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Sample records for nucleic acid assays

  1. 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.

  2. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Enterovirus nucleic acid assay. 866.3225 Section... nucleic acid assay. (a) Identification. An enterovirus nucleic acid assay is a device that consists of... Special Controls Guidance Document: Nucleic Acid Amplification Assay for the Detection of Enterovirus...

  3. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Enterovirus nucleic acid assay. 866.3225 Section... nucleic acid assay. (a) Identification. An enterovirus nucleic acid assay is a device that consists of... Special Controls Guidance Document: Nucleic Acid Amplification Assay for the Detection of Enterovirus...

  4. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Enterovirus nucleic acid assay. 866.3225 Section... nucleic acid assay. (a) Identification. An enterovirus nucleic acid assay is a device that consists of primers, probes, enzymes, and controls for the amplification and detection of enterovirus ribonucleic acid...

  5. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Enterovirus nucleic acid assay. 866.3225 Section... nucleic acid assay. (a) Identification. An enterovirus nucleic acid assay is a device that consists of primers, probes, enzymes, and controls for the amplification and detection of enterovirus ribonucleic acid...

  6. Non-instrumented nucleic acid amplification assay

    NASA Astrophysics Data System (ADS)

    Weigl, Bernhard H.; Domingo, Gonzalo; Gerlach, Jay; Tang, Dennis; Harvey, Darrel; Talwar, Nick; Fichtenholz, Alex; van Lew, Bill; LaBarre, Paul

    2008-02-01

    We have developed components of a diagnostic disposable platform that has the dual purpose of providing molecular diagnostics at the point of care (POC) as well as stabilizing specimens for further analysis via a centralized surveillance system. This diagnostic is targeted for use in low-resource settings by minimally trained health workers. The disposable device does not require any additional instrumentation and will be almost as rapid and simple to use as a lateral flow strip test - yet will offer the sensitivity and specificity of nucleic acid amplification tests (NAATs). The low-cost integrated device is composed of three functional components: (1) a sample-processing subunit that generates clean and stabilized DNA from raw samples containing nucleic acids, (2) a NA amplification subunit, and (3) visual amplicon detection sub-unit. The device integrates chemical exothermic heating, temperature stabilization using phase-change materials, and isothermal nucleic acid amplification. The aim of developing this system is to provide pathogen detection with NAAT-level sensitivity in low-resource settings where there is no access to instrumentation. If a disease occurs, patients would be tested with the disposable in the field. A nucleic acid sample would be preserved within the spent disposable which could be sent to a central laboratory facility for further analysis if needed.

  7. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Enterovirus nucleic acid assay. 866.3225 Section 866.3225 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3225 Enterovirus...

  8. 21 CFR 866.5910 - Quality control material for cystic fibrosis nucleic acid assays.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... nucleic acid assays. 866.5910 Section 866.5910 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... 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...

  9. 21 CFR 866.5910 - Quality control material for cystic fibrosis nucleic acid assays.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... nucleic acid assays. 866.5910 Section 866.5910 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... 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...

  10. 21 CFR 866.5910 - Quality control material for cystic fibrosis nucleic acid assays.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... nucleic acid assays. 866.5910 Section 866.5910 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... 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...

  11. A homogeneous nucleic acid hybridization assay based on strand displacement.

    PubMed Central

    Vary, C P

    1987-01-01

    A homogeneous nucleic acid hybridization assay which is conducted in solution and requires no separation steps is described. The assay is based on the concept of strand displacement. In the strand displacement assay, an RNA "signal strand" is hybridized within a larger DNA strand termed the "probe strand", which is, in turn, complementary to the target nucleic acid of interest. Hybridization of the target nucleic acid with the probe strand ultimately results in displacement of the RNA signal strand. Strand displacement, therefore, causes conversion of the RNA from double to single-stranded form. The single-strand specificity of polynucleotide phosphorylase (EC 2.7.7.8) allows discrimination between double-helical and single-stranded forms of the RNA signal strand. As displacement proceeds, free RNA signal strands are preferentially phosphorolyzed to component nucleoside diphosphates, including adenosine diphosphate. The latter nucleotide is converted to ATP by pyruvate kinase(EC 2.7.1.40). Luciferase catalyzed bioluminescence is employed to measure the ATP generated as a result of strand displacement. Images PMID:3309890

  12. 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 panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended...

  13. 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 panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended...

  14. 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 panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended...

  15. 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 panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended...

  16. 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 panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended...

  17. Simple Bulk Readout of Digital Nucleic Acid Quantification Assays.

    PubMed

    Morinishi, Leanna S; Blainey, Paul

    2015-09-24

    Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays. We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout. Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

  18. Bioluminescence regenerative cycle (BRC) system for nucleic acid quantification assays

    NASA Astrophysics Data System (ADS)

    Hassibi, Arjang; Lee, Thomas H.; Davis, Ronald W.; Pourmand, Nader

    2003-07-01

    A new label-free methodology for nucleic acid quantification has been developed where the number of pyrophosphate molecules (PPi) released during polymerization of the target nucleic acid is counted and correlated to DNA copy number. The technique uses the enzymatic complex of ATP-sulfurylase and firefly luciferase to generate photons from PPi. An enzymatic unity gain positive feedback is also implemented to regenerate the photon generation process and compensate any decay in light intensity by self regulation. Due to this positive feedback, the total number of photons generated by the bioluminescence regenerative cycle (BRC) can potentially be orders of magnitude higher than typical chemiluminescent processes. A system level kinetic model that incorporates the effects of contaminations and detector noise was used to show that the photon generation process is in fact steady and also proportional to the nucleic acid quantity. Here we show that BRC is capable of detecting quantities of DNA as low as 1 amol (10-18 mole) in 40μlit aqueous solutions, and this enzymatic assay has a controllable dynamic range of 5 orders of magnitude. The sensitivity of this technology, due to the excess number of photons generated by the regenerative cycle, is not constrained by detector performance, but rather by possible PPi or ATP (adenosine triphosphate) contamination, or background bioluminescence of the enzymatic complex.

  19. 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.

  20. Multicenter evaluation of the Verigene Clostridium difficile nucleic acid assay.

    PubMed

    Carroll, Karen C; Buchan, Blake W; Tan, Sokha; Stamper, Paul D; Riebe, Katherine M; Pancholi, Preeti; Kelly, Cheryl; Rao, Arundhati; Fader, Robert; Cavagnolo, Robert; Watson, Wendy; Goering, Richard V; Trevino, Ernest A; Weissfeld, Alice S; Ledeboer, Nathan A

    2013-12-01

    The Verigene Clostridium difficile Nucleic Acid test (Verigene CDF test) (Nanosphere, Northbrook, IL) is a multiplex qualitative PCR assay that utilizes a nanoparticle-based array hybridization method to detect C. difficile tcdA and tcdB in fecal specimens. In addition, the assay detects binary toxin gene sequences and the single base pair deletion at nucleotide 117 (Δ 117) in tcdC to provide a presumptive identification of the epidemic strain 027/NAP1/BI (referred to here as ribotype 027). This study compared the Verigene CDF test with anaerobic direct and enriched toxigenic culture on stool specimens from symptomatic patients among five geographically diverse laboratories within the United States. The Verigene CDF test was performed according to the manufacturer's instructions, and the reference methods performed by a central laboratory included direct culture onto cycloserine cefoxitin fructose agar (CCFA) and enriched culture using cycloserine cefoxitin mannitol broth with taurocholate and lysozyme. Recovered isolates were identified as C. difficile using gas liquid chromatography and were tested for toxin using a cell culture cytotoxicity neutralization assay. Strains belonging to ribotype 027 were determined by PCR ribotyping and bidirectional sequencing for Δ 117 in tcdC. A total of 1,875 specimens were evaluable. Of these, 275 specimens (14.7%) were culture positive by either direct or enriched culture methods. Compared to direct culture alone, the overall sensitivity, specificity, positive predictive value, and negative predictive value for the Verigene CDF test were 98.7%, 87.5%, 42%, and 99.9%, respectively. Compared to combined direct and enriched culture results, the sensitivity, specificity, positive predictive value, and negative predictive values of the Verigene CDF test were 90.9%, 92.5%, 67.6%, and 98.3%, respectively. Of the 250 concordantly culture-positive specimens, 59 (23.6%) were flagged as "hypervirulent"; 53 were confirmed as ribotype

  1. 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...

  2. 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...

  3. Nonenzymatic catalytic signal amplification for nucleic acid hybridization assays

    NASA Technical Reports Server (NTRS)

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

  4. Detection of North American eastern and western equine encephalitis viruses by nucleic acid amplification assays.

    PubMed

    Lambert, Amy J; Martin, Denise A; Lanciotti, Robert S

    2003-01-01

    We have developed nucleic acid sequence-based amplification (NASBA), standard reverse transcription PCR (RT-PCR), and TaqMan nucleic acid amplification assays for the rapid detection of North American eastern equine encephalitis (EEE) and western equine encephalitis (WEE) viral RNAs from samples collected in the field and clinical samples. The sensitivities of these assays have been compared to that of virus isolation. While all three types of nucleic acid amplification assays provide rapid detection of viral RNAs comparable to the isolation of viruses in Vero cells, the TaqMan assays for North American EEE and WEE viral RNAs are the most sensitive. We have shown these assays to be specific for North American EEE and WEE viral RNAs by testing geographically and temporally distinct strains of EEE and WEE viruses along with a battery of related and unrelated arthropodborne viruses. In addition, all three types of nucleic acid amplification assays have been used to detect North American EEE and WEE viral RNAs from mosquito and vertebrate tissue samples. The sensitivity, specificity, and rapidity of nucleic acid amplification demonstrate the usefulness of NASBA, standard RT-PCR, and TaqMan assays, in both research and diagnostic settings, to detect North American EEE and WEE viral RNAs.

  5. 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.

