacteremia, 9 (36.0%) were positive and 1 (1.5%) of 66 control samples was positive by NAAT. The NAAT shows promise, but modifications should focus on improving sensitivity.

  4. Performance of Nucleic Acid Amplification following Extraction of 5 Milliliters of Whole Blood for Diagnosis of Mycobacterium tuberculosis Bacteremia

    PubMed Central

    Tuohy, Marion J.; Morrissey, Anne B.; Ramadhani, Habib O.; Njau, Boniface N.; Maro, Venance P.; Reller, L. Barth; Procop, Gary W.

    2012-01-01

    To investigate the performance of a nucleic acid amplification test (NAAT) for the diagnosis of Mycobacterium tuberculosis bacteremia, 5-ml aliquots of blood were inoculated into bioMérieux mycobacterial (MB) bottles and incubated, and 5-ml aliquots of blood were extracted and tested by real-time PCR. Of 25 samples from patients with M. tuberculosis bacteremia, 9 (36.0%) were positive and 1 (1.5%) of 66 control samples was positive by NAAT. The NAAT shows promise, but modifications should focus on improving sensitivity. PMID:22031703

  5. Oligonucleoside alkyl or arylphosphonate derivatives capable of crosslinking with or cleaving nucleic acids

    DOEpatents

    Miller, Paul S.; Ts'o, Paul O.P.

    1999-06-15

    A composition for inactivating a target nucleic acid which comprises an oligonucleoside alkyl or arylphosphonate analogue which is complementary to the sequence of the target nucleic acid and includes a functional group which reacts with the target nucleic acid to render the target nucleic acid inactive or nonfunctional.

  6. Accelerated digestion of nucleic acids by pepsin from the stomach of chicken.

    PubMed

    Liu, Y; Zhang, Y; Guo, H; Wu, W; Dong, P; Liang, X

    2016-10-01

    Nucleic acids have become an important nutritional supplement in poultry feed; however, the digestion of nucleic acids in poultry is unclear. The objective of this study was to investigate the digestion of nucleic acids by chicken pepsin in vitro. The extracted pepsinogen from the stomach of the chicken was purified to homogeneity. Upon activation at pH 2.0, chicken pepsinogen was converted to its active form. Nucleic acids, including λ-DNA, salmon sperm DNA and single-strand DNA (ssDNA), can be used as substrates and digested into short-chain oligonucleotides by pepsin. Interestingly, the digestion of the nucleic acids was inhibited when pepsin was treated by alkaline solution (pH 8.0) or pepstatin A. Also, the digestion of the nucleic acids was not affected by the addition of haemoglobin or bovine serum albumin. The results suggested that nucleic acids could be digested by chicken pepsin. Thus pepsin may have a role in digesting nucleic acids in vivo. Nucleic acids added to poultry fed may be digested, starting from the stomach.

  7. Nucleic acid sequence detection using multiplexed oligonucleotide PCR

    DOEpatents

    Nolan, John P [Santa Fe, NM; White, P Scott [Los Alamos, NM

    2006-12-26

    Methods for rapidly detecting single or multiple sequence alleles in a sample nucleic acid are described. Provided are all of the oligonucleotide pairs capable of annealing specifically to a target allele and discriminating among possible sequences thereof, and ligating to each other to form an oligonucleotide complex when a particular sequence feature is present (or, alternatively, absent) in the sample nucleic acid. The design of each oligonucleotide pair permits the subsequent high-level PCR amplification of a specific amplicon when the oligonucleotide complex is formed, but not when the oligonucleotide complex is not formed. The presence or absence of the specific amplicon is used to detect the allele. Detection of the specific amplicon may be achieved using a variety of methods well known in the art, including without limitation, oligonucleotide capture onto DNA chips or microarrays, oligonucleotide capture onto beads or microspheres, electrophoresis, and mass spectrometry. Various labels and address-capture tags may be employed in the amplicon detection step of multiplexed assays, as further described herein.

  8. Macro-/Nano- Materials Based Ultrasensitive Lateral Flow Nucleic Acid Biosensors

    NASA Astrophysics Data System (ADS)

    Takalkar, Sunitha

    Ultrasensitive detection of nucleic acids plays a very important role in the field of molecular diagnosis for the detection of various diseases. Lateral flow biosensors (LFB) are convenient, easy-to-use, patient friendly forms of detection methods offering rapid and convenient clinical testing in close proximity to the patients thus drawing a lot of attention in different areas of research over the years. In comparison with the traditional immunoassays, the nucleic acid based lateral flow biosensors (NABLFB) has several advantages in terms of stability and interference capabilities. NABLFB utilizes nucleic acid probes as the bio-recognition element. The target analyte typically is the oligonucleotide like the DNA, mRNA, miRNA which are among the nucleic acid secretions by the tumor cells when it comes to detection of cancer. Traditionally gold nanoparticles (GNPs) have been used as labels for conjugating with the detection probes for the qualitative and semi quantitative analysis, the application of GNP-based LFB is limited by its low sensitivity. This dissertation describes the use of different nanomaterials and advanced detection technologies to enhance the sensitivities of the LFB based methods. Silica Nanorods decorated with GNP were synthesized and employed as labels for ultrasensitive detection of miRNA on the LFB. Owing to the biocompatibility and convenience in surface modification of SiNRs, they acted as good carriers to load numerous GNPs. The sensitivity of the GNP-SiNR-based LFSB was enhanced six times compared to the previous GNP-based LFSB. A fluorescent carbon nanoparticle (FCN) was first used as a tag to develop a lateral flow nucleic acid biosensor for ultrasensitive and quantitative detection of nucleic acid samples. Under optimal conditions, the FCN-based LFNAB was capable of detecting minimum 0.4 fM target DNA without complex operations and additional signal amplification. The carbon nanotube was used as a label and carrier of numerous enzyme and DNA molecules simultaneously thus resulting in the enormous amplification of the colorimetric signal. This CNT-enzyme label thus aided the ultra-sensitive detection of pancreatic cancer (PC) biomarker miRNA 210 and PC biomarker panel (miRNA 16, miRNA 21 and miRNA 196a). All these LFBs were also applied in the field of real sample detection.

  9. Detection of nucleic acid sequences by invader-directed cleavage

    DOEpatents

    Brow, Mary Ann D.; Hall, Jeff Steven Grotelueschen; Lyamichev, Victor; Olive, David Michael; Prudent, James Robert

    1999-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The 5' nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based by charge.

  10. Cleavage of nucleic acids

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor L.; Brow, Mary Ann D.; Dahlberg, James E.

    2007-12-11

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  11. Invasive cleavage of nucleic acids

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann D.; Dahlberg, James E.

    1999-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  12. Invasive cleavage of nucleic acids

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann D.; Dahlberg, James E.

    2002-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  13. Cleavage of nucleic acids

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow; Mary Ann D.; Dahlberg, James E.

    2010-11-09

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  14. Cleavage of nucleic acids

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann D.; Dahlberg, James E.

    2000-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  15. Nucleic acid detection assays

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann; Dahlberg, James E.

    2005-04-05

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  16. Oligonucleoside alkyl or arylphosphonate derivatives capable of crosslinking with or cleaving nucleic acids

    DOEpatents

    Miller, P.S.; Ts'o, P.O.P.

    1999-06-15

    A composition for inactivating a target nucleic acid which comprises an oligonucleoside alkyl or arylphosphonate analogue which is complementary to the sequence of the target nucleic acid is provided. It includes a functional group which reacts with the target nucleic acid to render the target nucleic acid inactive or nonfunctional. 16 figs.

  17. Evaluation of FTA paper and phenol for storage, extraction and molecular characterization of infectious bursal disease virus.

    PubMed

    Purvis, Linda B; Villegas, Pedro; Perozo, Francisco

    2006-12-01

    Infectious bursal disease virus (IBDV) is an important poultry pathogen and is distributed world wide that can cause immune suppression and lesions of the bursa of Fabricius. The main component of the virus, VP2, is not only responsible for the bird's immune response, but is important for the molecular identification of this virus as well. The nucleic acid of the virus must be adequately preserved to be analyzed by reverse-transcriptase PCR (RT-PCR) and sequenced for the molecular characterization of the field strain. Phenol inactivation has been the standard for IBDV tissue collection and international shipment; however, there have been some reports of interference with molecular detection capabilities when using phenol. Phenol is also a hazardous chemical and must be handled and shipped carefully. The ability to use the Flinders Technology Associates filter paper (FTA card) for inactivation of several avian pathogens has been proven previously, however no work has been published on its use in IBDV nucleic acid detection. Bursas from experimentally infected birds was imprinted on FTA cards, and then placed in phenol. Samples were evaluated and compared based on molecular detection capabilities between the two inactivation methods. The nucleic acid of the virus was detected in 85% of the FTA card inactivated samples compared to 71% in the phenol inactivated samples. Sequence analysis was performed on samples inactivated by both methods and no differences were found. When comparing the RNA stability at different temperatures, euthanized IBDV infected birds were held at two different temperatures before sampling. No differences were detected for FTA sampling; however, for tissues in phenol the nucleic acid was only detectable up to 2 h post-mortem in the tissues held at 4 degrees C prior to sampling. These findings indicate that the FTA card is an efficient and reliable alternative collection method for molecular detection and characterization of IBDV.

  18. RPA using a multiplexed cartridge for low cost point of care diagnostics in the field.

    PubMed

    Ereku, Luck Tosan; Mackay, Ruth E; Craw, Pascal; Naveenathayalan, Angel; Stead, Thomas; Branavan, Manorharanehru; Balachandran, Wamadeva

    2018-04-15

    A point of care device utilising Lab-on-a-Chip technologies that is applicable for biological pathogens was designed, fabricated and tested showing sample in to answer out capabilities. The purpose of the design was to develop a cartridge with the capability to perform nucleic acid extraction and purification from a sample using a chitosan membrane at an acidic pH. Waste was stored within the cartridge with the use of sodium polyacrylate to solidify or gelate the sample in a single chamber. Nucleic acid elution was conducted using the RPA amplification reagents (alkaline pH). Passive valves were used to regulate the fluid flow and a multiplexer was designed to distribute the fluid into six microchambers for amplification reactions. Cartridges were produced using soft lithography of silicone from 3D printed moulds, bonded to glass substrates. The isothermal technique, RPA is employed for amplification. This paper shows the results from two separate experiments: the first using the RPA control nucleic acid, the second showing successful amplification from Chlamydia Trachomatis. Endpoint analysis conducted for the RPA analysis was gel electrophoresis that showed 143 base pair DNA was amplified successfully for positive samples whilst negative samples did not show amplification. End point analysis for Chlamydia Trachomatis samples was fluorescence detection that showed successful detection of 1 copy/μL and 10 copies/μL spiked in a MES buffer. Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.

  19. On-Chip, Amplification-Free Quantification of Nucleic Acid for Point-of-Care Diagnosis

    NASA Astrophysics Data System (ADS)

    Yen, Tony Minghung

    This dissertation demonstrates three physical device concepts to overcome limitations in point-of-care quantification of nucleic acids. Enabling sensitive, high throughput nucleic acid quantification on a chip, outside of hospital and centralized laboratory setting, is crucial for improving pathogen detection and cancer diagnosis and prognosis. Among existing platforms, microarray have the advantages of being amplification free, low instrument cost, and high throughput, but are generally less sensitive compared to sequencing and PCR assays. To bridge this performance gap, this dissertation presents theoretical and experimental progress to develop a platform nucleic acid quantification technology that is drastically more sensitive than current microarrays while compatible with microarray architecture. The first device concept explores on-chip nucleic acid enrichment by natural evaporation of nucleic acid solution droplet. Using a micro-patterned super-hydrophobic black silicon array device, evaporative enrichment is coupled with nano-liter droplet self-assembly workflow to produce a 50 aM concentration sensitivity, 6 orders of dynamic range, and rapid hybridization time at under 5 minutes. The second device concept focuses on improving target copy number sensitivity, instead of concentration sensitivity. A comprehensive microarray physical model taking into account of molecular transport, electrostatic intermolecular interactions, and reaction kinetics is considered to guide device optimization. Device pattern size and target copy number are optimized based on model prediction to achieve maximal hybridization efficiency. At a 100-mum pattern size, a quantum leap in detection limit of 570 copies is achieved using black silicon array device with self-assembled pico-liter droplet workflow. Despite its merits, evaporative enrichment on black silicon device suffers from coffee-ring effect at 100-mum pattern size, and thus not compatible with clinical patient samples. The third device concept utilizes an integrated optomechanical laser system and a Cytop microarray device to reverse coffee-ring effect during evaporative enrichment at 100-mum pattern size. This method, named "laser-induced differential evaporation" is expected to enable 570 copies detection limit for clinical samples in near future. While the work is ongoing as of the writing of this dissertation, a clear research plan is in place to implement this method on microarray platform toward clinical sample testing for disease applications and future commercialization.

  20. Microfluidic "Pouch" Chips for Immunoassays and Nucleic Acid Amplification Tests.

    PubMed

    Mauk, Michael G; Liu, Changchun; Qiu, Xianbo; Chen, Dafeng; Song, Jinzhao; Bau, Haim H

    2017-01-01

    Microfluidic cassettes ("chips") for processing and analysis of clinical specimens and other sample types facilitate point-of-care (POC) immunoassays and nucleic acid based amplification tests. These single-use test chips can be self-contained and made amenable to autonomous operation-reducing or eliminating supporting instrumentation-by incorporating laminated, pliable "pouch" and membrane structures for fluid storage, pumping, mixing, and flow control. Materials and methods for integrating flexible pouch compartments and diaphragm valves into hard plastic (e.g., acrylic and polycarbonate) microfluidic "chips" for reagent storage, fluid actuation, and flow control are described. We review several versions of these pouch chips for immunoassay and nucleic acid amplification tests, and describe related fabrication techniques. These protocols thus offer a "toolbox" of methods for storage, pumping, and flow control functions in microfluidic devices.

  1. Visualization of nucleic acids with synthetic exciton-controlled fluorescent oligonucleotide probes.

    PubMed

    Wang, Dan Ohtan; Okamoto, Akimitsu

    2015-01-01

    Engineered probes to adapt new photochemical properties upon recognition of target nucleic acids offer powerful tools to DNA and RNA visualization technologies. Herein, we describe a rapid and effective visualization method of nucleic acids in both fixed and living cells with hybridization-sensitive fluorescent oligonucleotide probes. These probes are efficiently quenched in an aqueous environment due to the homodimeric, excitonic interactions between fluorophores but become highly fluorescent upon hybridization to DNA or RNA with complementary sequences. The fast hybridization kinetics and quick fluorescence activation of the new probes allow applications to simplify the conventional fluorescent in situ hybridization protocols and reduce the amount of time to process the samples. Furthermore, hybridization-sensitive fluorescence emission of the probes allows monitoring dynamic behaviors of RNA in living cells.

  2. Biostable L-DNAzyme for Sensing of Metal Ions in Biological Systems

    PubMed Central

    2015-01-01

    DNAzymes, an important type of metal ion-dependent functional nucleic acid, are widely applied in bioanalysis and biomedicine. However, the use of DNAzymes in practical applications has been impeded by the intrinsic drawbacks of natural nucleic acids, such as interferences from nuclease digestion and protein binding, as well as undesired intermolecular interactions with other nucleic acids. On the basis of reciprocal chiral substrate specificity, the enantiomer of D-DNAzyme, L-DNAzyme, could initiate catalytic cleavage activity with the same achiral metal ion as a cofactor. Meanwhile, by using the advantage of nonbiological L-DNAzyme, which is not subject to the interferences of biological matrixes, as recognition units, a facile and stable L-DNAzyme sensor was proposed for sensing metal ions in complex biological samples and live cells. PMID:26691677

  3. Rope-based oral fluid sampling for early detection of classical swine fever in domestic pigs at group level.

    PubMed

    Dietze, Klaas; Tucakov, Anna; Engel, Tatjana; Wirtz, Sabine; Depner, Klaus; Globig, Anja; Kammerer, Robert; Mouchantat, Susan

    2017-01-05

    Non-invasive sampling techniques based on the analysis of oral fluid specimen have gained substantial importance in the field of swine herd management. Methodological advances have a focus on endemic viral diseases in commercial pig production. More recently, these approaches have been adapted to non-invasive sampling of wild boar for transboundary animal disease detection for which these effective population level sampling methods have not been available. In this study, a rope-in-a-bait based oral fluid sampling technique was tested to detect classical swine fever virus nucleic acid shedding from experimentally infected domestic pigs. Separated in two groups treated identically, the course of the infection was slightly differing in terms of onset of the clinical signs and levels of viral ribonucleic acid detection in the blood and oral fluid. The technique was capable of detecting classical swine fever virus nucleic acid as of day 7 post infection coinciding with the first detection in conventional oropharyngeal swab samples from some individual animals. Except for day 7 post infection in the "slower onset group", the chances of classical swine fever virus nucleic acid detection in ropes were identical or higher as compared to the individual sampling. With the provided evidence, non-invasive oral fluid sampling at group level can be considered as additional cost-effective detection tool in classical swine fever prevention and control strategies. The proposed methodology is of particular use in production systems with reduced access to veterinary services such as backyard or scavenging pig production where it can be integrated in feeding or baiting practices.

  4. Enhanced analytical sensitivity of a quantitative PCR for CMV using a modified nucleic-acid extraction procedure.

    PubMed

    Ferreira-Gonzalez, A; Yanovich, S; Langley, M R; Weymouth, L A; Wilkinson, D S; Garrett, C T

    2000-01-01

    Accurate and rapid diagnosis of CMV disease in immunocompromised individuals remains a challenge. Quantitative polymerase chain reaction (QPCR) methods for detection of CMV in peripheral blood mononuclear cells (PBMC) have improved the positive and negative predictive value of PCR for diagnosis of CMV disease. However, detection of CMV in plasma has demonstrated a lower negative predictive value for plasma as compared with PBMC. To enhance the sensitivity of the QPCR assay for plasma specimens, plasma samples were centrifuged before nucleic-acid extraction and the extracted DNA resolubilized in reduced volume. Optimization of the nucleic-acid extraction focused on decreasing or eliminating the presence of inhibitors in the pelleted plasma. Quantitation was achieved by co-amplifying an internal quantitative standard (IS) with the same primer sequences as CMV. PCR products were detected by hybridization in a 96-well microtiter plate coated with a CMV or IS specific probe. The precision of the QPCR assay for samples prepared from untreated and from pelleted plasma was then assessed. The coefficient of variation for both types of samples was almost identical and the magnitude of the coefficient of variations was reduced by a factor of ten if the data were log transformed. Linearity of the QPCR assay extended over a 3.3-log range for both types of samples but the range of linearity for pelleted plasma was 20 to 40,000 viral copies/ml (vc/ml) in contrast to 300 to 400,000 vc/ml for plasma. Thus, centrifugation of plasma before nucleic-acid extraction and resuspension of extracted CMV DNA in reduced volume enhanced the analytical sensitivity approximately tenfold over the dynamic range of the assay. Copyright 2000 Wiley-Liss, Inc.

  5. Automation of diagnostic genetic testing: mutation detection by cyclic minisequencing.

    PubMed

    Alagrund, Katariina; Orpana, Arto K

    2014-01-01

    The rising role of nucleic acid testing in clinical decision making is creating a need for efficient and automated diagnostic nucleic acid test platforms. Clinical use of nucleic acid testing sets demands for shorter turnaround times (TATs), lower production costs and robust, reliable methods that can easily adopt new test panels and is able to run rare tests in random access principle. Here we present a novel home-brew laboratory automation platform for diagnostic mutation testing. This platform is based on the cyclic minisequecing (cMS) and two color near-infrared (NIR) detection. Pipetting is automated using Tecan Freedom EVO pipetting robots and all assays are performed in 384-well micro plate format. The automation platform includes a data processing system, controlling all procedures, and automated patient result reporting to the hospital information system. We have found automated cMS a reliable, inexpensive and robust method for nucleic acid testing for a wide variety of diagnostic tests. The platform is currently in clinical use for over 80 mutations or polymorphisms. Additionally to tests performed from blood samples, the system performs also epigenetic test for the methylation of the MGMT gene promoter, and companion diagnostic tests for analysis of KRAS and BRAF gene mutations from formalin fixed and paraffin embedded tumor samples. Automation of genetic test reporting is found reliable and efficient decreasing the work load of academic personnel.

  6. Method for promoting specific alignment of short oligonucleotides on nucleic acids

    DOEpatents

    Studier, F. William; Kieleczawa, Jan; Dunn, John J.

    1996-01-01

    Disclosed is a method for promoting specific alignment of short oligonucleotides on a nucleic acid polymer. The nucleic acid polymer is incubated in a solution containing a single-stranded DNA-binding protein and a plurality of oligonucleotides which are perfectly complementary to distinct but adjacent regions of a predetermined contiguous nucleotide sequence in the nucleic acid polymer. The plurality of oligonucleotides anneal to the nucleic acid polymer to form a contiguous region of double stranded nucleic acid. Specific application of the methods disclosed include priming DNA synthesis and template-directed ligation.

  7. High processivity polymerases

    DOEpatents

    Shamoo, Yousif; Sun, Siyang

    2014-06-10

    Chimeric proteins comprising a sequence nonspecific single-stranded nucleic-acid-binding domain joined to a catalytic nucleic-acid-modifying domain are provided. Methods comprising contacting a nucleic acid molecule with a chimeric protein, as well as systems comprising a nucleic acid molecule, a chimeric protein, and an aqueous solution are also provided. The joining of sequence nonspecific single-stranded nucleic-acid-binding domain and a catalytic nucleic-acid-modifying domain in chimeric proteins, among other things, may prevent the separation of the two domains due to their weak association and thereby enhances processivity while maintaining fidelity.

  8. Method for analyzing nucleic acids by means of a substrate having a microchannel structure containing immobilized nucleic acid probes

    DOEpatents

    Ramsey, J. Michael; Foote, Robert S.

    2003-12-09

    A method and apparatus for analyzing nucleic acids includes immobilizing nucleic probes at specific sites within a microchannel structure and moving target nucleic acids into proximity to the probes in order to allow hybridization and fluorescence detection of specific target sequences.

  9. Method for analyzing nucleic acids by means of a substrate having a microchannel structure containing immobilized nucleic acid probes

    DOEpatents

    Ramsey, J. Michael; Foote, Robert S.

    2002-01-01

    A method and apparatus for analyzing nucleic acids includes immobilizing nucleic probes at specific sites within a microchannel structure and moving target nucleic acids into proximity to the probes in order to allow hybridization and fluorescence detection of specific target sequences.

  10. Nucleic acid-binding polymers as anti-inflammatory agents

    PubMed Central

    Lee, Jaewoo; Sohn, Jang Wook; Zhang, Ying; Leong, Kam W.; Pisetsky, David; Sullenger, Bruce A.

    2011-01-01

    Dead and dying cells release nucleic acids. These extracellular RNAs and DNAs can be taken up by inflammatory cells and activate multiple nucleic acid-sensing toll-like receptors (TLR3, 7, 8, and 9). The inappropriate activation of these TLRs can engender a variety of inflammatory and autoimmune diseases. The redundancy of the TLR family encouraged us to seek materials that can neutralize the proinflammatory effects of any nucleic acid regardless of its sequence, structure or chemistry. Herein we demonstrate that certain nucleic acid-binding polymers can inhibit activation of all nucleic acid-sensing TLRs irrespective of whether they recognize ssRNA, dsRNA or hypomethylated DNA. Furthermore, systemic administration of such polymers can prevent fatal liver injury engendered by proinflammatory nucleic acids in an acute toxic shock model in mice. Therefore these polymers represent a novel class of anti-inflammatory agent that can act as molecular scavengers to neutralize the proinflammatory effects of various nucleic acids. PMID:21844380

  11. Miniaturized isothermal nucleic acid amplification, a review.

    PubMed

    Asiello, Peter J; Baeumner, Antje J

    2011-04-21

    Micro-Total Analysis Systems (µTAS) for use in on-site rapid detection of DNA or RNA are increasingly being developed. Here, amplification of the target sequence is key to increasing sensitivity, enabling single-cell and few-copy nucleic acid detection. The several advantages to miniaturizing amplification reactions and coupling them with sample preparation and detection on the same chip are well known and include fewer manual steps, preventing contamination, and significantly reducing the volume of expensive reagents. To-date, the majority of miniaturized systems for nucleic acid analysis have used the polymerase chain reaction (PCR) for amplification and those systems are covered in previous reviews. This review provides a thorough overview of miniaturized analysis systems using alternatives to PCR, specifically isothermal amplification reactions. With no need for thermal cycling, isothermal microsystems can be designed to be simple and low-energy consuming and therefore may outperform PCR in portable, battery-operated detection systems in the future. The main isothermal methods as miniaturized systems reviewed here include nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), and strand displacement amplification (SDA). Also, important design criteria for the miniaturized devices are discussed. Finally, the potential of miniaturization of some new isothermal methods such as the exponential amplification reaction (EXPAR), isothermal and chimeric primer-initiated amplification of nucleic acids (ICANs), signal-mediated amplification of RNA technology (SMART) and others is presented.

  12. Localization of Bovine Papillomavirus Nucleic Acid in Equine Sarcoids.

    PubMed

    Gaynor, A M; Zhu, K W; Dela Cruz, F N; Affolter, V K; Pesavento, P A

    2016-05-01

    Bovine papillomaviruses (BPV1/BPV2) have long been associated with equine sarcoids; deciphering their contribution has been difficult due to their ubiquitous presence on skin and in the environment, as well as the lack of decent techniques to interrogate their role in pathogenesis. We have developed and characterized an in situ hybridization (ISH) assay that uses a pool of probes complementary to portions of the E5, E6, and E7 genes. This assay is highly sensitive for direct visualization of viral transcript and nucleic acid in routinely processed histopathologic samples. We demonstrate here the visualization of BPV nucleic acid in 18 of 18 equine sarcoids, whereas no detectable viral DNA was present in 15 of 15 nonsarcoid controls by this technique. In nearly 90% (16/18) of the sarcoids, 50% or more of the fibroblastic cell nuclei distributed throughout the neoplasm had detectable hybridization. In the remaining 2 cases, fewer than half of the fibroblastic cells contained detectable hybridization, but viral nucleic acid was also detected in epithelial cells of the sebaceous glands, hair follicles and epidermis. A sensitive ISH assay is an indispensable addition to the molecular methods used to detect viral nucleic acid in tissue. We have used this technique to determine the specific cellular localization and distribution of BPV in a subset of equine sarcoids. © The Author(s) 2015.

  13. Self-assembling multimeric nucleic acid constructs

    DOEpatents

    Cantor, Charles R.; Niemeyer, Christof M.; Smith, Cassandra L.; Sano, Takeshi; Hnatowich, Donald J.; Rusckowski, Mary

    1999-10-12

    The invention is directed to constructs and compositions containing multimeric forms of nucleic acid. Multimeric nucleic acids comprise single-stranded nucleic acids attached via biotin to streptavidin and bound with a functional group. These constructs can be utilized in vivo to treat or identify diseased tissue or cells. Repeated administrations of multimeric nucleic acid compositions produce a rapid and specific amplification of nucleic acid constructs and their attached functional groups. For treatment purposes, functional groups may be toxins, radioisotopes, genes or enzymes. Diagnostically, labeled multimeric constructs may be used to identify specific targets in vivo or in vitro. Multimeric nucleic acids may also be used in nanotechnology and to create self-assembling polymeric aggregates such as membranes of defined porosity, microcircuits and many other products.

  14. Self-assembling multimeric nucleic acid constructs

    DOEpatents

    Cantor, Charles R.; Niemeyer, Christof M.; Smith, Cassandra L.; Sano, Takeshi; Hnatowich, Donald J.; Rusckowski, Mary

    1996-01-01

    The invention is directed to constructs and compositions containing multimeric forms of nucleic acid. Multimeric nucleic acids comprise single-stranded nucleic acids attached via biotin to streptavidin and bound with a functional group. These constructs can be utilized in vivo to treat or identify diseased tissue or cells. Repeated administrations of multimeric nucleic acid compositions produce a rapid and specific amplification of nucleic acid constructs and their attached functional groups. For treatment purposes, functional groups may be toxins, radioisotopes, genes or enzymes. Diagnostically, labeled multimeric constructs may be used to identify specific targets in vivo or in vitro. Multimeric nucleic acids may also be used in nanotechnology and to create self-assembling polymeric aggregates such as membranes of defined porosity, microcircuits and many other products.

  15. Shaping up nucleic acid computation.

    PubMed

    Chen, Xi; Ellington, Andrew D

    2010-08-01

    Nucleic acid-based nanotechnology has always been perceived as novel, but has begun to move from theoretical demonstrations to practical applications. In particular, the large address spaces available to nucleic acids can be exploited to encode algorithms and/or act as circuits and thereby process molecular information. In this review we not only revisit several milestones in the field of nucleic acid-based computation, but also highlight how the prospects for nucleic acid computation go beyond just a large address space. Functional nucleic acid elements (aptamers, ribozymes, and deoxyribozymes) can serve as inputs and outputs to the environment, and can act as logical elements. Into the future, the chemical dynamics of nucleic acids may prove as useful as hybridization for computation. Copyright © 2010 Elsevier Ltd. All rights reserved.

  16. Self-assembling multimeric nucleic acid constructs

    DOEpatents

    Cantor, C.R.; Niemeyer, C.M.; Smith, C.L.; Sano, Takeshi; Hnatowich, D.J.; Rusckowski, M.

    1996-10-01

    The invention is directed to constructs and compositions containing multimeric forms of nucleic acid. Multimeric nucleic acids comprise single-stranded nucleic acids attached via biotin to streptavidin and bound with a functional group. These constructs can be utilized in vivo to treat or identify diseased tissue or cells. Repeated administrations of multimeric nucleic acid compositions produce a rapid and specific amplification of nucleic acid constructs and their attached functional groups. For treatment purposes, functional groups may be toxins, radioisotopes, genes or enzymes. Diagnostically, labeled multimeric constructs may be used to identify specific targets in vivo or in vitro. Multimeric nucleic acids may also be used in nanotechnology and to create self-assembling polymeric aggregates such as membranes of defined porosity, microcircuits and many other products. 5 figs.

  17. Magnetic particles for in vitro molecular diagnosis: From sample preparation to integration into microsystems.

    PubMed

    Tangchaikeeree, Tienrat; Polpanich, Duangporn; Elaissari, Abdelhamid; Jangpatarapongsa, Kulachart

    2017-10-01

    Colloidal magnetic particles (MPs) have been developed in association with molecular diagnosis for several decades. MPs have the great advantage of easy manipulation using a magnet. In nucleic acid detection, these particles can act as a capture support for rapid and simple biomolecule separation. The surfaces of MPs can be modified by coating with various polymer materials to provide functionalization for different applications. The use of MPs enhances the sensitivity and specificity of detection due to the specific activity on the surface of the particles. Practical applications of MPs demonstrate greater efficiency than conventional methods. Beyond traditional detection, MPs have been successfully adopted as a smart carrier in microfluidic and lab-on-a-chip biosensors. The versatility of MPs has enabled their integration into small single detection units. MPs-based biosensors can facilitate rapid and highly sensitive detection of very small amounts of a sample. In this review, the application of MPs to the detection of nucleic acids, from sample preparation to analytical readout systems, is described. State-of-the-art integrated microsystems containing microfluidic and lab-on-a-chip biosensors for the nucleic acid detection are also addressed. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Quantitative thermodynamic predication of interactions between nucleic acid and non-nucleic acid species using Microsoft excel.

    PubMed

    Zou, Jiaqi; Li, Na

    2013-09-01

    Proper design of nucleic acid sequences is crucial for many applications. We have previously established a thermodynamics-based quantitative model to help design aptamer-based nucleic acid probes by predicting equilibrium concentrations of all interacting species. To facilitate customization of this thermodynamic model for different applications, here we present a generic and easy-to-use platform to implement the algorithm of the model with Microsoft(®) Excel formulas and VBA (Visual Basic for Applications) macros. Two Excel spreadsheets have been developed: one for the applications involving only nucleic acid species, the other for the applications involving both nucleic acid and non-nucleic acid species. The spreadsheets take the nucleic acid sequences and the initial concentrations of all species as input, guide the user to retrieve the necessary thermodynamic constants, and finally calculate equilibrium concentrations for all species in various bound and unbound conformations. The validity of both spreadsheets has been verified by comparing the modeling results with the experimental results on nucleic acid sequences reported in the literature. This Excel-based platform described here will allow biomedical researchers to rationalize the sequence design of nucleic acid probes using the thermodynamics-based modeling even without relevant theoretical and computational skills. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  19. Method of increasing conversion of a fatty acid to its corresponding dicarboxylic acid

    DOEpatents

    Craft, David L.; Wilson, C. Ron; Eirich, Dudley; Zhang, Yeyan

    2004-09-14

    A nucleic acid sequence including a CYP promoter operably linked to nucleic acid encoding a heterologous protein is provided to increase transcription of the nucleic acid. Expression vectors and host cells containing the nucleic acid sequence are also provided. The methods and compositions described herein are especially useful in the production of polycarboxylic acids by yeast cells.

  20. Lab-on-a-chip nucleic-acid analysis towards point-of-care applications

    NASA Astrophysics Data System (ADS)

    Kopparthy, Varun Lingaiah

    Recent infectious disease outbreaks, such as Ebola in 2013, highlight the need for fast and accurate diagnostic tools to combat the global spread of the disease. Detection and identification of the disease-causing viruses and bacteria at the genetic level is required for accurate diagnosis of the disease. Nucleic acid analysis systems have shown promise in identifying diseases such as HIV, anthrax, and Ebola in the past. Conventional nucleic acid analysis systems are still time consuming, and are not suitable for point-ofcare applications. Miniaturized nucleic acid systems has shown great promise for rapid analysis, but they have not been commercialized due to several factors such as footprint, complexity, portability, and power consumption. This dissertation presents the development of technologies and methods for a labon-a-chip nucleic acid analysis towards point-of-care applications. An oscillatory-flow PCR methodology in a thermal gradient is developed which provides real-time analysis of nucleic-acid samples. Oscillating flow PCR was performed in the microfluidic device under thermal gradient in 40 minutes. Reverse transcription PCR (RT-PCR) was achieved in the system without an additional heating element for incubation to perform reverse transcription step. A novel method is developed for the simultaneous pattering and bonding of all-glass microfluidic devices in a microwave oven. Glass microfluidic devices were fabricated in less than 4 minutes. Towards an integrated system for the detection of amplified products, a thermal sensing method is studied for the optimization of the sensor output. Calorimetric sensing method is characterized to identify design considerations and optimal parameters such as placement of the sensor, steady state response, and flow velocity for improved performance. An understanding of these developed technologies and methods will facilitate the development of lab-on-a-chip systems for point-of-care analysis.

  1. Differential display detects host nucleic acid motifs altered in scrapie-infected brain.

    PubMed

    Lathe, Richard; Harris, Alyson

    2009-09-25

    The transmissible spongiform encephalopathies (TSEs) including scrapie have been attributed to an infectious protein or prion. Infectivity is allied to conversion of the endogenous nucleic-acid-binding protein PrP to an infectious modified form known as PrP(sc). The protein-only theory does not easily explain the enigmatic properties of the agent including strain variation. It was previously suggested that a short nucleic acid, perhaps host-encoded, might contribute to the pathoetiology of the TSEs. No candidate host molecules that might explain transmission of strain differences have yet been put forward. Differential display is a robust technique for detecting nucleic acid differences between two populations. We applied this technique to total nucleic acid preparations from scrapie-infected and control brain. Independent RNA preparations from eight normal and eight scrapie-infected (strain 263K) hamster brains were randomly amplified and visualized in parallel. Though the nucleic acid patterns were generally identical in scrapie-infected versus control brain, some rare bands were differentially displayed. Molecular species consistently overrepresented (or underrepresented) in all eight infected brain samples versus all eight controls were excised from the display, sequenced, and assembled into contigs. Only seven ros contigs (RNAs over- or underrepresented in scrapie) emerged, representing <4 kb from the transcriptome. All contained highly stable regions of secondary structure. The most abundant scrapie-only ros sequence was homologous to a repetitive transposable element (LINE; long interspersed nuclear element). Other ros sequences identified cellular RNA 7SL, clathrin heavy chain, visinin-like protein-1, and three highly specific subregions of ribosomal RNA (ros1-3). The ribosomal ros sequences accurately corresponded to LINE; retrotransposon insertion sites in ribosomal DNA (p<0.01). These differential motifs implicate specific host RNAs in the pathoetiology of the TSEs.

  2. Nonenzymatic catalytic signal amplification for nucleic acid hybridization assays

    NASA Technical Reports Server (NTRS)

    Fan, Wenhong (Inventor); Han, Jie (Inventor); Cassell, Alan M. (Inventor)

    2006-01-01

    Devices, methods, and kits for amplifying the signal from hybridization reactions between nucleic acid probes and their cognate targets are presented. The devices provide partially-duplexed, immobilized probe complexes, spatially separate from and separately addressable from immobilized docking strands. Cognate target acts catalytically to transfer probe from the site of probe complex immobilization to the site of immobilized docking strand, generating a detectable signal. The methods and kits of the present invention may be used to identify the presence of cognate target in a fluid sample.

  3. Utility of Helicobacter spp. associated GFD markers for detecting avian fecal pollution in natural waters of two continents.

    PubMed

    Ahmed, W; Harwood, V J; Nguyen, K; Young, S; Hamilton, K; Toze, S

    2016-01-01

    Avian fecal droppings may negatively impact environmental water quality due to the presence of high concentrations of fecal indicator bacteria (FIB) and zoonotic pathogens. This study was aimed at evaluating the performance characteristics and utility of a Helicobacter spp. associated GFD marker by screening 265 fecal and wastewater samples from a range of avian and non-avian host groups from two continents (Brisbane, Australia and Florida, USA). The host-prevalence and -specificity of this marker among fecal and wastewater samples tested from Brisbane were 0.58 and 0.94 (maximum value of 1.00). These values for the Florida fecal samples were 0.30 (host-prevalence) and 1.00 (host-specificity). The concentrations of the GFD markers in avian and non-avian fecal nucleic acid samples were measured at a test concentration of 10 ng of nucleic acid at Brisbane and Florida laboratories using the quantitative PCR (qPCR) assay. The mean concentrations of the GFD marker in avian fecal nucleic acid samples (5.2 × 10(3) gene copies) were two orders of magnitude higher than non-avian fecal nucleic acid samples (8.6 × 10(1) gene copies). The utility of this marker was evaluated by testing water samples from the Brisbane River, Brisbane and a freshwater creek in Florida. Among the 18 water samples tested from the Brisbane River, 83% (n = 18) were positive for the GFD marker, and the concentrations ranged from 6.0 × 10(1)-3.2 × 10(2) gene copies per 100 mL water. In all, 92% (n = 25) water samples from the freshwater creek in Florida were also positive for the GFD marker with concentrations ranging from 2.8 × 10(1)-1.3 × 10(4) gene copies per 100 mL water. Based on the results, it can be concluded that the GFD marker is highly specific to avian host groups, and could be used as a reliable marker to detect the presence and amount of avian fecal pollution in environmental waters. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  4. Enhancing the sensitivity of immunoassay procedures by use of antibodies directed to the product of a reaction between probe labels and assay substrates

    DOEpatents

    Erlanger, B.F.; Chen, B.X.

    1997-07-22

    The subject invention provides an antibody which specifically binds to the product of a reaction between a labeling substance and a substrate. The subject invention also provides a method of making an immunogen used to produce the antibody of the subject invention. The invention further provides methods of using the subject antibody for detecting an antigen of interest in a sample, for example detecting a protein comprising an amino acid sequence of interest and detecting a nucleic acid molecule comprising a nucleic acid sequence of interest. 8 figs.

  5. Enhancing the sensitivity of immunoassay procedures by use of antibodies directed to the product of a reaction between probe labels and assay substrates

    DOEpatents

    Erlanger, Bernard F.; Chen, Bi-Xing

    1997-01-01

    The subject invention provides an antibody which specifically binds to the product of a reaction between a labeling substance and a substrate. The subject invention also provides a method of making an immunogen used to produce the antibody of the subject invention. The invention further provides methods of using the subject antibody for detecting an antigen of interest in a sample, for example detecting a protein comprising an amino acid sequence of interest and detecting a nucleic acid molecule comprising a nucleic acid sequence of interest.

  6. Nucleic Acid Immunity.

    PubMed

    Hartmann, G

    2017-01-01

    Organisms throughout biology need to maintain the integrity of their genome. From bacteria to vertebrates, life has established sophisticated mechanisms to detect and eliminate foreign genetic material or to restrict its function and replication. Tremendous progress has been made in the understanding of these mechanisms which keep foreign or unwanted nucleic acids from viruses or phages in check. Mechanisms reach from restriction-modification systems and CRISPR/Cas in bacteria and archaea to RNA interference and immune sensing of nucleic acids, altogether integral parts of a system which is now appreciated as nucleic acid immunity. With inherited receptors and acquired sequence information, nucleic acid immunity comprises innate and adaptive components. Effector functions include diverse nuclease systems, intrinsic activities to directly restrict the function of foreign nucleic acids (e.g., PKR, ADAR1, IFIT1), and extrinsic pathways to alert the immune system and to elicit cytotoxic immune responses. These effects act in concert to restrict viral replication and to eliminate virus-infected cells. The principles of nucleic acid immunity are highly relevant for human disease. Besides its essential contribution to antiviral defense and restriction of endogenous retroelements, dysregulation of nucleic acid immunity can also lead to erroneous detection and response to self nucleic acids then causing sterile inflammation and autoimmunity. Even mechanisms of nucleic acid immunity which are not established in vertebrates are relevant for human disease when they are present in pathogens such as bacteria, parasites, or helminths or in pathogen-transmitting organisms such as insects. This review aims to provide an overview of the diverse mechanisms of nucleic acid immunity which mostly have been looked at separately in the past and to integrate them under the framework nucleic acid immunity as a basic principle of life, the understanding of which has great potential to advance medicine. © 2017 Elsevier Inc. All rights reserved.

  7. Isothermal amplification detection of nucleic acids by a double-nicked beacon.

    PubMed

    Shi, Chao; Zhou, Meiling; Pan, Mei; Zhong, Guilin; Ma, Cuiping

    2016-03-01

    Isothermal and rapid amplification detection of nucleic acids is an important technology in environmental monitoring, foodborne pathogen detection, and point-of-care clinical diagnostics. Here we have developed a novel method of isothermal signal amplification for single-stranded DNA (ssDNA) detection. The ssDNA target could be used as an initiator, coupled with a double-nicked molecular beacon, to originate amplification cycles, achieving cascade signal amplification. In addition, the method showed good specificity and strong anti-jamming capability. Overall, it is a one-pot and isothermal strand displacement amplification method without the requirement of a stepwise procedure, which greatly simplifies the experimental procedure and decreases the probability of contamination of samples. With its advantages, the method would be very useful to detect nucleic acids in point-of-care or field use. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Use of FTA cards for the storage of breast carcinoma nucleic acid on fine-needle aspiration samples.

    PubMed

    Peluso, Anna Lucia; Cascone, Anna Maria; Lucchese, Lucrezia; Cozzolino, Immacolata; Ieni, Antonio; Mignogna, Chiara; Pepe, Stefano; Zeppa, Pio

    2015-10-01

    The preservation and storage of nucleic acids is important for DNA molecular techniques. The material obtained by fine-needle aspiration (FNA) is often scanty and can not be wasted. FTA cards are filter papers that immobilize and stabilize nucleic acids and can be stored at room temperature. The current study evaluated whether nucleic acids of breast carcinoma cells, obtained by FNA in a clinical setting, may be collected, stored, and preserved on FTA cards. Thirty breast carcinoma, 5 non-Hodgkin lymphoma (NHL), and 5 benign reactive lymph node (RLN) cell samples obtained by FNA were stored at -80 °C and on FTA cards. DNA extraction and polymerase chain reaction were performed on cells at -80 °C and on 2 punched disks of FTA cards. Fifty nanograms of extracted DNA from both sample types were used to amplify the Janus Kinase 2 (JAK2) gene. The mean value of DNA extracted from breast carcinoma cells was 28.19 ng/µL for that stored at -80 °C and 3.28 ng/µL for that stored on FTA cards. Agarose gel analysis demonstrated expected bands of DNA in 29 cases (97%) with both methods. The mean value of DNA extracted from NHL and RLN samples was 37.54 ng/µL and 4.28 ng/µL, respectively, and agarose gel analysis demonstrated bands of high molecular weight DNA in both methods. Significant differences in DNA yield were found between storage at -80 °C and FTA cards (P<.0001), but no differences were detected between 260/280 nm ratios in breast carcinoma and NHL/RLN samples. FTA cards can be conveniently used for the storage of breast carcinoma cells obtained by FNA, thus providing a reliable alternative to traditional methods. © 2015 American Cancer Society.