  6. Standard in vitro assays for protein-nucleic acid interactions--gel shift assays for RNA and DNA binding.

    PubMed

    Mitchell, Sarah F; Lorsch, Jon R

    2014-01-01

    The characterization of protein-nucleic acid interactions is necessary for the study of a wide variety of biological processes. One straightforward and widely used approach to this problem is the electrophoretic mobility shift assay (EMSA), in which the binding of a nucleic acid to one or more proteins changes its mobility through a nondenaturing gel matrix. Usually, the mobility of the nucleic acid is reduced, but examples of increased mobility do exist. This type of assay can be used to investigate the affinity of the interaction between the protein and nucleic acid, the specificity of the interaction, the minimal binding site, and the kinetics of the interaction. One particular advantage of EMSA is the ability to analyze multiple proteins, or protein complexes, binding to nucleic acids. This assay is relatively quick and easy and utilizes equipment available in most laboratories; however, there are many variables that can only be determined empirically; therefore, optimization is necessary and can be highly dependent upon the system. The protocol described here is for the poly(A)-binding protein (PABP) binding to an unstructured RNA probe of 43 bases. While this may be a useful protocol for some additional assays, it is recommended that both reaction conditions and gel running conditions be tailored to the individual interaction to be probed. © 2014 Elsevier Inc. All rights reserved.

  7. Functional nucleic acid-based sensors for heavy metal ion assays.

    PubMed

    Zhu, Guichi; Zhang, Chun-yang

    2014-12-21

    Heavy metal contaminants such as lead ions (Pb(2+)), mercury ions (Hg(2+)) and silver ions (Ag(+)) can cause significant harm to humans and generate enduring bioaccumulation in ecological systems. Even though a variety of methods have been developed for Pb(2+), Hg(2+) and Ag(+) assays, most of them are usually laborious and time-consuming with poor sensitivity. Due to their unique advantages of excellent catalytic properties and high affinity for heavy metal ions, functional nucleic acids such as DNAzymes and aptamers show great promise in the development of novel sensors for heavy metal ion assays. In this review, we summarize the development of functional nucleic acid-based sensors for the detection of Pb(2+), Hg(2+) and Ag(+), and especially focus on two categories including the direct assay and the amplification-based assay. We highlight the emerging trends in the development of sensitive and selective sensors for heavy metal ion assays as well.

  8. Development and Evaluation of a Calibrator Material for Nucleic Acid-Based Assays for Diagnosing Aspergillosis

    PubMed Central

    Abdul-Ali, Deborah; Loeffler, Juergen; White, P. Lewis; Wickes, Brian; Herrera, Monica L.; Alexander, Barbara D.; Baden, Lindsey R.; Clancy, Cornelius; Denning, David; Nguyen, M. Hong; Sugrue, Michele; Wheat, L. Joseph; Wingard, John R.; Donnelly, J. Peter; Barnes, Rosemary; Patterson, Thomas F.; Caliendo, Angela M.

    2013-01-01

    Twelve laboratories evaluated candidate material for an Aspergillus DNA calibrator. The DNA material was quantified using limiting-dilution analysis; the mean concentration was determined to be 1.73 × 1010 units/ml. The calibrator can be used to standardize aspergillosis diagnostic assays which detect and/or quantify nucleic acid. PMID:23616459

  9. Medical Devices; Immunology and Microbiology Devices; Classification of Trichomonas Vaginalis Nucleic Acid Assay. Final order.

    PubMed

    2015-08-04

    The Food and Drug Administration (FDA) is classifying a Trichomonas vaginalis nucleic acid assay into class II (special controls). The Agency is classifying the device into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of the device.

  10. Development of a Quantitative BRET Affinity Assay for Nucleic Acid-Protein Interactions

    PubMed Central

    Vickers, Timothy A.; Crooke, Stanley T.

    2016-01-01

    Protein-nucleic acid interactions play a crucial role in the regulation of diverse biological processes. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination, and RNA processing continues to be a crucial aspect of understanding of cell biology and the mechanisms of disease. In addition, proteins have been demonstrated to interact with antisense oligonucleotide therapeutics in a sequence and chemistry dependent manner, influencing ASO potency and distribution in cells and in vivo. While many assays have been developed to measure protein-nucleic acid interactions, many suffer from lack of throughput and sensitivity, or challenges with protein purification and scalability. In this report we present a new BRET assay for the analysis of DNA-protein interactions which makes use of an extremely bright luciferase as a tag for the binding protein, along with a long-wavelength fluorophore conjugated to the nucleic acid. The resulting assay is high throughput, sensitive, does not require protein purification, and even allows for quantitative characterization of these interactions within the biologically relevant context of whole cells. PMID:27571227

  11. Development of a Quantitative BRET Affinity Assay for Nucleic Acid-Protein Interactions.

    PubMed

    Vickers, Timothy A; Crooke, Stanley T

    2016-01-01

    Protein-nucleic acid interactions play a crucial role in the regulation of diverse biological processes. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination, and RNA processing continues to be a crucial aspect of understanding of cell biology and the mechanisms of disease. In addition, proteins have been demonstrated to interact with antisense oligonucleotide therapeutics in a sequence and chemistry dependent manner, influencing ASO potency and distribution in cells and in vivo. While many assays have been developed to measure protein-nucleic acid interactions, many suffer from lack of throughput and sensitivity, or challenges with protein purification and scalability. In this report we present a new BRET assay for the analysis of DNA-protein interactions which makes use of an extremely bright luciferase as a tag for the binding protein, along with a long-wavelength fluorophore conjugated to the nucleic acid. The resulting assay is high throughput, sensitive, does not require protein purification, and even allows for quantitative characterization of these interactions within the biologically relevant context of whole cells.

  12. Final Report Nucleic Acid System - PCR, Multiplex Assays and Sample Preparation Project

    SciTech Connect

    Koopman, R.P.; Langlois, R.G.; Nasarabadi, S.; Benett, W.J.; Richards, J.B.; Hadley, D.R.; Miles, R.R.; Brown, S.B.; Stratton, P.L.; Milanovich, F.P.

    2001-04-20

    The objective of this project was to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction). This entailed not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This project had two principal deliverables: (1) design, construct, test and deliver a 24 chamber, multiplex capable suitcase sized PCR instrument, and (2) develop and reduce to practice a multiplex assay for the detection of PCR product by flow cytometry. In addition, significant resources were allocated to test and evaluation of the Hand-held Advanced Nucleic Acid Analyzer (HANAA). This project helps provide the signature and intelligence gathering community the ability to perform, on-site or remote, rapid analysis of environmental or like samples for the presence of a suite of biological warfare pathogens.

  13. Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus

    PubMed Central

    Abd El Galil, Khaled H.; El Sokkary, M. A.; Kheira, S. M.; Salazar, Andre M.; Yates, Marylynn V.; Chen, Wilfred; Mulchandani, Ashok

    2005-01-01

    A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water. PMID:16269748

  14. An electrochemical clamp assay for direct, rapid analysis of circulating nucleic acids in serum

    NASA Astrophysics Data System (ADS)

    Das, Jagotamoy; Ivanov, Ivaylo; Montermini, Laura; Rak, Janusz; Sargent, Edward H.; Kelley, Shana O.

    2015-07-01

    The analysis of cell-free nucleic acids (cfNAs), which are present at significant levels in the blood of cancer patients, can reveal the mutational spectrum of a tumour without the need for invasive sampling of the tissue. However, this requires differentiation between the nucleic acids that originate from healthy cells and the mutated sequences shed by tumour cells. Here we report an electrochemical clamp assay that directly detects mutated sequences in patient serum. This is the first successful detection of cfNAs without the need for enzymatic amplification, a step that normally requires extensive sample processing and is prone to interference. The new chip-based assay reads out the presence of mutations within 15 minutes using a collection of oligonucleotides that sequester closely related sequences in solution, and thus allow only the mutated sequence to bind to a chip-based sensor. We demonstrate excellent levels of sensitivity and specificity and show that the clamp assay accurately detects mutated sequences in a collection of samples taken from lung cancer and melanoma patients.

  15. Interaction of nucleic acids with Coomassie Blue G-250 in the Bradford assay.

    PubMed

    Wenrich, Broc R; Trumbo, Toni A

    2012-09-15

    The Bradford assay has been used reliably for decades to quantify protein in solution. The analyte is incubated in acidic solution of Coomassie Blue G-250 dye, during which reversible ionic and nonionic binding interactions form. Bradford assay color yields were determined for salmon, bovine, shrimp, and kiwi fruit genomic DNA; baker's yeast RNA; bovine serum albumin (BSA); and hen egg lysozyme. Pure DNA and RNA bound the dye, with color yields of 0.0017 mg⁻¹ cm⁻¹ and 0.0018 mg⁻¹ cm⁻¹, respectively. The nucleic acid-Coomassie Blue response was significant, at roughly 9% of that for BSA and 18% of that for lysozyme. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Final Report Nucleic Acid System - Hybrid PCR and Multiplex Assay Project Phase 2

    SciTech Connect

    Koopman, R P; Langlois, R G; Nasarabadi, S; Benett, W J; Colston, B W; Johnson, D C; Brown, S B; Stratton, P L; Milanovich, F P

    2002-04-17

    This report covers phase 2 (year 2) of the Nucleic Acid System--Hybrid PCR and Multiplex Assay project. The objective of the project is to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction) in a multiplex mode using flow cytometry. The Hybrid instrument consists of a flow-through PCR module capable of handling a multiplexed PCR assay, a hybridizing module capable of hybridizing multiplexed PCR amplicons and beads, and a flow cytometer module for bead-based identification, all controlled by a single computer. Multiplex immunoassay using bead-based Luminex flow cytometry is available, allowing rapid screening for many agents. PCR is highly specific and complements and verifies immunoassay. It can also be multiplexed and detection provided using the bead-based Luminex flow cytometer. This approach allows full access to the speed and 100-fold multiplex capability of flow cytometry for rapid screening as well as the accuracy and specificity of PCR. This project has two principal activities: (1) Design, build and test a prototype hybrid PCR/flow cytometer with the basic capabilities for rapid, broad spectrum detection and identification, and (2) Develop and evaluate multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products. This project requires not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This involves development and evaluation of multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products.

  17. Paper-based fluorescence resonance energy transfer assay for directly detecting nucleic acids and proteins.

    PubMed

    Li, Hua; Fang, Xueen; Cao, Hongmei; Kong, Jilie

    2016-06-15

    Paper-based fluorescence resonance energy transfer assay (FRET) is gaining great interest in detecting macro-biological molecule. It is difficult to achieve conveniently and fast detection for macro-biological molecule. Herein, a graphene oxide (GO)-based paper chip (glass fiber) integrated with fluorescence labeled single-stranded DNA (ssDNA) for fast, inexpensive and direct detection of biological macromolecules (proteins and nucleic acids) has been developed. In this paper, we employed the Cy3/FAM-labeled ssDNA as the reporter and the GO as quencher and the original glass fiber paper as data acquisition substrates. The chip which was designed and fabricated by a cutting machine is a miniature biosensor that monitors fluorescence recovery from resonance energy transfer. The hybridization assays and fluorescence detection were all simplified, and the surface of the chip did not require immobilization or washing. A Nikon Eclipse was employed as excited resource and a commercial digital camera was employed for capturing digital images. This paper-based microfluidics chip has been applied in the detection of proteins and nucleic acids. The biosensing capability meets many potential requirements for disease diagnosis and biological analysis.

  18. Immunochemical Assays and Nucleic-Acid Detection Techniques for Clinical Diagnosis of Prostate Cancer

    PubMed Central

    Kanyong, Prosper; Rawlinson, Sean; Davis, James

    2016-01-01

    Prostate cancer (PCa) is a significant cause of morbidity and mortality and the most common cancer in men in Europe, North America, and some parts of Africa. The established methods for detecting PCa are normally based on tests using Prostate Specific Antigen (PSA) in blood, Prostate cancer antigen 3 (PCA3) in urine and tissue Alpha-methylacyl-CoA racemase (AMACR) as tumour markers in patient samples. Prior to the introduction of PSA in clinics, prostatic acid phosphatase (PAP) was the most widely used biomarker. An early diagnosis of PCa through the detection of these biomarkers requires the availability of simple, reliable, cost-effective and robust techniques. Immunoassays and nucleic acid detection techniques have experienced unprecedented growth in recent years and seem to be the most promising analytical tools. This growth has been driven in part by the surge in demand for near-patient-testing systems in clinical diagnosis. This article reviews immunochemical assays, and nucleic-acid detection techniques that have been used to clinically diagnose PCa. PMID:26958088

  19. Immunochemical Assays and Nucleic-Acid Detection Techniques for Clinical Diagnosis of Prostate Cancer.

    PubMed

    Kanyong, Prosper; Rawlinson, Sean; Davis, James

    2016-01-01

    Prostate cancer (PCa) is a significant cause of morbidity and mortality and the most common cancer in men in Europe, North America, and some parts of Africa. The established methods for detecting PCa are normally based on tests using Prostate Specific Antigen (PSA) in blood, Prostate cancer antigen 3 (PCA3) in urine and tissue Alpha-methylacyl-CoA racemase (AMACR) as tumour markers in patient samples. Prior to the introduction of PSA in clinics, prostatic acid phosphatase (PAP) was the most widely used biomarker. An early diagnosis of PCa through the detection of these biomarkers requires the availability of simple, reliable, cost-effective and robust techniques. Immunoassays and nucleic acid detection techniques have experienced unprecedented growth in recent years and seem to be the most promising analytical tools. This growth has been driven in part by the surge in demand for near-patient-testing systems in clinical diagnosis. This article reviews immunochemical assays, and nucleic-acid detection techniques that have been used to clinically diagnose PCa.

  20. Quantitative rRNA-targeted solution-based hybridization assay using peptide nucleic acid molecular beacons.

    PubMed

    Li, Xu; Morgenroth, Eberhard; Raskin, Lutgarde

    2008-12-01

    The potential of a solution-based hybridization assay using peptide nucleic acid (PNA) molecular beacon (MB) probes to quantify 16S rRNA of specific populations in RNA extracts of environmental samples was evaluated by designing PNA MB probes for the genera Dechloromonas and Dechlorosoma. In a kinetic study with 16S rRNA from pure cultures, the hybridization of PNA MB to target 16S rRNA exhibited a higher final hybridization signal and a lower apparent rate constant than the hybridizations to nontarget 16S rRNAs. A concentration of 10 mM NaCl in the hybridization buffer was found to be optimal for maximizing the difference between final hybridization signals from target and nontarget 16S rRNAs. Hybridization temperatures and formamide concentrations in hybridization buffers were optimized to minimize signals from hybridizations of PNA MB to nontarget 16S rRNAs. The detection limit of the PNA MB hybridization assay was determined to be 1.6 nM of 16S rRNA. To establish proof for the application of PNA MB hybridization assays in complex systems, target 16S rRNA from Dechlorosoma suillum was spiked at different levels to RNA isolated from an environmental (bioreactor) sample, and the PNA MB assay enabled effective quantification of the D. suillum RNA in this complex mixture. For another environmental sample, the quantitative results from the PNA MB hybridization assay were compared with those from clone libraries.

  1. Intraoperative diagnosis of sentinel lymph node metastases in breast cancer treatment with one-step nucleic acid amplification assay (OSNA)

    PubMed Central

    Szychta, Paweł; Westfal, Bogusław; Maciejczyk, Rafał; Smolarz, Beata; Romanowicz, Hanna; Krawczyk, Tomasz

    2016-01-01

    Introduction The aim of the study was to evaluate the clinical usefulness of a one-step nucleic acid amplification assay (OSNA) for intraoperative detection of metastases to sentinel lymph nodes (SLNs) in comparison to examination of frozen sections, and to summarize the results of previous studies. Material and methods We enrolled 98 patients aged 58.13 ±10.74 years treated surgically for breast cancer, and 99 biopsies of SLNs were followed by analysis of 105 SLNs. The central 1 mm slice of SLN was used for examination of frozen sections, whereas 2 outer slices of SLNs were analyzed intraoperatively with OSNA. Detection of isolated tumor cells (ITC), micrometastases or macrometastases with OSNA extended surgery to axillary lymph node dissection. Congruency of results was assessed between OSNA and examination of frozen sections. Results One-step nucleic acid amplification assay detected metastases in 29/105 SLNs in surgery of 27/99 breasts, including ITC in 3/29 SLNs, micrometastases in 12/29 and macrometastases in 14/29. One-step nucleic acid amplification assay detected significantly more metastases to SLNs than examination of frozen sections (p < 0.0001). All 8 inconsistent results were positive in OSNA and negative in examination of frozen sections; ITC were identified in 2/8 SLNs and micrometastases in 6/8 SLNs. Sensitivity for OSNA was calculated as 100%, specificity as 90.47%, and κ was 79.16%. Conclusions One-step nucleic acid amplification assay analysis allows rapid and quantitative detection of mRNA CK19 with high specificity and a low rate of false positives. One-step nucleic acid amplification assay is a reliable tool for intraoperative diagnosis of whole SLNs during surgery of breast cancer. One-step nucleic acid amplification assay minimizes the need for secondary surgery and avoids delays in the adjuvant treatment. PMID:27904514

  2. Evaluation of three-dimensional microchannel glass biochips for multiplexed nucleic acid fluorescence hybridization assays.

    PubMed

    Benoit, V; Steel, A; Torres, M; Yu, Y Y; Yang, H; Cooper, J

    2001-06-01

    Three-dimensional, flow-through microchannel glass substrates have a potential for enhanced performance, including increased sensitivity and dynamic range, over traditional planar substrates used in medium-density microarray platforms. This paper presents a methodology for the implementation of multiplexed nucleic acid hybridization fluorescence assays on microchannel glass substrates. Fluorescence detection was achieved, in a first instance, using conventional low-magnification microscope objective lenses, as imaging optics whose depth-of-field characteristics match the thickness of the microchannel glass chip. The optical properties of microchannel glass were shown, through experimental results and simulations, to be compatible with the quantitative detection of heterogeneous hybridization events taking place along the microchannel sidewalls, with detection limits for oligonucleotide targets in the low-attomole range.

  3. Comparison of manual and automated nucleic acid isolation methods for HBV-DNA and HCV-RNA assays.

    PubMed

    Yagmur, Gulhan; Altun, Hatice Uludag; Gökahmetoglu, Selma; Basok, Ela

    2015-09-01

    In the diagnosis and monitoring of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, it is important to use methods that can provide rapid and reliable results. The present study aimed to compare the automated and manual extraction methods during the nucleic acid isolation phase for HBV-DNA and HCV-RNA assays. The study included 93 serum samples, 49 of which were for the HBV-DNA assay and 44 for the HCV-RNA assay. DNA and RNA isolation from the samples was performed manually with a "QIAmpMin Elute Kit" (Qiagen, Germany) and the automated isolation system, NucliSens easyMAG (BioMérieux, France). All the extraction products were amplified using the iCycler device (Bio-Rad, USA). With both methods, compliance was found in 21 (42.8%) samples in the HBV-DNA assay; nine (18.3%) samples had a higher amount of viral nucleic acid with the manual method, whereas 19 samples (38.7%) were found to have a higher amount of nucleic acid with the automated system. For the HCV-RNA assay, total compliance was found in 31 (70.4%) samples; 12 (27.2%) samples had a higher amount of viral nucleic acid with the manual method whereas one sample (2.2%) was found to have a higher amount of nucleic acid with the automated system. It was concluded that the NucliSens easyMAG automated isolation system can be used with confidence for nucleic acid extraction due to its higher sensitivity, providing results in a shorter time, and assured standardization.