  9. Detection of Shigella spp. nucleic acids in the synovial tissue of Tunisian rheumatoid arthritis patients and other forms of arthritis by quantitative real-time polymerase chain reaction.

    PubMed

    Siala, Mariam; Rihl, Markus; Sellami, Hanen; Znazen, Abir; Sassi, Nadia; Laadhar, Lilia; Gdoura, Radhouane; Belghuith, Imen; Mrabet, Dalila; Baklouti, Sofien; Sellami, Slaheddine; Sibilia, Jean; Fourati, Hela; Hammami, Adnene; Cheour, Ilhem

    2018-06-01

    Enterobacterial components in the joints of patients are believed to contribute to a perpetuating inflammation leading to a reactive arthritis (ReA), a condition in which microbial agents cannot be recovered from the joint. At present, it is unclear whether nucleic acids from Shigella spp. are playing a pathogenic role in causing not only ReA but also other forms of arthritis. Quantitative real-time polymerase chain reaction assay (qPCR) is the method of choice for the identification of bacteria within the synovium. The aim of our study was to detect the presence of Shigella spp. nucleic acids in the synovial tissue (ST) of Tunisian arthritis patients. We investigated 57 ST samples from rheumatoid arthritis (RA) n = 38, undifferentiated oligoarthritis (UOA) n = 12, and spondyloarthritis (SpA) n = 7 patients; 5 ST samples from healthy individuals were used as controls. Shigella spp. DNA and mRNA transcripts encoding the virulence gene A (VirA) were examined using an optimized qPCR with newly designed primers and probes. Using qPCR, Shigella spp. DNA was found in 37/57 (65%) ST samples (24/38, i.e., 63.2% of RA, 8/12, i.e., 67% of UOA, and 5/7, i.e., 71.4% of SpA patients). Paired DNA and mRNA were extracted from 39 ST samples, whose VirA cDNA was found in 29/39 (74.4%) patients. qPCR did not yield any nucleic acids in the five healthy control ST samples. The qPCR assay was sensitive and showed a good intra- and inter-run reproducibility. These preliminary findings generated by an optimized, highly sensitive PCR assay underline a potential role of past gastrointestinal infections. In Tunisian patients, a bacterial etiology involving Shigella spp. in the manifestation of arthritic disorders including RA might be more common than expected.

  10. Real-time assays with molecular beacons and other fluorescent nucleic acid hybridization probes.

    PubMed

    Marras, Salvatore A E; Tyagi, Sanjay; Kramer, Fred Russell

    2006-01-01

    A number of formats for nucleic acid hybridization have been developed to identify DNA and RNA sequences that are involved in cellular processes and that aid in the diagnosis of genetic and infectious diseases. The introduction of hybridization probes with interactive fluorophore pairs has enabled the development of homogeneous hybridization assays for the direct identification of nucleic acids. A change in the fluorescence of these probes indicates the presence of a target nucleic acid, and there is no need to separate unbound probes from hybridized probes. The advantages of homogeneous hybridization assays are their speed and simplicity. In addition, homogeneous assays can be combined with nucleic acid amplification, enabling the detection of rare target nucleic acids. These assays can be followed in real time, providing quantitative determination of target nucleic acids over a broad range of concentrations.

  11. Applications of synchrotron-based spectroscopic techniques in studying nucleic acids and nucleic acid-functionalized nanomaterials

    PubMed Central

    Wu, Peiwen; Yu, Yang; McGhee, Claire E.; Tan, Li Huey

    2014-01-01

    In this review, we summarize recent progresses in the application of synchrotron-based spectroscopic techniques for nucleic acid research that takes advantage of high-flux and high-brilliance electromagnetic radiation from synchrotron sources. The first section of the review focuses on the characterization of the structure and folding processes of nucleic acids using different types of synchrotron-based spectroscopies, such as X-ray absorption spectroscopy, X-ray emission spectroscopy, X-ray photoelectron spectroscopy, synchrotron radiation circular dichroism, X-ray footprinting and small-angle X-ray scattering. In the second section, the characterization of nucleic acid-based nanostructures, nucleic acid-functionalized nanomaterials and nucleic acid-lipid interactions using these spectroscopic techniques is summarized. Insights gained from these studies are described and future directions of this field are also discussed. PMID:25205057

  12. Applications of synchrotron-based spectroscopic techniques in studying nucleic acids and nucleic acid-functionalized nanomaterials

    DOE PAGES

    Wu, Peiwen; Yu, Yang; McGhee, Claire E.; ...

    2014-09-10

    In this paper, we summarize recent progress in the application of synchrotron-based spectroscopic techniques for nucleic acid research that takes advantage of high-flux and high-brilliance electromagnetic radiation from synchrotron sources. The first section of the review focuses on the characterization of the structure and folding processes of nucleic acids using different types of synchrotron-based spectroscopies, such as X-ray absorption spectroscopy, X-ray emission spectroscopy, X-ray photoelectron spectroscopy, synchrotron radiation circular dichroism, X-ray footprinting and small-angle X-ray scattering. In the second section, the characterization of nucleic acid-based nanostructures, nucleic acid-functionalized nanomaterials and nucleic acid-lipid interactions using these spectroscopic techniques is summarized. Insightsmore » gained from these studies are described and future directions of this field are also discussed.« less

  13. 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Respiratory viral panel multiplex nucleic acid... § 866.3980 Respiratory viral panel multiplex nucleic acid assay. (a) Identification. A respiratory viral... simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or...

  14. 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Respiratory viral panel multiplex nucleic acid... § 866.3980 Respiratory viral panel multiplex nucleic acid assay. (a) Identification. A respiratory viral... simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or...

  15. 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Respiratory viral panel multiplex nucleic acid... § 866.3980 Respiratory viral panel multiplex nucleic acid assay. (a) Identification. A respiratory viral... simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or...

  16. 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Respiratory viral panel multiplex nucleic acid... § 866.3980 Respiratory viral panel multiplex nucleic acid assay. (a) Identification. A respiratory viral... simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or...

  17. 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Respiratory viral panel multiplex nucleic acid... § 866.3980 Respiratory viral panel multiplex nucleic acid assay. (a) Identification. A respiratory viral... simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or...

  18. The value of cell-free DNA for molecular pathology.

    PubMed

    Stewart, Caitlin M; Kothari, Prachi D; Mouliere, Florent; Mair, Richard; Somnay, Saira; Benayed, Ryma; Zehir, Ahmet; Weigelt, Britta; Dawson, Sarah-Jane; Arcila, Maria E; Berger, Michael F; Tsui, Dana Wy

    2018-04-01

    Over the past decade, advances in molecular biology and genomics techniques have revolutionized the diagnosis and treatment of cancer. The technological advances in tissue profiling have also been applied to the study of cell-free nucleic acids, an area of increasing interest for molecular pathology. Cell-free nucleic acids are released from tumour cells into the surrounding body fluids and can be assayed non-invasively. The repertoire of genomic alterations in circulating tumour DNA (ctDNA) is reflective of both primary tumours and distant metastatic sites, and ctDNA can be sampled multiple times, thereby overcoming the limitations of the analysis of single biopsies. Furthermore, ctDNA can be sampled regularly to monitor response to treatment, to define the evolution of the tumour genome, and to assess the acquisition of resistance and minimal residual disease. Recently, clinical ctDNA assays have been approved for guidance of therapy, which is an exciting first step in translating cell-free nucleic acid research tests into clinical use for oncology. In this review, we discuss the advantages of cell-free nucleic acids as analytes in different body fluids, including blood plasma, urine, and cerebrospinal fluid, and their clinical applications in solid tumours and haematological malignancies. We will also discuss practical considerations for clinical deployment, such as preanalytical factors and regulatory requirements. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  19. Nanoplasmonic molecular ruler for nuclease activity and DNA footprinting

    DOEpatents

    Chen, Fanqing Frank; Liu, Gang L; Lee, Luke P

    2013-10-29

    This invention provides a nanoplasmonic molecular ruler, which can perform label-free and real-time monitoring of nucleic acid (e.g., DNA) length changes and perform nucleic acid footprinting. In various embodiments the ruler comprises a nucleic acid attached to a nanoparticle, such that changes in the nucleic acid length are detectable using surface plasmon resonance. The nanoplasmonic ruler provides a fast and convenient platform for mapping nucleic acid-protein interactions, for nuclease activity monitoring, and for other footprinting related methods.

  20. [Qualitative analysis of bis-(3'-5')-cyclic dimeric adenosine monophosphate of Porphyromonas gingivalis by high performance liquid chromatography coupled with mass spectrometry].

    PubMed

    Yongmei, Tan; Xiaojun, Yang; Juan, Du; Wanghong, Zhao; Xiaodan, Chen; Jin, Hou

    2016-06-01

    To test whether Porphyromonas gingivalis (P. gingivalis) could produce bacterial signal molecule, bis-(3'-5')-cyclic dimeric adenosine monophosphate (c-di-AMP) and lay the foundation for explorations of its roles in life metabolism and periodontitis immunity of P. gingivalis. P. gingivalis standard strain ATCC33277 was used as the experimental strain to extract nucleic acids from the bacteria. Then, c-di-AMP was detected using high performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS). Subsequently, HPLC was used to validate the sample further. Based on the signal/noise (S/N) for 3 : 1, the limit of determination of HPLC-MS/MS for peak time of c-di-AMP standard substances was 7.49 min and nucleic acid extractions from P. gingivalis was 8.82 min (S/N > 3). Further confirmation of HPLC showed that nucleic acid extractions from both P. gingivalis and c-di-AMP standard substances pre- sented goal absorbent peaks at 15.7 min, with the same ultraviolet absorbent spectrum. The nucleic acid extrac- tions from P. gingivalis contained c-di-AMP, which shows that P. gingivalis could produce c-di-AMP.

  1. Use of CYP52A2A promoter to increase gene expression in yeast

    DOEpatents

    Craft, David L.; Wilson, C. Ron; Eirich, Dudley; Zhang, Yeyan

    2004-01-06

    A nucleic acid sequence including a CYP promoter operably linked to nucleic acid encoding a heterologous protein is provided to increase transcription of the nucleic acid. Expression vectors and host cells containing the nucleic acid sequence are also provided. The methods and compositions described herein are especially useful in the production of polycarboxylic acids by yeast cells.

  2. Lipid and polymeric carrier-mediated nucleic acid delivery

    PubMed Central

    Zhu, Lin; Mahato, Ram I

    2010-01-01

    Importance of the field Nucleic acids such as plasmid DNA, antisense oligonucleotide, and RNA interference (RNAi) molecules, have a great potential to be used as therapeutics for the treatment of various genetic and acquired diseases. To design a successful nucleic acid delivery system, the pharmacological effect of nucleic acids, the physiological condition of the subjects or sites, and the physicochemical properties of nucleic acid and carriers have to be thoroughly examined. Areas covered in this review The commonly used lipids, polymers and corresponding delivery systems are reviewed in terms of their characteristics, applications, advantages and limitations. What the reader will gain This article aims to provide an overview of biological barriers and strategies to overcome these barriers by properly designing effective synthetic carriers for nucleic acid delivery. Take home message A thorough understanding of biological barriers and the structure–activity relationship of lipid and polymeric carriers is the key for effective nucleic acid therapy. PMID:20836625

  3. Electrophoretic Mobility Shift Assay (EMSA) for Detecting Protein-Nucleic Acid Interactions

    PubMed Central

    Hellman, Lance M.; Fried, Michael G.

    2009-01-01

    The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this article, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided. PMID:17703195

  4. Sensitive life detection strategies for low-biomass environments: optimizing extraction of nucleic acids adsorbing to terrestrial and Mars analogue minerals.

    PubMed

    Direito, Susana O L; Marees, Andries; Röling, Wilfred F M

    2012-07-01

    The adsorption of nucleic acids to mineral matrixes can result in low extraction yields and negatively influences molecular microbial ecology studies, in particular for low-biomass environments on Earth and Mars. We determined the recovery of nucleic acids from a range of minerals relevant to Earth and Mars. Clay minerals, but also other silicates and nonsilicates, showed very low recovery (< 1%). Consequently, optimization of DNA extraction was directed towards clays. The high temperatures and acidic conditions used in some methods to dissolve mineral matrices proved to destruct DNA. The most efficient method comprised a high phosphate solution (P/EtOH; 1 M phosphate, 15% ethanol buffer at pH 8) introduced at the cell-lysing step in DNA extraction, to promote chemical competition with DNA for adsorption sites. This solution increased DNA yield from clay samples spiked with known quantities of cells up to nearly 100-fold. DNA recovery was also enhanced from several mineral samples retrieved from an aquifer, while maintaining reproducible DGGE profiles. DGGE profiles were obtained for a clay sample for which no profile could be generated with the standard DNA isolation protocol. Mineralogy influenced microbial community composition. The method also proved suitable for the recovery of low molecular weight DNA (< 1.5 kb). © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  5. Refinement of Generalized Born Implicit Solvation Parameters for Nucleic Acids and their Complexes with Proteins

    PubMed Central

    Nguyen, Hai; Pérez, Alberto; Bermeo, Sherry; Simmerling, Carlos

    2016-01-01

    The Generalized Born (GB) implicit solvent model has undergone significant improvements in accuracy for modeling of proteins and small molecules. However, GB still remains a less widely explored option for nucleic acid simulations, in part because fast GB models are often unable to maintain stable nucleic acid structures, or they introduce structural bias in proteins, leading to difficulty in application of GB models in simulations of protein-nucleic acid complexes. Recently, GB-neck2 was developed to improve the behavior of protein simulations. In an effort to create a more accurate model for nucleic acids, a similar procedure to the development of GB-neck2 is described here for nucleic acids. The resulting parameter set significantly reduces absolute and relative energy error relative to Poisson Boltzmann for both nucleic acids and nucleic acid-protein complexes, when compared to its predecessor GB-neck model. This improvement in solvation energy calculation translates to increased structural stability for simulations of DNA and RNA duplexes, quadruplexes, and protein-nucleic acid complexes. The GB-neck2 model also enables successful folding of small DNA and RNA hairpins to near native structures as determined from comparison with experiment. The functional form and all required parameters are provided here and also implemented in the AMBER software. PMID:26574454

  6. Cellular nucleic acid binding protein binds G-rich single-stranded nucleic acids and may function as a nucleic acid chaperone.

    PubMed

    Armas, Pablo; Nasif, Sofía; Calcaterra, Nora B

    2008-02-15

    Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression. Copyright 2007 Wiley-Liss, Inc.

  7. Synthetic oligonucleotide antigens modified with locked nucleic acids detect disease specific antibodies

    NASA Astrophysics Data System (ADS)

    Samuelsen, Simone V.; Solov'Yov, Ilia A.; Balboni, Imelda M.; Mellins, Elizabeth; Nielsen, Christoffer Tandrup; Heegaard, Niels H. H.; Astakhova, Kira

    2016-10-01

    New techniques to detect and quantify antibodies to nucleic acids would provide a significant advance over current methods, which often lack specificity. We investigate the potential of novel antigens containing locked nucleic acids (LNAs) as targets for antibodies. Particularly, employing molecular dynamics we predict optimal nucleotide composition for targeting DNA-binding antibodies. As a proof of concept, we address a problem of detecting anti-DNA antibodies that are characteristic of systemic lupus erythematosus, a chronic autoimmune disease with multiple manifestations. We test the best oligonucleotide binders in surface plasmon resonance studies to analyze binding and kinetic aspects of interactions between antigens and target DNA. These DNA and LNA/DNA sequences showed improved binding in enzyme-linked immunosorbent assay using human samples of pediatric lupus patients. Our results suggest that the novel method is a promising tool to create antigens for research and point-of-care monitoring of anti-DNA antibodies.

  8. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6

    PubMed Central

    Gootenberg, Jonathan S.; Abudayyeh, Omar O.; Kellner, Max J.; Joung, Julia; Collins, James J.; Zhang, Feng

    2018-01-01

    Rapid detection of nucleic acids is integral for clinical diagnostics and biotechnological applications. We recently developed a platform termed SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) that combines isothermal pre-amplification with Cas13 to detect single molecules of RNA or DNA. Through characterization of CRISPR enzymology and application development, we report here four advances integrated into SHERLOCKv2: 1) 4-channel single reaction multiplexing using orthogonal CRISPR enzymes; 2) quantitative measurement of input down to 2 aM; 3) 3.5-fold increase in signal sensitivity by combining Cas13 with Csm6, an auxilary CRISPR-associated enzyme; and 4) lateral flow read-out. SHERLOCKv2 can detect Dengue or Zika virus ssRNA as well as mutations in patient liquid biopsy samples via lateral flow, highlighting its potential as a multiplexable, portable, rapid, and quantitative detection platform of nucleic acids. PMID:29449508

  9. Nucleic Acid-Induced Resistance to Viral Infection

    PubMed Central

    Takano, Kouichi; Warren, Joel; Jensen, Keith E.; Neal, Alan L.

    1965-01-01

    Takano, Kouichi (Chas. Pfizer & Co., Inc., Terre Haute, Ind.), Joel Warren, Keith E. Jensen, and Alan L. Neal. Nucleic acid resistance to viral infection. J. Bacteriol. 90:1542–1547. 1965.—Administration of nonviral nucleic acids to mice increased their resistance to a subsequent infection with influenza or encephalomyocarditis viruses. Injection of ribonucleic acid or deoxyribonucleic acid by peripheral routes did not modify susceptibility to intranasal infection. Lung tissue extracts from animals previously treated with yeast nucleic acid inhibited the growth of vaccinia and influenza viruses. The protective effect of exogenous nucleic acids persisted in mice for several days, but gradually diminished to undetectable levels. PMID:4285332

  10. Preventing disease transmission by deceased tissue donors by testing blood for viral nucleic acid.

    PubMed

    Strong, D Michael; Nelson, Karen; Pierce, Marge; Stramer, Susan L

    2005-01-01

    Nucleic acid testing (NAT) has reduced the risk of transmitting infectious disease through blood transfusion. Currently NAT for HIV-1 and HCV are FDA licensed and performed by nearly all blood collection facilities, but HBV NAT is performed under an investigational study protocol. Residual risk estimates indicate that NAT could potentially reduce disease transmission through transplanted tissue. However, tissue donor samples obtained post-mortem have the potential to produce an invalid NAT result due to inhibition of amplification reactions by hemolysis and other factors. The studies reported here summarize the development of protocols to allow NAT of deceased donor samples with reduced rates of invalid results. Using these protocols, inventories from two tissue centers were tested with greater than 99% of samples producing a valid test result.

  11. Artificial mismatch hybridization

    DOEpatents

    Guo, Zhen; Smith, Lloyd M.

    1998-01-01

    An improved nucleic acid hybridization process is provided which employs a modified oligonucleotide and improves the ability to discriminate a control nucleic acid target from a variant nucleic acid target containing a sequence variation. The modified probe contains at least one artificial mismatch relative to the control nucleic acid target in addition to any mismatch(es) arising from the sequence variation. The invention has direct and advantageous application to numerous existing hybridization methods, including, applications that employ, for example, the Polymerase Chain Reaction, allele-specific nucleic acid sequencing methods, and diagnostic hybridization methods.

  12. Transgenic cells with increased plastoquinone levels and methods of use

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

    Sayre, Richard T.; Subramanian, Sowmya; Cahoon, Edgar

    Disclosed herein are transgenic cells expressing a heterologous nucleic acid encoding a prephenate dehydrogenase (PDH) protein, a heterologous nucleic acid encoding a homogentisate solanesyl transferase (HST) protein, a heterologous nucleic acid encoding a deoxyxylulose phosphate synthase (DXS) protein, or a combination of two or more thereof. In particular examples, the disclosed transgenic cells have increased plastoquinone levels. Also disclosed are methods of increasing cell growth rates or production of biomass by cultivating transgenic cells expressing a heterologous nucleic acid encoding a PDH protein, a heterologous nucleic acid encoding an HST protein, a heterologous nucleic acid encoding a DXS protein, ormore » a combination of two or more thereof under conditions sufficient to produce cell growth or biomass.« less

  13. Enhanced conformational sampling of nucleic acids by a new Hamiltonian replica exchange molecular dynamics approach.

    PubMed

    Curuksu, Jeremy; Zacharias, Martin

    2009-03-14

    Although molecular dynamics (MD) simulations have been applied frequently to study flexible molecules, the sampling of conformational states separated by barriers is limited due to currently possible simulation time scales. Replica-exchange (Rex)MD simulations that allow for exchanges between simulations performed at different temperatures (T-RexMD) can achieve improved conformational sampling. However, in the case of T-RexMD the computational demand grows rapidly with system size. A Hamiltonian RexMD method that specifically enhances coupled dihedral angle transitions has been developed. The method employs added biasing potentials as replica parameters that destabilize available dihedral substates and was applied to study coupled dihedral transitions in nucleic acid molecules. The biasing potentials can be either fixed at the beginning of the simulation or optimized during an equilibration phase. The method was extensively tested and compared to conventional MD simulations and T-RexMD simulations on an adenine dinucleotide system and on a DNA abasic site. The biasing potential RexMD method showed improved sampling of conformational substates compared to conventional MD simulations similar to T-RexMD simulations but at a fraction of the computational demand. It is well suited to study systematically the fine structure and dynamics of large nucleic acids under realistic conditions including explicit solvent and ions and can be easily extended to other types of molecules.

  14. [Comparison of MPure-12 Automatic Nucleic Acid Purification and Chelex-100 Method].

    PubMed

    Shen, X; Li, M; Wang, Y L; Chen, Y L; Lin, Y; Zhao, Z M; Que, T Z

    2017-04-01

    To explore the forensic application value of MPure-12 automatic nucleic acid purification （MPure-12 Method） for DNA extraction by extracting and typing DNA from bloodstains and various kinds of biological samples with different DNA contents. Nine types of biological samples, such as bloodstains, semen stains, and saliva were collected. DNA were extracted using MPure-12 method and Chelex-100 method, followed by PCR amplification and electrophoresis for obtaining STR-profiles. The samples such as hair root, chutty, butt, muscular tissue, saliva stain, bloodstain and semen stain were typed successfully by MPure-12 method. Partial alleles were lacked in the samples of saliva, and the genotyping of contact swabs was unsatisfactory. Additional, all of the bloodstains （20 μL, 15 μL, 10 μL, 5 μL, 1 μL） showed good typing results using Chelex-100 method. But the loss of alleles occurred in 1 μL blood volume by MPure-12 method. MPure-12 method is suitable for DNA extraction of a certain concentration blood samples．Chelex-100 method may be better for the extraction of trace blood samples．This instrument used in nucleic acid extraction has the advantages of simplicity of operator, rapidity, high extraction efficiency, high rate of reportable STR-profiles and lower man-made pollution. Copyright© by the Editorial Department of Journal of Forensic Medicine

  15. Principles of nucleic acid hybridization and comparison with monoclonal antibody technology for the diagnosis of infectious diseases.

    PubMed Central

    Edberg, S. C.

    1985-01-01

    Until the 1980s the diagnosis of specific etiologic agents of infectious diseases rested with their isolation in vitro and identification by analysis of their phenotypic characteristics. In the 1970s the concept of a microbial species evolved from phenotypic analysis to nucleic acid homology. Currently, nucleic acid sequences specific for a given species are being isolated and amplified and utilized not only to identify the pathogen after it has been grown in vitro but also elucidate it directly in biological material. The procedures for making nucleic acid hybridization probes are analogous to the generation of monoclonal antibody tests. Currently, research and development are centered in choosing the particular nucleic acid to analyze, establishing the most efficient vector system for amplifying the nucleic acid, generating an efficient means of selecting the particular nucleic acid fragment specific for the microorganism, and in measuring the hybridization reaction. While immunological techniques have been utilized in the clinical laboratory for over thirty years, the means of detecting nucleic acid hybridization reactions are just beginning to be usable in the clinical diagnostic laboratory. Much of nucleic acid hybridization research is proprietary, and a particular challenge is to develop a means whereby information can be used for the progress of science as a whole when generated by private ownership. Images FIG. 4 PMID:3004048

  16. Simplified Microarray Technique for Identifying mRNA in Rare Samples

    NASA Technical Reports Server (NTRS)

    Almeida, Eduardo; Kadambi, Geeta

    2007-01-01

    Two simplified methods of identifying messenger ribonucleic acid (mRNA), and compact, low-power apparatuses to implement the methods, are at the proof-of-concept stage of development. These methods are related to traditional methods based on hybridization of nucleic acid, but whereas the traditional methods must be practiced in laboratory settings, these methods could be practiced in field settings. Hybridization of nucleic acid is a powerful technique for detection of specific complementary nucleic acid sequences, and is increasingly being used for detection of changes in gene expression in microarrays containing thousands of gene probes. A traditional microarray study entails at least the following six steps: 1. Purification of cellular RNA, 2. Amplification of complementary deoxyribonucleic acid [cDNA] by polymerase chain reaction (PCR), 3. Labeling of cDNA with fluorophores of Cy3 (a green cyanine dye) and Cy5 (a red cyanine dye), 4. Hybridization to a microarray chip, 5. Fluorescence scanning the array(s) with dual excitation wavelengths, and 6. Analysis of the resulting images. This six-step procedure must be performed in a laboratory because it requires bulky equipment.

  17. dbAMEPNI: a database of alanine mutagenic effects for protein–nucleic acid interactions

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

    Liu, Ling; Xiong, Yi; Gao, Hongyun

    Protein–nucleic acid interactions play essential roles in various biological activities such as gene regulation, transcription, DNA repair and DNA packaging. Understanding the effects of amino acid substitutions on protein–nucleic acid binding affinities can help elucidate the molecular mechanism of protein–nucleic acid recognition. Until now, no comprehensive and updated database of quantitative binding data on alanine mutagenic effects for protein–nucleic acid interactions is publicly accessible. Thus, we developed a new database of Alanine Mutagenic Effects for Protein-Nucleic Acid Interactions (dbAMEPNI). dbAMEPNI is a manually curated, literature-derived database, comprising over 577 alanine mutagenic data with experimentally determined binding affinities for protein–nucleic acidmore » complexes. Here, it contains several important parameters, such as dissociation constant (Kd), Gibbs free energy change (ΔΔG), experimental conditions and structural parameters of mutant residues. In addition, the database provides an extended dataset of 282 single alanine mutations with only qualitative data (or descriptive effects) of thermodynamic information.« less

  18. dbAMEPNI: a database of alanine mutagenic effects for protein–nucleic acid interactions

    DOE PAGES

    Liu, Ling; Xiong, Yi; Gao, Hongyun; ...

    2018-04-02

    Protein–nucleic acid interactions play essential roles in various biological activities such as gene regulation, transcription, DNA repair and DNA packaging. Understanding the effects of amino acid substitutions on protein–nucleic acid binding affinities can help elucidate the molecular mechanism of protein–nucleic acid recognition. Until now, no comprehensive and updated database of quantitative binding data on alanine mutagenic effects for protein–nucleic acid interactions is publicly accessible. Thus, we developed a new database of Alanine Mutagenic Effects for Protein-Nucleic Acid Interactions (dbAMEPNI). dbAMEPNI is a manually curated, literature-derived database, comprising over 577 alanine mutagenic data with experimentally determined binding affinities for protein–nucleic acidmore » complexes. Here, it contains several important parameters, such as dissociation constant (Kd), Gibbs free energy change (ΔΔG), experimental conditions and structural parameters of mutant residues. In addition, the database provides an extended dataset of 282 single alanine mutations with only qualitative data (or descriptive effects) of thermodynamic information.« less

  19. Introduction of structural affinity handles as a tool in selective nucleic acid separations

    NASA Technical Reports Server (NTRS)

    Willson, III, Richard Coale (Inventor); Cano, Luis Antonio (Inventor)

    2011-01-01

    The method is used for separating nucleic acids and other similar constructs. It involves selective introduction, enhancement, or stabilization of affinity handles such as single-strandedness in the undesired (or desired) nucleic acids as compared to the usual structure (e.g., double-strandedness) of the desired (or undesired) nucleic acids. The undesired (or desired) nucleic acids are separated from the desired (or undesired) nucleic acids due to capture by methods including but not limited to immobilized metal affinity chromatography, immobilized single-stranded DNA binding (SSB) protein, and immobilized oligonucleotides. The invention is useful to: remove contaminating genomic DNA from plasmid DNA; remove genomic DNA from plasmids, BACs, and similar constructs; selectively separate oligonucleotides and similar DNA fragments from their partner strands; purification of aptamers, (deoxy)-ribozymes and other highly structured nucleic acids; Separation of restriction fragments without using agarose gels; manufacture recombinant Taq polymerase or similar products that are sensitive to host genomic DNA contamination; and other applications.

  20. Selective Attachment of Nucleic Acid Molecules to Patterned Self-Assembled Surfaces.

    DTIC Science & Technology

    1994-12-01

    of different sequence is accomplished by placement of 8 liquid portions of nucleic acids at the desired position on the 9 filter. This method is...acids are selectively 24 bound from regions to which nucleic acids are excluded, other than 25 by placement of liquid aliquots (generally >1 Al) of...is typically non-covalent (i.e., ionic 16 bonding, or, less often, hydrogen bonding). Advantageously, non- 17 covalent bonding of nucleic acid

  1. European Science Notes, Volume 40, Number 7.

    DTIC Science & Technology

    1986-07-01

    for example, University of i.e., the details of protein-protein, Gbttingen--have departments of biochem- protein-nucleic acid , and nucleic acid ...istry but do not award degrees in bio- nucleic acid interactions and their reg- chemistry.) The Institute for Biochem- ulation is still to be resolved. A...tertiary structure acids structure and function; protein/ of the 5S rRNA molecule--the folding of nucleic- acid interactions; molecular the entire molecule of

  2. Reactivity of Nucleic Acid Radicals

    PubMed Central

    Greenberg, Marc M.

    2016-01-01

    Nucleic acid oxidation plays a vital role in the etiology and treatment of diseases, as well as aging. Reagents that oxidize nucleic acids are also useful probes of the biopolymers’ structure and folding. Radiation scientists have contributed greatly to our understanding of nucleic acid oxidation using a variety of techniques. During the past two decades organic chemists have applied the tools of synthetic and mechanistic chemistry to independently generate and study the reactive intermediates produced by ionizing radiation and other nucleic acid damaging agents. This approach has facilitated resolving mechanistic controversies and lead to the discovery of new reactive processes. PMID:28529390

  3. Cytosolic nucleic acid sensors and innate immune regulation.

    PubMed

    Ori, Daisuke; Murase, Motoya; Kawai, Taro

    2017-03-04

    During viral and bacterial infections, pathogen-derived cytosolic nucleic acids are recognized by the intracellular RNA sensors retinoic acid-inducible gene I and melanoma-differentiated gene 5 and intracellular DNA sensors, including cyclic-di-GMP-AMP synthase, absent in melanoma 2, interferon (IFN)-gamma inducible protein 16, polymerase III, and so on. Binding of intracellular nucleic acids to these sensors activates downstream signaling cascades, resulting in the production of type I IFNs and pro-inflammatory cytokines to induce appropriate systematic immune responses. While these sensors also recognize endogenous nucleic acids and activate immune responses, they can discriminate between self- and non-self-nucleic acids. However, dysfunction of these sensors or failure of regulatory mechanisms causes aberrant activation of immune response and autoimmune disorders. In this review, we focus on how intracellular immune sensors recognize exogenous nucleic acids and activate the innate immune system, and furthermore, how autoimmune diseases result from dysfunction of these sensors.

  4. Full genome virus detection in fecal samples using sensitive nucleic acid preparation, deep sequencing, and a novel iterative sequence classification algorithm.

    PubMed

    Cotten, Matthew; Oude Munnink, Bas; Canuti, Marta; Deijs, Martin; Watson, Simon J; Kellam, Paul; van der Hoek, Lia

    2014-01-01

    We have developed a full genome virus detection process that combines sensitive nucleic acid preparation optimised for virus identification in fecal material with Illumina MiSeq sequencing and a novel post-sequencing virus identification algorithm. Enriched viral nucleic acid was converted to double-stranded DNA and subjected to Illumina MiSeq sequencing. The resulting short reads were processed with a novel iterative Python algorithm SLIM for the identification of sequences with homology to known viruses. De novo assembly was then used to generate full viral genomes. The sensitivity of this process was demonstrated with a set of fecal samples from HIV-1 infected patients. A quantitative assessment of the mammalian, plant, and bacterial virus content of this compartment was generated and the deep sequencing data were sufficient to assembly 12 complete viral genomes from 6 virus families. The method detected high levels of enteropathic viruses that are normally controlled in healthy adults, but may be involved in the pathogenesis of HIV-1 infection and will provide a powerful tool for virus detection and for analyzing changes in the fecal virome associated with HIV-1 progression and pathogenesis.

  5. Full Genome Virus Detection in Fecal Samples Using Sensitive Nucleic Acid Preparation, Deep Sequencing, and a Novel Iterative Sequence Classification Algorithm

    PubMed Central

    Cotten, Matthew; Oude Munnink, Bas; Canuti, Marta; Deijs, Martin; Watson, Simon J.; Kellam, Paul; van der Hoek, Lia

    2014-01-01

    We have developed a full genome virus detection process that combines sensitive nucleic acid preparation optimised for virus identification in fecal material with Illumina MiSeq sequencing and a novel post-sequencing virus identification algorithm. Enriched viral nucleic acid was converted to double-stranded DNA and subjected to Illumina MiSeq sequencing. The resulting short reads were processed with a novel iterative Python algorithm SLIM for the identification of sequences with homology to known viruses. De novo assembly was then used to generate full viral genomes. The sensitivity of this process was demonstrated with a set of fecal samples from HIV-1 infected patients. A quantitative assessment of the mammalian, plant, and bacterial virus content of this compartment was generated and the deep sequencing data were sufficient to assembly 12 complete viral genomes from 6 virus families. The method detected high levels of enteropathic viruses that are normally controlled in healthy adults, but may be involved in the pathogenesis of HIV-1 infection and will provide a powerful tool for virus detection and for analyzing changes in the fecal virome associated with HIV-1 progression and pathogenesis. PMID:24695106

  6. Simple System for Isothermal DNA Amplification Coupled to Lateral Flow Detection

    PubMed Central

    Roskos, Kristina; Hickerson, Anna I.; Lu, Hsiang-Wei; Ferguson, Tanya M.; Shinde, Deepali N.; Klaue, Yvonne; Niemz, Angelika

    2013-01-01

    Infectious disease diagnosis in point-of-care settings can be greatly improved through integrated, automated nucleic acid testing devices. We have developed an early prototype for a low-cost system which executes isothermal DNA amplification coupled to nucleic acid lateral flow (NALF) detection in a mesofluidic cartridge attached to a portable instrument. Fluid handling inside the cartridge is facilitated through one-way passive valves, flexible pouches, and electrolysis-driven pumps, which promotes a compact and inexpensive instrument design. The closed-system disposable prevents workspace amplicon contamination. The cartridge design is based on standard scalable manufacturing techniques such as injection molding. Nucleic acid amplification occurs in a two-layer pouch that enables efficient heat transfer. We have demonstrated as proof of principle the amplification and detection of Mycobacterium tuberculosis (M.tb) genomic DNA in the cartridge, using either Loop Mediated Amplification (LAMP) or the Exponential Amplification Reaction (EXPAR), both coupled to NALF detection. We envision that a refined version of this cartridge, including upstream sample preparation coupled to amplification and detection, will enable fully-automated sample-in to answer-out infectious disease diagnosis in primary care settings of low-resource countries with high disease burden. PMID:23922706

  7. Quantitative Investigation of Protein-Nucleic Acid Interactions by Biosensor Surface Plasmon Resonance.

    PubMed

    Wang, Shuo; Poon, Gregory M K; Wilson, W David

    2015-01-01

    Biosensor-surface plasmon resonance (SPR) technology has emerged as a powerful label-free approach for the study of nucleic acid interactions in real time. The method provides simultaneous equilibrium and kinetic characterization for biomolecular interactions with low sample requirements and without the need for external probes. A detailed and practical guide for protein-DNA interaction analyses using biosensor-SPR methods is presented. Details of SPR technology and basic fundamentals are described with recommendations on the preparation of the SPR instrument, sensor chips and samples, experimental design, quantitative and qualitative data analyses and presentation. A specific example of the interaction of a transcription factor with DNA is provided with results evaluated by both kinetic and steady-state SPR methods.

  8. Study on the resonance light scattering spectrum of berberine-cetyltrimethylammonium bromide system and the determination of nucleic acids at nanogram levels

    NASA Astrophysics Data System (ADS)

    Liu, Rutao; Yang, Jinghe; Wu, Xia; Sun, Changxia

    2002-02-01

    The interaction of berberine with nucleic acid in the presence of cetyltrimethylammonium bromide (CTMAB) in aqueous solution has been studied by spectrophotometry and resonance light scattering (RLS) spectroscopy. At pH 7.30, the RLS signals of berberine were greatly enhanced by nucleic acid in the region of 300-600 nm characterized by four peaks at 324.0, 386.5, 416.5 and 465.0 nm. The binding properties were examined by using a Scatchard plot based on the measurement of enhanced RLS data at 416.5 nm. Under optimum conditions, the increase of RLS intensity of this system at 416.5 nm is proportional to the concentration of nucleic acid. The linear range is 7.5×10 -9-7.5×10 -5 g ml -1 for calf thymus DNA, 7.5×10 -9-2.5×10 -5 g ml -1 for herring sperm DNA, and 5.0×10 -9-2.5×10 -5 g ml -1 for yeast RNA. The detection limits (S/N=3) are 2.1 ng ml -1 for calf thymus DNA, 6.5 ng ml -1 for herring sperm DNA and 3.5 ng ml -1 for yeast RNA, respectively. Three synthetic samples were analyzed satisfactorily.

  9. Effectiveness of a flow-based device using riboflavin photochemistry in damaging blood-borne viral nucleic acids.

    PubMed

    Zhu, Liguo; Tong, Hongli; Wang, Shufang; Yu, Yang; Liu, Zhong; Li, Changqing; Wang, Deqing

    2018-05-03

    Effectiveness of a flow-based treatment device using riboflavin photochemistry was demonstrated by cytopathic effect method using indicator viruses. However, inactivation efficacy against real blood-borne viruses needs to be evaluated, especially at nucleic acid level. Special plasma samples with varying concentrations of blood-borne virus were selected using a strict blood selection procedure and were treated with device treatment (DT). Nucleic acid test (NAT) using polymerase chain reaction fluorescence method was used to detect virus copies. The NAT value of 4325 in plasma with high Hepatitis B Virus (HBV) concentrations decreased to 1330 with DT. After 100-fold dilution, the NAT value was below the NAT detection limits with DT compared with 23.0 that without DT. The NAT value of 61.9 in plasma with medium HBV concentrations decreased to 37.8 with DT, and after 10-fold dilution, the NAT value was below the NAT detection limits with DT compared with below 20 that without DT. The Ct values of plasma with low concentrations of blood-borne viruses were below the NAT detection limits with DT. There was a dose effect with DT which was effective in blood-borne viruses damaging nucleic acids to a level below the NAT detection limits. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. Revealing Nucleic Acid Mutations Using Förster Resonance Energy Transfer-Based Probes

    PubMed Central

    Junager, Nina P. L.; Kongsted, Jacob; Astakhova, Kira

    2016-01-01

    Nucleic acid mutations are of tremendous importance in modern clinical work, biotechnology and in fundamental studies of nucleic acids. Therefore, rapid, cost-effective and reliable detection of mutations is an object of extensive research. Today, Förster resonance energy transfer (FRET) probes are among the most often used tools for the detection of nucleic acids and in particular, for the detection of mutations. However, multiple parameters must be taken into account in order to create efficient FRET probes that are sensitive to nucleic acid mutations. In this review; we focus on the design principles for such probes and available computational methods that allow for their rational design. Applications of advanced, rationally designed FRET probes range from new insights into cellular heterogeneity to gaining new knowledge of nucleic acid structures directly in living cells. PMID:27472344

  11. RNA-guided transcriptional regulation

    DOEpatents

    Church, George M.; Mali, Prashant G.; Esvelt, Kevin M.

    2016-02-23

    Methods of modulating expression of a target nucleic acid in a cell are provided including introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to DNA, wherein the DNA includes the target nucleic acid, introducing into the cell a second foreign nucleic acid encoding a nuclease-null Cas9 protein that binds to the DNA and is guided by the one or more RNAs, introducing into the cell a third foreign nucleic acid encoding a transcriptional regulator protein or domain, wherein the one or more RNAs, the nuclease-null Cas9 protein, and the transcriptional regulator protein or domain are expressed, wherein the one or more RNAs, the nuclease-null Cas9 protein and the transcriptional regulator protein or domain co-localize to the DNA and wherein the transcriptional regulator protein or domain regulates expression of the target nucleic acid.

  12. Crystallization and X-ray diffraction analysis of an 'all-locked' nucleic acid duplex derived from a tRNA(Ser) microhelix.

    PubMed

    Behling, Katja; Eichert, André; Fürste, Jens P; Betzel, Christian; Erdmann, Volker A; Förster, Charlotte

    2009-08-01

    Modified nucleic acids are of great interest with respect to their nuclease resistance and enhanced thermostability. In therapeutical and diagnostic applications, such molecules can substitute for labile natural nucleic acids that are targeted against particular diseases or applied in gene therapy. The so-called 'locked nucleic acids' contain modified sugar moieties such as 2'-O,4'-C-methylene-bridged beta-D-ribofuranose and are known to be very stable nucleic acid derivatives. The structure of locked nucleic acids in single or multiple LNA-substituted natural nucleic acids and in LNA-DNA or LNA-RNA heteroduplexes has been well investigated, but the X-ray structure of an ;all-locked' nucleic acid double helix has not been described to date. Here, the crystallization and X-ray diffraction data analysis of an 'all-locked' nucleic acid helix, which was designed as an LNA originating from a tRNA(Ser) microhelix RNA structure, is presented. The crystals belonged to space group C2, with unit-cell parameters a = 77.91, b = 40.74, c = 30.06 A, beta = 91.02 degrees . A high-resolution and a low-resolution data set were recorded, with the high-resolution data showing diffraction to 1.9 A resolution. The crystals contained two double helices per asymmetric unit, with a Matthews coefficient of 2.48 A(3) Da(-1) and a solvent content of 66.49% for the merged data.

  13. Nucleic acid-functionalized transition metal nanosheets for biosensing applications

    PubMed Central

    Mo, Liuting; Li, Juan; Liu, Qiaoling; Qiu, Liping; Tan, Weihong

    2017-01-01

    In clinical diagnostics, as well as food and environmental safety practices, biosensors are powerful tools for monitoring biological or biochemical processes. Two-dimensional (2D) transition metal nanomaterials, including transition metal chalcogenides (TMCs) and transition metal oxides (TMOs), are receiving growing interest for their use in biosensing applications based on such unique properties as high surface area and fluorescence quenching abilities. Meanwhile, nucleic acid probes based on Watson-Crick base-pairing rules are also being widely applied in biosensing based on their excellent recognition capability. In particular, the emergence of functional nucleic acids in the 1980s, especially aptamers, has substantially extended the recognition capability of nucleic acids to various targets, ranging from small organic molecules and metal ions to proteins and cells. Based on π-π stacking interaction between transition metal nanosheets and nucleic acids, biosensing systems can be easily assembled. Therefore, the combination of 2D transition metal nanomaterials and nucleic acids brings intriguing opportunities in bioanalysis and biomedicine. In this review, we summarize recent advances of nucleic acid-functionalized transition metal nanosheets in biosensing applications. The structure and properties of 2D transition metal nanomaterials are first discussed, emphasizing the interaction between transition metal nanosheets and nucleic acids. Then, the applications of nucleic acid-functionalized transition metal nanosheet-based biosensors are discussed in the context of different signal transducing mechanisms, including optical and electrochemical approaches. Finally, we provide our perspectives on the current challenges and opportunities in this promising field. PMID:27020066

  14. Nucleic acid-functionalized transition metal nanosheets for biosensing applications.

    PubMed

    Mo, Liuting; Li, Juan; Liu, Qiaoling; Qiu, Liping; Tan, Weihong

    2017-03-15

    In clinical diagnostics, as well as food and environmental safety practices, biosensors are powerful tools for monitoring biological or biochemical processes. Two-dimensional (2D) transition metal nanomaterials, including transition metal chalcogenides (TMCs) and transition metal oxides (TMOs), are receiving growing interest for their use in biosensing applications based on such unique properties as high surface area and fluorescence quenching abilities. Meanwhile, nucleic acid probes based on Watson-Crick base-pairing rules are also being widely applied in biosensing based on their excellent recognition capability. In particular, the emergence of functional nucleic acids in the 1980s, especially aptamers, has substantially extended the recognition capability of nucleic acids to various targets, ranging from small organic molecules and metal ions to proteins and cells. Based on π-π stacking interaction between transition metal nanosheets and nucleic acids, biosensing systems can be easily assembled. Therefore, the combination of 2D transition metal nanomaterials and nucleic acids brings intriguing opportunities in bioanalysis and biomedicine. In this review, we summarize recent advances of nucleic acid-functionalized transition metal nanosheets in biosensing applications. The structure and properties of 2D transition metal nanomaterials are first discussed, emphasizing the interaction between transition metal nanosheets and nucleic acids. Then, the applications of nucleic acid-functionalized transition metal nanosheet-based biosensors are discussed in the context of different signal transducing mechanisms, including optical and electrochemical approaches. Finally, we provide our perspectives on the current challenges and opportunities in this promising field. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. The implementation of nucleic acid amplification technology testing for living tissue donors.

    PubMed

    Westby, J; Lomas, R J; Kearney, J N

    2010-05-01

    There is a significant requirement within the United Kingdom for tissue grafts from living donors. To ensure safety, blood samples from these donors are tested for pathogens at donation, and at 180 days post donation. Nucleic acid amplification technology (NAT) permits more sensitive detection of pathogens in blood samples than serum antigen testing. NAT testing can be applied to samples from living tissue donors to eliminate the need to re-test these donors 180 days post-donation before grafts can be implanted. This has major financial and operational advantages for a tissue bank, and this manuscript describes how NAT testing was assessed and implemented by NHSBT Tissue Services. When compared to traditional serum antigen testing, NAT testing was more cost effective, more convenient for donors and resulted in a greater proportion of donated grafts being made available for transplant.

  16. Introductory Remarks

    NASA Astrophysics Data System (ADS)

    Lu, Yi; Li, Yingfu

    The emergence of a large number of natural and artificial functional nucleic acids (FNAs; aptamers and nucleic acid enzymes, collectively termed functional nucleic acids in this book) has generated tremendous enthusiasm and new opportunities for molecular scientists from diverse disciplines to devise new concepts and applications. In this volume, we have assembled some leading experts to provide a timely account of recent progress in sensing and other analytical applications that explore functional nucleic acids.

  17. Interactive fluorophore and quencher pairs for labeling fluorescent nucleic acid hybridization probes.

    PubMed

    Marras, Salvatore A E

    2008-03-01

    The use of fluorescent nucleic acid hybridization probes that generate a fluorescence signal only when they bind to their target enables real-time monitoring of nucleic acid amplification assays. Real-time nucleic acid amplification assays markedly improves the ability to obtain qualitative and quantitative results. Furthermore, these assays can be carried out in sealed tubes, eliminating carryover contamination. Fluorescent nucleic acid hybridization probes are available in a wide range of different fluorophore and quencher pairs. Multiple hybridization probes, each designed for the detection of a different nucleic acid sequence and each labeled with a differently colored fluorophore, can be added to the same nucleic acid amplification reaction, enabling the development of high-throughput multiplex assays. In order to develop robust, highly sensitive and specific real-time nucleic acid amplification assays it is important to carefully select the fluorophore and quencher labels of hybridization probes. Selection criteria are based on the type of hybridization probe used in the assay, the number of targets to be detected, and the type of apparatus available to perform the assay. This article provides an overview of different aspects of choosing appropriate labels for the different types of fluorescent hybridization probes used with different types of spectrofluorometric thermal cyclers currently available.

  18. Self-assembling nucleic acid delivery vehicles via linear, water-soluble, cyclodextrin-containing polymers.

    PubMed

    Davis, M E; Pun, S H; Bellocq, N C; Reineke, T M; Popielarski, S R; Mishra, S; Heidel, J D

    2004-01-01

    Non-viral (synthetic) nucleic acid delivery systems have the potential to provide for the practical application of nucleic acid-based therapeutics. We have designed and prepared a tunable, non-viral nucleic acid delivery system that self-assembles with nucleic acids and centers around a new class of polymeric materials; namely, linear, water-soluble cyclodextrin-containing polymers. The relationships between polymer structure and gene delivery are illustrated, and the roles of the cyclodextrin moieties for minimizing toxicity and forming inclusion complexes in the self-assembly processes are highlighted. This vehicle is the first example of a polymer-based gene delivery system formed entirely by self-assembly.

  19. Enhancing the sensitivity of immunoassay procedures by use of antibodies directed to the product of a reaction between probe labels and assay substrates

    DOEpatents

    Erlanger, Bernard F.; Chen, Bi-Xing

    1999-01-01

    The subject invention provides an antibody which specifically binds to the product of a reaction between a labeling substance and a substrate. The subject invention also provides a method of making an immunogen used to produce the antibody of the subject invention. The invention further provides methods of using the subject antibody for detecting an antigen of interest in a sample, for example, detecting a protein comprising an amino acid sequence of interest and detecting a nucleic acid molecule comprising a nucleic acid sequence of interest, detecting a polypeptide such as those expressed by infectious agents, fungi or parasites.

  20. Enhancing the sensitivity of immunoassay procedures by use of antibodies directed to the product of a reaction between probe labels and assay substrates

    DOEpatents

    Erlanger, B.F.; Chen, B.

    1999-07-20

    The subject invention provides an antibody which specifically binds to the product of a reaction between a labeling substance and a substrate. The subject invention also provides a method of making an immunogen used to produce the antibody of the subject invention. The invention further provides methods of using the subject antibody for detecting an antigen of interest in a sample, for example, detecting a protein comprising an amino acid sequence of interest and detecting a nucleic acid molecule comprising a nucleic acid sequence of interest, detecting a polypeptide such as those expressed by infectious agents, fungi or parasites. 25 figs.

  1. EGVII endoglucanase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2014-02-25

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

  2. EGVII endoglucanase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2006-05-16

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

  3. EGVI endoglucanase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

    2008-04-01

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6, and the corresponding EGVI amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

  4. EGVI endoglucanase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2010-10-12

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6, and the corresponding EGVI amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

  5. EGVIII endoglucanase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2006-05-23

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl8, and the corresponding EGVIII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVIII, recombinant EGVIII proteins and methods for producing the same.

  6. EGVI endoglucanase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2010-10-05

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6, and the corresponding EGVI amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

  7. EGVI endoglucanase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2006-06-06

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6, and the corresponding EGVI amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

  8. EGVII endoglucanase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

    2009-05-05

    The present invention provides an endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

  9. EGVII endoglucanase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2013-07-16

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

  10. EGVII endoglucanase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

    2012-02-14

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

  11. EGVII endoglucanase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2015-04-14

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

  12. Prospects for nucleic acid-based therapeutics against hepatitis C virus.

    PubMed

    Lee, Chang Ho; Kim, Ji Hyun; Lee, Seong-Wook

    2013-12-21

    In this review, we discuss recent advances in nucleic acid-based therapeutic technologies that target hepatitis C virus (HCV) infection. Because the HCV genome is present exclusively in RNA form during replication, various nucleic acid-based therapeutic approaches targeting the HCV genome, such as ribozymes, aptamers, siRNAs, and antisense oligonucleotides, have been suggested as potential tools against HCV. Nucleic acids are potentially immunogenic and typically require a delivery tool to be utilized as therapeutics. These limitations have hampered the clinical development of nucleic acid-based therapeutics. However, despite these limitations, nucleic acid-based therapeutics has clinical value due to their great specificity, easy and large-scale synthesis with chemical methods, and pharmaceutical flexibility. Moreover, nucleic acid therapeutics are expected to broaden the range of targetable molecules essential for the HCV replication cycle, and therefore they may prove to be more effective than existing therapeutics, such as interferon-α and ribavirin combination therapy. This review focuses on the current status and future prospects of ribozymes, aptamers, siRNAs, and antisense oligonucleotides as therapeutic reagents against HCV.

  13. Single-stranded nucleic acids promote SAMHD1 complex formation.

    PubMed

    Tüngler, Victoria; Staroske, Wolfgang; Kind, Barbara; Dobrick, Manuela; Kretschmer, Stefanie; Schmidt, Franziska; Krug, Claudia; Lorenz, Mike; Chara, Osvaldo; Schwille, Petra; Lee-Kirsch, Min Ae

    2013-06-01

    SAM domain and HD domain-containing protein 1 (SAMHD1) is a dGTP-dependent triphosphohydrolase that degrades deoxyribonucleoside triphosphates (dNTPs) thereby limiting the intracellular dNTP pool. Mutations in SAMHD1 cause Aicardi-Goutières syndrome (AGS), an inflammatory encephalopathy that mimics congenital viral infection and that phenotypically overlaps with the autoimmune disease systemic lupus erythematosus. Both disorders are characterized by activation of the antiviral cytokine interferon-α initiated by immune recognition of self nucleic acids. Here we provide first direct evidence that SAMHD1 associates with endogenous nucleic acids in situ. Using fluorescence cross-correlation spectroscopy, we demonstrate that SAMHD1 specifically interacts with ssRNA and ssDNA and establish that nucleic acid-binding and formation of SAMHD1 complexes are mutually dependent. Interaction with nucleic acids and complex formation do not require the SAM domain, but are dependent on the HD domain and the C-terminal region of SAMHD1. We finally demonstrate that mutations associated with AGS exhibit both impaired nucleic acid-binding and complex formation implicating that interaction with nucleic acids is an integral aspect of SAMHD1 function.

  14. Nucleic acid in-situ hybridization detection of infectious agents

    NASA Astrophysics Data System (ADS)

    Thompson, Curtis T.

    2000-04-01

    Limitations of traditional culture methods and newer polymerase chain reaction (PCR)-based methods for detection and speciation of infectious agents demonstrate the need for more rapid and better diagnostics. Nucleic acid hybridization is a detection technology that has gained wide acceptance in cancer and prenatal cytogenetics. Using a modification of the nucleic acid hybridization technique known as fluorescence in-situ hybridization, infectious agents can be detected in a variety of specimens with high sensitivity and specificity. The specimens derive from all types of human and animal sources including body fluids, tissue aspirates and biopsy material. Nucleic acid hybridization can be performed in less than one hour. The result can be interpreted either using traditional fluorescence microscopy or automated platforms such as micro arrays. This paper demonstrates proof of concept for nucleic acid hybridization detection of different infectious agents. Interpretation within a cytologic and histologic context is possible with fluorescence microscopic analysis, thereby providing confirmatory evidence of hybridization. With careful probe selection, nucleic acid hybridization promises to be a highly sensitive and specific practical diagnostic alternative to culture, traditional staining methods, immunohistochemistry and complicated nucleic acid amplification tests.

  15. Comparative Evaluation of Three Homogenization Methods for Isolating Middle East Respiratory Syndrome Coronavirus Nucleic Acids From Sputum Samples for Real-Time Reverse Transcription PCR.

    PubMed

    Sung, Heungsup; Yong, Dongeun; Ki, Chang Seok; Kim, Jae Seok; Seong, Moon Woo; Lee, Hyukmin; Kim, Mi Na

    2016-09-01

    Real-time reverse transcription PCR (rRT-PCR) of sputum samples is commonly used to diagnose Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Owing to the difficulty of extracting RNA from sputum containing mucus, sputum homogenization is desirable prior to nucleic acid isolation. We determined optimal homogenization methods for isolating viral nucleic acids from sputum. We evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and N-acetyl-L-cysteine and sodium citrate (NALC) treatment. Sputum samples were spiked with inactivated MERS-CoV culture isolates. RNA was extracted from pretreated, spiked samples using the easyMAG system (bioMérieux, France). Extracted RNAs were then subjected to rRT-PCR for MERS-CoV diagnosis (DiaPlex Q MERS-coronavirus, SolGent, Korea). While analyzing 15 spiked sputum samples prepared in technical duplicate, false-negative results were obtained with five (16.7%) and four samples (13.3%), respectively, by using the PBS and NALC methods. The range of threshold cycle (Ct) values observed when detecting upE in sputum samples was 31.1-35.4 with the PK-DNase method, 34.7-39.0 with the PBS method, and 33.9-38.6 with the NALC method. Compared with the control, which were prepared by adding a one-tenth volume of 1:1,000 diluted viral culture to PBS solution, the ranges of Ct values obtained by the PBS and NALC methods differed significantly from the mean control Ct of 33.2 (both P<0.0001). The PK-DNase method is suitable for homogenizing sputum samples prior to RNA extraction.

  16. Comparative Evaluation of Three Homogenization Methods for Isolating Middle East Respiratory Syndrome Coronavirus Nucleic Acids From Sputum Samples for Real-Time Reverse Transcription PCR

    PubMed Central

    Yong, Dongeun; Ki, Chang-Seok; Kim, Jae-Seok; Seong, Moon-Woo; Lee, Hyukmin

    2016-01-01

    Background Real-time reverse transcription PCR (rRT-PCR) of sputum samples is commonly used to diagnose Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Owing to the difficulty of extracting RNA from sputum containing mucus, sputum homogenization is desirable prior to nucleic acid isolation. We determined optimal homogenization methods for isolating viral nucleic acids from sputum. Methods We evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and N-acetyl-L-cysteine and sodium citrate (NALC) treatment. Sputum samples were spiked with inactivated MERS-CoV culture isolates. RNA was extracted from pretreated, spiked samples using the easyMAG system (bioMérieux, France). Extracted RNAs were then subjected to rRT-PCR for MERS-CoV diagnosis (DiaPlex Q MERS-coronavirus, SolGent, Korea). Results While analyzing 15 spiked sputum samples prepared in technical duplicate, false-negative results were obtained with five (16.7%) and four samples (13.3%), respectively, by using the PBS and NALC methods. The range of threshold cycle (Ct) values observed when detecting upE in sputum samples was 31.1–35.4 with the PK-DNase method, 34.7–39.0 with the PBS method, and 33.9–38.6 with the NALC method. Compared with the control, which were prepared by adding a one-tenth volume of 1:1,000 diluted viral culture to PBS solution, the ranges of Ct values obtained by the PBS and NALC methods differed significantly from the mean control Ct of 33.2 (both P<0.0001). Conclusions The PK-DNase method is suitable for homogenizing sputum samples prior to RNA extraction. PMID:27374711

  17. 7 CFR 331.1 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... acids. (1) Molecules that are constructed by joining nucleic acid molecules and that can replicate in a living cell (i.e., recombinant nucleic acids); or (2) Molecules that result from the replication of those.... Synthetic nucleic acids. (1) Molecules that are chemically or by other means synthesized or amplified...

  18. 7 CFR 331.1 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... acids. (1) Molecules that are constructed by joining nucleic acid molecules and that can replicate in a living cell (i.e., recombinant nucleic acids); or (2) Molecules that result from the replication of those.... Synthetic nucleic acids. (1) Molecules that are chemically or by other means synthesized or amplified...

  19. Nucleic Acid-Based Nanodevices in Biological Imaging.

    PubMed

    Chakraborty, Kasturi; Veetil, Aneesh T; Jaffrey, Samie R; Krishnan, Yamuna

    2016-06-02

    The nanoscale engineering of nucleic acids has led to exciting molecular technologies for high-end biological imaging. The predictable base pairing, high programmability, and superior new chemical and biological methods used to access nucleic acids with diverse lengths and in high purity, coupled with computational tools for their design, have allowed the creation of a stunning diversity of nucleic acid-based nanodevices. Given their biological origin, such synthetic devices have a tremendous capacity to interface with the biological world, and this capacity lies at the heart of several nucleic acid-based technologies that are finding applications in biological systems. We discuss these diverse applications and emphasize the advantage, in terms of physicochemical properties, that the nucleic acid scaffold brings to these contexts. As our ability to engineer this versatile scaffold increases, its applications in structural, cellular, and organismal biology are clearly poised to massively expand.

  20. Evidence of biological activity in Hawaiian subsurface basalts

    NASA Astrophysics Data System (ADS)

    Fisk, M. R.; Storrie-Lombardi, M. C.; Douglas, S.; Popa, R.; McDonald, G.; di Meo-Savoie, C.

    2003-12-01

    The Hawaii Scientific Drilling Program (HSDP) cored and recovered igneous rock from the surface to a depth of 3109 m near Hilo, Hawaii. Much of the deeper parts of the hole is composed of hyaloclastite (fractured basalt glass that has been cemented in situ with secondary minerals). Some hyaloclastite units have been altered in a manner attributed to microorganisms in volcanic rocks. Samples from one such unit (1336 m to 1404 m below sea level) were examined to test the hypothesis that the alteration was associated with microorganisms. Deep ultraviolet native fluorescence and resonance Raman spectroscopy indicate that nucleic acids and aromatic amino acids are present in clay inside spherical cavities (vesicles) within basalt glass. Chemical mapping shows that phosphorus and carbon were enriched at the boundary between the clay and volcanic glass of the vesicles. Environmental scanning electron microscopy (ESEM) reveals two to three micrometer coccoid structures in these same boundaries. ESEM-linked energy dispersive spectroscopy demonstrated carbon, phosphorous, chloride, and magnesium in these bodies significantly differing from unoccupied neighboring regions of basalt. These observations taken together indicate the presence of microorganisms at the boundary between primary volcanic glass and secondary clays. Amino acids and nucleic acids were extracted from bulk samples of the hyaloclastite unit. Amino acid abundance was low, and if the amino acids are derived from microorganisms in the rock, then there are less than 100,000 cells per gram of rock. Most nucleic acid sequences extracted from the unit were closely related to sequences of Crenarchaeota collected from the subsurface of the ocean floor.

  1. Macromolecular Competition Titration Method: Accessing Thermodynamics of the Unmodified Macromolecule–Ligand Interactions Through Spectroscopic Titrations of Fluorescent Analogs

    PubMed Central

    Bujalowski, Wlodzimierz; Jezewska, Maria J.

    2011-01-01

    Analysis of thermodynamically rigorous binding isotherms provides fundamental information about the energetics of the ligand–macromolecule interactions and often an invaluable insight about the structure of the formed complexes. The Macromolecular Competition Titration (MCT) method enables one to quantitatively obtain interaction parameters of protein–nucleic acid interactions, which may not be available by other methods, particularly for the unmodified long polymer lattices and specific nucleic acid substrates, if the binding is not accompanied by adequate spectroscopic signal changes. The method can be applied using different fluorescent nucleic acids or fluorophores, although the etheno-derivatives of nucleic acid are especially suitable as they are relatively easy to prepare, have significant blue fluorescence, their excitation band lies far from the protein absorption spectrum, and the modification eliminates the possibility of base pairing with other nucleic acids. The MCT method is not limited to the specific size of the reference nucleic acid. Particularly, a simple analysis of the competition titration experiments is described in which the fluorescent, short fragment of nucleic acid, spanning the exact site-size of the protein–nucleic acid complex, and binding with only a 1:1 stoichiometry to the protein, is used as a reference macromolecule. Although the MCT method is predominantly discussed as applied to studying protein–nucleic acid interactions, it can generally be applied to any ligand–macromolecule system by monitoring the association reaction using the spectroscopic signal originating from the reference macromolecule in the presence of the competing macromolecule, whose interaction parameters with the ligand are to be determined. PMID:21195223

  2. Modular development of a prototype point of care molecular diagnostic platform for sexually transmitted infections.

    PubMed

    Branavan, Manoharanehru; Mackay, Ruth E; Craw, Pascal; Naveenathayalan, Angel; Ahern, Jeremy C; Sivanesan, Tulasi; Hudson, Chris; Stead, Thomas; Kremer, Jessica; Garg, Neha; Baker, Mark; Sadiq, Syed T; Balachandran, Wamadeva

    2016-08-01

    This paper presents the design of a modular point of care test platform that integrates a proprietary sample collection device directly with a microfluidic cartridge. Cell lysis, within the cartridge, is conducted using a chemical method and nucleic acid purification is done on an activated cellulose membrane. The microfluidic device incorporates passive mixing of the lysis-binding buffers and sample using a serpentine channel. Results have shown extraction efficiencies for this new membrane of 69% and 57% compared to the commercial Qiagen extraction method of 85% and 59.4% for 0.1ng/µL and 100ng/µL salmon sperm DNA respectively spiked in phosphate buffered solution. Extraction experiments using the serpentine passive mixer cartridges incorporating lysis and nucleic acid purification showed extraction efficiency around 80% of the commercial Qiagen kit. Isothermal amplification was conducted using thermophillic helicase dependant amplification and recombinase polymerase amplification. A low cost benchtop real-time isothermal amplification platform has been developed capable of running six amplifications simultaneously. Results show that the platform is capable of detecting 1.32×10(6) of sample DNA through thermophillic helicase dependant amplification and 1×10(5) copy numbers Chlamydia trachomatis genomic DNA within 10min through recombinase polymerase nucleic acid amplification tests. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  3. Universal digital high-resolution melt: a novel approach to broad-based profiling of heterogeneous biological samples.

    PubMed

    Fraley, Stephanie I; Hardick, Justin; Masek, Billie J; Jo Masek, Billie; Athamanolap, Pornpat; Rothman, Richard E; Gaydos, Charlotte A; Carroll, Karen C; Wakefield, Teresa; Wang, Tza-Huei; Yang, Samuel

    2013-10-01

    Comprehensive profiling of nucleic acids in genetically heterogeneous samples is important for clinical and basic research applications. Universal digital high-resolution melt (U-dHRM) is a new approach to broad-based PCR diagnostics and profiling technologies that can overcome issues of poor sensitivity due to contaminating nucleic acids and poor specificity due to primer or probe hybridization inaccuracies for single nucleotide variations. The U-dHRM approach uses broad-based primers or ligated adapter sequences to universally amplify all nucleic acid molecules in a heterogeneous sample, which have been partitioned, as in digital PCR. Extensive assay optimization enables direct sequence identification by algorithm-based matching of melt curve shape and Tm to a database of known sequence-specific melt curves. We show that single-molecule detection and single nucleotide sensitivity is possible. The feasibility and utility of U-dHRM is demonstrated through detection of bacteria associated with polymicrobial blood infection and microRNAs (miRNAs) associated with host response to infection. U-dHRM using broad-based 16S rRNA gene primers demonstrates universal single cell detection of bacterial pathogens, even in the presence of larger amounts of contaminating bacteria; U-dHRM using universally adapted Lethal-7 miRNAs in a heterogeneous mixture showcases the single copy sensitivity and single nucleotide specificity of this approach.

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

    PubMed Central

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

    2013-01-01

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

  5. BGL7 beta-glucosidase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Ward, Michael

    2013-01-29

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl7, and the corresponding BGL7 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL7, recombinant BGL7 proteins and methods for producing the same.

  6. BGL6 .beta.-glucosidase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Ward, Michael

    2012-10-02

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

  7. BGL5 .beta.-glucosidase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2006-02-28

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl5, and the corresponding BGL5 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL5, recombinant BGL5 proteins and methods for producing the same.

  8. BGL5 .beta.-glucosidase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

    2008-03-18

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl5, and the corresponding BGL5 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL5, recombinant BGL5 proteins and methods for producing the same.

  9. BGL6 beta-glucosidase and nucleic acids encoding the same

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

    Dunn-Coleman, Nigel; Ward, Michael

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

  10. BGL6 beta-glucosidase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Ward, Michael

    2014-03-04

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

  11. BGL7 beta-glucosidase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Ward, Michael

    2015-04-14

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl7, and the corresponding BGL7 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL7, recombinant BGL7 proteins and methods for producing the same.

  12. BGL7 beta-glucosidase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Ward, Michael

    2014-03-25

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl7, and the corresponding BGL7 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL7, recombinant BGL7 proteins and methods for producing the same.

  13. BGL6 beta-glucosidase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Ward, Michael

    2015-08-11

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

  14. BGL3 beta-glucosidase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2007-09-25

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl3, and the corresponding BGL3 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL3, recombinant BGL3 proteins and methods for producing the same.

  15. BGL3 beta-glucosidase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

    2008-04-01

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl3, and the corresponding BGL3 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL3, recombinant BGL3 proteins and methods for producing the same.

  16. BGL4 beta-glucosidase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

    2011-12-06

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl4, and the corresponding BGL4 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL4, recombinant BGL4 proteins and methods for producing the same.

  17. BGL4 .beta.-glucosidase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2006-05-16

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl4, and the corresponding BGL4 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL4, recombinant BGL4 proteins and methods for producing the same.

  18. BGL3 beta-glucosidase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

    2011-06-14

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl3, and the corresponding BGL3 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL3, recombinant BGL3 proteins and methods for producing the same.

  19. BGL6 beta-glucosidase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel [Los Gatos, CA; Ward, Michael [San Francisco, CA

    2009-09-01

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

  20. BGL3 beta-glucosidase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2012-10-30

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl3, and the corresponding BGL3 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL3, recombinant BGL3 proteins and methods for producing the same.

  1. BGL4 beta-glucosidase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

    2008-01-22

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl4, and the corresponding BGL4 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL4, recombinant BGL4 proteins and methods for producing the same.

  2. A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers

    NASA Astrophysics Data System (ADS)

    Du, Yan; Hughes, Randall A.; Bhadra, Sanchita; Jiang, Yu Sherry; Ellington, Andrew D.; Li, Bingling

    2015-06-01

    Strand exchange nucleic acid circuitry can be used to transduce isothermal nucleic acid amplification products into signals that can be readable on an off-the-shelf glucometer. Loop-mediated isothermal amplification (LAMP) is limited by the accumulation of non-specific products, but nucleic acid circuitry can be used to probe and distinguish specific amplicons. By combining this high temperature isothermal amplification method with a thermostable invertase, we can directly transduce Middle-East respiratory syndrome coronavirus and Zaire Ebolavirus templates into glucose signals, with a sensitivity as low as 20-100 copies/μl, equating to atto-molar (or low zepto-mole). Virus from cell lysates and synthetic templates could be readily amplified and detected even in sputum or saliva. An OR gate that coordinately triggered on viral amplicons further guaranteed fail-safe virus detection. The method describes has potential for accelerating point-of-care applications, in that biological samples could be applied to a transducer that would then directly interface with an off-the-shelf, approved medical device.

  3. miRNA detection at single-cell resolution using microfluidic LNA flow-FISH

    DOE PAGES

    Wu, Meiye; Piccini, Matthew Ernest; Koh, Chung -Yan; ...

    2014-08-20

    Flow cytometry in combination with fluorescent in situ hybridization (flow-FISH) is a powerful technique that can be utilized to rapidly detect nucleic acids at single-cell resolution without the need for homogenization or nucleic acid extraction. Here, we describe a microfluidic-based method which enables the detection of microRNAs or miRNAs in single intact cells by flow-FISH using locked nucleic acid (LNA)-containing probes. Our method can be applied to all RNA species including mRNA and small noncoding RNA and is suitable for multiplexing with protein immunostaining in the same cell. For demonstration of our method, this chapter details the detection of miR155more » and CD69 protein in PMA and ionomycin-stimulated Jurkat cells. Here, we also include instructions on how to set up a microfluidic chip sample preparation station to prepare cells for imaging and analysis on a commercial flow cytometer or a custom-built micro-flow cytometer.« less

  4. Monte Carlo Modeling-Based Digital Loop-Mediated Isothermal Amplification on a Spiral Chip for Absolute Quantification of Nucleic Acids.

    PubMed

    Xia, Yun; Yan, Shuangqian; Zhang, Xian; Ma, Peng; Du, Wei; Feng, Xiaojun; Liu, Bi-Feng

    2017-03-21

    Digital loop-mediated isothermal amplification (dLAMP) is an attractive approach for absolute quantification of nucleic acids with high sensitivity and selectivity. Theoretical and numerical analysis of dLAMP provides necessary guidance for the design and analysis of dLAMP devices. In this work, a mathematical model was proposed on the basis of the Monte Carlo method and the theories of Poisson statistics and chemometrics. To examine the established model, we fabricated a spiral chip with 1200 uniform and discrete reaction chambers (9.6 nL) for absolute quantification of pathogenic DNA samples by dLAMP. Under the optimized conditions, dLAMP analysis on the spiral chip realized quantification of nucleic acids spanning over 4 orders of magnitude in concentration with sensitivity as low as 8.7 × 10 -2 copies/μL in 40 min. The experimental results were consistent with the proposed mathematical model, which could provide useful guideline for future development of dLAMP devices.

  5. Nucleic acid-induced antiviral immunity in invertebrates: an evolutionary perspective.

    PubMed

    Wang, Pei-Hui; Weng, Shao-Ping; He, Jian-Guo

    2015-02-01

    Nucleic acids derived from viral pathogens are typical pathogen associated molecular patterns (PAMPs). In mammals, the recognition of viral nucleic acids by pattern recognition receptors (PRRs), which include Toll-like receptors (TLRs) and retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), induces the release of inflammatory cytokines and type I interferons (IFNs) through the activation of nuclear factor κB (NF-κB) and interferon regulatory factor (IRF) 3/7 pathways, triggering the host antiviral state. However, whether nucleic acids can induce similar antiviral immunity in invertebrates remains ambiguous. Several studies have reported that nucleic acid mimics, especially dsRNA mimic poly(I:C), can strongly induce non-specific antiviral immune responses in insects, shrimp, and oyster. This behavior shows multiple similarities to the hallmarks of mammalian IFN responses. In this review, we highlight the current understanding of nucleic acid-induced antiviral immunity in invertebrates. We also discuss the potential recognition and regulatory mechanisms that confer non-specific antiviral immunity on invertebrate hosts. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Compatible solute influence on nucleic acids: Many questions but few answers

    PubMed Central

    Kurz, Matthias

    2008-01-01

    Compatible solutes are small organic osmolytes including but not limited to sugars, polyols, amino acids, and their derivatives. They are compatible with cell metabolism even at molar concentrations. A variety of organisms synthesize or take up compatible solutes for adaptation to extreme environments. In addition to their protective action on whole cells, compatible solutes display significant effects on biomolecules in vitro. These include stabilization of native protein and nucleic acid structures. They are used as additives in polymerase chain reactions to increase product yield and specificity, but also in other nucleic acid and protein applications. Interactions of compatible solutes with nucleic acids and protein-nucleic acid complexes are much less understood than the corresponding interactions of compatible solutes with proteins. Although we may begin to understand solute/nucleic acid interactions there are only few answers to the many questions we have. I summarize here the current state of knowledge and discuss possible molecular mechanisms and thermodynamics. PMID:18522725

  7. Small molecule-mediated duplex formation of nucleic acids with 'incompatible' backbones.

    PubMed

    Cafferty, Brian J; Musetti, Caterina; Kim, Keunsoo; Horowitz, Eric D; Krishnamurthy, Ramanarayanan; Hud, Nicholas V

    2016-04-07

    Proflavine, a known intercalator of DNA and RNA, promotes duplex formation by nucleic acids with natural and non-natural backbones that otherwise form duplexes with low thermal stability, and even some that show no sign of duplex formation in the absence of proflavine. These findings demonstrate the potential for intercalators to be used as cofactors for the assembly of rationally designed nucleic acid structures, and could provide fundamental insights regarding intercalation of natural nucleic acid duplexes.

  8. Detection and prevention of mycoplasma hominis infection

    DOEpatents

    DelVecchio, Vito G.; Gallia, Gary L.; McCleskey, Ferne K.

    1997-01-21

    The present invention is directed to a rapid and sensitive method for detecting Mycoplasma hominis using M. hominis-specific probes, oligonucleotides or antibodies. In particular a target sequence can be amplified by in vitro nucleic acid amplification techniques, detected by nucleic acid hybridization using the subject probes and oligonucleotides or detected by immunoassay using M. hominis-specific antibodies. M. hominis-specific nucleic acids which do not recognize or hybridize to genomic nucleic acid of other Mycoplasma species are also provided.

  9. Adsorption and isolation of nucleic acids on cellulose magnetic beads using a three-dimensional printed microfluidic chip

    PubMed Central

    Zhang, Lei; Deraney, Rachel N.; Tripathi, Anubhav

    2015-01-01

    While advances in genomics have enabled sensitive and highly parallel detection of nucleic acid targets, the isolation and extraction of the nucleic acids remain a critical bottleneck in the workflow. We present here a simple 3D printed microfluidic chip that allows for the vortex and centrifugation free extraction of nucleic acids. This novel microfluidic chip utilizes the presence of a water and oil interface to filter out the lysate contaminants. The pure nucleic acids, while bound on cellulose particles, are magnetically moved across the oil layer. We demonstrated efficient and rapid extraction of spiked Human Papillomavirus (HPV) 18 plasmids in specimen transport medium, in under 15 min. An overall extraction efficiency of 61% is observed across a range of HPV plasmid concentrations (5 × 101 to 5 × 106 copies/100 μl). The magnetic, interfacial, and viscous drag forces inside the microgeometries of the chip are modeled. We have also developed a kinetics model for the adsorption of nucleic acids on cellulose functionalized superparamagnetic beads. We also clarify here the role of carrier nucleic acids in the adsorption and isolation of nucleic acids. Based on the various mechanistic insights detailed here, customized microfluidic devices can be designed to meet the range of current and emerging point of care diagnostics needs. PMID:26734116

  10. Assessment of the presence of mucosal human papillomaviruses in malignant melanomas using combined fluorescent in situ hybridization and chemiluminescent immunohistochemistry.

    PubMed

    Ambretti, S; Venturoli, S; Mirasoli, M; La Placa, M; Bonvicini, F; Cricca, M; Zerbini, M; Roda, A; Musiani, M

    2007-01-01

    The vast majority of studies aimed at detecting human papillomavirus (HPV) DNA in skin cancer have used sensitive polymerase chain reaction (PCR) methods but the PCR technique, despite its high sensitivity, is not suitable to ascertain whether (i) the presence of HPV can be related only to few cells harbouring the virus, (ii) the presence of HPV is due to a tumour surface contamination and (iii) the presence of HPV is localized in cancer cells, rather than in normal keratinocytes present in the tumour biopsy. In a recent work we have found mucosal high-risk (HR) HPV genotypes in primary melanoma by PCR. To localize mucosal HR-HPV nucleic acids and tumoural melanocytic marker in the same sections of primary melanoma samples in order to understand the relationship between HPVs and melanoma cells. We have developed a very sensitive method that combines an enzyme-amplified fluorescent in situ hybridization (ISH) for the detection of HPV nucleic acids (types 16 and 18) with a chemiluminescent immunohistochemistry (IHC) method for the detection of the tumoural melanocytic marker HMB-45 sequentially in the same section. Digital images of fluorescent ISH and chemiluminescent IHC were separately recorded, assigned different colours and merged using specific software for image analysis. The combined fluorescent ISH and chemiluminescent IHC demonstrated a sharp colocalization (in the range 60-80%) of HPV nucleic acids and melanoma marker inside the same sections of melanoma biopsies, with a strong specificity and sensitivity. The strong colocalization of mucosal HR-HPV nucleic acids and HMB-45 melanocytic marker emphasized that viral nucleic acids were specifically present in melanoma cells and supported a possible active role of HPV in malignant melanoma.

  11. A duplex PCR-based assay for measuring the amount of bacterial contamination in a nucleic acid extract from a culture of free-living protists.

    PubMed

    Marron, Alan O; Akam, Michael; Walker, Giselle

    2013-01-01

    Cultures of heterotrophic protists often require co-culturing with bacteria to act as a source of nutrition. Such cultures will contain varying levels of intrinsic bacterial contamination that can interfere with molecular research and cause problems with the collection of sufficient material for sequencing. Measuring the levels of bacterial contamination for the purposes of molecular biology research is non-trivial, and can be complicated by the presence of a diverse bacterial flora, or by differences in the relative nucleic acid yield per bacterial or eukaryotic cell. Here we describe a duplex PCR-based assay that can be used to measure the levels of contamination from marine bacteria in a culture of loricate choanoflagellates. By comparison to a standard culture of known target sequence content, the assay can be used to quantify the relative proportions of bacterial and choanoflagellate material in DNA or RNA samples extracted from a culture. We apply the assay to compare methods of purifying choanoflagellate cultures prior to DNA extraction, to determine their effectiveness in reducing bacterial contamination. Together with measurements of the total nucleic acid concentration, the assay can then be used as the basis for determining the absolute amounts of choanoflagellate DNA or RNA present in a sample. The assay protocol we describe here is a simple and relatively inexpensive method of measuring contamination levels in nucleic acid samples. This provides a new way to establish quantification and purification protocols for molecular biology and genomics in novel heterotrophic protist species. Guidelines are provided to develop a similar protocol for use with any protistan culture. This assay method is recommended where qPCR equipment is unavailable, where qPCR is not viable because of the nature of the bacterial contamination or starting material, or where prior sequence information is insufficient to develop qPCR protocols.

  12. Suitability of Commercial Transport Media for Biological Pathogens under Nonideal Conditions

    PubMed Central

    Hubbard, Kyle; Pellar, Gregory; Emanuel, Peter

    2011-01-01

    There is extensive data to support the use of commercial transport media as a stabilizer for known clinical samples; however, there is little information to support their use outside of controlled conditions specified by the manufacturer. Furthermore, there is no data to determine the suitability of said media for biological pathogens, specifically those of interest to the US military. This study evaluates commercial off-the-shelf (COTS) transport media based on sample recovery, viability, and quality of nucleic acids and peptides for nonpathogenic strains of Bacillus anthracis, Yersinia pestis, and Venezuelan equine encephalitis virus, in addition to ricin toxin. Samples were stored in COTS, PBST, or no media at various temperatures over an extended test period. The results demonstrate that COTS media, although sufficient for the preservation of nucleic acid and proteinaceous material, are not capable of maintaining an accurate representation of biothreat agents at the time of collection. PMID:22121364

  13. Multi-chamber nucleic acid amplification and detection device

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

    Dugan, Lawrence

    A nucleic acid amplification and detection device includes an amplification cartridge with a plurality of reaction chambers for containing an amplification reagent and a visual detection reagent, and a plurality of optically transparent view ports for viewing inside the reaction chambers. The cartridge also includes a sample receiving port which is adapted to receive a fluid sample and fluidically connected to distribute the fluid sample to the reaction chamber, and in one embodiment, a plunger is carried by the cartridge for occluding fluidic communication to the reaction chambers. The device also includes a heating apparatus having a heating element whichmore » is activated by controller to generate heat when a trigger event is detected. The heating apparatus includes a cartridge-mounting section which positioned a cartridge in thermal communication with the heating element so that visual changes to the contents of the reaction chambers are viewable through the view ports.« less

  14. Quantification of HIV-1 DNA using real-time recombinase polymerase amplification.

    PubMed

    Crannell, Zachary Austin; Rohrman, Brittany; Richards-Kortum, Rebecca

    2014-06-17

    Although recombinase polymerase amplification (RPA) has many advantages for the detection of pathogenic nucleic acids in point-of-care applications, RPA has not yet been implemented to quantify sample concentration using a standard curve. Here, we describe a real-time RPA assay with an internal positive control and an algorithm that analyzes real-time fluorescence data to quantify HIV-1 DNA. We show that DNA concentration and the onset of detectable amplification are correlated by an exponential standard curve. In a set of experiments in which the standard curve and algorithm were used to analyze and quantify additional DNA samples, the algorithm predicted an average concentration within 1 order of magnitude of the correct concentration for all HIV-1 DNA concentrations tested. These results suggest that quantitative RPA (qRPA) may serve as a powerful tool for quantifying nucleic acids and may be adapted for use in single-sample point-of-care diagnostic systems.

  15. Measuring and mitigating inhibition during real-time, quantitative PCR analysis of viral nucleic acid extracts from large-volume environmental water samples

    USDA-ARS?s Scientific Manuscript database

    Naturally-occurring inhibitory compounds are a major concern during qPCR and RT-qPCR analysis of environmental samples, particularly large volume water samples. Here, a standardized method for measuring and mitigating sample inhibition in environmental water concentrates is described. Specifically, ...

  16. Artificial specific binders directly recovered from chemically modified nucleic acid libraries.

    PubMed

    Kasahara, Yuuya; Kuwahara, Masayasu

    2012-01-01

    Specific binders comprised of nucleic acids, that is, RNA/DNA aptamers, are attractive functional biopolymers owing to their potential broad application in medicine, food hygiene, environmental analysis, and biological research. Despite the large number of reports on selection of natural DNA/RNA aptamers, there are not many examples of direct screening of chemically modified nucleic acid aptamers. This is because of (i) the inferior efficiency and accuracy of polymerase reactions involving transcription/reverse-transcription of modified nucleotides compared with those of natural nucleotides, (ii) technical difficulties and additional time and effort required when using modified nucleic acid libraries, and (iii) ambiguous efficacies of chemical modifications in binding properties until recently; in contrast, the effects of chemical modifications on biostability are well studied using various nucleotide analogs. Although reports on the direct screening of a modified nucleic acid library remain in the minority, chemical modifications would be essential when further functional expansion of nucleic acid aptamers, in particular for medical and biological uses, is considered. This paper focuses on enzymatic production of chemically modified nucleic acids and their application to random screenings. In addition, recent advances and possible future research are also described.

  17. Scavenging nucleic acid debris to combat autoimmunity and infectious disease

    NASA Astrophysics Data System (ADS)

    Holl, Eda K.; Shumansky, Kara L.; Borst, Luke B.; Burnette, Angela D.; Sample, Christopher J.; Ramsburg, Elizabeth A.; Sullenger, Bruce A.

    2016-08-01

    Nucleic acid-containing debris released from dead and dying cells can be recognized as damage-associated molecular patterns (DAMPs) or pattern-associated molecular patterns (PAMPs) by the innate immune system. Inappropriate activation of the innate immune response can engender pathological inflammation and autoimmune disease. To combat such diseases, major efforts have been made to therapeutically target the pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs) that recognize such DAMPs and PAMPs, or the downstream effector molecules they engender, to limit inflammation. Unfortunately, such strategies can limit the ability of the immune system to combat infection. Previously, we demonstrated that nucleic acid-binding polymers can act as molecular scavengers and limit the ability of artificial nucleic acid ligands to activate PRRs. Herein, we demonstrate that nucleic acid scavengers (NASs) can limit pathological inflammation and nucleic acid-associated autoimmunity in lupus-prone mice. Moreover, we observe that such NASs do not limit an animal’s ability to combat viral infection, but rather their administration improves survival when animals are challenged with lethal doses of influenza. These results indicate that molecules that scavenge extracellular nucleic acid debris represent potentially safer agents to control pathological inflammation associated with a wide range of autoimmune and infectious diseases.

  18. A Theoretical Mechanism of Szilard Engine Function in Nucleic Acids and the Implications for Quantum Coherence in Biological Systems

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

    Matthew Mihelic, F.

    2010-12-22

    Nucleic acids theoretically possess a Szilard engine function that can convert the energy associated with the Shannon entropy of molecules for which they have coded recognition, into the useful work of geometric reconfiguration of the nucleic acid molecule. This function is logically reversible because its mechanism is literally and physically constructed out of the information necessary to reduce the Shannon entropy of such molecules, which means that this information exists on both sides of the theoretical engine, and because information is retained in the geometric degrees of freedom of the nucleic acid molecule, a quantum gate is formed through whichmore » multi-state nucleic acid qubits can interact. Entangled biophotons emitted as a consequence of symmetry breaking nucleic acid Szilard engine (NASE) function can be used to coordinate relative positioning of different nucleic acid locations, both within and between cells, thus providing the potential for quantum coherence of an entire biological system. Theoretical implications of understanding biological systems as such 'quantum adaptive systems' include the potential for multi-agent based quantum computing, and a better understanding of systemic pathologies such as cancer, as being related to a loss of systemic quantum coherence.« less

  19. A Theoretical Mechanism of Szilard Engine Function in Nucleic Acids and the Implications for Quantum Coherence in Biological Systems

    NASA Astrophysics Data System (ADS)

    Matthew Mihelic, F.