  4. Application of Peptide Nucleic Acid-based Assays Toward Detection of Somatic Mosaicism

    PubMed Central

    Hong, Christopher S; Yang, Chunzhang; Zhuang, Zhengping

    2016-01-01

    Peptide nucleic acids (PNAs) are synthetic oligonucleotides with many applications. Compared with DNA, PNAs bind their complementary DNA strand with higher specificity and strength, an attribute that can make it an effective polymerase chain reaction clamp. A growing body of work has demonstrated the utility of PNAs in detecting low levels of mutant DNA, particularly in the detection of circulating mutated tumor cells in the peripheral blood. The PNA-based assay has greater sensitivity than direct sequencing and is significantly more affordable and rapid than next-generation deep sequencing. We have previously demonstrated that PNAs can successfully detect somatic mosaicism in patients with suspected disease phenotypes. In this report, we detail our methodology behind PNA design and application. We describe our protocol for optimizing the PNA for sequencing use and for determining the sensitivity of the PNA-based assay. Lastly, we discuss the potential applications of our assay for future laboratory and clinical purposes and highlight the role of PNAs in the detection of somatic mosaicism. PMID:27115839

  5. Medical Devices; Immunology and Microbiology Devices; Classification of Gastrointestinal Microorganism Multiplex Nucleic Acid-Based Assay. Final order.

    PubMed

    2015-11-02

    The Food and Drug Administration (FDA) is classifying a gastrointestinal microorganism multiplex nucleic acid-based assay into class II (special controls). The Agency is classifying the device into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of the device.

  6. 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.

  7. Nucleic acid detection compositions

    DOEpatents

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

    2008-08-05

    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.

  8. 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.

  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. 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

  11. Systematic Evaluation of Different Nucleic Acid Amplification Assays for Cytomegalovirus Detection: Feasibility of Blood Donor Screening.

    PubMed

    Vollmer, T; Knabbe, C; Dreier, J

    2015-10-01

    Acute primary cytomegalovirus (CMV) infections, which commonly occur asymptomatically among blood donors, represent a significant risk for serious morbidity in immunocompromised patients (a major group of transfusion recipients). We implemented a routine CMV pool screening procedure for plasma for the identification of CMV DNA-positive donors, and we evaluated the sensitivities and performance of different CMV DNA amplification systems. Minipools (MPs) of samples from 18,405 individual donors (54,451 donations) were screened for CMV DNA using the RealStar CMV PCR assay (Altona Diagnostic Technologies), with a minimum detection limit of 11.14 IU/ml. DNA was extracted with a high-volume protocol (4.8 ml, Chemagic Viral 5K kit; PerkinElmer) for blood donor pool screening (MP-nucleic acid testing [NAT]) and with the Nuclisens easyMAG system (0.5 ml; bioMérieux) for individual donation (ID)-NAT. In total, six CMV DNA-positive donors (0.03%) were identified by routine CMV screening, with DNA concentrations ranging from 4.35 × 10(2) to 4.30 × 10(3) IU/ml. Five donors already showed seroconversion and detectable IgA, IgM, and/or IgG antibody titers (IgA(+)/IgM(+)/IgG(-) or IgA(+)/IgM(+)/IgG(+)), and one donor showed no CMV-specific antibodies. Comparison of three commercial assays, i.e., the RealStar CMV PCR kit, the Sentosa SA CMV quantitative PCR kit (Vela Diagnostics), and the CMV R-gene PCR kit (bioMérieux), for MP-NAT and ID-NAT showed comparably good analytical sensitivities, ranging from 10.23 to 11.14 IU/ml (MP-NAT) or from 37.66 to 57.94 IU/ml (ID-NAT). The clinical relevance of transfusion-associated CMV infections requires further investigation, and the evaluated methods present powerful basic tools providing sensitive possibilities for viral testing. The application of CMV MP-NAT facilitated the identification of one donor with a window-phase donation during acute primary CMV infection.

  12. Plasmon-enhanced fluorescence imaging with silicon-based silver chips for protein and nucleic acid assay.

    PubMed

    Yuan, Bing; Jiang, Xiangxu; Yao, Chu; Bao, Meimei; Liu, Jiaojiao; Dou, Yujiang; Xu, Yinze; He, Yao; Yang, Kai; Ma, Yuqiang

    2017-02-22

    Metal-enhanced fluorescence shows great potential for improving the sensitivity of fluoroscopy, which has been widely used in protein and nucleic acid detection for biosensor and bioassay applications. In comparison with the traditional glass-supported metal nanoparticles (MNPs), the introduction of a silicon substrate has been shown to provide an increased surface-enhanced Raman scattering (SERS) effect due to the coupling between the MNPs and the semiconducting silicon substrate. In this work, we further study the fluorescence-enhanced effect of the silicon-supported silver-island (Ag@Si) plasmonic chips. In particular, we investigate their practical application of improving the traditional immunoassay such as the biotin-streptavidin-based protein assay and the protein-/nucleic acid-labeled cell and tissue samples. The protein assay shows a wavelength-dependent enhancement effect of the Ag@Si chip, with an enhancement factor ranging from 1.2 (at 532 nm) to 57.3 (at 800 nm). Moreover, for the protein- and nucleic acid-labeled cell and tissue samples, the Ag@Si chip provides a fluorescence enhancement factor of 3.0-4.1 (at 800 nm) and a significant improvement in the signal/background ratio for the microscopy images. Such a ready accommodation of the fluorescence-enhanced effect for the immunoassay samples with simple manipulations indicates broad potential for applications of the Ag@Si chip not only in biological studies but also in the clinical field.

  13. Method for isolating nucleic acids

    SciTech Connect

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

    2015-09-29

    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 for a wide variety of applications including, sequencing or species population analysis.

  14. Nucleic acid detection methods

    DOEpatents

    Smith, C.L.; Yaar, R.; Szafranski, P.; Cantor, C.R.

    1998-05-19

    The invention relates to methods for rapidly determining the sequence and/or length a target sequence. The target sequence may be a series of known or unknown repeat sequences which are hybridized to an array of probes. The hybridized array is digested with a single-strand nuclease and free 3{prime}-hydroxyl groups extended with a nucleic acid polymerase. Nuclease cleaved heteroduplexes can be easily distinguish from nuclease uncleaved heteroduplexes by differential labeling. Probes and target can be differentially labeled with detectable labels. Matched target can be detected by cleaving resulting loops from the hybridized target and creating free 3-hydroxyl groups. These groups are recognized and extended by polymerases added into the reaction system which also adds or releases one label into solution. Analysis of the resulting products using either solid phase or solution. These methods can be used to detect characteristic nucleic acid sequences, to determine target sequence and to screen for genetic defects and disorders. Assays can be conducted on solid surfaces allowing for multiple reactions to be conducted in parallel and, if desired, automated. 18 figs.

  15. Nucleic Acid Detection Methods

    DOEpatents

    Smith, Cassandra L.; Yaar, Ron; Szafranski, Przemyslaw; Cantor, Charles R.

    1998-05-19

    The invention relates to methods for rapidly determining the sequence and/or length a target sequence. The target sequence may be a series of known or unknown repeat sequences which are hybridized to an array of probes. The hybridized array is digested with a single-strand nuclease and free 3'-hydroxyl groups extended with a nucleic acid polymerase. Nuclease cleaved heteroduplexes can be easily distinguish from nuclease uncleaved heteroduplexes by differential labeling. Probes and target can be differentially labeled with detectable labels. Matched target can be detected by cleaving resulting loops from the hybridized target and creating free 3-hydroxyl groups. These groups are recognized and extended by polymerases added into the reaction system which also adds or releases one label into solution. Analysis of the resulting products using either solid phase or solution. These methods can be used to detect characteristic nucleic acid sequences, to determine target sequence and to screen for genetic defects and disorders. Assays can be conducted on solid surfaces allowing for multiple reactions to be conducted in parallel and, if desired, automated.

  16. Instrument-free nucleic acid amplification assays for global health settings

    PubMed Central

    LaBarre, Paul; Boyle, David; Hawkins, Kenneth; Weigl, Bernhard

    2014-01-01

    Many infectious diseases that affect global health are most accurately diagnosed through nucleic acid amplification and detection. However, existing nucleic acid amplification tests are too expensive and complex for most low-resource settings. The small numbers of centralized laboratories that exist in developing countries tend to be in urban areas and primarily cater to the affluent. In contrast, rural area health care facilities commonly have only basic equipment and health workers have limited training and little ability to maintain equipment and handle reagents.1 Reliable electric power is a common infrastructure shortfall. In this paper, we discuss a practical approach to the design and development of non-instrumented molecular diagnostic tests that exploit the benefits of isothermal amplification strategies. We identify modular instrument-free technologies for sample collection, sample preparation, amplification, heating, and detection. By appropriately selecting and integrating these instrument-free modules, we envision development of an easy to use, infrastructure independent diagnostic test that will enable increased use of highly accurate molecular diagnostics at the point of care in low-resource settings. PMID:25089171

  17. Instrument-free nucleic acid amplification assays for global health settings

    NASA Astrophysics Data System (ADS)

    LaBarre, Paul; Boyle, David; Hawkins, Kenneth; Weigl, Bernhard

    2011-06-01

    Many infectious diseases that affect global health are most accurately diagnosed through nucleic acid amplification and detection. However, existing nucleic acid amplification tests are too expensive and complex for most low-resource settings. The small numbers of centralized laboratories that exist in developing countries tend to be in urban areas and primarily cater to the affluent. In contrast, rural area health care facilities commonly have only basic equipment and health workers have limited training and little ability to maintain equipment and handle reagents.1 Reliable electric power is a common infrastructure shortfall. In this paper, we discuss a practical approach to the design and development of non-instrumented molecular diagnostic tests that exploit the benefits of isothermal amplification strategies. We identify modular instrument-free technologies for sample collection, sample preparation, amplification, heating, and detection. By appropriately selecting and integrating these instrument-free modules, we envision development of an easy to use, infrastructure independent diagnostic test that will enable increased use of highly accurate molecular diagnostics at the point of care in low-resource settings.