    2010-12-01

    Nucleic acids theoretically possess a Szilard engine function that can convert the energy associated with the Shannon entropy of molecules for which they have coded recognition, into the useful work of geometric reconfiguration of the nucleic acid molecule. This function is logically reversible because its mechanism is literally and physically constructed out of the information necessary to reduce the Shannon entropy of such molecules, which means that this information exists on both sides of the theoretical engine, and because information is retained in the geometric degrees of freedom of the nucleic acid molecule, a quantum gate is formed through which multi-state nucleic acid qubits can interact. Entangled biophotons emitted as a consequence of symmetry breaking nucleic acid Szilard engine (NASE) function can be used to coordinate relative positioning of different nucleic acid locations, both within and between cells, thus providing the potential for quantum coherence of an entire biological system. Theoretical implications of understanding biological systems as such "quantum adaptive systems" include the potential for multi-agent based quantum computing, and a better understanding of systemic pathologies such as cancer, as being related to a loss of systemic quantum coherence.

  20. Nucleic acid based fluorescent sensor for mercury detection

    DOEpatents

    Lu, Yi; Liu, Juewen

    2013-02-05

    A nucleic acid enzyme comprises an oligonucleotide containing thymine bases. The nucleic acid enzyme is dependent on both Hg.sup.2+and a second ion as cofactors, to produce a product from a substrate. The substrate comprises a ribonucleotide, a deoxyribonucleotide, or both.

  1. IDENTIFYING POTENTIAL SOURCES OF BACKGROUND CONTAMINATION IN RT-PCR

    EPA Science Inventory

    Extraction of nucleic acids from low biomass samples, such as drinking water, is particularly sensitive to potential background contamination because the contaminating material is minimally diluted by the sample. The presence of bacterial DNA in Taq DNA polymerase is wel...

  2. Assessment of self taken swabs versus clinician taken swab cultures for diagnosing gonorrhoea in women: single centre, diagnostic accuracy study.

    PubMed

    Stewart, Catherine M W; Schoeman, Sarah A; Booth, Russell A; Smith, Susan D; Wilcox, Mark H; Wilson, Janet D

    2012-12-12

    To compare gonorrhoea detection by self taken vulvovaginal swabs (tested with nucleic acid amplification tests) with the culture of urethral and endocervical samples taken by clinicians. Prospective study of diagnostic accuracy. 1 sexual health clinic in an urban setting (Leeds Centre for Sexual Health, United Kingdom), between March 2009 and January 2010. Women aged 16 years or older, attending the clinic for sexually transmitted infection (STI) testing and consenting to perform a vulvovaginal swab themselves before routine examination. During examination, clinicians took urethral and endocervical samples for culture and an endocervical swab for nucleic acid amplification testing. Urethra and endocervix samples were analysed by gonococcal culture. Vulvovaginal swabs and endocervical swabs were analysed by the Aptima Combo 2 (AC2) assay; positive results from this assay were confirmed with a second nucleic acid amplification test. Positive confirmation of gonorrhoea. Of 3859 women with complete data and test results, 96 (2.5%) were infected with gonorrhoea (overall test sensitivities: culture 81%, endocervical swabs with AC2 96%, vulvovaginal swabs with AC2 99%). The AC2 assays were more sensitive than culture (P<0.001), but the endocervical and vulvovaginal assays did not differ significantly (P=0.375). Specificity of all Aptima Combo 2 tests was 100%. Of 1625 women who had symptoms suggestive of a bacterial STI, 56 (3.4%) had gonorrhoea (culture 84%, endocervical AC2 100%, vulvovaginal AC2 100%). The AC2 assays were more sensitive than culture (P=0.004), and the endocervical and vulvovaginal assays were equivalent to each other. Of 2234 women who did not have symptoms suggesting a bacterial STI, 40 (1.8%) had gonorrhoea (culture 78%, endocervical AC2 90%, vulvovaginal AC2 98%). The vulvovaginal swab was more sensitive than culture (P=0.008), but there was no difference between the endocervical and vulvovaginal AC2 assays (P=0.375) or between the endocervical AC2 assay and culture (P=0.125). The endocervical swab assay performed less well in women without symptoms of a bacterial STI than in those with symptoms (90% v 100%, P=0.028), whereas the vulvovaginal swab assay performed similarly (98% v 100%, P=0.42). Self taken vulvovaginal swabs analysed by nucleic acid amplification tests are significantly more sensitive at detecting gonorrhoea than culture of clinician taken urethral and endocervical samples, and are equivalent to endocervical swabs analysed by nucleic acid amplification tests. Self taken vulvovaginal swabs are the sample of choice in women without symptoms and have the advantage of being non-invasive. In women who need a clinical examination, either a clinician taken or self taken vulvovaginal swab is recommended.

  3. Evaluation of methods of determining humic acids in nucleic acid samples for molecular biological analysis.

    PubMed

    Wang, Yong; Fujii, Takeshi

    2011-01-01

    It is important in molecular biological analyses to evaluate contamination of co-extracted humic acids in DNA/RNA extracted from soil. We compared the sensitivity of various methods for measurement of humic acids, and influences of DNA/RNA and proteins on the measurement. Considering the results, we give suggestions as to choice of methods for measurement of humic acids in molecular biological analyses.

  4. Nucleic acid duplexes incorporating a dissociable covalent base pair

    NASA Technical Reports Server (NTRS)

    Gao, K.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1999-01-01

    We have used molecular modeling techniques to design a dissociable covalently bonded base pair that can replace a Watson-Crick base pair in a nucleic acid with minimal distortion of the structure of the double helix. We introduced this base pair into a potential precursor of a nucleic acid double helix by chemical synthesis and have demonstrated efficient nonenzymatic template-directed ligation of the free hydroxyl groups of the base pair with appropriate short oligonucleotides. The nonenzymatic ligation reactions, which are characteristic of base paired nucleic acid structures, are abolished when the covalent base pair is reduced and becomes noncoplanar. This suggests that the covalent base pair linking the two strands in the duplex is compatible with a minimally distorted nucleic acid double-helical structure.

  5. Promoters and proteins from Clostridium thermocellum and uses thereof

    DOEpatents

    Wu, J. H. David; Newcomb, Michael

    2012-11-13

    The present invention relates to an inducible and a high expression nucleic acid promoter isolated from Clostridium thermocellum. These promoters are useful for directing expression of a protein or polypeptide encoded by a nucleic acid molecule operably associated with the nucleic acid promoters. The present invention also relates to nucleic acid constructs including the C. thermocellum promoters, and expression vectors and hosts containing such nucleic acid constructs. The present invention also relates to protein isolated from Clostridium thermocellum, including a repressor protein. The present invention also provides methods of using the isolated promoters and proteins from Clostridium thermocellum, including methods for directing inducible in vitro and in vivo expression of a protein or polypeptide in a host, and methods of producing ethanol from a cellulosic biomass.

  6. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Dotson, William D.; Greenier, Jennifer; Ding, Hanshu

    2007-09-18

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated nucleic acids encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acids as well as methods for producing and using the polypeptides.

  7. 9 CFR 121.1 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    .... Recombinant nucleic acids. (1) Molecules that are constructed by joining nucleic acid molecules and that can... of the United States. Synthetic nucleic acids. (1) Molecules that are chemically or by other means synthesized or amplified, including those that are chemically or otherwise modified but can base pair with...

  8. 9 CFR 121.1 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    .... Recombinant nucleic acids. (1) Molecules that are constructed by joining nucleic acid molecules and that can... of the United States. Synthetic nucleic acids. (1) Molecules that are chemically or by other means synthesized or amplified, including those that are chemically or otherwise modified but can base pair with...

  9. A self-heating cartridge for molecular diagnostics.

    PubMed

    Liu, Changchun; Mauk, Michael G; Hart, Robert; Qiu, Xianbo; Bau, Haim H

    2011-08-21

    A disposable, water-activated, self-heating, easy-to-use, polymeric cartridge for isothermal nucleic acid amplification and visual fluorescent detection of the amplification products is described. The device is self-contained and does not require any special instruments to operate. The cartridge integrates chemical, water-triggered, exothermic heating with temperature regulation facilitated with a phase-change material (PCM) and isothermal nucleic acid amplification. The water flows into the exothermic reactor by wicking through a porous paper. The porous paper's characteristics control the rate of water supply, which in turn controls the rate of exothermic reaction. The PCM material enables the cartridge to maintain a desired temperature independent of ambient temperatures in the range between 20 °C and 40 °C. The utility of the cartridge is demonstrated by amplifying and detecting Escherichia coli DNA with loop mediated isothermal amplification (LAMP). The device can detect consistently as few as 10 target molecules in the sample. With proper modifications, the cartridge also can work with other isothermal nucleic acid amplification technologies for detecting nucleic acids associated with various pathogens borne in blood, saliva, urine, and other body fluids as well as in water and food. The device is suitable for use at home, in the field, and in poor-resource settings, where access to sophisticated laboratories is impractical, unaffordable, or nonexistent. This journal is © The Royal Society of Chemistry 2011

  10. Development of chemiluminescent probe hybridization, RT-PCR and nucleic acid cycle sequencing assays of Sabin type 3 isolates to identify base pair 472 Sabin type 3 mutants associated with vaccine associated paralytic poliomyelitis.

    PubMed

    Old, M O; Logan, L H; Maldonado, Y A

    1997-11-01

    Sabin type 3 polio vaccine virus is the most common cause of poliovaccine associated paralytic poliomyelitis. Vaccine associated paralytic poliomyelitis cases have been associated with Sabin type 3 revertants containing a single U to C substitution at bp 472 of Sabin type 3. A rapid method of identification of Sabin type 3 bp 472 mutants is described. An enterovirus group-specific probe for use in a chemiluminescent dot blot hybridization assay was developed to identify enterovirus positive viral lysates. A reverse transcription-polymerase chain reaction (RT-PCR) assay producing a 319 bp PCR product containing the Sabin type 3 bp 472 mutation site was then employed to identify Sabin type 3 isolates. Chemiluminescent nucleic acid cycle sequencing of the purified 319 bp PCR product was then employed to identify nucleic acid sequences at bp 472. The enterovirus group probe hybridization procedure and isolation of the Sabin type 3 PCR product were highly sensitive and specific; nucleic acid cycle sequencing corresponded to the known sequence of stock Sabin type 3 isolates. These methods will be used to identify the Sabin type 3 reversion rate from sequential stool samples of infants obtained after the first and second doses of oral poliovirus vaccine.

  11. RIG-I detects infection with live Listeria by sensing secreted bacterial nucleic acids

    PubMed Central

    Abdullah, Zeinab; Schlee, Martin; Roth, Susanne; Mraheil, Mobarak Abu; Barchet, Winfried; Böttcher, Jan; Hain, Torsten; Geiger, Sergej; Hayakawa, Yoshihiro; Fritz, Jörg H; Civril, Filiz; Hopfner, Karl-Peter; Kurts, Christian; Ruland, Jürgen; Hartmann, Gunther; Chakraborty, Trinad; Knolle, Percy A

    2012-01-01

    Immunity against infection with Listeria monocytogenes is not achieved from innate immune stimulation by contact with killed but requires viable Listeria gaining access to the cytosol of infected cells. It has remained ill-defined how such immune sensing of live Listeria occurs. Here, we report that efficient cytosolic immune sensing requires access of nucleic acids derived from live Listeria to the cytoplasm of infected cells. We found that Listeria released nucleic acids and that such secreted bacterial RNA/DNA was recognized by the cytosolic sensors RIG-I, MDA5 and STING thereby triggering interferon β production. Secreted Listeria nucleic acids also caused RIG-I-dependent IL-1β-production and inflammasome activation. The signalling molecule CARD9 contributed to IL-1β production in response to secreted nucleic acids. In conclusion, cytosolic recognition of secreted bacterial nucleic acids by RIG-I provides a mechanistic explanation for efficient induction of immunity by live bacteria. PMID:23064150

  12. Chlamydia and gonorrhoea contamination of clinic surfaces.

    PubMed

    Lewis, Natasha; Dube, Gail; Carter, Christine; Pitt, Rachel; Alexander, Sarah; Ison, Catherine A; Harding, Jan; Brown, Louise; Fryer, John; Hodson, James; Ross, Jonathan

    2012-10-01

    Nucleic acid amplification tests, with their ability to detect very small amounts of nucleic acid, have become the principle diagnostic tests for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) in many sexual health clinics. The aim of this study was to investigate the extent of surface contamination with CT and GC within a city centre sexual health clinic and to evaluate the potential for contamination of containers used for the collection of self-taken swabs. Surface contamination with CT and GC was assessed by systematically sampling 154 different sites within one clinic using transcription-mediated amplification (TMA), quantitative PCR and culture. The caps of containers used by patients to collect self-taken samples were also tested for CT and GC using TMA. Of the 154 sites sampled, 20 (13.0%) tested positive on TMA. Of these, five (3.2%) were positive for CT alone, 11 (7.1%) for GC alone and four (2.6%) for both CT and GC. The proportion of GC TMA-positive test results differed by gender, with 11 (18.3%) positive results from the male patient clinic area compared with one (1.6%) from the female area (p=0.002). Positive samples were obtained from a variety of locations in the clinic, but the patient toilets were more likely to be contaminated than examination rooms (p=0.015). Quantitative PCR and culture assays were negative for all samples. 46 caps of the containers used for self-taken swabs were negative for both CT and GC on TMA testing. Surface contamination with chlamydial and gonococcal rRNA can occur within sexual health clinics, but the quantity of nucleic acid detected is low and infection risk to patients and staff is small. There remains a potential risk of contamination of patient samples leading to false-positive results.

  13. Evaluation of six nucleic acid amplification tests used for diagnosis of Neisseria gonorrhoeae in Russia compared with an international strictly validated real-time porA pseudogene polymerase chain reaction.

    PubMed

    Shipitsyna, E; Zolotoverkhaya, E; Hjelmevoll, S O; Maximova, A; Savicheva, A; Sokolovsky, E; Skogen, V; Domeika, M; Unemo, M

    2009-11-01

    In Russia, laboratory diagnosis of gonorrhoea has been mainly based on microscopy only and, in some settings, relatively rare suboptimal culturing. In recent years, Russian developed and manufactured nucleic acid amplification tests (NAAT) have been implemented for routine diagnosis of Neisseria gonorrhoeae. However, these NAATs have never been validated to any international well-recognized diagnostic NAAT. This study aims to evaluate the performance characteristics of six Russian NAATs for N. gonorrhoeae diagnostics. In total, 496 symptomatic patients were included. Five polymerase chain reaction (PCR) assays and one real-time nucleic acid sequence based amplification (NASBA) assay, developed by three Russian companies, were evaluated on urogenital samples, i.e. cervical and first voided urine (FVU) samples from females (n = 319), urethral and FVU samples from males (n = 127), and extragenital samples, i.e. rectal and pharyngeal samples, from 50 additional female patients with suspicion of gonorrhoea. As reference method, an international strictly validated real-time porA pseudogene PCR was applied. The prevalence of N. gonorrhoeae was 2.7% and 16% among the patients providing urogenital and extragenital samples, respectively. The Russian NAATs and the reference method displayed high level of concordance (99.4-100%). The sensitivities, specificities, positive predictive values and negative predictive values of the Russian tests in different specimens were 66.7-100%, 100%, 100%, and 99.4-100%, respectively. Russian N. gonorrhoeae diagnostic NAATs comprise relatively good performance characteristics. However, larger studies are crucial and, beneficially, the Russian assays should also be evaluated to other international highly sensitive and specific, and ideally Food and Drug Administration approved, NAATs such as Aptima Combo 2 (Gen-Probe).

  14. Excretion and detection of SARS coronavirus and its nucleic acid from digestive system

    PubMed Central

    Wang, Xin-Wei; Li, Jin-Song; Guo, Ting-Kai; Zhen, Bei; Kong, Qing-Xin; Yi, Bin; Li, Zhong; Song, Nong; Jin, Min; Wu, Xiao-Ming; Xiao, Wen-Jun; Zhu, Xiu-Mei; Gu, Chang-Qing; Yin, Jing; Wei, Wei; Yao, Wei; Liu, Chao; Li, Jian-Feng; Ou, Guo-Rong; Wang, Min-Nian; Fang, Tong-Yu; Wang, Gui-Jie; Qiu, Yao-Hui; Wu, Huai-Huan; Chao, Fu-Huan; Li, Jun-Wen

    2005-01-01

    AIM: To study whether severe acute respiratory syndrome coronavirus (SARS-CoV) could be excreted from digestive system. METHODS: Cell culture and semi-nested RT-PCR were used to detect SARS-CoV and its RNA from 21 stool and urine samples, and a kind of electropositive filter media particles was used to concentrate the virus in 10 sewage samples from two hospitals receiving SARS patients in Beijing in China. RESULTS: It was demonstrated that there was no live SARS-CoV in all samples collected, but the RNA of SARS-CoV could be detected in seven stool samples from SARS patients with any one of the symptoms of fever, malaise, cough, or dyspnea, in 10 sewage samples before disinfection and 3 samples after disinfection from the two hospitals. The RNA could not be detected in urine and stool samples from patients recovered from SARS. CONCLUSION: Nucleic acid of SARS-CoV can be excreted through the stool of patients into sewage system, and the possibility of SARS-CoV transmitting through digestive system cannot be excluded. PMID:16038039

  15. Development and Initial Results of a Low Cost, Disposable, Point-of-Care Testing Device for Pathogen Detection

    PubMed Central

    Dugan, Lawrence C.; Baker, Brian R.; Hall, Sara B.; Ebert, Katja; Mioulet, Valerie; Madi, Mikidache; King, Donald P.

    2011-01-01

    Development of small footprint, disposable, fast, and inexpensive devices for pathogen detection in the field and clinic would benefit human and veterinary medicine by allowing evidence-based responses to future out breaks. We designed and tested an integrated nucleic acid extraction and amplification device employing a loop-mediated isothermal amplification (LAMP) or reverse transcriptase-LAMP assay. Our system provides a screening tool with polymerase-chain-reaction-level sensitivity and specificity for outbreak detection, response, and recovery. Time to result is ~90 min. The device utilizes a swab that collects sample and then transfers it to a disc of cellulose-based nucleic acid binding paper. The disc is positioned within a disposable containment tube with a manual loading port. In order to test for the presence of target pathogens, LAMP reagents are loaded through the tube’s port into contact with the sample containing cellulose disc. The reagents then are isothermally heated to 63°C for ~1 h to achieve sequence-specific target nucleic acid amplification. Due to the presence of a colorimetric dye, amplification induces visible color change in the reagents from purple to blue. As initial demonstrations, we detected methicillin resistant Staphylococcus aureus genomic DNA, as well as recombinant and live foot-and-mouth disease virus. PMID:21342806

  16. [Evaluation of the usefulness of various PCR method variations and nucleic acid hybridization for CMV infection in immunosuppressed patients].

    PubMed

    Siennicka, J; Trzcińska, A; Litwińska, B; Durlik, M; Seferyńska, I; Pałynyczko, G; Kańtoch, M

    2000-01-01

    In diagnosis of CMV infection various laboratory methods are used. The methods based on detection of viral nucleic acids have been introduced routinely in many laboratories. The aim of this study was to compare nucleic acid hybridisation method and various variants of PCR methods with respect to their ability to detect CMV DNA. The studied material comprised 60 blood samples from 19 patients including 13 renal transplant recipients and 6 with acute leukaemia. The samples were subjected to hybridisation (Murex Hybrid Capture System CMV DNA) and PCR carried out in 3 variants: with one pair of primers (single PCR), nested PCR and Digene SHARP System with detection of PCR product using a genetic probe in ELISA system. The sensitivity of the variants ranged from 10(0) particles of viral DNA in nested PCR to 10(2) in single PCR. The producer claimed the sensitivity of the hybridisation test to be 3 x 10(5) and it seems to be sufficient for detection of CMV infection. The obtained results show that sensitivity of hybridisation was comparable to that of single PCR and the possibility of obtaining quantitative results makes it superior, on efficacy of antiviral therapy, especially in monitoring CMV infection in immunossuppressed patients and in following the efficacy of antiviral treatment.

  17. Fully 3D printed integrated reactor array for point-of-care molecular diagnostics.

    PubMed

    Kadimisetty, Karteek; Song, Jinzhao; Doto, Aoife M; Hwang, Young; Peng, Jing; Mauk, Michael G; Bushman, Frederic D; Gross, Robert; Jarvis, Joseph N; Liu, Changchun

    2018-06-30

    Molecular diagnostics that involve nucleic acid amplification tests (NAATs) are crucial for prevention and treatment of infectious diseases. In this study, we developed a simple, inexpensive, disposable, fully 3D printed microfluidic reactor array that is capable of carrying out extraction, concentration and isothermal amplification of nucleic acids in variety of body fluids. The method allows rapid molecular diagnostic tests for infectious diseases at point of care. A simple leak-proof polymerization strategy was developed to integrate flow-through nucleic acid isolation membranes into microfluidic devices, yielding a multifunctional diagnostic platform. Static coating technology was adopted to improve the biocompatibility of our 3D printed device. We demonstrated the suitability of our device for both end-point colorimetric qualitative detection and real-time fluorescence quantitative detection. We applied our diagnostic device to detection of Plasmodium falciparum in plasma samples and Neisseria meningitides in cerebrospinal fluid (CSF) samples by loop-mediated, isothermal amplification (LAMP) within 50 min. The detection limits were 100 fg for P. falciparum and 50 colony-forming unit (CFU) for N. meningitidis per reaction, which are comparable to that of benchtop instruments. This rapid and inexpensive 3D printed device has great potential for point-of-care molecular diagnosis of infectious disease in resource-limited settings. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. Microchip Module for Blood Sample Preparation and Nucleic Acid Amplification Reactions

    PubMed Central

    Yuen, Po Ki; Kricka, Larry J.; Fortina, Paolo; Panaro, Nicholas J.; Sakazume, Taku; Wilding, Peter

    2001-01-01

    A computer numerical control-machined plexiglas-based microchip module was designed and constructed for the integration of blood sample preparation and nucleic acid amplification reactions. The microchip module is comprised of a custom-made heater-cooler for thermal cycling, a series of 254 μm × 254 μm microchannels for transporting human whole blood and reagents in and out of an 8–9 μL dual-purpose (cell isolation and PCR) glass-silicon microchip. White blood cells were first isolated from a small volume of human whole blood (<3 μL) in an integrated cell isolation–PCR microchip containing a series of 3.5-μm feature-sized “weir-type” filters, formed by an etched silicon dam spanning the flow chamber. A genomic target, a region in the human coagulation Factor V gene (226-bp), was subsequently directly amplified by microchip-based PCR on DNA released from white blood cells isolated on the filter section of the microchip mounted onto the microchip module. The microchip module provides a convenient means to simplify nucleic acid analyses by integrating two key steps in genetic testing procedures, cell isolation and PCR and promises to be adaptable for additional types of integrated assays. PMID:11230164

  19. Improved Analytical Sensitivity of Lateral Flow Assay using Sponge for HBV Nucleic Acid Detection.

    PubMed

    Tang, Ruihua; Yang, Hui; Gong, Yan; Liu, Zhi; Li, XiuJun; Wen, Ting; Qu, ZhiGuo; Zhang, Sufeng; Mei, Qibing; Xu, Feng

    2017-05-02

    Hepatitis B virus (HBV) infection is a serious public health problem, which can be transmitted through various routes (e.g., blood donation) and cause hepatitis, liver cirrhosis and liver cancer. Hence, it is necessary to do diagnostic screening for high-risk HBV patients in these transmission routes. Nowadays, protein-based technologies have been used for HBV testing, which however involve the issues of large sample volume, antibody instability and poor specificity. Nucleic acid hybridization-based lateral flow assay (LFA) holds great potential to address these limitations due to its low-cost, rapid, and simple features, but the poor analytical sensitivity of LFA restricts its application. In this study, we developed a low-cost, simple and easy-to-use method to improve analytical sensitivity by integrating sponge shunt into LFA to decrease the fluid flow rate. The thickness, length and hydrophobicity of the sponge shunt were sequentially optimized, and achieved 10-fold signal enhancement in nucleic acid testing of HBV as compared to the unmodified LFA. The enhancement was further confirmed by using HBV clinical samples, where we achieved the detection limit of 10 3 copies/ml as compared to 10 4 copies/ml in unmodified LFA. The improved LFA holds great potential for diseases diagnostics, food safety control and environment monitoring at point-of-care.

  20. Semiquantitative Nucleic Acid Test with Simultaneous Isotachophoretic Extraction and Amplification.

    PubMed

    Bender, Andrew T; Borysiak, Mark D; Levenson, Amanda M; Lillis, Lorraine; Boyle, David S; Posner, Jonathan D

    2018-06-19

    Nucleic acid amplification tests (NAATs) provide high diagnostic accuracy for infectious diseases and quantitative results for monitoring viral infections. The majority of NAATs require complex equipment, cold chain dependent reagents, and skilled technicians to perform the tests. This largely confines NAATs to centralized laboratories and can significantly delay appropriate patient care. Low-cost, point-of-care (POC) NAATs are especially needed in low-resource settings to provide patients with diagnosis and treatment planning in a single visit to improve patient care. In this work, we present a rapid POC NAAT with integrated sample preparation and amplification using electrokinetics and paper substrates. We use simultaneous isotachophoresis (ITP) and recombinase polymerase amplification (RPA) to rapidly extract, amplify, and detect target nucleic acids from serum and whole blood in a paper-based format. We demonstrate simultaneous ITP and RPA can consistently detect 5 copies per reaction in buffer and 10 000 copies per milliliter of human serum with no intermediate user steps. We also show preliminary extraction and amplification of DNA from whole blood samples. Our test is rapid (results in less than 20 min) and made from low-cost materials, indicating its potential for detecting infectious diseases and monitoring viral infections at the POC in low resource settings.

  1. BIOPHYSICAL PROPERTIES OF NUCLEIC ACIDS AT SURFACES RELEVANT TO MICROARRAY PERFORMANCE.

    PubMed

    Rao, Archana N; Grainger, David W

    2014-04-01

    Both clinical and analytical metrics produced by microarray-based assay technology have recognized problems in reproducibility, reliability and analytical sensitivity. These issues are often attributed to poor understanding and control of nucleic acid behaviors and properties at solid-liquid interfaces. Nucleic acid hybridization, central to DNA and RNA microarray formats, depends on the properties and behaviors of single strand (ss) nucleic acids (e.g., probe oligomeric DNA) bound to surfaces. ssDNA's persistence length, radius of gyration, electrostatics, conformations on different surfaces and under various assay conditions, its chain flexibility and curvature, charging effects in ionic solutions, and fluorescent labeling all influence its physical chemistry and hybridization under assay conditions. Nucleic acid (e.g., both RNA and DNA) target interactions with immobilized ssDNA strands are highly impacted by these biophysical states. Furthermore, the kinetics, thermodynamics, and enthalpic and entropic contributions to DNA hybridization reflect global probe/target structures and interaction dynamics. Here we review several biophysical issues relevant to oligomeric nucleic acid molecular behaviors at surfaces and their influences on duplex formation that influence microarray assay performance. Correlation of biophysical aspects of single and double-stranded nucleic acids with their complexes in bulk solution is common. Such analysis at surfaces is not commonly reported, despite its importance to microarray assays. We seek to provide further insight into nucleic acid-surface challenges facing microarray diagnostic formats that have hindered their clinical adoption and compromise their research quality and value as genomics tools.

  2. BIOPHYSICAL PROPERTIES OF NUCLEIC ACIDS AT SURFACES RELEVANT TO MICROARRAY PERFORMANCE

    PubMed Central

    Rao, Archana N.; Grainger, David W.

    2014-01-01

    Both clinical and analytical metrics produced by microarray-based assay technology have recognized problems in reproducibility, reliability and analytical sensitivity. These issues are often attributed to poor understanding and control of nucleic acid behaviors and properties at solid-liquid interfaces. Nucleic acid hybridization, central to DNA and RNA microarray formats, depends on the properties and behaviors of single strand (ss) nucleic acids (e.g., probe oligomeric DNA) bound to surfaces. ssDNA’s persistence length, radius of gyration, electrostatics, conformations on different surfaces and under various assay conditions, its chain flexibility and curvature, charging effects in ionic solutions, and fluorescent labeling all influence its physical chemistry and hybridization under assay conditions. Nucleic acid (e.g., both RNA and DNA) target interactions with immobilized ssDNA strands are highly impacted by these biophysical states. Furthermore, the kinetics, thermodynamics, and enthalpic and entropic contributions to DNA hybridization reflect global probe/target structures and interaction dynamics. Here we review several biophysical issues relevant to oligomeric nucleic acid molecular behaviors at surfaces and their influences on duplex formation that influence microarray assay performance. Correlation of biophysical aspects of single and double-stranded nucleic acids with their complexes in bulk solution is common. Such analysis at surfaces is not commonly reported, despite its importance to microarray assays. We seek to provide further insight into nucleic acid-surface challenges facing microarray diagnostic formats that have hindered their clinical adoption and compromise their research quality and value as genomics tools. PMID:24765522

  3. Nucleic Acid Amplification Testing in Suspected Child Sexual Abuse

    ERIC Educational Resources Information Center

    Esernio-Jenssen, Debra; Barnes, Marilyn

    2011-01-01

    The American Academy of Pediatrics recommends that site-specific cultures be obtained, when indicated, for sexually victimized children. Nucleic acid amplification testing is a highly sensitive and specific methodology for identifying sexually transmitted infections. Nucleic acid amplification tests are also less invasive than culture, and this…

  4. Hybridization of Environmental Microbial Community Nucleic Acids by GeoChip.

    PubMed

    Van Nostrand, Joy D; Yin, Huaqin; Wu, Liyou; Yuan, Tong; Zhou, Jizhong

    2016-01-01

    Functional gene arrays, like the GeoChip, allow for the study of tens of thousands of genes in a single assay. The GeoChip array (5.0) contains probes for genes involved in geochemical cycling (N, C, S, and P), metal homeostasis, stress response, organic contaminant degradation, antibiotic resistance, secondary metabolism, and virulence factors as well as genes specific for fungi, protists, and viruses. Here, we briefly describe GeoChip design strategies (gene selection and probe design) and discuss minimum quantity and quality requirements for nucleic acids. We then provide detailed protocols for amplification, labeling, and hybridization of samples to the GeoChip.

  5. Experimental Approaches to Microarray Analysis of Tumor Samples

    ERIC Educational Resources Information Center

    Furge, Laura Lowe; Winter, Michael B.; Meyers, Jacob I.; Furge, Kyle A.

    2008-01-01

    Comprehensive measurement of gene expression using high-density nucleic acid arrays (i.e. microarrays) has become an important tool for investigating the molecular differences in clinical and research samples. Consequently, inclusion of discussion in biochemistry, molecular biology, or other appropriate courses of microarray technologies has…

  6. Detection and quantification of Plasmodium falciparum in blood samples using quantitative nucleic acid sequence-based amplification.

    PubMed

    Schoone, G J; Oskam, L; Kroon, N C; Schallig, H D; Omar, S A

    2000-11-01

    A quantitative nucleic acid sequence-based amplification (QT-NASBA) assay for the detection of Plasmodium parasites has been developed. Primers and probes were selected on the basis of the sequence of the small-subunit rRNA gene. Quantification was achieved by coamplification of the RNA in the sample with one modified in vitro RNA as a competitor in a single-tube NASBA reaction. Parasite densities ranging from 10 to 10(8) Plasmodium falciparum parasites per ml could be demonstrated and quantified in whole blood. This is approximately 1,000 times more sensitive than conventional microscopy analysis of thick blood smears. Comparison of the parasite densities obtained by microscopy and QT-NASBA with 120 blood samples from Kenyan patients with clinical malaria revealed that for 112 of 120 (93%) of the samples results were within a 1-log difference. QT-NASBA may be especially useful for the detection of low parasite levels in patients with early-stage malaria and for the monitoring of the efficacy of drug treatment.

  7. Nucleic acids encoding metal uptake transporters and their uses

    DOEpatents

    Schroeder, Julian I.; Antosiewicz, Danuta M.; Schachtman, Daniel P.; Clemens, Stephan

    1999-01-01

    The invention provides LCT1 nucleic acids which encode metal ion uptake transporters. The invention also provides methods of modulating heavy metal and alkali metal uptake in plants. The methods involve producing transgenic plants comprising a recombinant expression cassette containing an LCT1 nucleic acid linked to a plant promoter.

  8. 21 CFR 866.5910 - Quality control material for cystic fibrosis nucleic acid assays.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5910 Quality control material for cystic fibrosis nucleic acid assays. (a... cystic fibrosis nucleic acid assays is a device intended to help monitor reliability of a test system by...

  9. Nucleic acids encoding mosaic clade M human immunodeficiency virus type 1 (HIV-1) envelope immunogens

    DOEpatents

    Korber, Bette T; Fischer, William; Liao, Hua-Xin; Haynes, Barton F; Letvin, Norman; Hahn, Beatrice H

    2015-04-21

    The present invention relates to nucleic acids encoding mosaic clade M HIV-1 Env polypeptides and to compositions and vectors comprising same. The nucleic acids of the invention are suitable for use in inducing an immune response to HIV-1 in a human.

  10. Methods and compositions for efficient nucleic acid sequencing

    DOEpatents

    Drmanac, Radoje

    2006-07-04

    Disclosed are novel methods and compositions for rapid and highly efficient nucleic acid sequencing based upon hybridization with two sets of small oligonucleotide probes of known sequences. Extremely large nucleic acid molecules, including chromosomes and non-amplified RNA, may be sequenced without prior cloning or subcloning steps. The methods of the invention also solve various current problems associated with sequencing technology such as, for example, high noise to signal ratios and difficult discrimination, attaching many nucleic acid fragments to a surface, preparing many, longer or more complex probes and labelling more species.

  11. Methods and compositions for efficient nucleic acid sequencing

    DOEpatents

    Drmanac, Radoje

    2002-01-01

    Disclosed are novel methods and compositions for rapid and highly efficient nucleic acid sequencing based upon hybridization with two sets of small oligonucleotide probes of known sequences. Extremely large nucleic acid molecules, including chromosomes and non-amplified RNA, may be sequenced without prior cloning or subcloning steps. The methods of the invention also solve various current problems associated with sequencing technology such as, for example, high noise to signal ratios and difficult discrimination, attaching many nucleic acid fragments to a surface, preparing many, longer or more complex probes and labelling more species.

  12. Nucleic acid detection system and method for detecting influenza

    DOEpatents

    Cai, Hong; Song, Jian

    2015-03-17

    The invention provides a rapid, sensitive and specific nucleic acid detection system which utilizes isothermal nucleic acid amplification in combination with a lateral flow chromatographic device, or DNA dipstick, for DNA-hybridization detection. The system of the invention requires no complex instrumentation or electronic hardware, and provides a low cost nucleic acid detection system suitable for highly sensitive pathogen detection. Hybridization to single-stranded DNA amplification products using the system of the invention provides a sensitive and specific means by which assays can be multiplexed for the detection of multiple target sequences.

  13. [Genotoxic modification of nucleic acid bases and biological consequences of it. Review and prospects of experimental and computational investigations

    NASA Technical Reports Server (NTRS)

    Poltev, V. I.; Bruskov, V. I.; Shuliupina, N. V.; Rein, R.; Shibata, M.; Ornstein, R.; Miller, J.

    1993-01-01

    The review is presented of experimental and computational data on the influence of genotoxic modification of bases (deamination, alkylation, oxidation) on the structure and biological functioning of nucleic acids. Pathways are discussed for the influence of modification on coding properties of bases, on possible errors of nucleic acid biosynthesis, and on configurations of nucleotide mispairs. The atomic structure of nucleic acid fragments with modified bases and the role of base damages in mutagenesis and carcinogenesis are considered.

  14. Hybridization-based biosensor containing hairpin probes and use thereof

    DOEpatents

    Miller, Benjamin L.; Strohsahl, Christopher M.

    2010-10-12

    A sensor chip that includes: a fluorescence quenching surface; a nucleic acid probe that contains first and second ends with the first end bound to the fluorescence quenching surface, and is characterized by being able to self-anneal into a hairpin conformation; and a first fluorophore bound to the second end of the first nucleic acid molecule. When the first nucleic acid molecule is in the hairpin conformation, the fluorescence quenching surface substantially quenches fluorescent emissions by the first fluorophore; and when the first nucleic acid molecule is in a non-hairpin conformation, fluorescent emissions by the fluorophore are substantially free of quenching by the fluorescence quenching surface. Various nucleic acid probes, methods of making the sensor chip, biological sensor devices that contain the sensor chip, and their methods of use are also disclosed.

  15. Nucleic acid duplexes incorporating a dissociable covalent base pair

    PubMed Central

    Gao, Kui; Orgel, Leslie E.

    1999-01-01

    We have used molecular modeling techniques to design a dissociable covalently bonded base pair that can replace a Watson-Crick base pair in a nucleic acid with minimal distortion of the structure of the double helix. We introduced this base pair into a potential precursor of a nucleic acid double helix by chemical synthesis and have demonstrated efficient nonenzymatic template-directed ligation of the free hydroxyl groups of the base pair with appropriate short oligonucleotides. The nonenzymatic ligation reactions, which are characteristic of base paired nucleic acid structures, are abolished when the covalent base pair is reduced and becomes noncoplanar. This suggests that the covalent base pair linking the two strands in the duplex is compatible with a minimally distorted nucleic acid double-helical structure. PMID:10611299

  16. Plants having modified response to ethylene by transformation with an ETR nucleic acid

    DOEpatents

    Meyerowitz, Elliott M.; Chang, Caren; Bleecker, Anthony B.

    2001-01-01

    The invention includes transformed plants having at least one cell transformed with a modified ETR nucleic acid. Such plants have a phenotype characterized by a decrease in the response of at least one transformed plant cell to ethylene as compared to a plant not containing the transformed plant cell. Tissue and/or temporal specificity for expression of the modified ETR nucleic acid is controlled by selecting appropriate expression regulation sequences to target the location and/or time of expression of the transformed nucleic acid. The plants are made by transforming at least one plant cell with an appropriate modified ETR nucleic acid, regenerating plants from one or more of the transformed plant cells and selecting at least one plant having the desired phenotype.

  17. Replica amplification of nucleic acid arrays

    DOEpatents

    Church, George M.; Mitra, Robi D.

    2010-08-31

    Disclosed are improved methods of making and using immobilized arrays of nucleic acids, particularly methods for producing replicas of such arrays. Included are methods for producing high density arrays of nucleic acids and replicas of such arrays, as well as methods for preserving the resolution of arrays through rounds of replication. Also included are methods which take advantage of the availability of replicas of arrays for increased sensitivity in detection of sequences on arrays. Improved methods of sequencing nucleic acids immobilized on arrays utilizing single copies of arrays and methods taking further advantage of the availability of replicas of arrays are disclosed. The improvements lead to higher fidelity and longer read lengths of sequences immobilized on arrays. Methods are also disclosed which improve the efficiency of multiplex PCR using arrays of immobilized nucleic acids.

  18. A Magnetic Nanoparticle Based Nucleic Acid Isolation and Purification Instrument for DNA Extraction of Escherichia Coli O157: H7.

    PubMed

    Chen, Yahui; Lin, Jianhan; Jiang, Qin; Chen, Qi; Zhang, Shengjun; Li, Li

    2016-03-01

    The objective of this study was to evaluate the performance of a nucleic acid isolation and purification instrument using Escherichia coli O157:H7 as the model. The instrument was developed with magnetic nanoparticles for efficiently capturing nucleic acids and an intelligent mechanical unit for automatically performing the whole nucleic acid extraction process. A commercial DNA extraction kit from Huier Nano Company was used as reference. Nucleic acids in 1 ml of E. coli O157: H7 at a concentration of 5 x 10(8) CFU/mL were extracted by using this instrument and the kit in parallel and then detected by an ultraviolet spectrophotometer to obtain A260 values and A260/A280 values for the determination of the extracted DNA's quantity and purity, respectively. The A260 values for the instrument and the kit were 0.78 and 0.61, respectively, and the A260/A280 values were 1.98 and 1.93. The coefficient of variations of these parallel tests ranged from 10.5% to 16.7%. The results indicated that this nucleic acid isolation and purification instrument could extract a comparable level of nucleic acid within 50 min compared to the commercial DNA extraction kit.

  19. Non-viral nucleic acid containing nanoparticles as cancer therapeutics.

    PubMed

    Kozielski, Kristen L; Rui, Yuan; Green, Jordan J

    2016-10-01

    The delivery of nucleic acids such as DNA and short interfering RNA (siRNA) is promising for the treatment of many diseases, including cancer, by enabling novel biological mechanisms of action. Non-viral nanoparticles are a promising class of nucleic acid carriers that can be designed to be safer and more versatile than traditional viral vectors. In this review, recent advances in the intracellular delivery of DNA and siRNA are described with a focus on non-viral nanoparticle-based delivery methods. Material properties that have enabled successful delivery are discussed as well as applications that have directly been applied to cancer therapy. Strategies to co-deliver different nucleic acids are highlighted, as are novel targets for nucleic acid co-delivery. The treatment of complex genetically-based diseases such as cancer can be enabled by safe and effective intracellular delivery of multiple nucleic acids. Non-viral nanoparticles can be fabricated to deliver multiple nucleic acids to the same cell simultaneously to prevent tumor cells from easily compensating for the knockdown or overexpression of one genetic target. The continued innovation of new therapeutic modalities and non-viral nanotechnologies to provide target-specific and personalized forms of gene therapy hold promise for genetic medicine to treat diseases like cancer in the clinic.

  20. Identification of Nucleic Acid Binding Sites on Translin-Associated Factor X (TRAX) Protein

    PubMed Central

    Gupta, Gagan Deep; Kumar, Vinay

    2012-01-01

    Translin and TRAX proteins play roles in very important cellular processes such as DNA recombination, spatial and temporal expression of mRNA, and in siRNA processing. Translin forms a homomeric nucleic acid binding complex and binds to ssDNA and RNA. However, a mutant translin construct that forms homomeric complex lacking nucleic acid binding activity is able to form fully active heteromeric translin-TRAX complex when co-expressed with TRAX. A substantial progress has been made in identifying translin sites that mediate its binding activity, while TRAX was thought not to bind DNA or RNA on its own. We here for the first time demonstrate nucleic acid binding to TRAX by crosslinking radiolabeled ssDNA to heteromeric translin-TRAX complex using UV-laser. The TRAX and translin, photochemically crosslinked with ssDNA, were individually detected on SDS-PAGE. We mutated two motifs in TRAX and translin, designated B2 and B3, to help define the nucleic acid binding sites in the TRAX sequence. The most pronounced effect was observed in the mutants of B3 motif that impaired nucleic acid binding activity of the heteromeric complexes. We suggest that both translin and TRAX are binding competent and contribute to the nucleic acid binding activity. PMID:22427937

  1. Non-viral nucleic acid containing nanoparticles as cancer therapeutics

    PubMed Central

    Kozielski, Kristen L.; Rui, Yuan

    2016-01-01

    Introduction The delivery of nucleic acids such as DNA and short interfering RNA (siRNA) is promising for the treatment of many diseases, including cancer, by enabling novel biological mechanisms of action. Non-viral nanoparticles are a promising class of nucleic acid carriers that can be designed to be safer and more versatile than traditional viral vectors. Areas covered In this review, recent advances in the intracellular delivery of DNA and siRNA are described with a focus on non-viral nanoparticle-based delivery methods. Material properties that have enabled successful delivery are discussed as well as applications that have directly been applied to cancer therapy. Strategies to co-deliver different nucleic acids are highlighted, as are novel targets for nucleic acid co-delivery. Expert opinion The treatment of complex genetically-based diseases such as cancer can be enabled by safe and effective intracellular delivery of multiple nucleic acids. Non-viral nanoparticles can be fabricated to deliver multiple nucleic acids to the same cell simultaneously to prevent tumor cells from easily compensating for the knockdown or overexpression of one genetic target. The continued innovation of new therapeutic modalities and non-viral nanotechnologies to provide target-specific and personalized forms of gene therapy hold promise for genetic medicine to treat diseases like cancer in the clinic. PMID:27248202

  2. Integrated DNA and RNA extraction and purification on an automated microfluidic cassette from bacterial and viral pathogens causing community-acquired lower respiratory tract infections.

    PubMed

    Van Heirstraeten, Liesbet; Spang, Peter; Schwind, Carmen; Drese, Klaus S; Ritzi-Lehnert, Marion; Nieto, Benjamin; Camps, Marta; Landgraf, Bryan; Guasch, Francesc; Corbera, Antoni Homs; Samitier, Josep; Goossens, Herman; Malhotra-Kumar, Surbhi; Roeser, Tina

    2014-05-07

    In this paper, we describe the development of an automated sample preparation procedure for etiological agents of community-acquired lower respiratory tract infections (CA-LRTI). The consecutive assay steps, including sample re-suspension, pre-treatment, lysis, nucleic acid purification, and concentration, were integrated into a microfluidic lab-on-a-chip (LOC) cassette that is operated hands-free by a demonstrator setup, providing fluidic and valve actuation. The performance of the assay was evaluated on viral and Gram-positive and Gram-negative bacterial broth cultures previously sampled using a nasopharyngeal swab. Sample preparation on the microfluidic cassette resulted in higher or similar concentrations of pure bacterial DNA or viral RNA compared to manual benchtop experiments. The miniaturization and integration of the complete sample preparation procedure, to extract purified nucleic acids from real samples of CA-LRTI pathogens to, and above, lab quality and efficiency, represent important steps towards its application in a point-of-care test (POCT) for rapid diagnosis of CA-LRTI.

  3. Application of Droplet Digital PCR to Validate Rift Valley Fever Vaccines.

    PubMed

    Ly, Hoai J; Lokugamage, Nandadeva; Ikegami, Tetsuro

    2016-01-01

    Droplet Digital™ polymerase chain reaction (ddPCR™) is a promising technique that quantitates the absolute concentration of nucleic acids in a given sample. This technique utilizes water-in-oil emulsion technology, a system developed by Bio-Rad Laboratories that partitions a single sample into thousands of nanoliter-sized droplets and counts nucleic acid molecules encapsulated in each individual particle as one PCR reaction. This chapter discusses the applications and methodologies of ddPCR for development of Rift Valley fever (RVF) vaccine, using an example that measures RNA copy numbers of a live-attenuated MP-12 vaccine from virus stocks, infected cells, or animal blood. We also discuss how ddPCR detects a reversion mutant of MP-12 from virus stocks accurately. The use of ddPCR improves the quality control of live-attenuated vaccines in the seed lot systems.

  4. Detection of Nucleic Acids in Complex Samples via Magnetic Microbead-assisted Catalyzed Hairpin Assembly and "DD-A" FRET.

    PubMed

    Fang, Hongmei; Xie, Nuli; Ou, Min; Huang, Jin; Li, Wenshan; Wang, Qing; Liu, Jianbo; Yang, Xiaohai; Wang, Kemin

    2018-05-21

    Nucleic acids, as one kind of significant biomarkers, have attracted tremendous attention and exhibited immense value in fundamental studies and clinical applications. In this work, we developed a fluorescent assay for detecting nucleic acids in complex samples based on magnetic microbead (MMB)-assisted catalyzed hairpin assembly (CHA) and donor donor-acceptor fluorescence resonance energy transfer ("DD-A" FRET) signaling mechanism. Three types of DNA hairpin probes were employed in this system, including Capture, H1 (double FAM-labelled probe as FRET donor) and H2 (TAMRA-labelled probe as FRET acceptor). Firstly, the Captures immobilized on MMBs bound to targets in complex samples, and the sequences in Captures that could trigger catalyzed hairpin assembly (CHA) were exposed. Then, target-enriched MMBs complexes were separated and resuspended in the reaction buffer containing H1 and H2. As a result, numerous H1-H2 duplexes were formed during CHA process, inducing an obvious FRET signal. In contrast, CHA could not be trigger and the FRET signal was weak while target was absent. With the aid of magnetic separation and "DD-A" FRET, it was demonstrated to effectively eliminate errors from background interference. Importantly, this strategy realized amplified detection in buffer, with detection limits of microRNA as low as 34 pM. Furthermore, this method was successfully applied to detect microRNA-21 in serum and cell culture media. The results showed that our method has the potential for biomedical research and clinical application.

  5. SERS-based inverse molecular sentinel (iMS) nanoprobes for multiplexed detection of microRNA cancer biomarkers in biological samples

    NASA Astrophysics Data System (ADS)

    Crawford, Bridget M.; Wang, Hsin-Neng; Fales, Andrew M.; Bowie, Michelle L.; Seewaldt, Victoria L.; Vo-Dinh, Tuan

    2017-02-01

    The development of sensitive and selective biosensing techniques is of great interest for clinical diagnostics. Here, we describe the development and application of a surface enhanced Raman scattering (SERS) sensing technology, referred to as "inverse Molecular Sentinel (iMS)" nanoprobes, for the detection of nucleic acid biomarkers in biological samples. This iMS nanoprobe involves the use of plasmonic-active nanostars as the sensing platform for a homogenous assay for multiplexed detection of nucleic acid biomarkers, including DNA, RNA and microRNA (miRNA). The "OFF-to-ON" signal switch is based on a non-enzymatic strand-displacement process and the conformational change of stem-loop (hairpin) oligonucleotide probes upon target binding. Here, we demonstrate the development of iMS nanoprobes for the detection of DNA sequences as well as a modified design of the nanoprobe for the detection of short (22-nt) microRNA sequences. The application of iMS nanoprobes to detect miRNAs in real biological samples was performed with total small RNA extracted from breast cancer cell lines. The multiplex capability of the iMS technique was demonstrated using a mixture of the two differently labeled nanoprobes to detect miR-21 and miR-34a miRNA biomarkers for breast cancer. The results of this study demonstrate the feasibility of applying the iMS technique for multiplexed detection of nucleic acid biomarkers, including short miRNAs molecules.

  6. Thermodynamics of Nucleic Acid ‘Shape Readout’ by an Aminosugar†

    PubMed Central

    Xi, Hongjuan; Davis, Erik; Ranjan, Nihar; Xue, Liang; Hyde-Volpe, David; Arya, Dev P.

    2012-01-01

    Recognition of nucleic acids is important for our understanding of nucleic acid structure as well as for our understanding of nucleic acid-protein interactions. In addition to the direct readout mechanisms of nucleic acids such as H-bonding, shape recognition of nucleic acids is being increasingly recognized to play an equally important role in DNA recognition. Competition Dialysis, UV, Flourescent Intercalator displacement (FID), Computational Docking, and calorimetry studies were conducted to study the interaction of neomycin with a variety of nucleic acid conformations (shapes). At pH 5.5, these results suggest: (1) Neomycin binds three RNA structures (16S A site rRNA, poly(rA)•poly(rA), and poly(rA)•poly(rU)) with high affinities, Ka~107M−1. (2) The binding of neomycin to A-form GC-rich oligomer d(A2G15C15T2)2 has comparable affinity to RNA structures. (3) The binding of neomycin to DNA•RNA hybrids shows a three-fold variance attributable to their structural differences (poly(dA) •poly(rU), Ka=9.4×106M−1 and poly(rA)•poly(dT), Ka=3.1×106M−1). (4) The interaction of neomycin with DNA triplex poly(dA)•2poly(dT) yields a binding affinity of Ka=2.4×105M−1. (5) Poly(dA-dT)2 showed the lowest association constant for all nucleic acids studied (Ka=<105). (6) Neomycin binds to G-quadruplexes with Ka~104-105M−1. (7) Computational studies show that the decrease in major groove width in the B to A transition correlates with increasing neomycin affinity. Neomycin’s affinity for various nucleic acid structures can be ranked as follows, RNAs and GC-rich d(A2G15C15T2)2 structures > poly(dA)•poly(rU) > poly(rA)•poly(dT) > T•A-T triplex , G-quadruplexes, B-form AT-rich or GC-rich DNA sequences. The results illustrate the first example of a small molecule based ‘shape readout’ of different nucleic acid conformations. PMID:21863895

  7. HeLa Nucleic Acid Contamination in The Cancer Genome Atlas Leads to the Misidentification of Human Papillomavirus 18

    PubMed Central

    Cantalupo, Paul G.; Katz, Joshua P.

    2015-01-01

    ABSTRACT We searched The Cancer Genome Atlas (TCGA) database for viruses by comparing non-human reads present in transcriptome sequencing (RNA-Seq) and whole-exome sequencing (WXS) data to viral sequence databases. Human papillomavirus 18 (HPV18) is an etiologic agent of cervical cancer, and as expected, we found robust expression of HPV18 genes in cervical cancer samples. In agreement with previous studies, we also found HPV18 transcripts in non-cervical cancer samples, including those from the colon, rectum, and normal kidney. However, in each of these cases, HPV18 gene expression was low, and single-nucleotide variants and positions of genomic alignments matched the integrated portion of HPV18 present in HeLa cells. Chimeric reads that match a known virus-cell junction of HPV18 integrated in HeLa cells were also present in some samples. We hypothesize that HPV18 sequences in these non-cervical samples are due to nucleic acid contamination from HeLa cells. This finding highlights the problems that contamination presents in computational virus detection pipelines. IMPORTANCE Viruses associated with cancer can be detected by searching tumor sequence databases. Several studies involving searches of the TCGA database have reported the presence of HPV18, a known cause of cervical cancer, in a small number of additional cancers, including those of the rectum, kidney, and colon. We have determined that the sequences related to HPV18 in non-cervical samples are due to nucleic acid contamination from HeLa cells. To our knowledge, this is the first report of the misidentification of viruses in next-generation sequencing data of tumors due to contamination with a cancer cell line. These results raise awareness of the difficulty of accurately identifying viruses in human sequence databases. PMID:25631090

  8. Arginine-based poly(ester amide) nanoparticle platform: From structure-property relationship to nucleic acid delivery.

    PubMed

    You, Xinru; Gu, Zhipeng; Huang, Jun; Kang, Yang; Chu, Chih-Chang; Wu, Jun

    2018-05-25

    Many different types of polycations have been vigorously studied for nucleic acid delivery, but a systematical investigation of the structure-property relationships of polycations for nucleic acid delivery is still lacking. In this study, a new library of biodegradable and biocompatible arginine-based poly(ester amide) (Arg-PEA) biomaterials was designed and synthesized with a tunable structure for such a comprehensive structure-property research. Nanoparticle (NP) complexes were formed through the electrostatic interactions between the polycationic Arg-PEAs and anionic nucleic acids. The following structure effects of the Arg-PEAs on the transfection efficiency of nucleic acids were investigated: 1) the linker/spacer length (length effect and odd-even effect); 2) salt type of arginine; 3) the side chain; 4) chain stiffness; 5) molecular weight (MW). The data obtained revealed that a slight change in the Arg-PEA structure could finely tune its physicochemical property such as hydrophobicity, and this could subsequently affect the nanoparticle size and zeta potential, which, in turn, regulate the transfection efficiency and silencing outcomes. A further study of the Arg-PEA/CpG oligodeoxynucleotide NP complexes indicated that the polymer structure could precisily regulate the immune response of CpG, thus providing a new potential nano-immunotherapy strategy. The in vitro data have further confirmed that the Arg-PEA NPs showed a satisfactory delivery performance for a variety of nucleic acids. Therefore, the data from the current study provide comprehensive information about the Arg-PEA structure-transfection property relationship; the tunable property of the library of Arg-PEA biomaterials can be one of the promising candidates for nucleic acid delivery and other biomedical applications. Polycations have being intensive utilized for nucleic acid delivery. However, there has not been elucidated about the relationship between polycation's structure and the physicochemical properties/biological function. In this timely report, an arginine based poly(ester amide) (Arg-PEA) library was prepared with finely tunable structure to systematically investigate the structure-property relationships of polycations for nucleic acid delivery. The results revealed that slight change of Arg-PEA structure could finely tune the physicochemical property (such as hydrophobicity), which subsequently affect the size and zeta potential of Arg-PEA/nucleic acid nanoparticles(NPs), and finally regulate the resulting transfection or silencing outcomes. Further study of Arg-PEA/CpG NPs indicated that the polymer structure could precisely regulate immuno response of CpG, providing new potential nano-immunotherapy strategy. In vitro evaluations confirmed that the NPs showed satisfied delivery performance for a variety types of nucleic acids. Therefore, these studies provide comprehensive information of Arg-PEA structure-property relationship, and the tunable properties of Arg-PEAs make them promising candidates for nucleic acid delivery and other biomedical applications. Overall, we have shown enough significance and novelty in terms of nucleic acid delivery, biomaterials, pharmaceutical science and nanomedicine. Copyright © 2018. Published by Elsevier Ltd.

  9. 21 CFR 866.5910 - Quality control material for cystic fibrosis nucleic acid assays.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Quality control material for cystic fibrosis... Test Systems § 866.5910 Quality control material for cystic fibrosis nucleic acid assays. (a) Identification. Quality control material for cystic fibrosis nucleic acid assays. A quality control material for...

  10. 21 CFR 866.5910 - Quality control material for cystic fibrosis nucleic acid assays.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Quality control material for cystic fibrosis... Test Systems § 866.5910 Quality control material for cystic fibrosis nucleic acid assays. (a) Identification. Quality control material for cystic fibrosis nucleic acid assays. A quality control material for...

  11. 21 CFR 866.5910 - Quality control material for cystic fibrosis nucleic acid assays.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Quality control material for cystic fibrosis... Test Systems § 866.5910 Quality control material for cystic fibrosis nucleic acid assays. (a) Identification. Quality control material for cystic fibrosis nucleic acid assays. A quality control material for...

  12. 21 CFR 866.5910 - Quality control material for cystic fibrosis nucleic acid assays.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Quality control material for cystic fibrosis... Test Systems § 866.5910 Quality control material for cystic fibrosis nucleic acid assays. (a) Identification. Quality control material for cystic fibrosis nucleic acid assays. A quality control material for...

  13. Magnetically enhanced nucleic acid delivery. Ten years of magnetofection-progress and prospects.

    PubMed

    Plank, Christian; Zelphati, Olivier; Mykhaylyk, Olga

    2011-11-01

    Nucleic acids carry the building plans of living systems. As such, they can be exploited to make cells produce a desired protein, or to shut down the expression of endogenous genes or even to repair defective genes. Hence, nucleic acids are unique substances for research and therapy. To exploit their potential, they need to be delivered into cells which can be a challenging task in many respects. During the last decade, nanomagnetic methods for delivering and targeting nucleic acids have been developed, methods which are often referred to as magnetofection. In this review we summarize the progress and achievements in this field of research. We discuss magnetic formulations of vectors for nucleic acid delivery and their characterization, mechanisms of magnetofection, and the application of magnetofection in viral and nonviral nucleic acid delivery in cell culture and in animal models. We summarize results that have been obtained with using magnetofection in basic research and in preclinical animal models. Finally, we describe some of our recent work and end with some conclusions and perspectives. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Reduction of Nucleic Acid Content in Candida Yeast Cells by Bovine Pancreatic Ribonuclease A Treatment

    PubMed Central

    Castro, A. C.; Sinskey, A. J.; Tannenbaum, S. R.

    1971-01-01

    Yeast as a source of protein for human consumption is limited by its relatively high nucleic acid content. In this study, we developed an enzymatic method of decreasing the nucleic acid content. Candida utilis cells, heat-shocked at 80 C for 30 sec, were treated with bovine pancreatic ribonuclease A. Maximum leakage of nucleic acid was observed when the incubation temperature was between 55 and 65 C, the pH of the system from 6.75 to 8.0, and the enzyme-to-cell ratio 1:10,000 on a weight-by-weight basis. Other factors, such as yeast strain, age of cells, and method of propagation, did not influence the susceptibility of the yeast cells to the action of ribonuclease. Buffers and monovalent cations had no inhibiting effects. Magnesium and calcium ions at concentrations greater than 0.001 m showed marked inhibition on the rate of nucleic acid leakage. This enzymatic method reduced the nucleic acid content of yeast cells from 7.5 to 9.0% to 1.5 to 2.0% with no significant concomitant loss of protein. PMID:5165838

  15. Flexibility of nucleic acids: From DNA to RNA

    NASA Astrophysics Data System (ADS)

    Lei, Bao; Xi, Zhang; Lei, Jin; Zhi-Jie, Tan

    2016-01-01

    The structural flexibility of nucleic acids plays a key role in many fundamental life processes, such as gene replication and expression, DNA-protein recognition, and gene regulation. To obtain a thorough understanding of nucleic acid flexibility, extensive studies have been performed using various experimental methods and theoretical models. In this review, we will introduce the progress that has been made in understanding the flexibility of nucleic acids including DNAs and RNAs, and will emphasize the experimental findings and the effects of salt, temperature, and sequence. Finally, we will discuss the major unanswered questions in understanding the flexibility of nucleic acids. Project supported by the National Basic Research Program of China (Grant No. 2011CB933600), the National Natural Science Foundation of China (Grant Nos. 11175132, 11575128, and 11374234), and the Program for New Century Excellent Talents, China (Grant No. NCET 08-0408).

  16. Nucleic acid polymeric properties and electrostatics: Directly comparing theory and simulation with experiment.

    PubMed

    Sim, Adelene Y L

    2016-06-01

    Nucleic acids are biopolymers that carry genetic information and are also involved in various gene regulation functions such as gene silencing and protein translation. Because of their negatively charged backbones, nucleic acids are polyelectrolytes. To adequately understand nucleic acid folding and function, we need to properly describe its i) polymer/polyelectrolyte properties and ii) associating ion atmosphere. While various theories and simulation models have been developed to describe nucleic acids and the ions around them, many of these theories/simulations have not been well evaluated due to complexities in comparison with experiment. In this review, I discuss some recent experiments that have been strategically designed for straightforward comparison with theories and simulation models. Such data serve as excellent benchmarks to identify limitations in prevailing theories and simulation parameters. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Heat Capacity Changes Associated with Nucleic Acid Folding

    PubMed Central

    Mikulecky, Peter J.; Feig, Andrew L.

    2008-01-01

    Whereas heat capacity changes (ΔCPs) associated with folding transitions are commonplace in the literature of protein folding, they have long been considered a minor energetic contributor in nucleic acid folding. Recent advances in the understanding of nucleic acid folding and improved technology for measuring the energetics of folding transitions have allowed a greater experimental window for measuring these effects. We present in this review a survey of current literature that confronts the issue of ΔCPs associated with nucleic acid folding transitions. This work helps to gather the molecular insights that can be gleaned from analysis of ΔCPs and points toward the challenges that will need to be overcome if the energetic contribution of ΔCP terms are to be put to use in improving free energy calculations for nucleic acid structure prediction. PMID:16429398

  18. Fluorogenic, catalytic, photochemical reaction for amplified detection of nucleic acids.

    PubMed

    Dutta, Subrata; Fülöp, Annabelle; Mokhir, Andriy

    2013-09-18

    Photochemical, nucleic acid-induced reactions, which are controlled by nontoxic red light, are well-suited for detection of nucleic acids in live cells, since they do not require any additives and can be spatially and temporally regulated. We have recently described the first reaction of this type, in which a phenylselenyl derivative of thymidine (5'-PhSeT-ODNa) is cleaved in the presence of singlet oxygen (Fülöp, A., Peng, X., Greenberg, M. M., Mokhir, A. (2010) A nucleic acid directed, red light-induced chemical reaction. Chem. Commun. 46, 5659-5661). The latter reagent is produced upon exposure of a photosensitizer 3'-PS-ODNb (PS = Indium(III)-pyropheophorbide-a-chloride: InPPa) to >630 nm light. In 2012 we reported on a fluorogenic version of this reaction (Dutta, S., Flottmann, B., Heilemann, M., Mokhir, A. (2012) Hybridization and reaction-based, fluorogenic nucleic acid probes. Chem. Commun. 47, 9664-9666), which is potentially applicable for the detection of nucleic acids in cells. Unfortunately, its yield does not exceed 25% and no catalytic turnover could be observed in the presence of substrate excess. This problem occurs due to the efficient, competing oxidation of the substrate containing an electron rich carbon-carbon double bonds (SCH═CHS) in the presence of singlet oxygen with formation of a noncleavable product (SCH═CHSO). Herein we describe a related, but substantially improved photochemical, catalytic transformation of a fluorogenic, organic substrate, which consists of 9,10-dialkoxyanthracene linked to fluorescein, with formation of a bright fluorescent dye. In highly dilute solution this reaction occurs only in the presence of a nucleic acid template. We developed three types of such a reaction and demonstrated that they are high yielding and generate over 7.7 catalytic turnovers, are sensitive to single mismatches in nucleic acid targets, and can be applied for determination of both the amount of nucleic acids and potentially their localization.

  19. Simultaneous detection and determination of mercury (II) and lead (II) ions through the achievement of novel functional nucleic acid-based biosensors.

    PubMed

    Khoshbin, Zahra; Housaindokht, Mohammad Reza; Verdian, Asma; Bozorgmehr, Mohammad Reza

    2018-09-30

    The serious threats of mercury (Hg 2+ ) and lead (Pb 2+ ) ions for the public health makes it important to achieve the detection methods of the ions with high affinity and specificity. Metal ions usually coexist in some environment and foodstuff or clinical samples. Therefore, it is very necessary to develop a fast and simple method for simultaneous monitoring the amount of metal ions, especially when Hg 2+ and Pb 2+ coexist. DNAzyme-based biosensors and aptasensors have been highly regarded for this purpose as two main groups of the functional nucleic acid (FNA)-based biosensors. In this review, we summarize the recent achievements of functional nucleic acid-based biosensors for the simultaneous detection of Hg 2+ and Pb 2+ ions in two main optical and electrochemical groups. The tremendous interest in utilizing the various nanomaterials is also highlighted in the fabrication of the FNA-based biosensors. Finally, some results are presented based on the advantages and disadvantages of the studied FNA-based biosensors to compare their validation. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. Investigation of interaction between magnetic silica particles and lambda phage DNA fragment.

    PubMed

    Smerkova, Kristyna; Dostalova, Simona; Vaculovicova, Marketa; Kynicky, Jindrich; Trnkova, Libuse; Kralik, Miroslav; Adam, Vojtech; Hubalek, Jaromir; Provaznik, Ivo; Kizek, Rene

    2013-12-01

    Nucleic acids belong to the most important molecules and therefore the understanding of their properties, function and behavior is crucial. Even though a range of analytical and biochemical methods have been developed for this purpose, one common step is essential for all of them - isolation of the nucleic acid from the from complex sample matrix. The use of magnetic particles for the separation of nucleic acids has many advantages over other isolation methods. In this study, an isolation procedure for extraction of DNA was optimized. Each step of the isolation process including washing, immobilization and elution was optimized and therefore the efficiency was increased from 1.7% to 28.7% and the total time was shortened from 75 to 30min comparing to the previously described method. Quantification of the particular parameter influence was performed by square-wave voltammetry using hanging drop mercury electrode. Further, we compared the optimized method with standard chloroform extraction and applied on isolation of DNA from Staphylococcus aureus and Escherichia coli. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Quantitative analysis of small molecule-nucleic acid interactions with a biosensor surface and surface plasmon resonance detection.

    PubMed

    Liu, Yang; Wilson, W David

    2010-01-01

    Surface plasmon resonance (SPR) technology with biosensor surfaces has become a widely-used tool for the study of nucleic acid interactions without any labeling requirements. The method provides simultaneous kinetic and equilibrium characterization of the interactions of biomolecules as well as small molecule-biopolymer binding. SPR monitors molecular interactions in real time and provides significant advantages over optical or calorimetic methods for systems with strong binding coupled to small spectroscopic signals and/or reaction heats. A detailed and practical guide for nucleic acid interaction analysis using SPR-biosensor methods is presented. Details of the SPR technology and basic fundamentals are described with recommendations on the preparation of the SPR instrument, sensor chips, and samples, as well as extensive information on experimental design, quantitative and qualitative data analysis and presentation. A specific example of the interaction of a minor-groove-binding agent with DNA is evaluated by both kinetic and steady-state SPR methods to illustrate the technique. Since the molecules that bind cooperatively to specific DNA sequences are attractive for many applications, a cooperative small molecule-DNA interaction is also presented.

  2. Solar thermal polymerase chain reaction for smartphone-assisted molecular diagnostics.

    PubMed

    Jiang, Li; Mancuso, Matthew; Lu, Zhengda; Akar, Gunkut; Cesarman, Ethel; Erickson, David

    2014-02-20

    Nucleic acid-based diagnostic techniques such as polymerase chain reaction (PCR) are used extensively in medical diagnostics due to their high sensitivity, specificity and quantification capability. In settings with limited infrastructure and unreliable electricity, however, access to such devices is often limited due to the highly specialized and energy-intensive nature of the thermal cycling process required for nucleic acid amplification. Here we integrate solar heating with microfluidics to eliminate thermal cycling power requirements as well as create a simple device infrastructure for PCR. Tests are completed in less than 30 min, and power consumption is reduced to 80 mW, enabling a standard 5.5 Wh iPhone battery to provide 70 h of power to this system. Additionally, we demonstrate a complete sample-to-answer diagnostic strategy by analyzing human skin biopsies infected with Kaposi's Sarcoma herpesvirus (KSHV/HHV-8) through the combination of solar thermal PCR, HotSHOT DNA extraction and smartphone-based fluorescence detection. We believe that exploiting the ubiquity of solar thermal energy as demonstrated here could facilitate broad availability of nucleic acid-based diagnostics in resource-limited areas.

  3. Solar thermal polymerase chain reaction for smartphone-assisted molecular diagnostics

    NASA Astrophysics Data System (ADS)

    Jiang, Li; Mancuso, Matthew; Lu, Zhengda; Akar, Gunkut; Cesarman, Ethel; Erickson, David

    2014-02-01

    Nucleic acid-based diagnostic techniques such as polymerase chain reaction (PCR) are used extensively in medical diagnostics due to their high sensitivity, specificity and quantification capability. In settings with limited infrastructure and unreliable electricity, however, access to such devices is often limited due to the highly specialized and energy-intensive nature of the thermal cycling process required for nucleic acid amplification. Here we integrate solar heating with microfluidics to eliminate thermal cycling power requirements as well as create a simple device infrastructure for PCR. Tests are completed in less than 30 min, and power consumption is reduced to 80 mW, enabling a standard 5.5 Wh iPhone battery to provide 70 h of power to this system. Additionally, we demonstrate a complete sample-to-answer diagnostic strategy by analyzing human skin biopsies infected with Kaposi's Sarcoma herpesvirus (KSHV/HHV-8) through the combination of solar thermal PCR, HotSHOT DNA extraction and smartphone-based fluorescence detection. We believe that exploiting the ubiquity of solar thermal energy as demonstrated here could facilitate broad availability of nucleic acid-based diagnostics in resource-limited areas.

  4. Chip-based sequencing nucleic acids

    DOEpatents

    Beer, Neil Reginald

    2014-08-26

    A system for fast DNA sequencing by amplification of genetic material within microreactors, denaturing, demulsifying, and then sequencing the material, while retaining it in a PCR/sequencing zone by a magnetic field. One embodiment includes sequencing nucleic acids on a microchip that includes a microchannel flow channel in the microchip. The nucleic acids are isolated and hybridized to magnetic nanoparticles or to magnetic polystyrene-coated beads. Microreactor droplets are formed in the microchannel flow channel. The microreactor droplets containing the nucleic acids and the magnetic nanoparticles are retained in a magnetic trap in the microchannel flow channel and sequenced.

  5. Metal resistant plants and phytoremediation of environmental contamination

    DOEpatents

    Meagher, Richard B.; Li, Yujing; Dhankher, Om P.

    2010-04-20

    The present disclosure provides a method of producing transgenic plants which are resistant to at least one metal ion by transforming the plant with a recombinant DNA comprising a nucleic acid encoding a bacterial arsenic reductase under the control of a plant expressible promoter, and a nucleic acid encoding a nucleotide sequence encoding a phytochelatin biosynthetic enzyme under the control of a plant expressible promoter. The invention also relates a method of phytoremediation of a contaminated site by growing in the site a transgenic plant expressing a nucleic acid encoding a bacterial arsenate reductase and a nucleic acid encoding a phytochelatin biosynthetic enzyme.

  6. Microfluidic device for acoustic cell lysis

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

    Branch, Darren W.; Cooley, Erika Jane; Smith, Gennifer Tanabe

    2015-08-04

    A microfluidic acoustic-based cell lysing device that can be integrated with on-chip nucleic acid extraction. Using a bulk acoustic wave (BAW) transducer array, acoustic waves can be coupled into microfluidic cartridges resulting in the lysis of cells contained therein by localized acoustic pressure. Cellular materials can then be extracted from the lysed cells. For example, nucleic acids can be extracted from the lysate using silica-based sol-gel filled microchannels, nucleic acid binding magnetic beads, or Nafion-coated electrodes. Integration of cell lysis and nucleic acid extraction on-chip enables a small, portable system that allows for rapid analysis in the field.

  7. Selection of fluorophore and quencher pairs for fluorescent nucleic acid hybridization probes.

    PubMed

    Marras, Salvatore A E

    2006-01-01

    With the introduction of simple and relatively inexpensive methods for labeling nucleic acids with nonradioactive labels, doors have been opened that enable nucleic acid hybridization probes to be used for research and development, as well as for clinical diagnostic applications. The use of fluorescent hybridization probes that generate a fluorescence signal only when they bind to their target enables real-time monitoring of nucleic acid amplification assays. The use of hybridization probes that bind to the amplification products in real-time markedly improves the ability to obtain quantitative results. Furthermore, real-time nucleic acid amplification assays can be carried out in sealed tubes, eliminating carryover contamination. Because fluorescent hybridization probes are available in a wide range of colors, multiple hybridization probes, each designed for the detection of a different nucleic acid sequence and each labeled with a differently colored fluorophore, can be added to the same nucleic acid amplification reaction, enabling the development of high-throughput multiplex assays. It is therefore important to carefully select the labels of hybridization probes, based on the type of hybridization probe used in the assay, the number of targets to be detected, and the type of apparatus available to perform the assay. This chapter outlines different aspects of choosing appropriate labels for the different types of fluorescent hybridization probes used with different types of spectrofluorometric thermal cyclers.

  8. Neutron Nucleic Acid Crystallography.

    PubMed

    Chatake, Toshiyuki

    2016-01-01

    The hydration shells surrounding nucleic acids and hydrogen-bonding networks involving water molecules and nucleic acids are essential interactions for the structural stability and function of nucleic acids. Water molecules in the hydration shells influence various conformations of DNA and RNA by specific hydrogen-bonding networks, which often contribute to the chemical reactivity and molecular recognition of nucleic acids. However, X-ray crystallography could not provide a complete description of structural information with respect to hydrogen bonds. Indeed, X-ray crystallography is a powerful tool for determining the locations of water molecules, i.e., the location of the oxygen atom of H2O; however, it is very difficult to determine the orientation of the water molecules, i.e., the orientation of the two hydrogen atoms of H2O, because X-ray scattering from the hydrogen atom is very small.Neutron crystallography is a specialized tool for determining the positions of hydrogen atoms. Neutrons are not diffracted by electrons, but are diffracted by atomic nuclei; accordingly, neutron scattering lengths of hydrogen and its isotopes are comparable to those of non-hydrogen atoms. Therefore, neutron crystallography can determine both of the locations and orientations of water molecules. This chapter describes the current status of neutron nucleic acid crystallographic research as well as the basic principles of neutron diffraction experiments performed on nucleic acid crystals: materials, crystallization, diffraction experiments, and structure determination.

  9. Logic gates and antisense DNA devices operating on a translator nucleic Acid scaffold.

    PubMed

    Shlyahovsky, Bella; Li, Yang; Lioubashevski, Oleg; Elbaz, Johann; Willner, Itamar

    2009-07-28

    A series of logic gates, "AND", "OR", and "XOR", are designed using a DNA scaffold that includes four "footholds" on which the logic operations are activated. Two of the footholds represent input-recognition strands, and these are blocked by complementary nucleic acids, whereas the other two footholds are blocked by nucleic acids that include the horseradish peroxidase (HRP)-mimicking DNAzyme sequence. The logic gates are activated by either nucleic acid inputs that hybridize to the respective "footholds", or by low-molecular-weight inputs (adenosine monophosphate or cocaine) that yield the respective aptamer-substrate complexes. This results in the respective translocation of the blocking nucleic acids to the footholds carrying the HRP-mimicking DNAzyme sequence, and the concomitant release of the respective DNAzyme. The released product-strands then self-assemble into the hemin/G-quadruplex-HRP-mimicking DNAzyme that biocatalyzes the formation of a colored product and provides an output signal for the different logic gates. The principle of the logic operation is, then, implemented as a possible paradigm for future nanomedicine. The nucleic acid inputs that bind to the blocked footholds result in the translocation of the blocking nucleic acids to the respective footholds carrying the antithrombin aptamer. The released aptamer inhibits, then, the hydrolytic activity of thrombin. The system demonstrates the regulation of a biocatalytic reaction by a translator system activated on a DNA scaffold.

  10. Fatty acid-producing hosts

    DOEpatents

    Pfleger, Brian F; Lennen, Rebecca M

    2013-12-31

    Described are hosts for overproducing a fatty acid product such as a fatty acid. The hosts include an exogenous nucleic acid encoding a thioesterase and, optionally, an exogenous nucleic acid encoding an acetyl-CoA carboxylase, wherein an acyl-CoA synthetase in the hosts are functionally delected. The hosts prefereably include the nucleic acid encoding the thioesterase at an intermediate copy number. The hosts are preferably recominantly stable and growth-competent at 37.degree. C. Methods of producing a fatty acid product comprising culturing such hosts at 37.degree. C. are also described.

  11. Oxidative Stress and Nucleic Acid Oxidation in Patients with Chronic Kidney Disease

    PubMed Central

    Sung, Chih-Chien; Hsu, Yu-Chuan; Lin, Yuh-Feng

    2013-01-01

    Patients with chronic kidney disease (CKD) have high cardiovascular mortality and morbidity and a high risk for developing malignancy. Excessive oxidative stress is thought to play a major role in elevating these risks by increasing oxidative nucleic acid damage. Oxidative stress results from an imbalance between reactive oxygen/nitrogen species (RONS) production and antioxidant defense mechanisms and can cause vascular and tissue injuries as well as nucleic acid damage in CKD patients. The increased production of RONS, impaired nonenzymatic or enzymatic antioxidant defense mechanisms, and other risk factors including gene polymorphisms, uremic toxins (indoxyl sulfate), deficiency of arylesterase/paraoxonase, hyperhomocysteinemia, dialysis-associated membrane bioincompatibility, and endotoxin in patients with CKD can inhibit normal cell function by damaging cell lipids, arachidonic acid derivatives, carbohydrates, proteins, amino acids, and nucleic acids. Several clinical biomarkers and techniques have been used to detect the antioxidant status and oxidative stress/oxidative nucleic acid damage associated with long-term complications such as inflammation, atherosclerosis, amyloidosis, and malignancy in CKD patients. Antioxidant therapies have been studied to reduce the oxidative stress and nucleic acid oxidation in patients with CKD, including alpha-tocopherol, N-acetylcysteine, ascorbic acid, glutathione, folic acid, bardoxolone methyl, angiotensin-converting enzyme inhibitor, and providing better dialysis strategies. This paper provides an overview of radical production, antioxidant defence, pathogenesis and biomarkers of oxidative stress in patients with CKD, and possible antioxidant therapies. PMID:24058721

  12. Red-shifted fluorescent proteins mPlum and mRaspberry and polynucleotides encoding the same

    DOEpatents

    Tsien, Roger Y [La Jolla, CA; Wang, Lei [San Diego, CA

    2008-07-01

    Methods using somatic hypermutation (SHM) for producing polypeptide and nucleic acid variants, and nucleic acids encoding such polypeptide variants are disclosed. Such variants may have desired properties. Also disclosed are novel polypeptides, such as improved fluorescent proteins, produced by the novel methods, and nucleic acids, vectors, and host cells comprising such vectors.

  13. Methods for engineering polypeptide variants via somatic hypermutation and polypeptide made thereby

    DOEpatents

    Tsien, Roger Y; Wang, Lei

    2015-01-13

    Methods using somatic hypermutation (SHM) for producing polypeptide and nucleic acid variants, and nucleic acids encoding such polypeptide variants are disclosed. Such variants may have desired properties. Also disclosed are novel polypeptides, such as improved fluorescent proteins, produced by the novel methods, and nucleic acids, vectors, and host cells comprising such vectors.

  14. [Clinical usefulness of urine-formed elements' information obtained from bacteria detection by flow cytometry method that uses nucleic acid staining].

    PubMed

    Nakagawa, Hiroko; Yuno, Tomoji; Itho, Kiichi

    2009-03-01

    Recently, specific detection method for Bacteria, by flow cytometry method using nucleic acid staining, was developed as a function of automated urine formed elements analyzer for routine urine testing. Here, we performed a basic study on this bacteria analysis method. In addition, we also have a comparison among urine sediment analysis, urine Gram staining and urine quantitative cultivation, the conventional methods performed up to now. As a result, the bacteria analysis with flow cytometry method that uses nucleic acid staining was excellent in reproducibility, and higher sensitivity compared with microscopic urinary sediment analysis. Based on the ROC curve analysis, which settled urine culture method as standard, cut-off level of 120/microL was defined and its sensitivity = 85.7%, specificity = 88.2%. In the analysis of scattergram, accompanied with urine culture method, among 90% of rod positive samples, 80% of dots were appeared in the area of 30 degrees from axis X. In addition, one case even indicated that analysis of bacteria by flow cytometry and scattergram of time series analysis might be helpful to trace the progress of causative bacteria therefore the information supposed to be clinically significant. Reporting bacteria information with nucleic acid staining flow cytometry method is expected to contribute to a rapid diagnostics and treatment of urinary tract infections. Besides, the contribution to screening examination of microbiology and clinical chemistry, will deliver a more efficient solution to urine analysis.

  15. Construction Strategy for an Internal Amplification Control for Real-Time Diagnostic Assays Using Nucleic Acid Sequence-Based Amplification: Development and Clinical Application

    PubMed Central

    Rodríguez-Lázaro, David; D'Agostino, Martin; Pla, Maria; Cook, Nigel

    2004-01-01

    An important analytical control in molecular amplification-based methods is an internal amplification control (IAC), which should be included in each reaction mixture. An IAC is a nontarget nucleic acid sequence which is coamplified simultaneously with the target sequence. With negative results for the target nucleic acid, the absence of an IAC signal indicates that amplification has failed. A general strategy for the construction of an IAC for inclusion in molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assays is presented. Construction proceeds in two phases. In the first phase, a double-stranded DNA molecule that contains nontarget sequences flanked by target sequences complementary to the NASBA primers is produced. At the 5′ end of this DNA molecule is a T7 RNA polymerase binding sequence. In the second phase of construction, RNA transcripts are produced from the DNA by T7 RNA polymerase. This RNA is the IAC; it is amplified by the target NASBA primers and is detected by a molecular beacon probe complementary to the internal nontarget sequences. As a practical example, an IAC for use in an assay for the detection of Mycobacterium avium subsp. paratuberculosis is described, its incorporation and optimization within the assay are detailed, and its application to spiked and natural clinical samples is shown to illustrate the correct interpretation of the diagnostic results. PMID:15583319

  16. Nuclease-resistant double-stranded DNA controls or standards for hepatitis B virus nucleic acid amplification assays

    PubMed Central

    2009-01-01

    Background Identical blood samples tested using different kits can give markedly different hepatitis B virus (HBV) DNA levels, which can cause difficulty in the interpretation of viral load. A universal double-stranded DNA control or standard that can be used in all commercial HBV DNA nucleic acid amplification assay kits is urgently needed. By aligning all HBV genotypes (A-H), we found that the surface antigen gene and precore-core gene regions of HBV are the most conserved regions among the different HBV genotypes. We constructed a chimeric fragment by overlapping extension polymerase chain reaction and obtained a 1,349-bp HBVC+S fragment. We then packaged the fragment into lambda phages using a traditional lambda phage cloning procedure. Results The obtained armored DNA was resistant to DNase I digestion and was stable, noninfectious to humans, and could be easily extracted using commercial kits. More importantly, the armored DNA may be used with all HBV DNA nucleic acid amplification assay kits. Conclusions The lambda phage packaging system can be used as an excellent expression platform for armored DNA. The obtained armored DNA possessed all characteristics of an excellent positive control or standard. In addition, this armored DNA is likely to be appropriate for all commercial HBV DNA nucleic acid amplification detection kits. Thus, the constructed armored DNA can probably be used as a universal positive control or standard in HBV DNA assays. PMID:20025781

  17. STUDIES OF THE MECHANISM OF ACTION OF URETHANE IN INITIATING PULMONARY ADENOMAS IN MICE

    PubMed Central

    Rogers, Stanfield

    1957-01-01

    The process of carcinogenesis following exposure of mice to urethane is demonstrated in the present work to be intimately related to nucleic acid synthesis. Injection of animals with a DNA hydrolysate immediately prior to a single exposure of the animals to urethane markedly reduced the number of pulmonary adenomas initiated. Aminopterin, known to interfere in nucleic acid synthesis (46), potentiated the carcinogenic action of urethane and this potentiation was blocked by injection of a DNA hydrolysate. Of the components and precursors of nucleic acids the pyrimidine series seemed especially concerned. Alterations in the utilization of oxaloacetate, ureidosuccinic acid, dihydro-orotic acid, orotic acid, cytidylic acid, and thymine appeared to be critical steps in the oncogenic process, following upon the primary disorder of cellular metabolism initiated by the carcinogen. All these substances except oxaloacetate profoundly reduced the number of tumors initiated by urethane. Oxaloacetate potentiated the carcinogenic effect. When these results are viewed together and in relation to known facts concerning nucleic acid synthesis they provide evidence suggesting that the point of action of the carcinogen is in the pathway of nucleic acid synthesis below orotic acid and perhaps at the level of ureidosuccinic acid. The potentiating influence of adenine, 4-amino-5-imidazole carboxamide, and aminopterin, the lack of effect of uracil, and the inhibitory influence of thymine together suggest that DNA rather than RNA is the nucleic acid critical to the oncogenic response of mice to urethane. PMID:13416469

  18. No Evidence for XMRV Nucleic Acids, Infectious Virus or Anti-XMRV Antibodies in Canadian Patients with Chronic Fatigue Syndrome

    PubMed Central

    Steffen, Imke; Tyrrell, D. Lorne; Stein, Eleanor; Montalvo, Leilani; Lee, Tzong-Hae; Zhou, Yanchen; Lu, Kai; Switzer, William M.; Tang, Shaohua; Jia, Hongwei; Hockman, Darren; Santer, Deanna M.; Logan, Michael; Landi, Amir; Law, John; Houghton, Michael; Simmons, Graham

    2011-01-01

    The gammaretroviruses xenotropic murine leukemia virus (MLV)-related virus (XMRV) and MLV have been reported to be more prevalent in plasma and peripheral blood mononuclear cells of chronic fatigue syndrome (CFS) patients than in healthy controls. Here, we report the complex analysis of whole blood and plasma samples from 58 CFS patients and 57 controls from Canada for the presence of XMRV/MLV nucleic acids, infectious virus, and XMRV/MLV-specific antibodies. Multiple techniques were employed, including nested and qRT-PCR, cell culture, and immunoblotting. We found no evidence of XMRV or MLV in humans and conclude that CFS is not associated with these gammaretroviruses. PMID:22114717

  19. Nucleic acid sensing and innate immunity: signaling pathways controlling viral pathogenesis and autoimmunity.

    PubMed

    Ahlers, Laura R H; Goodman, Alan G

    2016-09-01

    Innate immunity refers to the body's initial response to curb infection upon exposure to invading organisms. While the detection of pathogen-associated molecules is an ancient form of host defense, if dysfunctional, autoimmune disease may result. The innate immune response during pathogenic infection is initiated through the activation of receptors recognizing conserved molecular patterns, such as nucleic acids from a virus' genome or replicative cycle. Additionally, the host's own nucleic acids are capable of activating an immune response. Therefore, it follows that the nucleic acid-sensing pathways must be tightly controlled to avoid an autoimmune response from recognition of self, yet still be unimpeded to respond to viral infections. In this review, we will describe the nucleic acid sensing pathways and how they respond to virus infection. Moreover, we will discuss autoimmune diseases that develop when these pathways fail to signal properly and identify knowledge gaps that are prime for interrogation.