  18. Organ donor screening using parallel nucleic acid testing allows assessment of transmission risk and assay results in real time.

    PubMed

    Baleriola, C; Tu, E; Johal, H; Gillis, J; Ison, M G; Law, M; Coghlan, P; Rawlinson, W D

    2012-06-01

    Expansion of the donor pool may lead to utilization of donors with risk factors for viral infections. Donor laboratory screening relies on serological and nucleic acid testing (NAT). The increased sensitivity of NAT in low prevalence populations may result in false-positive results (FPR) and may cause unnecessary discard of organs.We developed a screening algorithm to deal, in real time, with potential FPR. Three NAT assays: COBAS AmpliScreen assay (CAS), AmpliPrep Total Nucleic Acid Isolation/CAS, and AmpliPrep/TaqMan assays, were validated and used in parallel for prospective screening of increased-risk donors (IRD), and the probability of FPR was calculated. The lower limit of detection of this algorithm was 9.79, 21.02, and 4.31 IU/mL for human immunodeficiency virus-1, hepatitis C virus, and hepatitis B virus, respectively, with an average turn-around-time of 7.67 h from sample receipt to result reporting. The probability that a donor is potentially infectious with two NAT concordant results was >90%. NAT screening of 35 IRD within 18 months resulted in transplantation of 102 additional organs that without screening would either not be used or used with restrictions in Australia. Using a parallel testing algorithm, real-time confirmation of seropositive donors allows use of organs from IRD and safer expansion of the donor pool.

  19. 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.

  20. Fabrication of Uniform DNA-Conjugated Hydrogel Microparticles via Replica Molding for Facile Nucleic Acid Hybridization Assays

    PubMed Central

    Lewis, Christina L.; Choi, Chang-Hyung; Lin, Yan; Lee, Chang-Soo; Yi, Hyunmin

    2010-01-01

    We identify and investigate several critical parameters in the fabrication of single-stranded DNA conjugated poly(ethylene glycol) (PEG) microparticles based on replica molding (RM) for highly uniform and robust nucleic acid hybridization assays. The effects of PEG-diacrylate, probe DNA, and photoinitiator concentrations on the overall fluorescence and target DNA penetration depth upon hybridization are examined. Fluorescence and confocal microscopy results illustrate high conjugation capacity of probe and target DNA, femtomole sensitivity, and sequence specificity. Combined these findings demonstrate a significant step toward simple, robust, and scalable procedures to manufacture highly uniform and high capacity hybridization assay particles in a well-controlled manner by exploiting many advantages that the batch processing-based RM technique offers. We envision that the results presented here may be readily applied to rapid and high throughput hybridization assays for a wide variety of applications in bioprocess monitoring, food safety, and biological threat detection. PMID:20527819

  1. Fabrication of uniform DNA-conjugated hydrogel microparticles via replica molding for facile nucleic acid hybridization assays.

    PubMed

    Lewis, Christina L; Choi, Chang-Hyung; Lin, Yan; Lee, Chang-Soo; Yi, Hyunmin

    2010-07-01

    We identify and investigate several critical parameters in the fabrication of single-stranded DNA conjugated poly(ethylene glycol) (PEG) microparticles based on replica molding (RM) for highly uniform and robust nucleic acid hybridization assays. The effects of PEG-diacrylate, probe DNA, and photoinitiator concentrations on the overall fluorescence and target DNA penetration depth upon hybridization are examined. Fluorescence and confocal microscopy results illustrate high conjugation capacity of the probe and target DNA, femtomole sensitivity, and sequence specificity. Combined, these findings demonstrate a significant step toward simple, robust, and scalable procedures to manufacture highly uniform and high-capacity hybridization assay particles in a well-controlled manner by exploiting many advantages that the batch processing-based RM technique offers. We envision that the results presented here may be readily applied to rapid and high-throughput hybridization assays for a wide variety of applications in bioprocess monitoring, food safety, and biological threat detection.

  2. Comparison of nucleic acid amplification assays with BD affirm VPIII for diagnosis of vaginitis in symptomatic women.

    PubMed

    Cartwright, Charles P; Lembke, Bryndon D; Ramachandran, Kalpana; Body, Barbara A; Nye, Melinda B; Rivers, Charles A; Schwebke, Jane R

    2013-11-01

    A commercially available, nonamplified, nucleic acid probe-based test system (BD Affirm VPIII) was compared with nucleic acid amplification (NAA)-based assays for determining the etiology of vaginitis in a cohort of 323 symptomatic women. First, a semiquantitative, multiplexed PCR assay (BV-PCR) and the Affirm VPIII Gardnerellavaginalis test were compared with a unified bacterial-vaginosis (BV) reference standard incorporating both Nugent Gram stain scores and Amsel clinical criteria. In the evaluable population of 305 patients, BV-PCR was 96.9% (191/197) sensitive and 92.6% specific (100/108) for BV, while Affirm VPIII was 90.1% sensitive (179/197) and 67.6% specific (73/108). Second, a multiplexed PCR assay detecting Candida albicans and Candida glabrata (CAN-PCR) was compared with the Affirm VPIII Candida test using a reference standard for vulvovaginal candidiasis (VVC) of yeast culture plus exclusion of alternate vaginitis etiologies. In the population evaluated (n = 102), CAN-PCR was 97.7% sensitive (42/43) and 93.2% specific (55/59) and Affirm VP III was 58.1% sensitive (25/43) and 100% specific (59/59) for VVC. Finally, the results of a commercial NAA test (GenProbe Aptima Trichomonas vaginalis assay; ATV) for T. vaginalis were compared with the Affirm VPIII Trichomonas vaginalis test. In the absence of an independent reference standard for trichomonal vaginitis (TV), a positive result in either assay was deemed to represent true infection. In the evaluable cohort of 388 patients, the sensitivity of ATV was 98.1% (53/54) versus 46.3% (25/54) for Affirm VPIII. The diagnostic accuracy of the combined NAA-based test construct was approximately 20 to 25% higher than that of the Affirm VPIII when modeled in populations with various prevalences of infectious vaginitis.

  3. Comparison of Nucleic Acid Amplification Assays with BD Affirm VPIII for Diagnosis of Vaginitis in Symptomatic Women

    PubMed Central

    Lembke, Bryndon D.; Ramachandran, Kalpana; Body, Barbara A.; Nye, Melinda B.; Rivers, Charles A.; Schwebke, Jane R.

    2013-01-01

    A commercially available, nonamplified, nucleic acid probe-based test system (BD Affirm VPIII) was compared with nucleic acid amplification (NAA)-based assays for determining the etiology of vaginitis in a cohort of 323 symptomatic women. First, a semiquantitative, multiplexed PCR assay (BV-PCR) and the Affirm VPIII Gardnerellavaginalis test were compared with a unified bacterial-vaginosis (BV) reference standard incorporating both Nugent Gram stain scores and Amsel clinical criteria. In the evaluable population of 305 patients, BV-PCR was 96.9% (191/197) sensitive and 92.6% specific (100/108) for BV, while Affirm VPIII was 90.1% sensitive (179/197) and 67.6% specific (73/108). Second, a multiplexed PCR assay detecting Candida albicans and Candida glabrata (CAN-PCR) was compared with the Affirm VPIII Candida test using a reference standard for vulvovaginal candidiasis (VVC) of yeast culture plus exclusion of alternate vaginitis etiologies. In the population evaluated (n = 102), CAN-PCR was 97.7% sensitive (42/43) and 93.2% specific (55/59) and Affirm VP III was 58.1% sensitive (25/43) and 100% specific (59/59) for VVC. Finally, the results of a commercial NAA test (GenProbe Aptima Trichomonas vaginalis assay; ATV) for T. vaginalis were compared with the Affirm VPIII Trichomonas vaginalis test. In the absence of an independent reference standard for trichomonal vaginitis (TV), a positive result in either assay was deemed to represent true infection. In the evaluable cohort of 388 patients, the sensitivity of ATV was 98.1% (53/54) versus 46.3% (25/54) for Affirm VPIII. The diagnostic accuracy of the combined NAA-based test construct was approximately 20 to 25% higher than that of the Affirm VPIII when modeled in populations with various prevalences of infectious vaginitis. PMID:23985917

  4. Nucleic acid sandwich hybridization assay with quantum dot-induced fluorescence resonance energy transfer for pathogen detection.

    PubMed

    Chou, Cheng-Chung; Huang, Yi-Han

    2012-12-04

    This paper reports a nucleic acid sandwich hybridization assay with a quantum dot (QD)-induced fluorescence resonance energy transfer (FRET) reporter system. Two label-free hemagglutinin H5 sequences (60-mer DNA and 630-nt cDNA fragment) of avian influenza viruses were used as the targets in this work. Two oligonucleotides (16 mers and 18 mers) that specifically recognize two separate but neighboring regions of the H5 sequences were served as the capturing and reporter probes, respectively. The capturing probe was conjugated to QD655 (donor) in a molar ratio of 10:1 (probe-to-QD), and the reporter probe was labeled with Alexa Fluor 660 dye (acceptor) during synthesis. The sandwich hybridization assay was done in a 20 μL transparent, adhesive frame-confined microchamber on a disposable, temperature-adjustable indium tin oxide (ITO) glass slide. The FRET signal in response to the sandwich hybridization was monitored by a homemade optical sensor comprising a single 400 nm UV light-emitting diode (LED), optical fibers, and a miniature 16-bit spectrophotometer. The target with a concentration ranging from 0.5 nM to 1 μM was successfully correlated with both QD emission decrease at 653 nm and dye emission increase at 690 nm. To sum up, this work is beneficial for developing a portable QD-based nucleic acid sensor for on-site pathogen detection.