  20. Gene Therapy for Advanced Melanoma: Selective Targeting and Therapeutic Nucleic Acids

    PubMed Central

    Viola, Joana R.; Rafael, Diana F.; Wagner, Ernst; Besch, Robert; Ogris, Manfred

    2013-01-01

    Despite recent advances, the treatment of malignant melanoma still results in the relapse of the disease, and second line treatment mostly fails due to the occurrence of resistance. A wide range of mutations are known to prevent effective treatment with chemotherapeutic drugs. Hence, approaches with biopharmaceuticals including proteins, like antibodies or cytokines, are applied. As an alternative, regimens with therapeutically active nucleic acids offer the possibility for highly selective cancer treatment whilst avoiding unwanted and toxic side effects. This paper gives a brief introduction into the mechanism of this devastating disease, discusses the shortcoming of current therapy approaches, and pinpoints anchor points which could be harnessed for therapeutic intervention with nucleic acids. We bring the delivery of nucleic acid nanopharmaceutics into perspective as a novel antimelanoma therapeutic approach and discuss the possibilities for melanoma specific targeting. The latest reports on preclinical and already clinical application of nucleic acids in melanoma are discussed. PMID:23634303

  1. Nucleic acid and nucleotide-mediated synthesis of inorganic nanoparticles

    NASA Astrophysics Data System (ADS)

    Berti, Lorenzo; Burley, Glenn A.

    2008-02-01

    Since the advent of practical methods for achieving DNA metallization, the use of nucleic acids as templates for the synthesis of inorganic nanoparticles (NPs) has become an active area of study. It is now widely recognized that nucleic acids have the ability to control the growth and morphology of inorganic NPs. These biopolymers are particularly appealing as templating agents as their ease of synthesis in conjunction with the possibility of screening nucleotide composition, sequence and length, provides the means to modulate the physico-chemical properties of the resulting NPs. Several synthetic procedures leading to NPs with interesting photophysical properties as well as studies aimed at rationalizing the mechanism of nucleic acid-templated NP synthesis are now being reported. This progress article will outline the current understanding of the nucleic acid-templated process and provides an up to date reference in this nascent field.

  2. Fluorescence enhancement of quercetin complexes by silver nanoparticles and its analytical application

    NASA Astrophysics Data System (ADS)

    Liu, Ping; Zhao, Liangliang; Wu, Xia; Huang, Fei; Wang, Minqin; Liu, Xiaodan

    2014-03-01

    It is found that the plasmon effect of silver nanoparticles (AgNPs) helps to enhance the fluorescence intensity of the quercetin (Qu) and nucleic acids system. Qu exhibited strong fluorescence enhancement when it bound to nucleic acids in the presence of AgNPs. Based on this, a sensitive method for the determination of nucleic acids was developed. The detection limits for the nucleic acids (S/N = 3) were reduced to the ng mL-1 level. The interaction mechanism of the AgNPs-fish sperm DNA (fsDNA)-Qu system was also investigated in this paper. This complex system of Qu and AgNPs was also successfully used for the detection of nucleic acids in agarose gel electrophoresis analysis. Preliminary results indicated that AgNPs also helped to improve sensitivity in the fluorescence image analysis of Qu combined with cellular contents in Arabidopsis thaliana protoplasts.

  3. Understanding Nucleic Acid–Ion Interactions

    PubMed Central

    Lipfert, Jan; Doniach, Sebastian; Das, Rhiju; Herschlag, Daniel

    2015-01-01

    Ions surround nucleic acids in what is referred to as an ion atmosphere. As a result, the folding and dynamics of RNA and DNA and their complexes with proteins and with each other cannot be understood without a reasonably sophisticated appreciation of these ions’ electrostatic interactions. However, the underlying behavior of the ion atmosphere follows physical rules that are distinct from the rules of site binding that biochemists are most familiar and comfortable with. The main goal of this review is to familiarize nucleic acid experimentalists with the physical concepts that underlie nucleic acid–ion interactions. Throughout, we provide practical strategies for interpreting and analyzing nucleic acid experiments that avoid pitfalls from oversimplified or incorrect models. We briefly review the status of theories that predict or simulate nucleic acid–ion interactions and experiments that test these theories. Finally, we describe opportunities for going beyond phenomenological fits to a next-generation, truly predictive understanding of nucleic acid–ion interactions. PMID:24606136

  4. Comparative studies of nucleic acid hybridization assay for Listeria in foods

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

    Klinger, J.D.; Johnson, A.; Croan, D.

    A nucleic acid hybridization assay has been developed for Listeria spp. in dairy foods and environmental samples. The assay is based on detection of unique Listeria 16S rRNA sequences by using a /sup 32/P-labeled synthetic DNA probe. Inclusivity and exclusivity of the probe were confirmed with 139 Listeria isolates representing all known species, and 73 non-Listeria bacterial strains. In this paper, we present results from our preliminary studies comparing the hybridization assay with conventional culture on a total of 575 specimens that represent a variety of inoculated and uninoculated foods and environmental samples. The assay, which is done in amore » filter manifold format after 2 days of cultural enrichment, requires a total assay time of less than 2.5 days. The false-negative rate for all sample groups tested using the GENE-TRAK hybridization assay was less than the rate for culture. Thus, the new assay allows rapid screening of the indicated product groups and provides reliable numerical results.« less

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

    PubMed Central

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

    2015-01-01

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

  6. Is it time to move to nucleic acid amplification tests screening for pharyngeal and rectal gonorrhoea in men who have sex with men to improve gonorrhoea control?

    PubMed

    Fairley, Christopher K; Chen, Marcus Y; Bradshaw, Catriona S; Tabrizi, Sepehr N

    2011-03-01

    The use of nucleic acid amplification tests (NAAT), as well as or in preference to culture for non-genital sites is now recommended both in Australia and overseas because of their greater sensitivity and improved specificity. A survey of 22 Australian sexual health clinics who each year test over 14500 men who have sex with men (MSM) show that culture remains the predominate method for detecting gonorrhoea at pharyngeal (64%) and rectal (73%) sites. This editorial discusses the potential disadvantages of using culture over NAAT in relation to optimal gonorrhoea control among MSM and advocates that significantly improved control would be achieved by moving to NAAT with the proviso that culture samples are taken wherever possible on NAAT-positive samples and from clients with urethritis to ensure continued surveillance for antimicrobial resistance.

  7. Rapid Threat Organism Recognition Pipeline

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

    Williams, Kelly P.; Solberg, Owen D.; Schoeniger, Joseph S.

    2013-05-07

    The RAPTOR computational pipeline identifies microbial nucleic acid sequences present in sequence data from clinical samples. It takes as input raw short-read genomic sequence data (in particular, the type generated by the Illumina sequencing platforms) and outputs taxonomic evaluation of detected microbes in various human-readable formats. This software was designed to assist in the diagnosis or characterization of infectious disease, by detecting pathogen sequences in nucleic acid sequence data from clinical samples. It has also been applied in the detection of algal pathogens, when algal biofuel ponds became unproductive. RAPTOR first trims and filters genomic sequence reads based on qualitymore » and related considerations, then performs a quick alignment to the human (or other host) genome to filter out host sequences, then performs a deeper search against microbial genomes. Alignment to a protein sequence database is optional. Alignment results are summarized and placed in a taxonomic framework using the Lowest Common Ancestor algorithm.« less

  8. DNA stable-isotope probing (DNA-SIP).

    PubMed

    Dunford, Eric A; Neufeld, Josh D

    2010-08-02

    DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

  9. Nonenzymatic synthesis of RNA and DNA oligomers on hexitol nucleic acid templates: the importance of the A structure

    NASA Technical Reports Server (NTRS)

    Kozlov, I. A.; Politis, P. K.; Van Aerschot, A.; Busson, R.; Herdewijn, P.; Orgel, L. E.; Bada, J. L. (Principal Investigator); Dolan, M. (Principal Investigator)

    1999-01-01

    Hexitol nucleic acid (HNA) is an analogue of DNA containing the standard nucleoside bases, but with a phosphorylated 1,5-anhydrohexitol backbone. HNA oligomers form duplexes having the nucleic acid A structure with complementary DNA or RNA oligomers. The HNA decacytidylate oligomer is an efficient template for the oligomerization of the 5'-phosphoroimidazolides of guanosine or deoxyguanosine. Comparison of the oligomerization efficiencies on HNA, RNA, and DNA decacytidylate templates under various conditions suggests strongly that only nucleic acid double helices with the A structure support efficient template-directed synthesis when 5'-phosphoroimidazolides of nucleosides are used as substrates.

  10. [Recombinase Polymerase Amplification and its Applications in Parasite Detection].

    PubMed

    ZHENG, Wen-bin; WU, Yao-dong; MA, Jian-gang; ZHU, Xing-quan; ZHOU, Dong-hui

    2015-10-01

    Recombinase polymerase amplification (RPA) is a recently -developed isothermal nucleic-acid-amplification technology that is based on the nucleic acid replication mechanism in T4 bacteriophage. With this technique, nucleic-acid templates can be amplified to measurable levels within 20 min at 37-42 °C. The. RPA process has high sensitivity and specificity, and is simple to operate, thus nucleic acids can be detected rapidly in non-laboratory conditions. Since its development in 2006, the RPA technique has been applied in agriculture, food safety, medicine, transgene detection, etc. In this review, we will give an overview on the research progress of RPA and its application in parasite detection.

  11. Integrated magnetic tweezers and single-molecule FRET for investigating the mechanical properties of nucleic acid.

    PubMed

    Long, Xi; Parks, Joseph W; Stone, Michael D

    2016-08-01

    Many enzymes promote structural changes in their nucleic acid substrates via application of piconewton forces over nanometer length scales. Magnetic tweezers (MT) is a single molecule force spectroscopy method widely used for studying the energetics of such mechanical processes. MT permits stable application of a wide range of forces and torques over long time scales with nanometer spatial resolution. However, in any force spectroscopy experiment, the ability to monitor structural changes in nucleic acids with nanometer sensitivity requires the system of interest to be held under high degrees of tension to improve signal to noise. This limitation prohibits measurement of structural changes within nucleic acids under physiologically relevant conditions of low stretching forces. To overcome this challenge, researchers have integrated a spatially sensitive fluorescence spectroscopy method, single molecule-FRET, with MT to allow simultaneous observation and manipulation of nanoscale structural transitions over a wide range of forces. Here, we describe a method for using this hybrid instrument to analyze the mechanical properties of nucleic acids. We expect that this method for analysis of nucleic acid structure will be easily adapted for experiments aiming to interrogate the mechanical responses of other biological macromolecules. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Recent Advances in Delivery of Drug-Nucleic Acid Combinations for Cancer Treatment

    PubMed Central

    Li, Jing; Wang, Yan; Zhu, Yu; Oupický, David

    2013-01-01

    Cancer treatment that uses a combination of approaches with the ability to affect multiple disease pathways has been proven highly effective in the treatment of many cancers. Combination therapy can include multiple chemotherapeutics or combinations of chemotherapeutics with other treatment modalities like surgery or radiation. However, despite the widespread clinical use of combination therapies, relatively little attention has been given to the potential of modern nanocarrier delivery methods, like liposomes, micelles, and nanoparticles, to enhance the efficacy of combination treatments. This lack of knowledge is particularly notable in the limited success of vectors for the delivery of combinations of nucleic acids with traditional small molecule drugs. The delivery of drug-nucleic acid combinations is particularly challenging due to differences in the physicochemical properties of the two types of agents. This review discusses recent advances in the development of delivery methods using combinations of small molecule drugs and nucleic acid therapeutics to treat cancer. This review primarily focuses on the rationale used for selecting appropriate drug-nucleic acid combinations as well as progress in the development of nanocarriers suitable for simultaneous delivery of drug-nucleic acid combinations. PMID:23624358

  13. Nucleic acid aptamer-based methods for diagnosis of infections.

    PubMed

    Park, Ki Soo

    2018-04-15

    Infectious diseases are a serious global problem, which not only take an enormous human toll but also incur tremendous economic losses. In combating infectious diseases, rapid and accurate diagnostic tests are required for pathogen identification at the point of care (POC). In this review, investigations of diagnostic strategies for infectious diseases that are based on aptamers, especially nucleic acid aptamers, oligonucleotides that have high affinities and specificities toward their targets, are described. Owing to their unique features including low cost of production, easy chemical modification, high chemical stability, reproducibility, and low levels of immunogenicity and toxicity, aptamers have been widely utilized as bio-recognition elements (bio-receptors) for the development of infection diagnostic systems. We discuss nucleic acid aptamer-based methods that have been developed for diagnosis of infections using a format that organizes discussion according to the target pathogenic analytes including toxins or proteins, whole cells and nucleic acids. Also included is, a summary of recent advances made in the sensitive detection of pathogenic bacteria utilizing the isothermal nucleic acid amplification method. Lastly, a nucleic acid aptamer-based POC system is described and future directions of studies in this area are discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Recent advances in delivery of drug-nucleic acid combinations for cancer treatment.

    PubMed

    Li, Jing; Wang, Yan; Zhu, Yu; Oupický, David

    2013-12-10

    Cancer treatment that uses a combination of approaches with the ability to affect multiple disease pathways has been proven highly effective in the treatment of many cancers. Combination therapy can include multiple chemotherapeutics or combinations of chemotherapeutics with other treatment modalities like surgery or radiation. However, despite the widespread clinical use of combination therapies, relatively little attention has been given to the potential of modern nanocarrier delivery methods, like liposomes, micelles, and nanoparticles, to enhance the efficacy of combination treatments. This lack of knowledge is particularly notable in the limited success of vectors for the delivery of combinations of nucleic acids with traditional small molecule drugs. The delivery of drug-nucleic acid combinations is particularly challenging due to differences in the physicochemical properties of the two types of agents. This review discusses recent advances in the development of delivery methods using combinations of small molecule drugs and nucleic acid therapeutics to treat cancer. This review primarily focuses on the rationale used for selecting appropriate drug-nucleic acid combinations as well as progress in the development of nanocarriers suitable for simultaneous delivery of drug-nucleic acid combinations. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Integrated magnetic tweezers and single-molecule FRET for investigating the mechanical properties of nucleic acid

    PubMed Central

    Long, Xi; Parks, Joseph W.; Stone, Michael D.

    2017-01-01

    Many enzymes promote structural changes in their nucleic acid substrates via application of piconewton forces over nanometer length scales. Magnetic tweezers (MT) is a single molecule force spectroscopy method widely used for studying the energetics of such mechanical processes. MT permits stable application of a wide range of forces and torques over long time scales with nanometer spatial resolution. However, in any force spectroscopy experiment, the ability to monitor structural changes in nucleic acids with nanometer sensitivity requires the system of interest to be held under high degrees of tension to improve signal to noise. This limitation prohibits measurement of structural changes within nucleic acids under physiologically relevant conditions of low stretching forces. To overcome this challenge, researchers have integrated a spatially sensitive fluorescence spectroscopy method, single molecule-FRET, with MT to allow simultaneous observation and manipulation of nanoscale structural transitions over a wide range of forces. Here, we describe a method for using this hybrid instrument to analyze the mechanical properties of nucleic acids. We expect that this method for analysis of nucleic acid structure will be easily adapted for experiments aiming to interrogate the mechanical responses of other biological macromolecules. PMID:27320203

  16. Nucleic acid-based nanoengineering: novel structures for biomedical applications

    PubMed Central

    Li, Hanying; LaBean, Thomas H.; Leong, Kam W.

    2011-01-01

    Nanoengineering exploits the interactions of materials at the nanometre scale to create functional nanostructures. It relies on the precise organization of nanomaterials to achieve unique functionality. There are no interactions more elegant than those governing nucleic acids via Watson–Crick base-pairing rules. The infinite combinations of DNA/RNA base pairs and their remarkable molecular recognition capability can give rise to interesting nanostructures that are only limited by our imagination. Over the past years, creative assembly of nucleic acids has fashioned a plethora of two-dimensional and three-dimensional nanostructures with precisely controlled size, shape and spatial functionalization. These nanostructures have been precisely patterned with molecules, proteins and gold nanoparticles for the observation of chemical reactions at the single molecule level, activation of enzymatic cascade and novel modality of photonic detection, respectively. Recently, they have also been engineered to encapsulate and release bioactive agents in a stimulus-responsive manner for therapeutic applications. The future of nucleic acid-based nanoengineering is bright and exciting. In this review, we will discuss the strategies to control the assembly of nucleic acids and highlight the recent efforts to build functional nucleic acid nanodevices for nanomedicine. PMID:23050076

  17. THE EFFECT OF $gamma$-RADIATION ON THE EXCHANGE OF NUCLEIC ACIDS IN THE FOOD RESERVES OF PLANTS (in Russian)

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

    Metlitskii, L.V.; Korableva, N.P.; Morozova, N.P.

    1962-03-01

    Two types of onion (Allium cera and Allium sativum), and potato tubers were subjected to doses of 5000 to 60,000 r. The changes in nucleic acid content in the embryonic and pulpy part of the plant were followed. A dose of 10,000 r to potato tubers results in a 50% decrease in the content of guanidine, adenine, cytidine, and uridine in the embryonic parts of the tissue. Observations show that the ordinary onion (Allium cera) is less resistant to radiation than the garlic (Allium sativum). A dose of 5000 r to the ordinary onion results in a 35% decrease inmore » content of nucleic acids in the merismatic parts of the plant, while a dose of 60,000 r caused hardly any change in the garlic. After 6 months storage there is a rise of 30 to 40% in nucleic acid content of the onion and potato bulbs, but the increase in nucleic acid content is much greater for the unirradiated bulbs. The retarding effect of gamma -irradiation on the growth of plants is caused by a disturbance in the nucleic acid exchange. (TTT).« less

  18. Recent Advances in Non-viral Vectors for Gene Delivery

    PubMed Central

    Guo, Xia; Huang, Leaf

    2011-01-01

    CONSPECTUS Non-viral vectors, typically based on cationic lipids or polymers, are preferred due to safety concerns with viral vectors. So far, non-viral vectors can proficiently transfect cells in culture, but obtaining efficient nanomedicines is far from evident. To overcome the hurdles associated with non-viral vectors is significant for improving delivery efficiency and therapeutic effect of nucleic acid. The drawbacks include the strong interaction of cationic delivery vehicles with blood components, uptake by the reticuloendothelial system (RES), toxicity, targeting ability of the carriers to the cells of interest, and so on. PEGylation is the predominant method used to reduce the binding of plasma proteins with non-viral vectors and minimize the clearance by RES after intravenous administration. The nanoparticles that are not rapidly cleared from the circulation accumulate in the tumors due to the enhanced permeability and retention effect, and the targeting ligands attached to the distal end of the PEGylated components allow binding to the receptors on the target cell surface. Neutral or anionic liposomes have been also developed for systemic delivery of nucleic acids in experimental animal model. Designing and synthesizing novel cationic lipids and polymers, and binding nucleic acid with peptides, targeting ligands, polymers, or environmentally sensitive moieties also attract many attentions for resolving the problems encountered by non-viral vectors. The application of inorganic nanoparticles in nucleic acid delivery is an emerging field, too. Recently, different classes of non-viral vectors appear to be converging and the features of different classes of non-viral vectors could be combined in one strategy. More hurdles associated with efficient nucleic acid delivery therefore might be expected to be overcome. In this account, we will focus on these novel non-viral vectors, which are classified into multifunctional hybrid nucleic acid vectors, novel membrane/core nanoparticles for nucleic acid delivery and ultrasound-responsive nucleic acid vectors. The systemic delivery studies are highlighted. Finally, we bring forward the prospect for nucleic acid delivery. We think a better understandings of the fate of the nanoparticles inside the cell and of the interactions between the parts of hybrid particles will lead to a delivery system suitable for clinical use. We also underscore the value of sustained release of nucleic acid and presume making vectors targeted to cells with sustained release in vivo should be an interesting research challenge. PMID:21870813

  19. 77 FR 68133 - Guidance for Industry: Use of Nucleic Acid Tests on Pooled and Individual Samples From Donors of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-15

    ... Blood and Blood Components, Including Source Plasma, To Reduce the Risk of Transmission of Hepatitis B... Components, including Source Plasma, to Reduce the Risk of Transmission of Hepatitis B Virus,'' dated October... (NAT) to screen blood donors for hepatitis B virus (HBV) deoxyribonucleic acid (DNA) and...

  20. Quality Assurance of Samples and Processes in the Spanish Renal Research Network (REDinREN) Biobank.

    PubMed

    Calleros-Basilio, Laura; Cortés, María Alicia; García-Jerez, Andrea; Luengo-Rodríguez, Alicia; Orozco-Agudo, Ana; Valdivielso, José Manuel; Rodríguez-Puyol, Diego; Rodríguez-Puyol, Manuel

    2016-12-01

    Biobanks are useful platforms to build bridges between basic, translational, and clinical research and clinical care. They are repositories of high-quality human biological samples ideal for evaluating their histological characteristics and also their genome, transcriptome, and proteome. The Spanish Renal Research Network Biobank contains more than 76,500 well-preserved frozen samples of a wide variety of kidney diseases, collected from 5450 patients seen by over 70 nephrology services throughout the Spanish territory. To determine and to report the results of the quality control of samples and processes conducted in our biobank, implemented in accordance with the requirements of the ISO 9001:2008 international standard. Two types of quality controls were performed: (1) systematic, that is, measurement of viable peripheral blood mononuclear cells (PBMCs) obtained and purity of nucleic acids and (2) ad-hoc, that is, viability of thawed PBMC, DNA extraction process reproducibility, and the integrity and functionality of nucleic acids, implemented on a routine basis. PBMC isolation by Ficoll yielded reproducible results and its cryopreserved viability was >90%. Acceptable A260/A280 ratios were obtained for the vast majority of the DNA (n = 2328) and RNA (n = 78) samples analyzed. DNA integrity was demonstrated by agarose gels and by β-globulin gene polymerase chain reaction (PCR) amplification of 1327 and 989 bp fragments. DNA of acceptable quality had at least three bands of β-globulin amplified obtained (n = 26/30). RNA integrity number (RIN) determinations obtained RIN numbers ≥7 (n = 87/96). The amplifiability of nucleic acids was confirmed by qPCR and RT-qPCR of β-actin and GAPDH genes. Long storage or delayed processing time did not affect the quality of the samples analyzed. The processes of DNA extraction also yielded reproducible results. These results clearly indicate that our PBMC, DNA, and RNA stored samples meet the required quality standards to be used for biomedical research, ensuring their long-term preservation.

  1. Detection of equine herpesvirus-4 and physiological stress patterns in young Thoroughbreds consigned to a South African auction sale.

    PubMed

    Badenhorst, Marcha; Page, Patrick; Ganswindt, Andre; Laver, Peter; Guthrie, Alan; Schulman, Martin

    2015-06-02

    The prevalence of equine herpesvirus types-1 and -4 (EHV-1 and -4) in South African Thoroughbreds at auction sales is currently undefined. Commingling of young Thoroughbreds from various populations together with physiological stress related to their transport and confinement at a sales complex, may be associated with shedding and transmission of EHV-1 and -4. This prospective cohort study sampled 90 young Thoroughbreds consigned from eight farms, originating from three provinces representative of the South African Thoroughbred breeding demographic to a sales complex. Nasal swabs for quantitative real-time polymerase chain reaction (qPCR) assay to detect EHV-1 and -4 nucleic acid and blood samples for enzyme-linked immunosorbent assay for EHV-1 and -4 antibodies were collected from all horses on arrival and departure. Additional nasal swabs for qPCR were obtained serially from those displaying pyrexia and, or nasal discharge. Daily faecal samples were used for determination of faecal glucocorticoid metabolite (FGM) concentrations as a measurement of physiological stress and these values were modelled to determine the factors best explaining FGM variability. EHV-4 nucleic acid was detected in 14.4 % and EHV-1 from none of the animals in the study population. Most (93.3 %) and very few (1.1 %) of this population showed antibodies indicating prior exposure to EHV-4 and EHV-1 respectively. Pyrexia and nasal discharge were poor predictors for detecting EHV-4 nucleic acid. The horses' FGM concentrations increased following arrival before decreasing for most of the remaining study period including the auction process. Model averaging showed that variation in FGM concentrations was best explained by days post-arrival and transport duration. In this study population, sales consignment was associated with limited detection of EHV-4 nucleic acid in nasal secretions, with most showing prior exposure to EHV-4 and very few to EHV-1. The physiological stress response shown by most reflected the combination of stressors associated with transport and arrival and these are key areas for future investigation into management practices to enhance health and welfare of young Thoroughbreds during sales consignment.

  2. Hepatitis B virus X protein (HBx)-induced abnormalities of nucleic acid metabolism revealed by (1)H-NMR-based metabonomics.

    PubMed

    Dan Yue; Zhang, Yuwei; Cheng, Liuliu; Ma, Jinhu; Xi, Yufeng; Yang, Liping; Su, Chao; Shao, Bin; Huang, Anliang; Xiang, Rong; Cheng, Ping

    2016-04-14

    Hepatitis B virus X protein (HBx) plays an important role in HBV-related hepatocarcinogenesis; however, mechanisms underlying HBx-mediated carcinogenesis remain unclear. In this study, an NMR-based metabolomics approach was applied to systematically investigate the effects of HBx on cell metabolism. EdU incorporation assay was conducted to examine the effects of HBx on DNA synthesis, an important feature of nucleic acid metabolism. The results revealed that HBx disrupted metabolism of glucose, lipids, and amino acids, especially nucleic acids. To understand the potential mechanism of HBx-induced abnormalities of nucleic acid metabolism, gene expression profiles of HepG2 cells expressing HBx were investigated. The results showed that 29 genes involved in DNA damage and DNA repair were differentially expressed in HBx-expressing HepG2 cells. HBx-induced DNA damage was further demonstrated by karyotyping, comet assay, Western blotting, immunofluorescence and immunohistochemistry analyses. Many studies have previously reported that DNA damage can induce abnormalities of nucleic acid metabolism. Thus, our results implied that HBx initially induces DNA damage, and then disrupts nucleic acid metabolism, which in turn blocks DNA repair and induces the occurrence of hepatocellular carcinoma (HCC). These findings further contribute to our understanding of the occurrence of HCC.

  3. A novel method for room temperature distribution and conservation of RNA and DNA reference materials for guaranteeing performance of molecular diagnostics in onco-hematology: A GBMHM study.

    PubMed

    Cayuela, Jean-Michel; Mauté, Carole; Fabre, Anne-Lise; Nibourel, Olivier; Dulucq, Stéphanie; Delabesse, Eric; Villarèse, Patrick; Hayette, Sandrine; Mozziconacci, Marie-Joelle; Macintyre, Elizabeth

    2015-10-01

    Performance of methods used for molecular diagnostics must be closely controlled by regular analysis of internal quality controls. However, conditioning, shipping and long lasting storage of nucleic acid controls remain problematic. Therefore, we evaluated the minicapsule-based innovative process developed by Imagene (Evry, France) for implementing DNA and RNA controls designed for clonality assessment of lymphoproliferations and BCR-ABL1 mRNA quantification, respectively. DNA samples were extracted from 12 cell lines selected for giving specific amplifications with most BIOMED-2 PCR tubes. RNA samples were extracted from 8 cell line mixtures expressing various BCR-ABL1 transcript levels. DNA and RNA were encapsulated by Imagene and shipped at room temperature to participating laboratories. Biologists were asked to report quality data of recovered nucleic acids as well as PCR results. Encapsulated nucleic acids samples were easily and efficiently recovered from minicapsules. The expected rearrangements at immunoglobulin, T-cell receptor and BCL2 loci were detected in DNA samples by all laboratories. Quality of RNA was consistent between laboratories and met the criteria requested for quantification of BCR-ABL1 transcripts. Expression levels measured by the 5 laboratories were within ±2 fold interval from the corresponding pre-encapsulation reference value. Moreover aging studies of encapsulated RNA simulating up to 100 years storage at room temperature show no bias in quantitative outcome. Therefore, Imagene minicapsules are suitable for storage and distribution at room temperature of genetic material designed for proficiency control of molecular diagnostic methods based on end point or real-time quantitative PCR. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  4. Iron mediates catalysis of nucleic acid processing enzymes: support for Fe(II) as a cofactor before the great oxidation event.

    PubMed

    Okafor, C Denise; Lanier, Kathryn A; Petrov, Anton S; Athavale, Shreyas S; Bowman, Jessica C; Hud, Nicholas V; Williams, Loren Dean

    2017-04-20

    Life originated in an anoxic, Fe2+-rich environment. We hypothesize that on early Earth, Fe2+ was a ubiquitous cofactor for nucleic acids, with roles in RNA folding and catalysis as well as in processing of nucleic acids by protein enzymes. In this model, Mg2+ replaced Fe2+ as the primary cofactor for nucleic acids in parallel with known metal substitutions of metalloproteins, driven by the Great Oxidation Event. To test predictions of this model, we assay the ability of nucleic acid processing enzymes, including a DNA polymerase, an RNA polymerase and a DNA ligase, to use Fe2+ in place of Mg2+ as a cofactor during catalysis. Results show that Fe2+ can indeed substitute for Mg2+ in catalytic function of these enzymes. Additionally, we use calculations to unravel differences in energetics, structures and reactivities of relevant Mg2+ and Fe2+ complexes. Computation explains why Fe2+ can be a more potent cofactor than Mg2+ in a variety of folding and catalytic functions. We propose that the rise of O2 on Earth drove a Fe2+ to Mg2+ substitution in proteins and nucleic acids, a hypothesis consistent with a general model in which some modern biochemical systems retain latent abilities to revert to primordial Fe2+-based states when exposed to pre-GOE conditions. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Evolution of functional nucleic acids in the presence of nonheritable backbone heterogeneity.

    PubMed

    Trevino, Simon G; Zhang, Na; Elenko, Mark P; Lupták, Andrej; Szostak, Jack W

    2011-08-16

    Multiple lines of evidence support the hypothesis that the early evolution of life was dominated by RNA, which can both transfer information from generation to generation through replication directed by base-pairing, and carry out biochemical activities by folding into functional structures. To understand how life emerged from prebiotic chemistry we must therefore explain the steps that led to the emergence of the RNA world, and in particular, the synthesis of RNA. The generation of pools of highly pure ribonucleotides on the early Earth seems unlikely, but the presence of alternative nucleotides would support the assembly of nucleic acid polymers containing nonheritable backbone heterogeneity. We suggest that homogeneous monomers might not have been necessary if populations of heterogeneous nucleic acid molecules could evolve reproducible function. For such evolution to be possible, function would have to be maintained despite the repeated scrambling of backbone chemistry from generation to generation. We have tested this possibility in a simplified model system, by using a T7 RNA polymerase variant capable of transcribing nucleic acids that contain an approximately 11 mixture of deoxy- and ribonucleotides. We readily isolated nucleotide-binding aptamers by utilizing an in vitro selection process that shuffles the order of deoxy- and ribonucleotides in each round. We describe two such RNA/DNA mosaic nucleic acid aptamers that specifically bind ATP and GTP, respectively. We conclude that nonheritable variations in nucleic acid backbone structure may not have posed an insurmountable barrier to the emergence of functionality in early nucleic acids.

  6. Comparative evaluation of three commercial systems for nucleic acid extraction from urine specimens.

    PubMed

    Tang, Yi-Wei; Sefers, Susan E; Li, Haijing; Kohn, Debra J; Procop, Gary W

    2005-09-01

    A nucleic acid extraction system that can handle small numbers of specimens with a short test turnaround time and short hands-on time is desirable for emergent testing. We performed a comparative validation on three systems: the MagNA Pure compact system (Compact), the NucliSens miniMAG extraction instrument (miniMAG), and the BioRobot EZ1 system (EZ1). A total of 75 urine specimens submitted for polyomavirus BK virus detection were used. The human beta-actin gene was detected on 75 (100%), 75 (100%), and 72 (96%) nucleic acid extracts prepared by the miniMAG, EZ1, and Compact, respectively. The miniMAG produced the highest quantity of nucleic acids and the best precision among the three systems. The agreement rate was 100% for BKV detection on nucleic acid extracts prepared by the three extraction systems. When a full panel of specimens was run, the hands-on time and test turnaround time were 105.7 and 121.1 min for miniMAG, 6.1 and 22.6 min for EZ1, and 7.4 and 33.7 min for Compact, respectively. The EZ1 and Compact systems processed automatic nucleic acid extraction properly, providing a good solution to the need for sporadic but emergent specimen detection. The miniMAG yielded the highest quantity of nucleic acids, suggesting that this system would be the best for specimens containing a low number of microorganisms of interest.

  7. Real-time polymerase chain reaction in transfusion medicine: applications for detection of bacterial contamination in blood products.

    PubMed

    Dreier, Jens; Störmer, Melanie; Kleesiek, Knut

    2007-07-01

    Bacterial contamination of blood components, particularly of platelet concentrates (PCs), represents the greatest infectious risk in blood transfusion. Although the incidence of platelet bacterial contamination is approximately 1 per 2,000 U, the urgent need for a method for the routine screening of PCs to improve safety for patients had not been considered for a long time. Besides the culturing systems, which will remain the criterion standard, rapid methods for sterility screening will play a more important role in transfusion medicine in the future. In particular, nucleic acid amplification techniques (NATs) are powerful potential tools for bacterial screening assays. The combination of excellent sensitivity and specificity, reduced contamination risk, ease of performance, and speed has made real-time polymerase chain reaction (PCR) technology an appealing alternative to conventional culture-based testing methods. When using real-time PCR for the detection of bacterial contamination, several points have to be considered. The main focus is the choice of the target gene; the assay format; the nucleic acid extraction method, depending on the sample type; and the evaluation of an ideal sampling strategy. However, several factors such as the availability of bacterial-derived nucleic acid amplification reagents, the impracticability, and the cost have limited the use of NATs until now. Attempts to reduce the presence of contaminating nucleic acids from reagents in real-time PCR have been described, but none of these approaches have proven to be very effective or to lower the sensitivity of the assay. Recently, a number of broad-range NAT assays targeting the 16S ribosomal DNA or 23S ribosomal RNA for the detection of bacteria based on real-time technology have been reported. This review will give a short survey of current approaches to and the limitations of the application of real-time PCR for bacterial detection in blood components, with emphasis on the bacterial contamination of PCs.

  8. Clinical validation of a novel diagnostic HIV-2 total nucleic acid qualitative assay using the Abbott m2000 platform: Implications for complementary HIV-2 nucleic acid testing for the CDC 4th generation HIV diagnostic testing algorithm.

    PubMed

    Chang, Ming; Wong, Audrey J S; Raugi, Dana N; Smith, Robert A; Seilie, Annette M; Ortega, Jose P; Bogusz, Kyle M; Sall, Fatima; Ba, Selly; Seydi, Moussa; Gottlieb, Geoffrey S; Coombs, Robert W

    2017-01-01

    The 2014 CDC 4th generation HIV screening algorithm includes an orthogonal immunoassay to confirm and discriminate HIV-1 and HIV-2 antibodies. Additional nucleic acid testing (NAT) is recommended to resolve indeterminate or undifferentiated HIV seroreactivity. HIV-2 NAT requires a second-line assay to detect HIV-2 total nucleic acid (TNA) in patients' blood cells, as a third of untreated patients have undetectable plasma HIV-2 RNA. To validate a qualitative HIV-2 TNA assay using peripheral blood mononuclear cells (PBMC) from HIV-2-infected Senegalese study participants. We evaluated the assay precision, sensitivity, specificity, and diagnostic performance of an HIV-2 TNA assay. Matched plasma and PBMC samples were collected from 25 HIV-1, 30 HIV-2, 8 HIV-1/-2 dual-seropositive and 25 HIV seronegative individuals. Diagnostic performance was evaluated by comparing the outcome of the TNA assay to the results obtained by the 4th generation HIV screening and confirmatory immunoassays. All PBMC from 30 HIV-2 seropositive participants tested positive for HIV-2 TNA including 23 patients with undetectable plasma RNA. Of the 30 matched plasma specimens, one was HIV non-reactive. Samples from 50 non-HIV-2 infected individuals were confirmed as non-reactive for HIV-2 Ab and negative for HIV-2 TNA. The agreement between HIV-2 TNA and the combined immunoassay results was 98.8% (79/80). Furthermore, HIV-2 TNA was detected in 7 of 8 PBMC specimens from HIV-1/HIV-2 dual-seropositive participants. Our TNA assay detected HIV-2 DNA/RNA in PBMC from serologically HIV-2 reactive, HIV indeterminate or HIV undifferentiated individuals with undetectable plasma RNA, and is suitable for confirming HIV-2 infection in the HIV testing algorithm. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Evaluation of a TaqMan Array Card for Detection of Central Nervous System Infections.

    PubMed

    Onyango, Clayton O; Loparev, Vladimir; Lidechi, Shirley; Bhullar, Vinod; Schmid, D Scott; Radford, Kay; Lo, Michael K; Rota, Paul; Johnson, Barbara W; Munoz, Jorge; Oneko, Martina; Burton, Deron; Black, Carolyn M; Neatherlin, John; Montgomery, Joel M; Fields, Barry

    2017-07-01

    Infections of the central nervous system (CNS) are often acute, with significant morbidity and mortality. Routine diagnosis of such infections is limited in developing countries and requires modern equipment in advanced laboratories that may be unavailable to a number of patients in sub-Saharan Africa. We developed a TaqMan array card (TAC) that detects multiple pathogens simultaneously from cerebrospinal fluid. The 21-pathogen CNS multiple-pathogen TAC (CNS-TAC) assay includes two parasites ( Balamuthia mandrillaris and Acanthamoeba ), six bacterial pathogens ( Streptococcus pneumonia e, Haemophilus influenzae , Neisseria meningitidis , Mycoplasma pneumoniae , Mycobacterium tuberculosis , and Bartonella ), and 13 viruses (parechovirus, dengue virus, Nipah virus, varicella-zoster virus, mumps virus, measles virus, lyssavirus, herpes simplex viruses 1 and 2, Epstein-Barr virus, enterovirus, cytomegalovirus, and chikungunya virus). The card also includes human RNase P as a nucleic acid extraction control and an internal manufacturer control, GAPDH (glyceraldehyde-3-phosphate dehydrogenase). This CNS-TAC assay can test up to eight samples for all 21 agents within 2.5 h following nucleic acid extraction. The assay was validated for linearity, limit of detection, sensitivity, and specificity by using either live viruses (dengue, mumps, and measles viruses) or nucleic acid material (Nipah and chikungunya viruses). Of 120 samples tested by individual real-time PCR, 35 were positive for eight different targets, whereas the CNS-TAC assay detected 37 positive samples across nine different targets. The CNS-TAC assays showed 85.6% sensitivity and 96.7% specificity. Therefore, the CNS-TAC assay may be useful for outbreak investigation and surveillance of suspected neurological disease. Copyright © 2017 American Society for Microbiology.

  10. Prospective evaluation of the Alere i Influenza A&B nucleic acid amplification versus Xpert Flu/RSV.

    PubMed

    Nguyen Van, J C; Caméléna, F; Dahoun, M; Pilmis, B; Mizrahi, A; Lourtet, J; Behillil, S; Enouf, V; Le Monnier, A

    2016-05-01

    The rapid and accurate detection of influenza virus in respiratory specimens is required for optimal management of patients with acute respiratory infections. Because of the variability of the symptoms and the numerous other causes of influenza-like illness, the diagnosis of influenza cannot be made on the basis of clinical criteria alone. Thus, rapid influenza diagnostic tests have been developed such as the Alere i Influenza A&B isothermal nucleic acid assay. We prospectively evaluated the performance of the Alere i Influenza A&B assay in comparison with our routine Xpert Flu/RSV assay. Positive samples were subtyped according to the protocol from the National Influenza Center (Paris, France). A total of 96 respiratory nasal swab samples were analyzed: with both methods, 38 were positive and 56 were negative. Samples were prospectively collected from January 20 to April 8, 2015, from patient (86 adult and 10 pediatric patients) presenting with an influenza-like illness through the French influenza season. In comparison with the Xpert Flu/RSV assay, the overall sensitivity and specificity of the Alere i Influenza A&B assay were 95% and 100%, respectively. Our results indicate that the Alere i Influenza A&B assay has a good overall analytical performance and a high degree of concordance with the PCR-based Xpert Flu/RSV assay. The Alere i Influenza A&B isothermal nucleic acid amplification test is a powerful tool for influenza detection due to its high sensitivity and specificity as well as its ability to generate results within 15min. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. MOLECULAR METHODS (E.G., MICROARRAYS) APPLIED TO PLANT GENOMES FOR ASSESSING GENETIC CHANGE AND ENVIRONMENTAL STRESS

    EPA Science Inventory

    This is a technical document that presents a detailed sample standard operating procedure (S.O.P.) for preparing plant nucleic acid samples for microarray analyses using commercial ¿chips¿ such as those sold by Affymetrix. It also presents the application of a commercially availa...

  12. Assembly of barcode-like nucleic acid nanostructures.

    PubMed

    Wang, Pengfei; Tian, Cheng; Li, Xiang; Mao, Chengde

    2014-10-15

    Barcode-like (BC) nanopatterns from programmed self-assembly of nucleic acids (DNA and RNA) are reported. BC nanostructures are generated by the introduction of open spaces at selected sites to an otherwise closely packed, plain, rectangle nucleic acid nanostructure. This strategy is applied to nanostructures assembled from both origami approach and single stranded tile approach. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Standardized Procedures for Use of Nucleic Acid-Based Tools

    EPA Science Inventory

    Groundwater and soil samples are frequently analyzed using molecular biological tools (MBTs) to detect unique genetic biomarkers associated with Dehalococcoides (Dhc) and other environmentally relevant microorganisms. The results of these analyses are increasingly used b...

  14. Stability of Zika virus in urine: Specimen processing considerations and implications for the detection of RNA targets in urine.

    PubMed

    Tan, Susanna K; Sahoo, Malaya K; Milligan, Stephen B; Taylor, Nathaniel; Pinsky, Benjamin A

    2017-10-01

    Detection of Zika virus (ZIKV) RNA in urine is of increasing interest for the diagnosis of ZIKV infection. Pre-analytical variables can significantly impact the stability of RNA in urine. To determine optimal specimen processing protocols that would maximize detection of ZIKV RNA in urine by real-time, reverse transcriptase PCR, we investigated the effect of temperature, initial ZIKV concentration, use of nucleic acid stabilizers, and time on ZIKV RNA levels. Urine samples from healthy donors were spiked with ZIKV using the Exact Diagnostics ® ZIKV Verification Panel, a commercially available panel composed of heat-inactivated ZIKV, at concentrations of 5.0 log 10 copies/mL (ZIKV-high) and 4.0 log 10 copies/mL (ZIKV-low). Samples were stored at room temperature, 4°C, or -80°C and frozen aliquots were exposed to no stabilizer (urine), Buffer ATL (Qiagen, Germantown, MD), or DNA/RNA Shield (Zymo Research, Irvine, CA). ZIKV RNA levels in urine declined steadily at room temperature, though was not significant by 48h (ZIKV-high, p=0.09; ZIKV-low, p=0.20). ZIKV RNA titers were consistently higher when stored at 4°C, suggesting that storage at 4°C can slow the progression of RNA degradation. Freezing urine samples at -80°C resulted in a significant loss of detectable ZIKV RNA in the ZIKV-low group. ZIKV RNA was detected in 5/6 replicates at 3days, 1/6 replicates at 10days, and 1/3 replicates at 30days, with findings reproducible on repeat testing. Presence of either nucleic acid stabilizer in urine corrected this effect, and resulted in recovery of ZIKV RNA in all replicates. Use of a nucleic acid stabilizer in the ZIKV-high group did not add incremental benefit for the detection or quantitation of ZIKV RNA. ZIKV RNA is prone to degradation in urine with loss of detectable virus even when specimens are frozen at -80°C for 10days. Detection of ZIKV-positive urine samples, particularly those containing low ZIKV titers may be aided with the addition of a nucleic acid stabilizer during urine specimen processing. Copyright © 2017. Published by Elsevier B.V.