  5. Nucleic Acid Sandwich Hybridization Assay with Quantum Dot-Induced Fluorescence Resonance Energy Transfer for Pathogen Detection

    PubMed Central

    Chou, Cheng-Chung; Huang, Yi-Han

    2012-01-01

    This paper reports a nucleic acid sandwich hybridization assay with a quantum dot (QD)-induced fluorescence resonance energy transfer (FRET) reporter system. Two label-free hemagglutinin H5 sequences (60-mer DNA and 630-nt cDNA fragment) of avian influenza viruses were used as the targets in this work. Two oligonucleotides (16 mers and 18 mers) that specifically recognize two separate but neighboring regions of the H5 sequences were served as the capturing and reporter probes, respectively. The capturing probe was conjugated to QD655 (donor) in a molar ratio of 10:1 (probe-to-QD), and the reporter probe was labeled with Alexa Fluor 660 dye (acceptor) during synthesis. The sandwich hybridization assay was done in a 20 μL transparent, adhesive frame-confined microchamber on a disposable, temperature-adjustable indium tin oxide (ITO) glass slide. The FRET signal in response to the sandwich hybridization was monitored by a homemade optical sensor comprising a single 400 nm UV light-emitting diode (LED), optical fibers, and a miniature 16-bit spectrophotometer. The target with a concentration ranging from 0.5 nM to 1 μM was successfully correlated with both QD emission decrease at 653 nm and dye emission increase at 690 nm. To sum up, this work is beneficial for developing a portable QD-based nucleic acid sensor for on-site pathogen detection. PMID:23211753

  6. Isothermal Amplification of Nucleic Acids.

    PubMed

    Zhao, Yongxi; Chen, Feng; Li, Qian; Wang, Lihua; Fan, Chunhai

    2015-11-25

    Isothermal amplification of nucleic acids is a simple process that rapidly and efficiently accumulates nucleic acid sequences at constant temperature. Since the early 1990s, various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). These isothermal amplification methods have been used for biosensing targets such as DNA, RNA, cells, proteins, small molecules, and ions. The applications of these techniques for in situ or intracellular bioimaging and sequencing have been amply demonstrated. Amplicons produced by isothermal amplification methods have also been utilized to construct versatile nucleic acid nanomaterials for promising applications in biomedicine, bioimaging, and biosensing. The integration of isothermal amplification into microsystems or portable devices improves nucleic acid-based on-site assays and confers high sensitivity. Single-cell and single-molecule analyses have also been implemented based on integrated microfluidic systems. In this review, we provide a comprehensive overview of the isothermal amplification of nucleic acids encompassing work published in the past two decades. First, different isothermal amplification techniques are classified into three types based on reaction kinetics. Then, we summarize the applications of isothermal amplification in bioanalysis, diagnostics, nanotechnology, materials science, and device integration. Finally, several challenges and perspectives in the field are discussed.

  7. Hybridization assay of insect antifreezing protein gene by novel multilayered porous silicon nucleic acid biosensor.

    PubMed

    Lv, Xiaoyi; Chen, Liangliang; Zhang, Hongyan; Mo, Jiaqing; Zhong, Furu; Lv, Changwu; Ma, Ji; Jia, Zhenhong

    2013-01-15

    A fabrication of a novel simple porous silicon polybasic photonic crystal with symmetrical structure has been reported as a nucleic acid biosensor for detecting antifreeze protein gene in insects (Microdera puntipennis dzhungarica), which would be helpful in the development of some new transgenic plants with tolerance of freezing stress. Compared to various porous silicon-based photonic configurations, porous silicon polytype layered structure is quite easy to prepare and shows more stability; moreover, polybasic photonic crystals with symmetrical structure exhibit interesting optical properties with a sharp resonance in the reflectance spectrum, giving a higher Q factor which causes higher sensitivity for sensing performance. In this experiment, DNA oligonucleotides were immobilized into the porous silicon pores using a standard crosslink chemistry method. The porous silicon polybasic symmetrical structure sensor possesses high specificity in performing controlled experiments with non-complementary DNA. The detection limit was found to be 21.3nM for DNA oligonucleotides. The fabricated multilayered porous silicon-based DNA biosensor has potential commercial applications in clinical chemistry for determination of an antifreeze protein gene or other genes.

  8. Analytical Performance and Clinical Utility of a Nucleic Acid Sequence-Based Amplification Assay for Detection of Cytomegalovirus Infection

    PubMed Central

    Witt, Donald J.; Kemper, M.; Stead, Andrew; Sillekens, P.; Ginocchio, Christine C.; Espy, Mark J.; Paya, Carlos V.; Smith, Thomas F.; Roeles, Frits; Caliendo, Angela M.

    2000-01-01

    A nucleic acid sequence-based amplification (NASBA) assay for qualitative detection of human cytomegalovirus (CMV) pp67 mRNA was evaluated in a multicenter study. Negative results were obtained for all specimens from 50 CMV-seronegative and 50 CMV-seropositive low-risk whole-blood donors. No interference with CMV mRNA amplification was observed in the testing of 288 specimens containing various potential interfering substances, nonspecifically reacting substances (including mRNA from other herpesviruses), and three anticoagulants. A total of 95% (50 of 51) of CMV-positive (cell culture- and antigenemia immunofluorescence [AG-IFA]-positive) clinical specimens were positive by the NASBA assay. Results from different operators over multiple testing days were consistent for each of four panel members containing different concentrations of CMV mRNA, indicating the reproducibility of the assay. The estimated 95% reliable upper detection limit of the assay was 600 mRNA copies; the lower limit of detection was less than 25 mRNA copies. The clinical utility of the assay was evaluated with longitudinally collected specimens from solid-organ transplant patients (n = 21). A total of 98% (81 of 83) of the specimens from CMV-negative patients were negative by the NASBA assay, while 90% (10 of 11) of patient specimens that were positive by cell culture or AG-IFA were positive by the NASBA assay. Positive NASBA assay results were obtained earlier than AG-IFA or cell culture results for 55% of the patients and at the same time for the remainder of the patients (45%). The overall agreement between the NASBA assay and current reference tests was 86% when active CMV infection was present. These studies indicate that the CMV pp67 mRNA NASBA assay has reproducible and sensitive performance characteristics that should enable more rapid diagnosis of CMV infection. PMID:11060058

  9. Calorimetry of Nucleic Acids.

    PubMed

    Rozners, Eriks; Pilch, Daniel S; Egli, Martin

    2015-12-01

    This unit describes the application of calorimetry to characterize the thermodynamics of nucleic acids, specifically, the two major calorimetric methodologies that are currently employed: differential scanning (DSC) and isothermal titration calorimetry (ITC). DSC is used to study thermally induced order-disorder transitions in nucleic acids. A DSC instrument measures, as a function of temperature (T), the excess heat capacity (C(p)(ex)) of a nucleic acid solution relative to the same amount of buffer solution. From a single curve of C(p)(ex) versus T, one can derive the following information: the transition enthalpy (ΔH), entropy (ΔS), free energy (ΔG), and heat capacity (ΔCp); the state of the transition (two-state versus multistate); and the average size of the molecule that melts as a single thermodynamic entity (e.g., the duplex). ITC is used to study the hybridization of nucleic acid molecules at constant temperature. In an ITC experiment, small aliquots of a titrant nucleic acid solution (strand 1) are added to an analyte nucleic acid solution (strand 2), and the released heat is monitored. ITC yields the stoichiometry of the association reaction (n), the enthalpy of association (ΔH), the equilibrium association constant (K), and thus the free energy of association (ΔG). Once ΔH and ΔG are known, ΔS can also be derived. Repetition of the ITC experiment at a number of different temperatures yields the ΔCp for the association reaction from the temperature dependence of ΔH.

  10. Medical devices; immunology and microbiology devices; classification of multiplex nucleic acid assay for identification of microorganisms and resistance markers from positive blood cultures. Final order.

    PubMed

    2015-05-27

    The Food and Drug Administration (FDA) is classifying multiplex nucleic acid assay for identification of microorganisms and resistance markers from positive blood cultures into class II (special controls). The special controls that will apply to this device are identified in this order and will be part of the codified language for the multiplex nucleic acid assay for identification of microorganisms and resistance markers from positive blood cultures. The Agency is classifying the device into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of the device.

  11. Toward a multiplexed solid-phase nucleic acid hybridization assay using quantum dots as donors in fluorescence resonance energy transfer.

    PubMed

    Algar, W Russ; Krull, Ulrich J

    2009-05-15

    Solid-phase assays using immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET) have been developed for the selective detection of nucleic acids. QDs were immobilized on optical fibers and conjugated with probe oligonucleotides. Hybridization with acceptor labeled target oligonucleotides generated FRET-sensitized acceptor fluorescence that was used as the analytical signal. A sandwich assay was also introduced and avoided the need for target labeling. Green and red emitting CdSe/ZnS QDs were used as donors with Cy3 and Alexa Fluor 647 acceptors, respectively. Quantitative measurements were made via spectrofluorimetry or fluorescence microscopy. Detection limits as low as 1 nM were obtained, and the discrimination of single nucleotide polymorphisms (SNPs) with contrast ratios as high as 31:1 was possible. The assays retained their selectivity and at least 50% of their signal when tested in bovine serum and against a large background of noncomplementary genomic DNA. Mixed films of the two colors of QD and two probe oligonucleotide sequences were prepared for multiplexed solid-phase hybridization assays. It was possible to simultaneously detect two target sequences with retention of selectivity, including SNP discrimination. This research provides an important precedent and framework for the future development of QD-based bioassays and biosensors.

  12. Novel multiplex PCR assay using locked nucleic acid (LNA)-based universal primers for the simultaneous detection of five swine viruses.