  15. Adsorption of nucleic acid bases and amino acids on single-walled carbon and boron nitride nanotubes: a first-principles study.

    PubMed

    Zheng, Jiaxin; Song, Wei; Wang, Lu; Lu, Jing; Luo, Guangfu; Zhou, Jing; Qin, Rui; Li, Hong; Gao, Zhengxiang; Lai, Lin; Li, Guangping; Mei, Wai Ning

    2009-11-01

    We study the adsorptions of nucleic acid bases adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) and four amino acids phenylalanine, tyrosine, tryptophan, alanine on the single-walled carbon nanotubes (SWCNTs) and boron nitride nanotubes (SWBNNTs) by using density functional theory. We find that the aromatic content plays a critical role in the adsorption. The adsorptions of nucleic acid bases and amino acids on the (7, 7) SWBNNT are stronger than those on the (7, 7) SWCNT. Oxidative treatment of SWCNTs favors the adsorption of biomolecules on nanotubes.

  16. The third annual BRDS on research and development of nucleic acid-based nanomedicines

    PubMed Central

    Chaudhary, Amit Kumar

    2017-01-01

    The completion of human genome project, decrease in the sequencing cost, and correlation of genome sequencing data with specific diseases led to the exponential rise in the nucleic acid-based therapeutic approaches. In the third annual Biopharmaceutical Research and Development Symposium (BRDS) held at the Center for Drug Discovery and Lozier Center for Pharmacy Sciences and Education at the University of Nebraska Medical Center (UNMC), we highlighted the remarkable features of the nucleic acid-based nanomedicines, their significance, NIH funding opportunities on nanomedicines and gene therapy research, challenges and opportunities in the clinical translation of nucleic acids into therapeutics, and the role of intellectual property (IP) in drug discovery and development. PMID:27848223

  17. Method and apparatus for purifying nucleic acids and performing polymerase chain reaction assays using an immiscible fluid

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

    Koh, Chung-Yan; Light, Yooli Kim; Piccini, Matthew Ernest

    Embodiments of the present invention are directed toward devices, systems, and methods for purifying nucleic acids to conduct polymerase chain reaction (PCR) assays. In one example, a method includes generating complexes of silica beads and nucleic acids in a lysis buffer, transporting the complexes through an immiscible fluid to remove interfering compounds from the complexes, further transporting the complexes into a density medium containing components required for PCR where the nucleic acids disassociate from the silica beads, and thermocycling the contents of the density medium to achieve PCR. Signal may be detected from labeling agents in the components required formore » PCR.« less

  18. Recent advances in peptide nucleic acid for cancer bionanotechnology.

    PubMed

    Wu, Jun-Chen; Meng, Qing-Chun; Ren, Hong-Mei; Wang, Hong-Tao; Wu, Jie; Wang, Qi

    2017-06-01

    Peptide nucleic acid (PNA) is an oligomer, in which the phosphate backbone has been replaced by a pseudopeptide backbone that is meant to mimic DNA. Peptide nucleic acids are of the utmost importance in the biomedical field because of their ability to hybridize with neutral nucleic acids and their special chemical and biological properties. In recent years, PNAs have emerged in nanobiotechnology for cancer diagnosis and therapy due to their high affinity and sequence selectivity toward corresponding DNA and RNA. In this review, we summarize the recent progresses that have been made in cancer detection and therapy with PNA biotechnology. In addition, we emphasize nanoparticle PNA-based strategies for the efficient delivery of drugs in anticancer therapies.

  19. Generation of Synthetic Copolymer Libraries by Combinatorial Assembly on Nucleic Acid Templates.

    PubMed

    Kong, Dehui; Yeung, Wayland; Hili, Ryan

    2016-07-11

    Recent advances in nucleic acid-templated copolymerization have expanded the scope of sequence-controlled synthetic copolymers beyond the molecular architectures witnessed in nature. This has enabled the power of molecular evolution to be applied to synthetic copolymer libraries to evolve molecular function ranging from molecular recognition to catalysis. This Review seeks to summarize different approaches available to generate sequence-defined monodispersed synthetic copolymer libraries using nucleic acid-templated polymerization. Key concepts and principles governing nucleic acid-templated polymerization, as well as the fidelity of various copolymerization technologies, will be described. The Review will focus on methods that enable the combinatorial generation of copolymer libraries and their molecular evolution for desired function.

  20. 78 FR 69693 - Draft Guidance for Industry: Recommendations for Premarket Notification (510(k)) Submissions for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-20

    ...] Draft Guidance for Industry: Recommendations for Premarket Notification (510(k)) Submissions for Nucleic... ``Guidance for Industry: Recommendations for Premarket Notification (510(k)) Submissions for Nucleic Acid... submitters and FDA reviewers in preparing and reviewing 510(k) submissions for nucleic acid-based HLA test...

  1. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

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

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extractsmore » are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.« less

  2. Enhancing and targeting nucleic acid delivery by magnetic force.

    PubMed

    Plank, Christian; Anton, Martina; Rudolph, Carsten; Rosenecker, Joseph; Krötz, Florian

    2003-08-01

    Insufficient contact of inherently highly active nucleic acid delivery systems with target cells is a primary reason for their often observed limited efficacy. Physical methods of targeting can overcome this limitation and reduce the risk of undesired side effects due to non-target site delivery. The authors and others have developed a novel means of physical targeting, exploiting magnetic force acting on nucleic acid vectors associated with magnetic particles in order to mediate the rapid contact of vectors with target cells. Here, the principles of magnetic drug and nucleic acid delivery are reviewed, and the facts and potentials of the technique for research and therapeutic applications are discussed. Magnetically enhanced nucleic acid delivery - magnetofection - is universally applicable to viral and non-viral vectors, is extraordinarily rapid, simple and yields saturation level transfection at low dose in vitro. The method is useful for site-specific vector targeting in vivo. Exploiting the full potential of the technique requires an interdisciplinary research effort in magnetic field physics, magnetic particle chemistry, pharmaceutical formulation and medical application.

  3. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

    DOEpatents

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Ward, Thomas E.

    2016-03-22

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

  4. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

    DOEpatents

    Thompson, David N; Apel, William A; Thompson, Vicki S; Ward, Thomas E

    2013-07-23

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

  5. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

    DOEpatents

    Thompson, David N; Apel, William A; Thompson, Vicki S; Ward, Thomas E

    2014-04-08

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

  6. Extracellular nucleic acids of the marine bacterium Rhodovulum sulfidophilum and recombinant RNA production technology using bacteria.

    PubMed

    Kikuchi, Yo; Umekage, So

    2018-02-01

    Extracellular nucleic acids of high molecular weight are detected ubiquitously in seawater. Recent studies have indicated that these nucleic acids are, at least in part, derived from active production by some bacteria. The marine bacterium Rhodovulum sulfidophilum is one of those bacteria. Rhodovulumsulfidophilum is a non-sulfur phototrophic marine bacterium that is known to form structured communities of cells called flocs, and to produce extracellular nucleic acids in culture media. Recently, it has been revealed that this bacterium produces gene transfer agent-like particles and that this particle production may be related to the extracellular nucleic acid production mechanism. This review provides a summary of recent physiological and genetic studies of these phenomena and also introduces a new method for extracellular production of artificial and biologically functional RNAs using this bacterium. In addition, artificial RNA production using Escherichia coli, which is related to this topic, will also be described. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. [Identification of hepatitis B virus YMDD point mutation using peptide nucleic acid clamping PCR].

    PubMed

    Zhang, Yingying; He, Haitang; Yang, Jie; Hou, Jinlin

    2013-06-01

    To establish a peptide nucleic acid clamping PCR assay for detecting hepatitis B virus (HBV) drug resistance mutation. RtM204I (ATT) mutant, rtM204V (GTG) mutant and rtM204 (ATG) wild-type plasmids mixed at different ratios were detected for mutations by PNA clamping PCR assay and direct sequencing, and the sensitivity and specificity of the two methods were compared. Serum samples from 85 patients with chronic HBV infection were detected for drug resistance using the two methods. The sensitivity of PNA-PCR assay was 0.001% in a 10(5)-fold excess of wild-type HBV DNA with a detection limit of 10(1) copies. The sensitivity of direct sequencing was 10% with a detection limit of 10(4) copies. Mutants were detected in 73 of the 85 serum samples (85.9%), including YIDD in 40 samples, YVDD in 23 samples, and YIDD+YVDD in 10 samples. The agreement of PNA-PCR assay with direct sequencing was only 40% (34/85, YIDD in 21 samples, YVDD in 11 samples, and YIDD+YVDD in 2 samples). Neither of the two methods yielded positive results for the negative control samples, suggesting their good specificity. PNA-PCR assay appears to be a more sensitive and rapid assay for detection of HBV genotypic resistance.

  8. Molecular testing for clinical diagnosis and epidemiological investigations of intestinal parasitic infections.

    PubMed

    Verweij, Jaco J; Stensvold, C Rune

    2014-04-01

    Over the past few decades, nucleic acid-based methods have been developed for the diagnosis of intestinal parasitic infections. Advantages of nucleic acid-based methods are numerous; typically, these include increased sensitivity and specificity and simpler standardization of diagnostic procedures. DNA samples can also be stored and used for genetic characterization and molecular typing, providing a valuable tool for surveys and surveillance studies. A variety of technologies have been applied, and some specific and general pitfalls and limitations have been identified. This review provides an overview of the multitude of methods that have been reported for the detection of intestinal parasites and offers some guidance in applying these methods in the clinical laboratory and in epidemiological studies.

  9. Molecular Testing for Clinical Diagnosis and Epidemiological Investigations of Intestinal Parasitic Infections

    PubMed Central

    Stensvold, C. Rune

    2014-01-01

    SUMMARY Over the past few decades, nucleic acid-based methods have been developed for the diagnosis of intestinal parasitic infections. Advantages of nucleic acid-based methods are numerous; typically, these include increased sensitivity and specificity and simpler standardization of diagnostic procedures. DNA samples can also be stored and used for genetic characterization and molecular typing, providing a valuable tool for surveys and surveillance studies. A variety of technologies have been applied, and some specific and general pitfalls and limitations have been identified. This review provides an overview of the multitude of methods that have been reported for the detection of intestinal parasites and offers some guidance in applying these methods in the clinical laboratory and in epidemiological studies. PMID:24696439

  10. Primaquine: Modes of Action and Mechanisms of Drug Resistance.

    DTIC Science & Technology

    1975-06-30

    on in vitro protein synthesis, nucleic acid synthesis in vitro and in isolated nuclei, in vitro lipid synthesis, andmembrane transport and permeability...vitro protein synthesis, nucleic acid synthesis in vitro and in isolated nuclei, in vitro lipid synthesis, and membrane transport and permeability. In...protein synthesis. 7 III. The effects of primaquine on nucleic acid synthesis in isolated nuclei. 7 IV. The effects of primaquine on DNA and RNA syntheses

  11. Use of linalool synthase in genetic engineering of scent production

    DOEpatents

    Pichersky, E.

    1998-12-15

    A purified S-linalool synthase polypeptide from Clarkia breweri is disclosed as is the recombinant polypeptide and nucleic acid sequences encoding the polypeptide. Also disclosed are antibodies immunoreactive with the purified peptide and with recombinant versions of the polypeptide. Methods of using the nucleic acid sequences, as well as methods of enhancing the smell and the flavor of plants expressing the nucleic acid sequences are also disclosed. 5 figs.

  12. Use of linalool synthase in genetic engineering of scent production

    DOEpatents

    Pichersky, Eran

    1998-01-01

    A purified S-linalool synthase polypeptide from Clarkia breweri is disclosed as is the recombinant polypeptide and nucleic acid sequences encoding the polypeptide. Also disclosed are antibodies immunoreactive with the purified peptide and with recombinant versions of the polypeptide. Methods of using the nucleic acid sequences, as well as methods of enhancing the smell and the flavor of plants expressing the nucleic acid sequences are also disclosed.

  13. Phytoagents for Cancer Management: Regulation of Nucleic Acid Oxidation, ROS, and Related Mechanisms

    PubMed Central

    Shyur, Lie-Fen

    2013-01-01

    Accumulation of oxidized nucleic acids causes genomic instability leading to senescence, apoptosis, and tumorigenesis. Phytoagents are known to reduce the risk of cancer development; whether such effects are through regulating the extent of nucleic acid oxidation remains unclear. Here, we outlined the role of reactive oxygen species in nucleic acid oxidation as a driving force in cancer progression. The consequential relationship between genome instability and cancer progression highlights the importance of modulation of cellular redox level in cancer management. Current epidemiological and experimental evidence demonstrate the effects and modes of action of phytoagents in nucleic acid oxidation and provide rationales for the use of phytoagents as chemopreventive or therapeutic agents. Vitamins and various phytoagents antagonize carcinogen-triggered oxidative stress by scavenging free radicals and/or activating endogenous defence systems such as Nrf2-regulated antioxidant genes or pathways. Moreover, metal ion chelation by phytoagents helps to attenuate oxidative DNA damage caused by transition metal ions. Besides, the prooxidant effects of some phytoagents pose selective cytotoxicity on cancer cells and shed light on a new strategy of cancer therapy. The “double-edged sword” role of phytoagents as redox regulators in nucleic acid oxidation and their possible roles in cancer prevention or therapy are discussed in this review. PMID:24454991

  14. Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes.

    PubMed

    Greenough, Lucia; Schermerhorn, Kelly M; Mazzola, Laurie; Bybee, Joanna; Rivizzigno, Danielle; Cantin, Elizabeth; Slatko, Barton E; Gardner, Andrew F

    2016-01-29

    Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3'-5' exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Termite enzymes and uses thereof for in vitro conversion of lignin-containing materials to fermentable products

    DOEpatents

    Scharf, Michael E; Boucias, Drion G; Tartar, Aurelien; Coy, Monique R; Zhou, Xuguo; Salem, Tamer Ibrahim Zaki; Jadhao, Sanjay B; Wheeler, Marsha M

    2013-05-21

    The disclosure provides isolated nucleic acid molecules derived from the gut of the termite R flavipes, recombinant nucleic acid molecules comprising a vector and an isolated heterologous nucleic acid molecule operably inserted therein, whereby, when transformed into an appropriate host cell system, the heterologous nucleic acid sequence is expressed as a polypeptide having an activity similar to that when expressed in the gut of the termite R. flavipes. The recombinant nucleic acid molecules can comprise more than one heterologous nucleic acid molecule such that more than one polypeptide may be expressed by the host system. The expressed polypeptides may be substantially purified, or used in a substantially unpurified form, to be admixed with a lignocellulose source to be converted to a fermentable product such as a sugar or a mixture of sugars. One aspect of the present disclosure, therefore, encompasses methods of converting a lignified plant material to a fermentable product, the method comprising obtaining a series of isolated polypeptides of a termite, wherein the series of polypeptides cooperate to convert a plant lignocellulose to a fermentable product; and incubating the series of polypeptides with a source of lignified plant material, under conditions allowing the polypeptides to cooperatively produce a fermentable product from the lignified plant material.

  16. Weigners fixative-an alternative to formalin fixation for histology with improved preservation of nucleic acids.

    PubMed

    Klopfleisch, R; von Deetzen, M; Weiss, A Th; Weigner, J; Weigner, F; Plendl, J; Gruber, A D

    2013-01-01

    Formalin fixation and paraffin embedding (FFPE) is the standard method for tissue storage in histopathology. However, FFPE has disadvantages in terms of user health, environment, and nucleic acid integrity. Weigners fixative has been suggested as an alternative for embalming cadavers in human and veterinary anatomy. The present study tested the applicability of Weigners for histology and immunohistochemistry and the preservation of nucleic acids. To this end, a set of organs was fixed for 2 days and up to 6 months in Weigners (WFPE) or formalin. WFPE tissues from the skin, brain, lymphatic tissues, liver, and muscle had good morphologic preservation, comparable to formalin fixation. The quality of kidney and lung samples was inferior to FFPE material due to less accentuated nuclear staining and retention of proteinaceous interstitial fluids. Azan, Turnbull blue, toluidin, and immunohistochemical stainings for CD79a, cytokeratin, vimentin, and von Willebrand factor led to comparable results with both fixates. Of note, immunohistochemical detection of CD3 was possible after 6 months in WFPE but not in FFPE tissues. mRNA, miRNA, and DNA from WFPE tissues had superior quality and allowed for amplification of miRNA, 400-bp-long mRNA, and 1000-bp-long DNA fragments after 6 months of fixation in WFPE. In summary, Weigners fixative is a nonhazardous alternative to formalin, which provides a good morphologic preservation of most organs, a similar sensitivity for protein detection, and a superior preservation of nucleic acids. Weigners may therefore be a promising alternative to cryopreservation and may be embraced by people affected by formalin allergies.

  17. Determination of helix orientations in a flexible DNA by multi-frequency EPR spectroscopy.

    PubMed

    Grytz, C M; Kazemi, S; Marko, A; Cekan, P; Güntert, P; Sigurdsson, S Th; Prisner, T F

    2017-11-15

    Distance measurements are performed between a pair of spin labels attached to nucleic acids using Pulsed Electron-Electron Double Resonance (PELDOR, also called DEER) spectroscopy which is a complementary tool to other structure determination methods in structural biology. The rigid spin label Ç, when incorporated pairwise into two helical parts of a nucleic acid molecule, allows the determination of both the mutual orientation and the distance between those labels, since Ç moves rigidly with the helix to which it is attached. We have developed a two-step protocol to investigate the conformational flexibility of flexible nucleic acid molecules by multi-frequency PELDOR. In the first step, a library with a broad collection of conformers, which are in agreement with topological constraints, NMR restraints and distances derived from PELDOR, was created. In the second step, a weighted structural ensemble of these conformers was chosen, such that it fits the multi-frequency PELDOR time traces of all doubly Ç-labelled samples simultaneously. This ensemble reflects the global structure and the conformational flexibility of the two-way DNA junction. We demonstrate this approach on a flexible bent DNA molecule, consisting of two short helical parts with a five adenine bulge at the center. The kink and twist motions between both helical parts were quantitatively determined and showed high flexibility, in agreement with a Förster Resonance Energy Transfer (FRET) study on a similar bent DNA motif. The approach presented here should be useful to describe the relative orientation of helical motifs and the conformational flexibility of nucleic acid structures, both alone and in complexes with proteins and other molecules.

  18. Structural aspects of catalytic mechanisms of endonucleases and their binding to nucleic acids

    NASA Astrophysics Data System (ADS)

    Zhukhlistova, N. E.; Balaev, V. V.; Lyashenko, A. V.; Lashkov, A. A.

    2012-05-01

    Endonucleases (EC 3.1) are enzymes of the hydrolase class that catalyze the hydrolytic cleavage of deoxyribonucleic and ribonucleic acids at any region of the polynucleotide chain. Endonucleases are widely used both in biotechnological processes and in veterinary medicine as antiviral agents. Medical applications of endonucleases in human cancer therapy hold promise. The results of X-ray diffraction studies of the spatial organization of endonucleases and their complexes and the mechanism of their action are analyzed and generalized. An analysis of the structural studies of this class of enzymes showed that the specific binding of enzymes to nucleic acids is characterized by interactions with nitrogen bases and the nucleotide backbone, whereas the nonspecific binding of enzymes is generally characterized by interactions only with the nucleic-acid backbone. It should be taken into account that the specificity can be modulated by metal ions and certain low-molecular-weight organic compounds. To test the hypotheses about specific and nonspecific nucleic-acid-binding proteins, it is necessary to perform additional studies of atomic-resolution three-dimensional structures of enzyme-nucleic-acid complexes by methods of structural biology.

  19. Novel high-performance purification protocol of recombinant CNBP suitable for biochemical and biophysical characterization.

    PubMed

    Challier, Emilse; Lisa, María-Natalia; Nerli, Bibiana B; Calcaterra, Nora B; Armas, Pablo

    2014-01-01

    Cellular nucleic acid binding protein (CNBP) is a highly conserved multi-zinc knuckle protein that enhances c-MYC expression, is related to certain human muscular diseases and is required for proper rostral head development. CNBP binds to single-stranded DNA (ssDNA) and RNA and acts as nucleic acid chaperone. Despite the advances made concerning CNBP biological roles, a full knowledge about the structure-function relationship has not yet been achieved, likely due to difficulty in obtaining pure and tag-free CNBP. Here, we report a fast, simple, reproducible, and high-performance expression and purification protocol that provides recombinant tag-free CNBP from Escherichia coli cultures. We determined that tag-free CNBP binds its molecular targets with higher affinity than tagged-CNBP. Furthermore, fluorescence spectroscopy revealed the presence of a unique and conserved tryptophan, which is exposed to the solvent and involved, directly or indirectly, in nucleic acid binding. Size-exclusion HPLC revealed that CNBP forms homodimers independently of nucleic acid binding and coexist with monomers as non-interconvertible forms or in slow equilibrium. Circular dichroism spectroscopy showed that CNBP has a secondary structure dominated by random-coil and β-sheet coincident with the sequence-predicted repetitive zinc knuckles motifs, which folding is required for CNBP structural stability and biochemical activity. CNBP structural stability increased in the presence of single-stranded nucleic acid targets similar to other unstructured nucleic acid chaperones. Altogether, data suggest that CNBP is a flexible protein with interspersed structured zinc knuckles, and acquires a more rigid structure upon nucleic acid binding. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Solar thermal polymerase chain reaction for smartphone-assisted molecular diagnostics

    PubMed Central

    Jiang, Li; Mancuso, Matthew; Lu, Zhengda; Akar, Gunkut; Cesarman, Ethel; Erickson, David

    2014-01-01

    Nucleic acid-based diagnostic techniques such as polymerase chain reaction (PCR) are used extensively in medical diagnostics due to their high sensitivity, specificity and quantification capability. In settings with limited infrastructure and unreliable electricity, however, access to such devices is often limited due to the highly specialized and energy-intensive nature of the thermal cycling process required for nucleic acid amplification. Here we integrate solar heating with microfluidics to eliminate thermal cycling power requirements as well as create a simple device infrastructure for PCR. Tests are completed in less than 30 min, and power consumption is reduced to 80 mW, enabling a standard 5.5 Wh iPhone battery to provide 70 h of power to this system. Additionally, we demonstrate a complete sample-to-answer diagnostic strategy by analyzing human skin biopsies infected with Kaposi's Sarcoma herpesvirus (KSHV/HHV-8) through the combination of solar thermal PCR, HotSHOT DNA extraction and smartphone-based fluorescence detection. We believe that exploiting the ubiquity of solar thermal energy as demonstrated here could facilitate broad availability of nucleic acid-based diagnostics in resource-limited areas. PMID:24553130

  1. A mathematical analysis of multiple-target SELEX.

    PubMed

    Seo, Yeon-Jung; Chen, Shiliang; Nilsen-Hamilton, Marit; Levine, Howard A

    2010-10-01

    SELEX (Systematic Evolution of Ligands by Exponential Enrichment) is a procedure by which a mixture of nucleic acids can be fractionated with the goal of identifying those with specific biochemical activities. One combines the mixture with a specific target molecule and then separates the target-NA complex from the resulting reactions. The target-NA complex is separated from the unbound NA by mechanical means (such as by filtration), the NA is eluted from the complex, amplified by PCR (polymerase chain reaction), and the process repeated. After several rounds, one should be left with the nucleic acids that best bind to the target. The problem was first formulated mathematically in Irvine et al. (J. Mol. Biol. 222:739-761, 1991). In Levine and Nilsen-Hamilton (Comput. Biol. Chem. 31:11-25, 2007), a mathematical analysis of the process was given. In Vant-Hull et al. (J. Mol. Biol. 278:579-597, 1998), multiple target SELEX was considered. It was assumed that each target has a single nucleic acid binding site that permits occupation by no more than one nucleic acid. Here, we revisit Vant-Hull et al. (J. Mol. Biol. 278:579-597, 1998) using the same assumptions. The iteration scheme is shown to be convergent and a simplified algorithm is given. Our interest here is in the behavior of the multiple target SELEX process as a discrete "time" dynamical system. Our goal is to characterize the limiting states and their dependence on the initial distribution of nucleic acid and target fraction components. (In multiple target SELEX, we vary the target component fractions, but not their concentrations, as fixed and the initial pool of nucleic acids as a variable starting condition). Given N nucleic acids and a target consisting of M subtarget component species, there is an M × N matrix of affinities, the (i,j) entry corresponding to the affinity of the jth nucleic acid for the ith subtarget. We give a structure condition on this matrix that is equivalent to the following statement: For any initial pool of nucleic acids such that all N species are represented, the dynamical system defined by the multiple target SELEX process will converge to a unique subset of nucleic acids, each of whose concentrations depend only upon the total nucleic acid concentration, the initial fractional target distribution (both of which are assumed to be the same from round to round), and the overall limiting association constant. (The overall association constant is the equilibrium constant for the system of MN reactions when viewed as a composite single reaction). This condition is equivalent to the statement that every member of a certain family of chemical potentials at infinite target dilution can have at most one critical point. (The condition replaces the statement for single target SELEX that the dynamical system generated via the process always converges to a pool that contains only the nucleic acid that binds best to the target). This suggests that the effectiveness of multiple target SELEX as a separation procedure may not be as useful as single target SELEX unless the thermodynamic properties of these chemical potentials are well understood.

  2. A RAPID METHOD FOR THE EXTRACTION OF FUNGAL DNA FROM ENVIRONMENTAL SAMPLES: EVALUATION IN THE QUANTITATIVE ANALYSIS OF MEMNONIELLA ECHINATA CONIDIA USING REAL TIME DETECTION OF PCR PRODUCTS

    EPA Science Inventory

    New technologies are creating the potential for using nucleic acid sequence detection to perform routine microbiological analyses of environmental samples. Our laboratory has recently reported on the development of a method for the quantitative detection of Stachybotrys chartarum...

  3. Nucleic Acid-Based Nanoconstructs

    Cancer.gov

    Focuses on the design, synthesis, characterization, and development of spherical nucleic acid constructs as effective nanotherapeutic, single-entity agents for the treatment of glioblastoma multiforme and prostate cancers.

  4. A survey of advancements in nucleic acid-based logic gates and computing for applications in biotechnology and biomedicine.

    PubMed

    Wu, Cuichen; Wan, Shuo; Hou, Weijia; Zhang, Liqin; Xu, Jiehua; Cui, Cheng; Wang, Yanyue; Hu, Jun; Tan, Weihong

    2015-03-04

    Nucleic acid-based logic devices were first introduced in 1994. Since then, science has seen the emergence of new logic systems for mimicking mathematical functions, diagnosing disease and even imitating biological systems. The unique features of nucleic acids, such as facile and high-throughput synthesis, Watson-Crick complementary base pairing, and predictable structures, together with the aid of programming design, have led to the widespread applications of nucleic acids (NA) for logic gate and computing in biotechnology and biomedicine. In this feature article, the development of in vitro NA logic systems will be discussed, as well as the expansion of such systems using various input molecules for potential cellular, or even in vivo, applications.

  5. A Survey of Advancements in Nucleic Acid-based Logic Gates and Computing for Applications in Biotechnology and biomedicine

    PubMed Central

    Wu, Cuichen; Wan, Shuo; Hou, Weijia; Zhang, Liqin; Xu, Jiehua; Cui, Cheng; Wang, Yanyue; Hu, Jun

    2015-01-01

    Nucleic acid-based logic devices were first introduced in 1994. Since then, science has seen the emergence of new logic systems for mimicking mathematical functions, diagnosing disease and even imitating biological systems. The unique features of nucleic acids, such as facile and high-throughput synthesis, Watson-Crick complementary base pairing, and predictable structures, together with the aid of programming design, have led to the widespread applications of nucleic acids (NA) for logic gating and computing in biotechnology and biomedicine. In this feature article, the development of in vitro NA logic systems will be discussed, as well as the expansion of such systems using various input molecules for potential cellular, or even in vivo, applications. PMID:25597946

  6. Towards XNA nanotechnology: new materials from synthetic genetic polymers

    PubMed Central

    Pinheiro, Vitor B.; Holliger, Philipp

    2014-01-01

    Nucleic acids display remarkable properties beyond information storage and propagation. The well-understood base pairing rules have enabled nucleic acids to be assembled into nanostructures of ever increasing complexity. Although nanostructures can be constructed using other building blocks, including peptides and lipids, it is the capacity to evolve that sets nucleic acids apart from all other nanoscale building materials. Nonetheless, the poor chemical and biological stability of DNA and RNA constrain their applications. Recent advances in nucleic acid chemistry and polymerase engineering enable the synthesis, replication, and evolution of a range of synthetic genetic polymers (XNAs) with improved chemical and biological stability. We discuss the impact of this technology on the generation of XNA ligands, enzymes, and nanostructures with tailor-made chemistry. PMID:24745974

  7. The increasing application of multiplex nucleic acid detection tests to the diagnosis of syndromic infections.

    PubMed

    Gray, J; Coupland, L J

    2014-01-01

    On 14 January 2013, the US Food and Drug Administration (FDA) announced permission for a multiplex nucleic acid test, the xTAG® Gastrointestinal Pathogen Panel (GPP) (Luminex Corporation, USA), which simultaneously detects 11 common viral, bacterial and parasitic causes of infectious gastroenteritis, to be marketed in the USA. This announcement reflects the current move towards the development and commercialization of detection technologies based on nucleic acid amplification techniques for diagnosis of syndromic infections. We discuss the limitations and advantages of nucleic acid amplification techniques and the recent advances in Conformité Européene - in-vitro diagnostic (CE-IVD)-approved multiplex real-time PCR kits for the simultaneous detection of multiple targets within the clinical diagnostics market.

  8. Compositions and methods for detecting single nucleotide polymorphisms

    DOEpatents

    Yeh, Hsin-Chih; Werner, James; Martinez, Jennifer S.

    2016-11-22

    Described herein are nucleic acid based probes and methods for discriminating and detecting single nucleotide variants in nucleic acid molecules (e.g., DNA). The methods include use of a pair of probes can be used to detect and identify polymorphisms, for example single nucleotide polymorphism in DNA. The pair of probes emit a different fluorescent wavelength of light depending on the association and alignment of the probes when hybridized to a target nucleic acid molecule. Each pair of probes is capable of discriminating at least two different nucleic acid molecules that differ by at least a single nucleotide difference. The methods can probes can be used, for example, for detection of DNA polymorphisms that are indicative of a particular disease or condition.

  9. Nucleic acids for the rational design of reaction circuits.

    PubMed

    Padirac, Adrien; Fujii, Teruo; Rondelez, Yannick

    2013-08-01

    Nucleic acid-based circuits are rationally designed in vitro assemblies that can perform complex preencoded programs. They can be used to mimic in silico computations. Recent works emphasized the modularity and robustness of these circuits, which allow their scaling-up. Another new development has led to dynamic, time-responsive systems that can display emergent behaviors like oscillations. These are closely related to biological architectures and provide an in vitro model of in vivo information processing. Nucleic acid circuits have already been used to handle various processes for technological or biotechnological purposes. Future applications of these chemical smart systems will benefit from the rapidly growing ability to design, construct, and model nucleic acid circuits of increasing size. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. Compaction agent clarification of microbial lysates

    NASA Technical Reports Server (NTRS)

    DeWalt, Brad W.; Murphy, Jason C.; Fox, George E.; Willson, Richard C.

    2003-01-01

    Recombinant proteins are often purified from microbial lysates containing high concentrations of nucleic acids. Pre-purification steps such as nuclease addition or precipitation with polyethyleneimine or ammonium sulfate are normally required to reduce viscosity and to eliminate competing polyanions before anion exchange chromatography. We report that small polycationic compaction agents such as spermine selectively precipitate nucleic acids during or after Escherichia coli lysis, allowing DNA and RNA to be pelleted with the insoluble cell debris. Analysis by spectrophotometry and protein assay confirmed a significant reduction in the concentration of nucleic acids present, with preservation of protein. Lysate viscosity is greatly reduced, facilitating subsequent processing. We have used 5mM spermine to remove nucleic acids from E. coli lysate in the purification of a hexahistidine-tagged HIV reverse transcriptase.

  11. Plants having modified response to ethylene

    DOEpatents

    Meyerowitz, Elliott M.; Chang, Caren; Bleecker, Anthony B.

    1997-01-01

    The invention includes transformed plants having at least one cell transformed with a modified ETR nucleic acid. Such plants have a phenotype characterized by a decrease in the response of at least one transformed plant cell to ethylene as compared to a plant not containing the transformed plant cell. Tissue and/or temporal specificity for expression of the modified ETR nucleic acid is controlled by selecting appropriate expression regulation sequences to target the location and/or time of expression of the transformed nucleic acid. The plants are made by transforming at least one plant cell with an appropriate modified ETR nucleic acid, regenerating plants from one or more of the transformed plant cells and selecting at least one plant having the desired phenotype.

  12. Plants having modified response to ethylene

    DOEpatents

    Meyerowitz, E.M.; Chang, C.; Bleecker, A.B.

    1998-10-20

    The invention includes transformed plants having at least one cell transformed with a modified ETR nucleic acid. Such plants have a phenotype characterized by a decrease in the response of at least one transformed plant cell to ethylene as compared to a plant not containing the transformed plant cell. Tissue and/or temporal specificity for expression of the modified ETR nucleic acid is controlled by selecting appropriate expression regulation sequences to target the location and/or time of expression of the transformed nucleic acid. The plants are made by transforming at least one plant cell with an appropriate modified ETR nucleic acid, regenerating plants from one or more of the transformed plant cells and selecting at least one plant having the desired phenotype. 67 figs.

  13. Plants having modified response to ethylene

    DOEpatents

    Meyerowitz, Elliot M.; Chang, Caren; Bleecker, Anthony B.

    1998-01-01

    The invention includes transformed plants having at least one cell transformed with a modified ETR nucleic acid. Such plants have a phenotype characterized by a decrease in the response of at least one transformed plant cell to ethylene as compared to a plant not containing the transformed plant cell. Tissue and/or temporal specificity for expression of the modified ETR nucleic acid is controlled by selecting appropriate expression regulation sequences to target the location and/or time of expression of the transformed nucleic acid. The plants are made by transforming at least one plant cell with an appropriate modified ETR nucleic acid, regenerating plants from one or more of the transformed plant cells and selecting at least one plant having the desired phenotype.

  14. Plants having modified response to ethylene

    DOEpatents

    Meyerowitz, E.M.; Chang, C.; Bleecker, A.B.

    1997-11-18

    The invention includes transformed plants having at least one cell transformed with a modified ETR nucleic acid. Such plants have a phenotype characterized by a decrease in the response of at least one transformed plant cell to ethylene as compared to a plant not containing the transformed plant cell. Tissue and/or temporal specificity for expression of the modified ETR nucleic acid is controlled by selecting appropriate expression regulation sequences to target the location and/or time of expression of the transformed nucleic acid. The plants are made by transforming at least one plant cell with an appropriate modified ETR nucleic acid, regenerating plants from one or more of the transformed plant cells and selecting at least one plant having the desired phenotype. 31 figs.

  15. Inorganic nanoparticles as nucleic acid transporters into eukaryotic cells

    NASA Astrophysics Data System (ADS)

    Amirkhanov, R. N.; Zarytova, V. F.; Zenkova, M. A.

    2017-02-01

    The review is concerned with inorganic nanoparticles (gold, titanium dioxide, silica, iron oxides, calcium phosphate) used as nucleic acid transporters into mammalian cells. Methods for the synthesis of nanoparticles and approaches to surface modification through covalent or noncovalent attachment of low- or high-molecular-weight compounds are considered. The data available from the literature on biological action of nucleic acids delivered into the cells by nanoparticles and on the effect of nanoparticles and their conjugates and complexes on the cell survival are summarized. Pathways of cellular internalization of nanoparticles and the mechanism of their excretion, as well as the ways of release of nucleic acids from their complexes with nanoparticles after the cellular uptake are described. The bibliography includes 161 references.

  16. NUCLEIC ACID CONCENTRATION AND RADIOSENSITIVITY OF THE SCORPION ANDROCTONUS AMOREUXI AUD. AND SAV (in French)

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

    Pascaud, X.; Niaussat, P.

    1963-01-01

    The concentration of desoxyribonucleic acid and of ribonucleic acid in the soft tissues was determined for the two invertebrates of the arid zone, the scorpion Androctonus amoreuxi Aud. and Sav. and the tenebrionide Pimelia angulata expiata Peyer. The radiosensitivity to gamma rays had been previously determined: LD/sub 50/30// days is 100,000 r for Androctonus and 40,000 for Pimelia. The mean rate of nucleic acids determined in the scorpion was relatively low. A possible relation between the high radioresistance of the scorpion and the low nucleic acid concentration was discussed. (J.S.R.)

  17. Digital PCR on a SlipChip.

    PubMed

    Shen, Feng; Du, Wenbin; Kreutz, Jason E; Fok, Alice; Ismagilov, Rustem F

    2010-10-21

    This paper describes a SlipChip to perform digital PCR in a very simple and inexpensive format. The fluidic path for introducing the sample combined with the PCR mixture was formed using elongated wells in the two plates of the SlipChip designed to overlap during sample loading. This fluidic path was broken up by simple slipping of the two plates that removed the overlap among wells and brought each well in contact with a reservoir preloaded with oil to generate 1280 reaction compartments (2.6 nL each) simultaneously. After thermal cycling, end-point fluorescence intensity was used to detect the presence of nucleic acid. Digital PCR on the SlipChip was tested quantitatively by using Staphylococcus aureus genomic DNA. As the concentration of the template DNA in the reaction mixture was diluted, the fraction of positive wells decreased as expected from the statistical analysis. No cross-contamination was observed during the experiments. At the extremes of the dynamic range of digital PCR the standard confidence interval determined using a normal approximation of the binomial distribution is not satisfactory. Therefore, statistical analysis based on the score method was used to establish these confidence intervals. The SlipChip provides a simple strategy to count nucleic acids by using PCR. It may find applications in research applications such as single cell analysis, prenatal diagnostics, and point-of-care diagnostics. SlipChip would become valuable for diagnostics, including applications in resource-limited areas after integration with isothermal nucleic acid amplification technologies and visual readout.

  18. Comparing viral metagenomics methods using a highly multiplexed human viral pathogens reagent

    PubMed Central

    Li, Linlin; Deng, Xutao; Mee, Edward T.; Collot-Teixeira, Sophie; Anderson, Rob; Schepelmann, Silke; Minor, Philip D.; Delwart, Eric

    2014-01-01

    Unbiased metagenomic sequencing holds significant potential as a diagnostic tool for the simultaneous detection of any previously genetically described viral nucleic acids in clinical samples. Viral genome sequences can also inform on likely phenotypes including drug susceptibility or neutralization serotypes. In this study, different variables of the laboratory methods often used to generate viral metagenomics libraries on the efficiency of viral detection and virus genome coverage were compared. A biological reagent consisting of 25 different human RNA and DNA viral pathogens was used to estimate the effect of filtration and nuclease digestion, DNA/RNA extraction methods, pre-amplification and the use of different library preparation kits on the detection of viral nucleic acids. Filtration and nuclease treatment led to slight decreases in the percentage of viral sequence reads and number of viruses detected. For nucleic acid extractions silica spin columns improved viral sequence recovery relative to magnetic beads and Trizol extraction. Pre-amplification using random RT-PCR while generating more viral sequence reads resulted in detection of fewer viruses, more overlapping sequences, and lower genome coverage. The ScriptSeq library preparation method retrieved more viruses and a greater fraction of their genomes than the TruSeq and Nextera methods. Viral metagenomics sequencing was able to simultaneously detect up to 22 different viruses in the biological reagent analyzed including all those detected by qPCR. Further optimization will be required for the detection of viruses in biologically more complex samples such as tissues, blood, or feces. PMID:25497414

  19. High-volume extraction of nucleic acids by magnetic bead technology for ultrasensitive detection of bacteria in blood components.

    PubMed

    Störmer, Melanie; Kleesiek, Knut; Dreier, Jens

    2007-01-01

    Nucleic acid isolation, the most technically demanding and laborious procedure performed in molecular diagnostics, harbors the potential for improvements in automation. A recent development is the use of magnetic beads covered with nucleic acid-binding matrices. We adapted this technology with a broad-range 23S rRNA real-time reverse transcription (RT)-PCR assay for fast and sensitive detection of bacterial contamination of blood products. We investigated different protocols for an automated high-volume extraction method based on magnetic-separation technology for the extraction of bacterial nucleic acids from platelet concentrates (PCs). We added 2 model bacteria, Staphylococcus epidermidis and Escherichia coli, to a single pool of apheresis-derived, single-donor platelets and assayed the PCs by real-time RT-PCR analysis with an improved primer-probe system and locked nucleic acid technology. Co-amplification of human beta(2)-microglobulin mRNA served as an internal control (IC). We used probit analysis to calculate the minimum concentration of bacteria that would be detected with 95% confidence. For automated magnetic bead-based extraction technology with the real-time RT-PCR, the 95% detection limit was 29 x 10(3) colony-forming units (CFU)/L for S. epidermidis and 22 x 10(3) CFU/L for E. coli. No false-positive results occurred, either due to nucleic acid contamination of reagents or externally during testing of 1030 PCs. High-volume nucleic acid extraction improved the detection limit of the assay. The improvement of the primer-probe system and the integration of an IC make the RT-PCR assay appropriate for bacteria screening of platelets.