    PubMed

    Chen, Ru; Gao, Xiao-Bo; Yu, Xiao-Lu; Song, Chang-Xu; Qiu, Yang

    2016-02-01

    A novel multiplex PCR assay using non-homologous oligonucleotides with locked nucleic acid (LNA) modifications as universal primers was developed and validated for the simultaneous detection of five swine viruses. The assay utilizes five virus-specific primer pairs modified at the 5' end through the addition of the universal primer sequence. In the reaction, small amounts of target templates with the 5' tail were generated and subsequently amplified through the extension of a LNA universal primer set. To validate the specificity of this assay, 27 viral target strains and 12 non-target pathogens were tested. The lower limit of detection of viral nucleic acids was 1.1-1.9 pg per reaction or 11-32 pg in a five-plex viral nucleic acid mixture. The LNA mPCR assay displayed higher analytical sensitivity and efficiency for the detection of plasmid standards compared with the conventional assay, which uses standard primers without the 5' tail. A total of 207 field samples were tested using both assays. The LNA mPCR assay provided numerically higher detection rates for all pathogens in independent samples. Moreover, the LNA mPCR assay had significantly higher detection rates in independent samples compared with the conventional assay.

  13. Paper-based solid-phase nucleic acid hybridization assay using immobilized quantum dots as donors in fluorescence resonance energy transfer.

    PubMed

    Noor, M Omair; Shahmuradyan, Anna; Krull, Ulrich J

    2013-02-05

    A paper-based solid-phase assay is presented for transduction of nucleic acid hybridization using immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). The surface of paper was modified with imidazole groups to immobilize QD-probe oligonucleotide conjugates that were assembled in solution. Green-emitting QDs (gQDs) were FRET-paired with Cy3 acceptor. Hybridization of Cy3-labeled oligonucleotide targets provided the proximity required for FRET-sensitized emission from Cy3, which served as an analytical signal. The assay exhibited rapid transduction of nucleic acid hybridization within minutes. Without any amplification steps, the limit of detection of the assay was found to be 300 fmol with the upper limit of the dynamic range at 5 pmol. The implementation of glutathione-coated QDs for the development of nucleic acid hybridization assay integrated on a paper-based platform exhibited excellent resistance to nonspecific adsorption of oligonucleotides and showed no reduction in the performance of the assay in the presence of large quantities of noncomplementary DNA. The selectivity of nucleic acid hybridization was demonstrated by single-nucleotide polymorphism (SNP) detection at a contrast ratio of 19 to 1. The reuse of paper over multiple cycles of hybridization and dehybridization was possible, with less than 20% reduction in the performance of the assay in five cycles. This work provides an important framework for the development of paper-based solid-phase QD-FRET nucleic acid hybridization assays that make use of a ratiometric approach for detection and analysis.

  14. Nucleic Acid Amplification Based Diagnostic of Lyme (Neuro-)borreliosis – Lost in the Jungle of Methods, Targets, and Assays?

    PubMed Central

    Nolte, Oliver

    2012-01-01

    Laboratory based diagnosis of infectious diseases usually relies on culture of the disease causing micro-organism, followed by identification and susceptibility testing. Since Borrelia burgdorferi sensu lato, the etiologic agent of Lyme disease or Lyme borreliosis, requires very specific culture conditions (e.g. specific liquid media, long term cul-ture) traditional bacteriology is often not done on a routine basis. Instead, confirmation of the clinical diagnosis needs ei-ther indirect techniques (like serology or measurement of cellular activity in the presence of antigens) or direct but culture independent techniques, like microscopy or nucleic acid amplification techniques (NAT), with polymerase chain reaction (PCR) being the most frequently applied NAT method in routine laboratories. NAT uses nucleic acids of the disease causing micro-organism as template for amplification, isolated from various sources of clinical specimens. Although the underlying principle, adoption of the enzymatic process running during DNA duplication prior to prokaryotic cell division, is comparatively easy, a couple of ‘pitfalls’ is associated with the technique itself as well as with interpretation of the results. At present, no commercial, CE-marked and sufficiently validated PCR assay is available. A number of homebrew assays have been published, which are different in terms of target (i.e. the gene targeted by the amplification primers), method (nested PCR, PCR followed by hybridization, real-time PCR) and validation criteria. Inhibitory compounds may lead to false negative results, if no appropriate internal control is included. Carry-over of amplicons, insufficient handling and workflow and/or insufficiently validated targets/primers may result in false positive results. Different targets may yield different analytical sensitivity, depending, among other factors, of the redundancy of a target gene in the genome. Per-formance characteristics (e.g. analytical sensitivity and

  15. Nucleic Acid Amplification Based Diagnostic of Lyme (Neuro-)borreliosis - Lost in the Jungle of Methods, Targets, and Assays?

    PubMed

    Nolte, Oliver

    2012-01-01

    Laboratory based diagnosis of infectious diseases usually relies on culture of the disease causing micro-organism, followed by identification and susceptibility testing. Since Borrelia burgdorferi sensu lato, the etiologic agent of Lyme disease or Lyme borreliosis, requires very specific culture conditions (e.g. specific liquid media, long term cul-ture) traditional bacteriology is often not done on a routine basis. Instead, confirmation of the clinical diagnosis needs ei-ther indirect techniques (like serology or measurement of cellular activity in the presence of antigens) or direct but culture independent techniques, like microscopy or nucleic acid amplification techniques (NAT), with polymerase chain reaction (PCR) being the most frequently applied NAT method in routine laboratories. NAT uses nucleic acids of the disease causing micro-organism as template for amplification, isolated from various sources of clinical specimens. Although the underlying principle, adoption of the enzymatic process running during DNA duplication prior to prokaryotic cell division, is comparatively easy, a couple of 'pitfalls' is associated with the technique itself as well as with interpretation of the results. At present, no commercial, CE-marked and sufficiently validated PCR assay is available. A number of homebrew assays have been published, which are different in terms of target (i.e. the gene targeted by the amplification primers), method (nested PCR, PCR followed by hybridization, real-time PCR) and validation criteria. Inhibitory compounds may lead to false negative results, if no appropriate internal control is included. Carry-over of amplicons, insufficient handling and workflow and/or insufficiently validated targets/primers may result in false positive results. Different targets may yield different analytical sensitivity, depending, among other factors, of the redundancy of a target gene in the genome. Per-formance characteristics (e.g. analytical sensitivity and

  16. Composition for nucleic acid sequencing

    DOEpatents

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

    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.

  17. Nanostructured luminescently labeled nucleic acids.

    PubMed

    Kricka, Larry J; Fortina, Paolo; Park, Jason Y

    2017-03-01

    Important and emerging trends at the interface of luminescence, nucleic acids and nanotechnology are: (i) the conventional luminescence labeling of nucleic acid nanostructures (e.g. DNA tetrahedron); (ii) the labeling of bulk nucleic acids (e.g. single-stranded DNA, double-stranded DNA) with nanostructured luminescent labels (e.g. copper nanoclusters); and (iii) the labeling of nucleic acid nanostructures (e.g. origami DNA) with nanostructured luminescent labels (e.g. silver nanoclusters). This review surveys recent advances in these three different approaches to the generation of nanostructured luminescently labeled nucleic acids, and includes both direct and indirect labeling methods. Copyright © 2016 John Wiley & Sons, Ltd.

  18. New Real-Time PCR Assay Using Locked Nucleic Acid Probes To Assess Prevalence of ParC Mutations in Fluoroquinolone-Susceptible Streptococcus pneumoniae Isolates from France

    PubMed Central

    Decousser, Jean-Winoc; Methlouthi, Imen; Pina, Patrick; Collignon, Anne; Allouch, Pierre

    2006-01-01

    A real-time PCR assay with locked nucleic acid probes was developed to screen mutations at codons 79 and 83 of the Streptococcus pneumoniae parC gene. Only silent mutations were detected among 236 French invasive fluoroquinolone-susceptible strains. This test could be useful for some high-risk patients or in national surveys. PMID:16569894

  19. Immunomodulatory spherical nucleic acids.

    PubMed

    Radovic-Moreno, Aleksandar F; Chernyak, Natalia; Mader, Christopher C; Nallagatla, Subbarao; Kang, Richard S; Hao, Liangliang; Walker, David A; Halo, Tiffany L; Merkel, Timothy J; Rische, Clayton H; Anantatmula, Sagar; Burkhart, Merideth; Mirkin, Chad A; Gryaznov, Sergei M

    2015-03-31

    Immunomodulatory nucleic acids have extraordinary promise for treating disease, yet clinical progress has been limited by a lack of tools to safely increase activity in patients. Immunomodulatory nucleic acids act by agonizing or antagonizing endosomal toll-like receptors (TLR3, TLR7/8, and TLR9), proteins involved in innate immune signaling. Immunomodulatory spherical nucleic acids (SNAs) that stimulate (immunostimulatory, IS-SNA) or regulate (immunoregulatory, IR-SNA) immunity by engaging TLRs have been designed, synthesized, and characterized. Compared with free oligonucleotides, IS-SNAs exhibit up to 80-fold increases in potency, 700-fold higher antibody titers, 400-fold higher cellular responses to a model antigen, and improved treatment of mice with lymphomas. IR-SNAs exhibit up to eightfold increases in potency and 30% greater reduction in fibrosis score in mice with nonalcoholic steatohepatitis (NASH). Given the clinical potential of SNAs due to their potency, defined chemical nature, and good tolerability, SNAs are attractive new modalities for developing immunotherapies.

  20. Immunomodulatory spherical nucleic acids

    PubMed Central

    Radovic-Moreno, Aleksandar F.; Chernyak, Natalia; Mader, Christopher C.; Nallagatla, Subbarao; Kang, Richard S.; Hao, Liangliang; Walker, David A.; Halo, Tiffany L.; Merkel, Timothy J.; Rische, Clayton H.; Anantatmula, Sagar; Burkhart, Merideth; Mirkin, Chad A.; Gryaznov, Sergei M.