  20. Quantifying Functional Group Interactions that Determine Urea Effects on Nucleic Acid Helix Formation

    PubMed Central

    Guinn, Emily J.; Schwinefus, Jeffrey J.; Cha, Hyo Keun; McDevitt, Joseph L.; Merker, Wolf E.; Ritzer, Ryan; Muth, Gregory W.; Engelsgjerd, Samuel W.; Mangold, Kathryn E.; Thompson, Perry J.; Kerins, Michael J.; Record, Thomas

    2013-01-01

    Urea destabilizes helical and folded conformations of nucleic acids and proteins, as well as protein-nucleic acid complexes. To understand these effects, extend previous characterizations of interactions of urea with protein functional groups, and thereby develop urea as a probe of conformational changes in protein and nucleic acid processes, we obtain chemical potential derivatives (μ23 = dμ2/dm3) quantifying interactions of urea (component 3) with nucleic acid bases, base analogs, nucleosides and nucleotide monophosphates (component 2) using osmometry and hexanol-water distribution assays. Dissection of these μ23 yields interaction potentials quantifying interactions of urea with unit surface areas of nucleic acid functional groups (heterocyclic aromatic ring, ring methyl, carbonyl and phosphate O, amino N, sugar (C,O)); urea interacts favorably with all these groups, relative to interactions with water. Interactions of urea with heterocyclic aromatic rings and attached methyl groups (as on thymine) are particularly favorable, as previously observed for urea-homocyclic aromatic ring interactions. Urea m-values determined for double helix formation by DNA dodecamers near 25°C are in the range 0.72 to 0.85 kcal mol−1 m−1 and exhibit little systematic dependence on nucleobase composition (17–42% GC). Interpretation of these results using the urea interaction potentials indicates that extensive (60–90%) stacking of nucleobases in the separated strands in the transition region is required to explain the m-value. Results for RNA and DNA dodecamers obtained at higher temperatures, and literature data, are consistent with this conclusion. This demonstrates the utility of urea as a quantitative probe of changes in surface area (ΔASA) in nucleic acid processes. PMID:23510511

  1. NALDB: nucleic acid ligand database for small molecules targeting nucleic acid

    PubMed Central

    Kumar Mishra, Subodh; Kumar, Amit

    2016-01-01

    Nucleic acid ligand database (NALDB) is a unique database that provides detailed information about the experimental data of small molecules that were reported to target several types of nucleic acid structures. NALDB is the first ligand database that contains ligand information for all type of nucleic acid. NALDB contains more than 3500 ligand entries with detailed pharmacokinetic and pharmacodynamic information such as target name, target sequence, ligand 2D/3D structure, SMILES, molecular formula, molecular weight, net-formal charge, AlogP, number of rings, number of hydrogen bond donor and acceptor, potential energy along with their Ki, Kd, IC50 values. All these details at single platform would be helpful for the development and betterment of novel ligands targeting nucleic acids that could serve as a potential target in different diseases including cancers and neurological disorders. With maximum 255 conformers for each ligand entry, our database is a multi-conformer database and can facilitate the virtual screening process. NALDB provides powerful web-based search tools that make database searching efficient and simplified using option for text as well as for structure query. NALDB also provides multi-dimensional advanced search tool which can screen the database molecules on the basis of molecular properties of ligand provided by database users. A 3D structure visualization tool has also been included for 3D structure representation of ligands. NALDB offers an inclusive pharmacological information and the structurally flexible set of small molecules with their three-dimensional conformers that can accelerate the virtual screening and other modeling processes and eventually complement the nucleic acid-based drug discovery research. NALDB can be routinely updated and freely available on bsbe.iiti.ac.in/bsbe/naldb/HOME.php. Database URL: http://bsbe.iiti.ac.in/bsbe/naldb/HOME.php PMID:26896846

  2. Photochemistry of nucleic acid bases and their thio- and aza-analogues in solution.

    PubMed

    Pollum, Marvin; Martínez-Fernández, Lara; Crespo-Hernández, Carlos E

    2015-01-01

    The steady-state and time-resolved photochemistry of the natural nucleic acid bases and their sulfur- and nitrogen-substituted analogues in solution is reviewed. Emphasis is given to the experimental studies performed over the last 3-5 years that showcase topical areas of scientific inquiry and those that require further scrutiny. Significant progress has been made toward mapping the radiative and nonradiative decay pathways of nucleic acid bases. There is a consensus that ultrafast internal conversion to the ground state is the primary relaxation pathway in the nucleic acid bases, whereas the mechanism of this relaxation and the level of participation of the (1)πσ*, (1) nπ*, and (3)ππ* states are still matters of debate. Although impressive research has been performed in recent years, the microscopic mechanism(s) by which the nucleic acid bases dissipate excess vibrational energy to their environment, and the role of the N-glycosidic group in this and in other nonradiative decay pathways, are still poorly understood. The simple replacement of a single atom in a nucleobase with a sulfur or nitrogen atom severely restricts access to the conical intersections responsible for the intrinsic internal conversion pathways to the ground state in the nucleic acid bases. It also enhances access to ultrafast and efficient inter-system crossing pathways that populate the triplet manifold in yields close to unity. Determining the coupled nuclear and electronic pathways responsible for the significantly different photochemistry in these nucleic acid base analogues serves as a convenient platform to examine the current state of knowledge regarding the photodynamic properties of the DNA and RNA bases from both experimental and computational perspectives. Further investigations should also aid in forecasting the prospective use of sulfur- and nitrogen-substituted base analogues in photochemotherapeutic applications.

  3. The in Silico Insight into Carbon Nanotube and Nucleic Acid Bases Interaction.

    PubMed

    Karimi, Ali Asghar; Ghalandari, Behafarid; Tabatabaie, Seyed Saleh; Farhadi, Mohammad

    2016-05-01

    To explore practical applications of carbon nanotubes (CNTs) in biomedical fields the properties of their interaction with biomolecules must be revealed. Recent years, the interaction of CNTs with biomolecules is a subject of research interest for practical applications so that previous research explored that CNTs have complementary structure properties with single strand DNA (ssDNA). Hence, the quantum mechanics (QM) method based on ab initio was used for this purpose. Therefore values of binding energy, charge distribution, electronic energy and other physical properties of interaction were studied for interaction of nucleic acid bases and SCNT. In this study, the interaction between nucleic acid bases and a (4, 4) single-walled carbon nanotube (SCNT) were investigated through calculations within quantum mechanics (QM) method at theoretical level of Hartree-Fock (HF) method using 6-31G basis set. Hence, the physical properties such as electronic energy, total dipole moment, charge distributions and binding energy of nucleic acid bases interaction with SCNT were investigated based on HF method. It has been found that the guanine base adsorption is bound stronger to the outer surface of nanotube in comparison to the other bases, consistent with the recent theoretical studies. In the other words, the results explored that guanine interaction with SCNT has optimum level of electronic energy so that their interaction is stable. Also, the calculations illustrated that SCNT interact to nucleic acid bases by noncovalent interaction because of charge distribution an electrostatic area is created in place of interaction. Consequently, small diameter SCNT interaction with nucleic acid bases is noncovalent. Also, the results revealed that small diameter SCNT interaction especially SCNT (4, 4) with nucleic acid bases can be useful in practical application area of biomedical fields such detection and drug delivery.

  4. Discrimination of Self and Non-Self Ribonucleic Acids

    PubMed Central

    Gebhardt, Anna; Laudenbach, Beatrice T.

    2017-01-01

    Most virus infections are controlled through the innate and adaptive immune system. A surprisingly limited number of so-called pattern recognition receptors (PRRs) have the ability to sense a large variety of virus infections. The reason for the broad activity of PRRs lies in the ability to recognize viral nucleic acids. These nucleic acids lack signatures that are present in cytoplasmic cellular nucleic acids and thereby marking them as pathogen-derived. Accumulating evidence suggests that these signatures, which are predominantly sensed by a class of PRRs called retinoic acid-inducible gene I (RIG-I)-like receptors and other proteins, are not unique to viruses but rather resemble immature forms of cellular ribonucleic acids generated by cellular polymerases. RIG-I-like receptors, and other cellular antiviral proteins, may therefore have mainly evolved to sense nonprocessed nucleic acids typically generated by primitive organisms and pathogens. This capability has not only implications on induction of antiviral immunity but also on the function of cellular proteins to handle self-derived RNA with stimulatory potential. PMID:28475460

  5. Urinary biomarkers of exposure and of oxidative damage in children exposed to low airborne concentrations of benzene.

    PubMed

    Andreoli, R; Spatari, G; Pigini, D; Poli, D; Banda, I; Goldoni, M; Riccelli, M G; Petyx, M; Protano, C; Vitali, M; Barbaro, M; Mutti, A

    2015-10-01

    The aim of this work was to evaluate the oxidative damage to nucleic acids in children (5-11 years) associated with exposure to environmental pollutants and tobacco smoke (ETS). For each subject, urinary sampling was done twice (evening and next morning) to measure by tandem LC-MS-MS such oxidated products of nucleic acids as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), 8-oxo-7,8-dihydroguanosine (8-oxoGuo), and 8-oxo-7,8-dihydroguanine (8-oxoGua). Methyl tert-butyl ether (U-MTBE), benzene (U-Benz), and its metabolites (t,t-muconic and S-phenylmercapturic acids, t,t-MA and S-PMA, respectively) were determined as biomarkers of exposure to air pollution, and cotinine as a biomarker of exposure to ETS. Biomarkers of exposure (S-PMA and U-MTBE) and of DNA oxidation (8-oxodGuo) were dependent on the urbanization and industrialization levels and increased in the evening sample as compared to next morning (p<0.05). In both evening and next morning samples, 8-oxodGuo and 8-oxoGuo correlated with each other (r=0.596 and r=0.537, respectively, p<0.01) and with biomarkers of benzene exposure, particularly S-PMA (r=0.59 and r=0.45 for 8-oxodGuo and r=0.411 and r=0.383 for 8-oxoGuo, p<0.01). No such correlations were observed for U-MTBE and cotinine. Multiple linear regression analyses showed that 8-oxodGuo was positively associated with S-PMA at both sampling times (β=0.18 and β=0.14 for evening and next morning sampling, respectively; p<0.02) and weakly with U-MTBE (β=0.07, p=0.020) only in the evening urines. These results suggest that the selected biomarkers of exposure to benzene, particularly S-PMA, are good tracers of exposure to complex mixtures of oxidative pollutants and that the associated oxidative damage to nucleic acids is detectable even at very low levels of exposure. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Prokaryotic Argonaute proteins: novel genome-editing tools?

    PubMed

    Hegge, Jorrit W; Swarts, Daan C; van der Oost, John

    2018-01-01

    Argonaute proteins constitute a highly diverse family of nucleic acid-guided proteins. They were first discovered in eukaryotes as key proteins in RNA interference systems, but homologous prokaryotic Argonaute proteins (pAgos) have also been found in archaea and bacteria. In this Progress article, we focus on long pAgo variants, a class of pAgos that are involved in nucleic acid-guided host defence against invading nucleic acids, and discuss the potential of pAgos in genome editing.

  7. Amplification of trace amounts of nucleic acids

    DOEpatents

    Church, George M [Brookline, MA; Zhang, Kun [Brighton, MA

    2008-06-17

    Methods of reducing background during amplification of small amounts of nucleic acids employ careful analysis of sources of low level contamination. Ultraviolet light can be used to reduce nucleic acid contaminants in reagents and equipment. "Primer-dimer" background can be reduced by judicious design of primers. We have shown clean signal-to-noise with as little as starting material as one single human cell (.about.6 picogram), E. coli cell (.about.5 femtogram) or Prochlorococcus cell (.about.3 femtogram).

  8. Parallel nucleic acid recognition by the LNA (locked nucleic acid) stereoisomers beta-L-LNA and alpha-D-LNA; studies in the mirror image world.

    PubMed

    Christensen, Nanna K; Bryld, Torsten; Sørensen, Mads D; Arar, Khalil; Wengel, Jesper; Nielsen, Poul

    2004-02-07

    Two LNA (locked nucleic acid) stereoisomers (beta-L-LNA and alpha-D-LNA) are evaluated in the mirror-image world, that is by the study of two mixed sequences of LNA and alpha-L-LNA and their L-DNA and L-RNA complements. Both are found to display high-affinity RNA-recognition by the formation of duplexes with parallel strand orientation.

  9. 5'to 3' nucleic acid synthesis using 3'-photoremovable protecting group

    DOEpatents

    Pirrung, Michael C.; Shuey, Steven W.; Bradley, Jean-Claude

    1999-01-01

    The present invention relates, in general, to a method of synthesizing a nucleic acid, and, in particular, to a method of effecting 5' to 3' nucleic acid synthesis. The method can be used to prepare arrays of oligomers bound to a support via their 5' end. The invention also relates to a method of effecting mutation analysis using such arrays. The invention further relates to compounds and compositions suitable for use in such methods.

  10. No evidence of persisting measles virus in peripheral blood mononuclear cells from children with autism spectrum disorder.

    PubMed

    D'Souza, Yasmin; Fombonne, Eric; Ward, Brian J

    2006-10-01

    Despite epidemiologic evidence to the contrary, claims of an association between measles-mumps-rubella vaccination and the development of autism have persisted. Such claims are based primarily on the identification of measles virus nucleic acids in tissues and body fluids by polymerase chain reaction. We sought to determine whether measles virus nucleic acids persist in children with autism spectrum disorder compared with control children. Peripheral blood mononuclear cells were isolated from 54 children with autism spectrum disorder and 34 developmentally normal children, and up to 4 real-time polymerase chain reaction assays and 2 nested polymerase chain reaction assays were performed. These assays targeted the nucleoprotein, fusion, and hemagglutinin genes of measles virus using previously published primer pairs with detection by SYBR green I. Our own real-time assay targeted the fusion gene using novel primers and an internal fluorescent probe. Positive reactions were evaluated rigorously, and amplicons were sequenced. Finally, anti-measles antibody titers were measured by enzyme immunoassay. The real-time assays based on previously published primers gave rise to a large number of positive reactions in both autism spectrum disorder and control samples. Almost all of the positive reactions in these assays were eliminated by evaluation of melting curves and amplicon band size. The amplicons for the remaining positive reactions were cloned and sequenced. No sample from either autism spectrum disorder or control groups was found to contain nucleic acids from any measles virus gene. In the nested polymerase chain reaction and in-house assays, none of the samples yielded positive results. Furthermore, there was no difference in anti-measles antibody titers between the autism and control groups. There is no evidence of measles virus persistence in the peripheral blood mononuclear cells of children with autism spectrum disorder.

  11. Activity and Phylogenetic Diversity of Bacterial Cells with High and Low Nucleic Acid Content and Electron Transport System Activity in an Upwelling Ecosystem

    PubMed Central

    Longnecker, K.; Sherr, B. F.; Sherr, E. B.

    2005-01-01

    We evaluated whether bacteria with higher cell-specific nucleic acid content (HNA) or an active electron transport system, i.e., positive for reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), were responsible for the bulk of bacterioplankton metabolic activity. We also examined whether the phylogenetic diversity of HNA and CTC-positive cells differed from the diversity of Bacteria with low nucleic acid content (LNA). Bacterial assemblages were sampled both in eutrophic shelf waters and in mesotrophic offshore waters in the Oregon coastal upwelling region. Cytometrically sorted HNA, LNA, and CTC-positive cells were assayed for their cell-specific [3H]leucine incorporation rates. Phylogenetic diversity in sorted non-radioactively labeled samples was assayed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes. Cell-specific rates of leucine incorporation of HNA and CTC-positive cells were on average only slightly greater than the cell-specific rates of LNA cells. HNA cells accounted for most bacterioplankton substrate incorporation due to high abundances, while the low abundances of CTC-positive cells resulted in only a small contribution by these cells to total bacterial activity. The proportion of the total bacterial leucine incorporation attributable to LNA cells was higher in offshore regions than in shelf waters. Sequence data obtained from DGGE bands showed broadly similar phylogenetic diversity across HNA, LNA, and CTC-positive cells, with between-sample and between-region variability in the distribution of phylotypes. Our results suggest that LNA bacteria are not substantially different from HNA bacteria in either cell-specific rates of substrate incorporation or phylogenetic composition and that they can be significant contributors to bacterial metabolism in the sea. PMID:16332746

  12. Activity and phylogenetic diversity of bacterial cells with high and low nucleic acid content and electron transport system activity in an upwelling ecosystem.

    PubMed

    Longnecker, K; Sherr, B F; Sherr, E B

    2005-12-01

    We evaluated whether bacteria with higher cell-specific nucleic acid content (HNA) or an active electron transport system, i.e., positive for reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), were responsible for the bulk of bacterioplankton metabolic activity. We also examined whether the phylogenetic diversity of HNA and CTC-positive cells differed from the diversity of Bacteria with low nucleic acid content (LNA). Bacterial assemblages were sampled both in eutrophic shelf waters and in mesotrophic offshore waters in the Oregon coastal upwelling region. Cytometrically sorted HNA, LNA, and CTC-positive cells were assayed for their cell-specific [3H]leucine incorporation rates. Phylogenetic diversity in sorted non-radioactively labeled samples was assayed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes. Cell-specific rates of leucine incorporation of HNA and CTC-positive cells were on average only slightly greater than the cell-specific rates of LNA cells. HNA cells accounted for most bacterioplankton substrate incorporation due to high abundances, while the low abundances of CTC-positive cells resulted in only a small contribution by these cells to total bacterial activity. The proportion of the total bacterial leucine incorporation attributable to LNA cells was higher in offshore regions than in shelf waters. Sequence data obtained from DGGE bands showed broadly similar phylogenetic diversity across HNA, LNA, and CTC-positive cells, with between-sample and between-region variability in the distribution of phylotypes. Our results suggest that LNA bacteria are not substantially different from HNA bacteria in either cell-specific rates of substrate incorporation or phylogenetic composition and that they can be significant contributors to bacterial metabolism in the sea.

  13. Future microfluidic and nanofluidic modular platforms for nucleic acid liquid biopsy in precision medicine

    PubMed Central

    Egatz-Gomez, Ana; Wang, Ceming; Klacsmann, Flora; Pan, Zehao; Marczak, Steve; Wang, Yunshan; Sun, Gongchen; Senapati, Satyajyoti; Chang, Hsueh-Chia

    2016-01-01

    Nucleic acid biomarkers have enormous potential in non-invasive diagnostics and disease management. In medical research and in the near future in the clinics, there is a great demand for accurate miRNA, mRNA, and ctDNA identification and profiling. They may lead to screening of early stage cancer that is not detectable by tissue biopsy or imaging. Moreover, because their cost is low and they are non-invasive, they can become a regular screening test during annual checkups or allow a dynamic treatment program that adjusts its drug and dosage frequently. We briefly review a few existing viral and endogenous RNA assays that have been approved by the Federal Drug Administration. These tests are based on the main nucleic acid detection technologies, namely, quantitative reverse transcription polymerase chain reaction (PCR), microarrays, and next-generation sequencing. Several of the challenges that these three technologies still face regarding the quantitative measurement of a panel of nucleic acids are outlined. Finally, we review a cluster of microfluidic technologies from our group with potential for point-of-care nucleic acid quantification without nucleic acid amplification, designed to overcome specific limitations of current technologies. We suggest that integration of these technologies in a modular design can offer a low-cost, robust, and yet sensitive/selective platform for a variety of precision medicine applications. PMID:27190565

  14. Museums and disease: using tissue archive and museum samples to study pathogens.

    PubMed

    Tsangaras, Kyriakos; Greenwood, Alex D

    2012-01-20

    Molecular studies of archival and fossil samples have traditionally focused on the nucleic acids derived from the host species. However, there has recently been an increase in ancient DNA research on the identification and characterization of infectious agents within the hosts. The study of pathogens from the past provides great opportunities for discovering the causes of historical infection events, characterizing host-microorganism co-evolution and directly investigating the evolution of specific pathogens. Several research teams have been able to isolate and characterize a variety of different bacterial, parasite and viral microorganisms. However, this emerging field is not without obstacles. The diagenetic processes that make ancient DNA research generally difficult are also impediments to ancient pathogen research and perhaps more so given that their DNA may represent an even rarer proportion of the remaining nucleic acids in a fossil sample than host DNA. However, studies performed under controlled conditions and following stringent ancient DNA protocols can and have yielded reliable and often surprising results. This article reviews the advantages, problems, and failures of ancient microbiological research. Copyright © 2011 Elsevier GmbH. All rights reserved.

  15. Signal Amplification Technologies for the Detection of Nucleic Acids: from Cell-Free Analysis to Live-Cell Imaging.

    PubMed

    Fozooni, Tahereh; Ravan, Hadi; Sasan, Hosseinali

    2017-12-01

    Due to their unique properties, such as programmability, ligand-binding capability, and flexibility, nucleic acids can serve as analytes and/or recognition elements for biosensing. To improve the sensitivity of nucleic acid-based biosensing and hence the detection of a few copies of target molecule, different modern amplification methodologies, namely target-and-signal-based amplification strategies, have already been developed. These recent signal amplification technologies, which are capable of amplifying the signal intensity without changing the targets' copy number, have resulted in fast, reliable, and sensitive methods for nucleic acid detection. Working in cell-free settings, researchers have been able to optimize a variety of complex and quantitative methods suitable for deploying in live-cell conditions. In this study, a comprehensive review of the signal amplification technologies for the detection of nucleic acids is provided. We classify the signal amplification methodologies into enzymatic and non-enzymatic strategies with a primary focus on the methods that enable us to shift away from in vitro detecting to in vivo imaging. Finally, the future challenges and limitations of detection for cellular conditions are discussed.

  16. Noncanonical structures and their thermodynamics of DNA and RNA under molecular crowding: beyond the Watson-Crick double helix.

    PubMed

    Sugimoto, Naoki

    2014-01-01

    How does molecular crowding affect the stability of nucleic acid structures inside cells? Water is the major solvent component in living cells, and the properties of water in the highly crowded media inside cells differ from that in buffered solution. As it is difficult to measure the thermodynamic behavior of nucleic acids in cells directly and quantitatively, we recently developed a cell-mimicking system using cosolutes as crowding reagents. The influences of molecular crowding on the structures and thermodynamics of various nucleic acid sequences have been reported. In this chapter, we discuss how the structures and thermodynamic properties of nucleic acids differ under various conditions such as highly crowded environments, compartment environments, and in the presence of ionic liquids, and the major determinants of the crowding effects on nucleic acids are discussed. The effects of molecular crowding on the activities of ribozymes and riboswitches on noncanonical structures of DNA- and RNA-like quadruplexes that play important roles in transcription and translation are also described. © 2014 Elsevier Inc. All rights reserved.

  17. Nucleic acid-based electrochemical nanobiosensors.

    PubMed

    Abi, Alireza; Mohammadpour, Zahra; Zuo, Xiaolei; Safavi, Afsaneh

    2018-04-15

    The detection of biomarkers using sensitive and selective analytical devices is critically important for the early stage diagnosis and treatment of diseases. The synergy between the high specificity of nucleic acid recognition units and the great sensitivity of electrochemical signal transductions has already shown promise for the development of efficient biosensing platforms. Yet nucleic-acid based electrochemical biosensors often rely on target amplification strategies (e.g., polymerase chain reactions) to detect analytes at clinically relevant concentration ranges. The complexity and time-consuming nature of these amplification methods impede moving nucleic acid-based electrochemical biosensors from laboratory-based to point-of-care test settings. Fortunately, advancements in nanotechnology have provided growing evidence that the recruitment of nanoscaled materials and structures can enhance the biosensing performance (particularly in terms of sensitivity and response time) to the level suitable for use in point-of-care diagnostic tools. This Review highlights the significant progress in the field of nucleic acid-based electrochemical nanobiosensing with the focus on the works published during the last five years. Copyright © 2017. Published by Elsevier B.V.

  18. PubDNA Finder: a web database linking full-text articles to sequences of nucleic acids.

    PubMed

    García-Remesal, Miguel; Cuevas, Alejandro; Pérez-Rey, David; Martín, Luis; Anguita, Alberto; de la Iglesia, Diana; de la Calle, Guillermo; Crespo, José; Maojo, Víctor

    2010-11-01

    PubDNA Finder is an online repository that we have created to link PubMed Central manuscripts to the sequences of nucleic acids appearing in them. It extends the search capabilities provided by PubMed Central by enabling researchers to perform advanced searches involving sequences of nucleic acids. This includes, among other features (i) searching for papers mentioning one or more specific sequences of nucleic acids and (ii) retrieving the genetic sequences appearing in different articles. These additional query capabilities are provided by a searchable index that we created by using the full text of the 176 672 papers available at PubMed Central at the time of writing and the sequences of nucleic acids appearing in them. To automatically extract the genetic sequences occurring in each paper, we used an original method we have developed. The database is updated monthly by automatically connecting to the PubMed Central FTP site to retrieve and index new manuscripts. Users can query the database via the web interface provided. PubDNA Finder can be freely accessed at http://servet.dia.fi.upm.es:8080/pubdnafinder

  19. Amplified electrochemical detection of nucleic acid hybridization via selective preconcentration of unmodified gold nanoparticles.

    PubMed

    Li, Yuan; Tian, Rui; Zheng, Xingwang; Huang, Rongfu

    2016-08-31

    The common drawback of optical methods for rapid detection of nucleic acid by exploiting the differential affinity of single-/double-stranded nucleic acids for unmodified gold nanoparticles (AuNPs) is its relatively low sensitivity. In this article, on the basis of selective preconcentration of AuNPs unprotected by single-stranded DNA (ssDNA) binding, a novel electrochemical strategy for nucleic acid sequence identification assay has been developed. Through detecting the redox signal mediated by AuNPs on 1, 6-hexanedithiol blocked gold electrode, the proposed method is able to ensure substantial signal amplification and a low background current. This strategy is demonstrated for quantitative analysis of the target microRNA (let-7a) in human breast adenocarcinoma cells, and a detection limit of 16 fM is readily achieved with desirable specificity and sensitivity. These results indicate that the selective preconcentration of AuNPs for electrochemical signal readout can offer a promising platform for the detection of specific nucleic acid sequence. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Visual detection of Ebola virus using reverse transcription loop-mediated isothermal amplification combined with nucleic acid strip detection.

    PubMed

    Xu, Changping; Wang, Hualei; Jin, Hongli; Feng, Na; Zheng, Xuexing; Cao, Zengguo; Li, Ling; Wang, Jianzhong; Yan, Feihu; Wang, Lina; Chi, Hang; Gai, Weiwei; Wang, Chong; Zhao, Yongkun; Feng, Yan; Wang, Tiecheng; Gao, Yuwei; Lu, Yiyu; Yang, Songtao; Xia, Xianzhu

    2016-05-01

    Ebola virus (species Zaire ebolavirus) (EBOV) is highly virulent in humans. The largest recorded outbreak of Ebola hemorrhagic fever in West Africa to date was caused by EBOV. Therefore, it is necessary to develop a detection method for this virus that can be easily distributed and implemented. In the current study, we developed a visual assay that can detect EBOV-associated nucleic acids. This assay combines reverse transcription loop-mediated isothermal amplification and nucleic acid strip detection (RT-LAMP-NAD). Nucleic acid amplification can be achieved in a one-step process at a constant temperature (58 °C, 35 min), and the amplified products can be visualized within 2-5 min using a nucleic acid strip detection device. The assay is capable of detecting 30 copies of artificial EBOV glycoprotein (GP) RNA and RNA encoding EBOV GP from 10(2) TCID50 recombinant viral particles per ml with high specificity. Overall, the RT-LAMP-NAD method is simple and has high sensitivity and specificity; therefore, it is especially suitable for the rapid detection of EBOV in African regions.

  1. Non-Viral Nucleic Acid Delivery Strategies to the Central Nervous System

    PubMed Central

    Tan, James-Kevin Y.; Sellers, Drew L.; Pham, Binhan; Pun, Suzie H.; Horner, Philip J.

    2016-01-01

    With an increased prevalence and understanding of central nervous system (CNS) injuries and neurological disorders, nucleic acid therapies are gaining promise as a way to regenerate lost neurons or halt disease progression. While more viral vectors have been used clinically as tools for gene delivery, non-viral vectors are gaining interest due to lower safety concerns and the ability to deliver all types of nucleic acids. Nevertheless, there are still a number of barriers to nucleic acid delivery. In this focused review, we explore the in vivo challenges hindering non-viral nucleic acid delivery to the CNS and the strategies and vehicles used to overcome them. Advantages and disadvantages of different routes of administration including: systemic injection, cerebrospinal fluid injection, intraparenchymal injection and peripheral administration are discussed. Non-viral vehicles and treatment strategies that have overcome delivery barriers and demonstrated in vivo gene transfer to the CNS are presented. These approaches can be used as guidelines in developing synthetic gene delivery vectors for CNS applications and will ultimately bring non-viral vectors closer to clinical application. PMID:27847462

  2. Accuracy assessment of the linear Poisson-Boltzmann equation and reparametrization of the OBC generalized Born model for nucleic acids and nucleic acid-protein complexes.

    PubMed

    Fogolari, Federico; Corazza, Alessandra; Esposito, Gennaro

    2015-04-05

    The generalized Born model in the Onufriev, Bashford, and Case (Onufriev et al., Proteins: Struct Funct Genet 2004, 55, 383) implementation has emerged as one of the best compromises between accuracy and speed of computation. For simulations of nucleic acids, however, a number of issues should be addressed: (1) the generalized Born model is based on a linear model and the linearization of the reference Poisson-Boltmann equation may be questioned for highly charged systems as nucleic acids; (2) although much attention has been given to potentials, solvation forces could be much less sensitive to linearization than the potentials; and (3) the accuracy of the Onufriev-Bashford-Case (OBC) model for nucleic acids depends on fine tuning of parameters. Here, we show that the linearization of the Poisson Boltzmann equation has mild effects on computed forces, and that with optimal choice of the OBC model parameters, solvation forces, essential for molecular dynamics simulations, agree well with those computed using the reference Poisson-Boltzmann model. © 2015 Wiley Periodicals, Inc.

  3. PrPC has nucleic acid chaperoning properties similar to the nucleocapsid protein of HIV-1.

    PubMed

    Derrington, Edmund; Gabus, Caroline; Leblanc, Pascal; Chnaidermann, Jonas; Grave, Linda; Dormont, Dominique; Swietnicki, Wieslaw; Morillas, Manuel; Marck, Daniel; Nandi, Pradip; Darlix, Jean-Luc

    2002-01-01

    The function of the cellular prion protein (PrPC) remains obscure. Studies suggest that PrPC functions in several processes including signal transduction and Cu2+ metabolism. PrPC has also been established to bind nucleic acids. Therefore we investigated the properties of PrPC as a putative nucleic acid chaperone. Surprisingly, PrPC possesses all the nucleic acid chaperoning properties previously specific to retroviral nucleocapsid proteins. PrPC appears to be a molecular mimic of NCP7, the nucleocapsid protein of HIV-1. Thus PrPC, like NCP7, chaperones the annealing of tRNA(Lys) to the HIV-1 primer binding site, the initial step of retrovirus replication. PrPC also chaperones the two DNA strand transfers required for production of a complete proviral DNA with LTRs. Concerning the functions of NCP7 during budding, PrPC also mimices NCP7 by dimerizing the HIV-1 genomic RNA. These data are unprecedented because, although many cellular proteins have been identified as nucleic acid chaperones, none have the properties of retroviral nucleocapsid proteins.

  4. Assessment for Melting Temperature Measurement of Nucleic Acid by HRM.

    PubMed

    Wang, Jing; Pan, Xiaoming; Liang, Xingguo

    2016-01-01

    High resolution melting (HRM), with a high sensitivity to distinguish the nucleic acid species with small variations, has been widely applied in the mutation scanning, methylation analysis, and genotyping. For the aim of extending HRM for the evaluation of thermal stability of nucleic acid secondary structures on sequence dependence, we investigated effects of the dye of EvaGreen, metal ions, and impurities (such as dNTPs) on melting temperature ( T m ) measurement by HRM. The accuracy of HRM was assessed as compared with UV melting method, and little difference between the two methods was found when the DNA T m was higher than 40°C. Both insufficiency and excessiveness of EvaGreen were found to give rise to a little bit higher T m , showing that the proportion of dye should be considered for precise T m measurement of nucleic acids. Finally, HRM method was also successfully used to measure T m s of DNA triplex, hairpin, and RNA duplex. In conclusion, HRM can be applied in the evaluation of thermal stability of nucleic acid (DNA or RNA) or secondary structural elements (even when dNTPs are present).

  5. A specific scenario for the origin of life and the genetic code based on peptide/oligonucleotide interdependence.

    PubMed

    Griffith, Robert W

    2009-12-01

    Among various scenarios that attempt to explain how life arose, the RNA world is currently the most widely accepted scientific hypothesis among biologists. However, the RNA world is logistically implausible and doesn't explain how translation arose and DNA became incorporated into living systems. Here I propose an alternative hypothesis for life's origin based on cooperation between simple nucleic acids, peptides and lipids. Organic matter that accumulated on the prebiotic Earth segregated into phases in the ocean based on density and solubility. Synthesis of complex organic monomers and polymerization reactions occurred within a surface hydrophilic layer and at its aqueous and atmospheric interfaces. Replication of nucleic acids and translation of peptides began at the emulsified interface between hydrophobic and aqueous layers. At the core of the protobiont was a family of short nucleic acids bearing arginine's codon and anticodon that added this amino acid to pre-formed peptides. In turn, the survival and replication of nucleic acid was aided by the peptides. The arginine-enriched peptides served to sequester and transfer phosphate bond energy and acted as cohesive agents, aggregating nucleic acids and keeping them at the interface.

  6. Structural aspects of catalytic mechanisms of endonucleases and their binding to nucleic acids

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

    Zhukhlistova, N. E.; Balaev, V. V.; Lyashenko, A. V.

    2012-05-15

    Endonucleases (EC 3.1) are enzymes of the hydrolase class that catalyze the hydrolytic cleavage of deoxyribonucleic and ribonucleic acids at any region of the polynucleotide chain. Endonucleases are widely used both in biotechnological processes and in veterinary medicine as antiviral agents. Medical applications of endonucleases in human cancer therapy hold promise. The results of X-ray diffraction studies of the spatial organization of endonucleases and their complexes and the mechanism of their action are analyzed and generalized. An analysis of the structural studies of this class of enzymes showed that the specific binding of enzymes to nucleic acids is characterized bymore » interactions with nitrogen bases and the nucleotide backbone, whereas the nonspecific binding of enzymes is generally characterized by interactions only with the nucleic-acid backbone. It should be taken into account that the specificity can be modulated by metal ions and certain low-molecular-weight organic compounds. To test the hypotheses about specific and nonspecific nucleic-acid-binding proteins, it is necessary to perform additional studies of atomic-resolution three-dimensional structures of enzyme-nucleic-acid complexes by methods of structural biology.« less

  7. 42 CFR 73.17 - Records.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... and/or synthetic nucleic acids, and organisms containing recombinant and/or synthetic nucleic acids... that all records and data bases created under this part are accurate, have controlled access, and that...

  8. 42 CFR 73.17 - Records.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... and/or synthetic nucleic acids, and organisms containing recombinant and/or synthetic nucleic acids... that all records and data bases created under this part are accurate, have controlled access, and that...

  9. Reactive Derivatives of Nucleic Acids and Their Components as Affinity Reagents

    NASA Astrophysics Data System (ADS)

    Knorre, Dmitrii G.; Vlasov, Valentin V.

    1985-09-01

    The review is devoted to derivatives of nucleic acids and their components — nucleotides, nucleoside triphosphates, and oligonucleotides carrying reactive groups. Such derivatives are important tools for the investigation of protein-nucleic acid interactions and the functional topography of complex protein and nucleoprotein structures and can give rise to the prospect of being able to influence in a highly selective manner living organisms, including the nucleic acids and the nucleoproteins of the genetic apparatus. The review considers the principal groups of such reagents, the methods of their synthesis, and their properties which determine the possibility of their use for the selective (affinity) modification of biopolymers. The general principles of the construction of affinity reagents and their applications are analysed in relation to nucleotide affinity reagents. The bibliography includes 121 references.

  10. Computation of statistical secondary structure of nucleic acids.

    PubMed Central

    Yamamoto, K; Kitamura, Y; Yoshikura, H

    1984-01-01

    This paper presents a computer analysis of statistical secondary structure of nucleic acids. For a given single stranded nucleic acid, we generated "structure map" which included all the annealing structures in the sequence. The map was transformed into "energy map" by rough approximation; here, the energy level of every pairing structure consisting of more than 2 successive nucleic acid pairs was calculated. By using the "energy map", the probability of occurrence of each annealed structure was computed, i.e., the structure was computed statistically. The basis of computation was the 8-queen problem in the chess game. The validity of our computer programme was checked by computing tRNA structure which has been well established. Successful application of this programme to small nuclear RNAs of various origins is demonstrated. PMID:6198622

  11. In vitro evolution of chemically-modified nucleic acid aptamers: Pros and cons, and comprehensive selection strategies.

    PubMed

    Lipi, Farhana; Chen, Suxiang; Chakravarthy, Madhuri; Rakesh, Shilpa; Veedu, Rakesh N

    2016-12-01

    Nucleic acid aptamers are single-stranded DNA or RNA oligonucleotide sequences that bind to a specific target molecule with high affinity and specificity through their ability to adopt 3-dimensional structure in solution. Aptamers have huge potential as targeted therapeutics, diagnostics, delivery agents and as biosensors. However, aptamers composed of natural nucleotide monomers are quickly degraded in vivo and show poor pharmacodynamic properties. To overcome this, chemically-modified nucleic acid aptamers are developed by incorporating modified nucleotides after or during the selection process by Systematic Evolution of Ligands by EXponential enrichment (SELEX). This review will discuss the development of chemically-modified aptamers and provide the pros and cons, and new insights on in vitro aptamer selection strategies by using chemically-modified nucleic acid libraries.

  12. In vitro evolution of chemically-modified nucleic acid aptamers: Pros and cons, and comprehensive selection strategies

    PubMed Central

    Chen, Suxiang; Chakravarthy, Madhuri; Rakesh, Shilpa; Veedu, Rakesh N.

    2016-01-01

    ABSTRACT Nucleic acid aptamers are single-stranded DNA or RNA oligonucleotide sequences that bind to a specific target molecule with high affinity and specificity through their ability to adopt 3-dimensional structure in solution. Aptamers have huge potential as targeted therapeutics, diagnostics, delivery agents and as biosensors. However, aptamers composed of natural nucleotide monomers are quickly degraded in vivo and show poor pharmacodynamic properties. To overcome this, chemically-modified nucleic acid aptamers are developed by incorporating modified nucleotides after or during the selection process by Systematic Evolution of Ligands by EXponential enrichment (SELEX). This review will discuss the development of chemically-modified aptamers and provide the pros and cons, and new insights on in vitro aptamer selection strategies by using chemically-modified nucleic acid libraries. PMID:27715478

  13. Sequence-specific label-free nucleic acid biosensor for the detection of the hepatitis C virus genotype 1a using a disposable pencil graphite electrode.

    PubMed

    Donmez, Soner; Arslan, Fatma; Arslan, Halit

    2016-05-01

    In this paper, we demonstrate a simple, sensitive, inexpensive, disposable and label-free electrochemical nucleic acid biosensor for the detection of the hepatitis C virus genotype 1a (HCV1a). The nucleic acid biosensor was designed with the amino-linked inosine-substituted 20-mer probes, which were immobilized onto a disposable pencil graphite electrode (PGE) by covalent linking. The proposed nucleic acid biosensor was linear in the range of 0.05 and 0.75 μM, exhibiting a limit of detection of 54.9 nM. The single-stranded synthetic PCR product analogs of HCV1a were also detected with satisfactory results under optimal conditions, showing the potential application of this biosensor.

  14. Hybrids of Nucleic Acids and Carbon Nanotubes for Nanobiotechnology.

    PubMed

    Umemura, Kazuo

    2015-03-12

    Recent progress in the combination of nucleic acids and carbon nanotubes (CNTs) has been briefly reviewed here. Since discovering the hybridization phenomenon of DNA molecules and CNTs in 2003, a large amount of fundamental and applied research has been carried out. Among thousands of papers published since 2003, approximately 240 papers focused on biological applications were selected and categorized based on the types of nucleic acids used, but not the types of CNTs. This survey revealed that the hybridization phenomenon is strongly affected by various factors, such as DNA sequences, and for this reason, fundamental studies on the hybridization phenomenon are important. Additionally, many research groups have proposed numerous practical applications, such as nanobiosensors. The goal of this review is to provide perspective on biological applications using hybrids of nucleic acids and CNTs.

  15. Beyond DNA origami: A look on the bright future of nucleic acid nanotechnology

    PubMed Central

    Michelotti, Nicole; Johnson-Buck, Alexander; Manzo, Anthony J.

    2012-01-01

    Nucleic acid nanotechnology exploits the programmable molecular recognition properties of natural and synthetic nucleic acids to assemble structures with nanometer-scale precision. In 2006, DNA origami transformed the field by providing a versatile platform for self-assembly of arbitrary shapes from one long DNA strand held in place by hundreds of short, site-specific (spatially addressable) DNA ”staples”. This revolutionary approach has led to the creation of a multitude of 2D and 3D scaffolds that form the basis for functional nanodevices. Not limited to nucleic acids, these nanodevices can incorporate other structural and functional materials, such as proteins and nanoparticles, making them broadly useful for current and future applications in emerging fields such as nanomedicine, nanoelectronics, and alternative energy. PMID:22131292

  16. Codes in the codons: construction of a codon/amino acid periodic table and a study of the nature of specific nucleic acid-protein interactions.

    PubMed

    Benyo, B; Biro, J C; Benyo, Z

    2004-01-01

    The theory of "codon-amino acid coevolution" was first proposed by Woese in 1967. It suggests that there is a stereochemical matching - that is, affinity - between amino acids and certain of the base triplet sequences that code for those amino acids. We have constructed a common periodic table of codons and amino acids, where the nucleic acid table showed perfect axial symmetry for codons and the corresponding amino acid table also displayed periodicity regarding the biochemical properties (charge and hydrophobicity) of the 20 amino acids and the position of the stop signals. The table indicates that the middle (2/sup nd/) amino acid in the codon has a prominent role in determining some of the structural features of the amino acids. The possibility that physical contact between codons and amino acids might exist was tested on restriction enzymes. Many recognition site-like sequences were found in the coding sequences of these enzymes and as many as 73 examples of codon-amino acid co-location were observed in the 7 known 3D structures (December 2003) of endonuclease-nucleic acid complexes. These results indicate that the smallest possible units of specific nucleic acid-protein interaction are indeed the stereochemically compatible codons and amino acids.

  17. Six-year pilot study on nucleic acid testing for blood donations in China.

    PubMed

    Ye, Xianlin; Yang, Baocheng; Zhu, Weigang; Zheng, Xin; Du, Peng; Zeng, Jingfeng; Li, Chengyao

    2013-10-01

    A six-year pilot study on nucleic acid testing for HBV, HCV and HIV-1 has been undertaken on sero-negative plasmas in mini-pool and individual donation testing at Shenzhen Blood Center. Of 307,740 sero-negative blood samples, 95 of 102 HBV DNA yields were confirmed positive, 80/95 (84.2%) were classified as occult HBV infection (OBI) and 15 (15.8%) as window period cases. Amongst OBIs, 45% carried anti-HBc only, 41.3% anti-HBc and anti-HBs and 13.7% anti-HBs only. HBV DNA yield was 1:3239. One HCV WP and one HIV-1 infected donations were detected. High residual risk was found in current blood donations screening in China. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Arrays of probes for positional sequencing by hybridization

    DOEpatents

    Cantor, Charles R [Boston, MA; Prezetakiewiczr, Marek [East Boston, MA; Smith, Cassandra L [Boston, MA; Sano, Takeshi [Waltham, MA

    2008-01-15

    This invention is directed to methods and reagents useful for sequencing nucleic acid targets utilizing sequencing by hybridization technology comprising probes, arrays of probes and methods whereby sequence information is obtained rapidly and efficiently in discrete packages. That information can be used for the detection, identification, purification and complete or partial sequencing of a particular target nucleic acid. When coupled with a ligation step, these methods can be performed under a single set of hybridization conditions. The invention also relates to the replication of probe arrays and methods for making and replicating arrays of probes which are useful for the large scale manufacture of diagnostic aids used to screen biological samples for specific target sequences. Arrays created using PCR technology may comprise probes with 5'- and/or 3'-overhangs.

  19. Uses of Ku70

    DOEpatents

    Li, Gloria C.; Cordon-Cardo, Carlos; Ouyang, Honghai

    2004-10-26

    This invention provides a method of diagnosing a predisposition to cancer in a subject comprising: (a) obtaining a nucleic acid sample from the subject; and; (b) determining whether one or more of the subject's Ku70 alleles or regulatory regions to those alleles are deleted or different from the wild type so as to reduce or eliminate the subject's expression of polypeptide having tumor suppressor activity. This invention also provides a method of assessing the severity of cancer in a subject comprising: (a) obtaining a nucleic acid sample from the subject; and (b) determining whether one or more of the subject's Ku70 alleles or regulatory regions to those alleles are deleted or different from the wild type so as to reduce or eliminate the subject's expression of polypeptide having tumor suppressor activity. This invention also provides a method of assessing the severity of cancer in a subject comprising: determining the subcellular localization of Ku70 in the subject, wherein an abnormal subcellular localization of Ku70 indicates a predisposition to cancer.

  20. Paper-based sample-to-answer molecular diagnostic platform for point-of-care diagnostics.

    PubMed

    Choi, Jane Ru; Tang, Ruihua; Wang, ShuQi; Wan Abas, Wan Abu Bakar; Pingguan-Murphy, Belinda; Xu, Feng

    2015-12-15

    Nucleic acid testing (NAT), as a molecular diagnostic technique, including nucleic acid extraction, amplification and detection, plays a fundamental role in medical diagnosis for timely medical treatment. However, current NAT technologies require relatively high-end instrumentation, skilled personnel, and are time-consuming. These drawbacks mean conventional NAT becomes impractical in many resource-limited disease-endemic settings, leading to an urgent need to develop a fast and portable NAT diagnostic tool. Paper-based devices are typically robust, cost-effective and user-friendly, holding a great potential for NAT at the point of care. In view of the escalating demand for the low cost diagnostic devices, we highlight the beneficial use of paper as a platform for NAT, the current state of its development, and the existing challenges preventing its widespread use. We suggest a strategy involving integrating all three steps of NAT into one single paper-based sample-to-answer diagnostic device for rapid medical diagnostics in the near future. Copyright © 2015 Elsevier B.V. All rights reserved.

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