    2015-01-01

    Immunomodulatory nucleic acids have extraordinary promise for treating disease, yet clinical progress has been limited by a lack of tools to safely increase activity in patients. Immunomodulatory nucleic acids act by agonizing or antagonizing endosomal toll-like receptors (TLR3, TLR7/8, and TLR9), proteins involved in innate immune signaling. Immunomodulatory spherical nucleic acids (SNAs) that stimulate (immunostimulatory, IS-SNA) or regulate (immunoregulatory, IR-SNA) immunity by engaging TLRs have been designed, synthesized, and characterized. Compared with free oligonucleotides, IS-SNAs exhibit up to 80-fold increases in potency, 700-fold higher antibody titers, 400-fold higher cellular responses to a model antigen, and improved treatment of mice with lymphomas. IR-SNAs exhibit up to eightfold increases in potency and 30% greater reduction in fibrosis score in mice with nonalcoholic steatohepatitis (NASH). Given the clinical potential of SNAs due to their potency, defined chemical nature, and good tolerability, SNAs are attractive new modalities for developing immunotherapies. PMID:25775582

  1. Fluorescence resonance energy transfer from pyrene to perylene labels for nucleic acid hybridization assays under homogeneous solution conditions

    PubMed Central

    Masuko, Masayuki; Ohuchi, Shohkichi; Sode, Koji; Ohtani, Hiroyuki; Shimadzu, Akira

    2000-01-01

    We characterized the fluorescence resonance energy transfer (FRET) from pyrene (donor) to perylene (acceptor) for nucleic acid assays under homogeneous solution conditions. We used the hybridization between a target 32mer and its complementary two sequential 16mer deoxyribonucleotides whose neighboring terminals were each respectively labeled with a pyrene and a perylene residue. A transfer efficiency of ~100% was attained upon the hybridization when observing perylene fluorescence at 459 nm with 347-nm excitation of a pyrene absorption peak. The Förster distance between two dye residues was 22.3 Å (the orientation factor of 2/3). We could change the distance between the residues by inserting various numbers of nucleotides into the center of the target, thus creating a gap between the dye residues on a hybrid. Assuming that the number of inserted nucleotides is proportional to the distance between the dye residues, the energy transfer efficiency versus number of inserted nucleotides strictly obeyed the Förster theory. The mean inter-nucleotide distance of the single-stranded portion was estimated to be 2.1 Å. Comparison between the fluorescent properties of a pyrene–perylene pair with those of a widely used fluorescein–rhodamine pair showed that the pyrene–perylene FRET is suitable for hybridization assays. PMID:10734211

  2. Impact of a rapid peptide nucleic acid fluorescence in situ hybridization assay on treatment of Candida infections.

    PubMed

    Heil, Emily L; Daniels, Lindsay M; Long, Dustin M; Rodino, Kyle G; Weber, David J; Miller, Melissa B

    2012-11-01

    The impact of a rapid peptide nucleic acid fluorescence in situ hybridization (PNA FISH) assay with an antimicrobial stewardship intervention on the treatment of Candida infections was studied. The utility of implementing the PNA FISH assay with an antimicrobial stewardship intervention in hospitalized patients with candidemia was evaluated by measuring the median time to Candida species identification, time to targeted therapy, and clinical outcomes, including time to culture clearance, hospital length of stay, and hospital mortality. Secondary objectives included determining the cost-effectiveness of the PNA FISH assay by assessing estimated antifungal drug costs (as average wholesale price) before (June 26, 2009-September 19, 2010) and after (September 20, 2010-June 13, 2011) test implementation and confirming test accuracy. For both groups, laboratory personnel notified the physician of the results of Gram's stain from blood culture. Time to targeted therapy significantly decreased after the implementation of the PNA FISH assay (p = 0.0016). The postimplementation group had a higher rate of culture clearance (p = 0.01). Median time to species identification was 0.2 day with the PNA FISH test versus 4 days with routine methods (p < 0.001). Accounting for the cost of the test itself and the cases in which patients were switched to more-expensive therapy on the basis of the test, we estimated that the PNA FISH test resulted in savings of approximately $415 per patient. Implementing a PNA FISH test to identify Candida species from yeast-positive blood cultures in conjunction with a pharmacy-driven antimicrobial stewardship protocol decreased the time to targeted antifungal therapy and the time to culture clearance.

  3. Development of Lentivirus-Based Reference Materials for Ebola Virus Nucleic Acid Amplification Technology-Based Assays.

    PubMed

    Mattiuzzo, Giada; Ashall, James; Doris, Kathryn S; MacLellan-Gibson, Kirsty; Nicolson, Carolyn; Wilkinson, Dianna E; Harvey, Ruth; Almond, Neil; Anderson, Robert; Efstathiou, Stacey; Minor, Philip D; Page, Mark

    2015-01-01

    The 2013-present Ebola virus outbreak in Western Africa has prompted the production of many diagnostic assays, mostly based on nucleic acid amplification technologies (NAT). The calibration and performance assessment of established assays and those under evaluation requires reference materials that can be used in parallel with the clinical sample to standardise or control for every step of the procedure, from extraction to the final qualitative/quantitative result. We have developed safe and stable Ebola virus RNA reference materials by encapsidating anti sense viral RNA into HIV-1-like particles. The lentiviral particles are replication-deficient and non-infectious due to the lack of HIV-1 genes and Envelope protein. Ebola virus genes were subcloned for encapsidation into two lentiviral preparations, one containing NP-VP35-GP and the other VP40 and L RNA. Each reference material was formulated as a high-titre standard for use as a calibrator for secondary or internal standards, and a 10,000-fold lower titre preparation to serve as an in-run control. The preparations have been freeze-dried to maximise stability. These HIV-Ebola virus RNA reference materials were suitable for use with in-house and commercial quantitative RT-PCR assays and with digital RT-PCR. The HIV-Ebola virus RNA reference materials are stable at up to 37°C for two weeks, allowing the shipment of the material worldwide at ambient temperature. These results support further evaluation of the HIV-Ebola virus RNA reference materials as part of an International collaborative study for the establishment of the 1st International Standard for Ebola virus RNA.

  4. The effect of primer-template mismatches on the detection and quantification of nucleic acids using the 5' nuclease assay.

    PubMed

    Stadhouders, Ralph; Pas, Suzan D; Anber, Jeer; Voermans, Jolanda; Mes, Ted H M; Schutten, Martin

    2010-01-01

    Real-time polymerase chain reaction (PCR) is the current method of choice for detection and quantification of nucleic acids, especially for molecular diagnostics. Complementarity between primers and template is often crucial for PCR applications, as mismatches can severely reduce priming efficiency. However, little quantitative data on the effect of these mismatches is available. We quantitatively investigated the effects of primer-template mismatches within the 3'-end primer region on real-time PCR using the 5'-nuclease assay. Our results show that single mismatches instigate a broad variety of effects, ranging from minor (<1.5 cycle threshold, eg, A-C, C-A, T-G, G-T) to severe impact (>7.0 cycle threshold, eg, A-A, G-A, A-G, C-C) on PCR amplification. A clear relationship between specific mismatch types, position, and impact was found, which remained consistent for DNA versus RNA amplifications and Taq/Moloney murine leukemia virus versus rTth based amplifications. The overall size of the impact among the various master mixes used differed substantially (up to sevenfold), and for certain master mixes a reverse or forward primer-specific impact was observed, emphasizing the importance of the experimental conditions used. Taken together these data suggest that mismatch impact follows a consistent pattern and enabled us to formulate several guidelines for predicting primer-template mismatch behavior when using specific 5-nuclease assay master mixes. Our study provides novel insight into mismatch behavior and should allow for more optimized development of real-time PCR assays involving primer-template mismatches.

  5. Screening metagenomic data for viruses using the E-Probe Diagnostic Nucleic Acid Assay (EDNA)

    USDA-ARS?s Scientific Manuscript database

    There are many plant pathogen-specific diagnostic assays, based on PCR and immune-detection. However, the ability to test for large numbers of pathogens simultaneously is lacking. Next generation sequencing (NGS) allows one to detect all organisms within a given sample, but has computational limitat...

  6. A sensitive colorimetric assay system for nucleic acid detection based on isothermal signal amplification technology.

    PubMed

    Hu, Bo; Guo, Jing; Xu, Ying; Wei, Hua; Zhao, Guojie; Guan, Yifu

    2017-08-01

    Rapid and accurate detection of microRNAs in biological systems is of great importance. Here, we report the development of a visual colorimetric assay which possesses the high amplification capabilities and high selectivity of the rolling circle amplification (RCA) method and the simplicity and convenience of gold nanoparticles used as a signal indicator. The designed padlock probe recognizes the target miRNA and is circularized, and then acts as the template to extend the target miRNA into a long single-stranded nucleotide chain of many tandem repeats of nucleotide sequences. Next, the RCA product is hybridized with oligonucleotides tagged onto gold nanoparticles. This interaction leads to the aggregation of gold nanoparticles, and the color of the system changes from wine red to dark blue according to the abundance of miRNA. A linear correlation between fluorescence and target oligonucleotide content was obtained in the range 0.3-300 pM, along with a detection limit of 0.13 pM (n = 7) and a RSD of 3.9% (30 pM, n = 9). The present approach provides a simple, rapid, and accurate visual colorimetric assay that allows sensitive biodetection and bioanalysis of DNA and RNA nucleotides of interest in biologically important samples. Graphical abstract The colorimetric assay system for analyzing target oligonucleotides.

  7. High affinity γPNA sandwich hybridization assay for rapid detection of short nucleic acid targets with single mismatch discrimination.

    PubMed

    Goldman, Johnathan M; Zhang, Li Ang; Manna, Arunava; Armitage, Bruce A; Ly, Danith H; Schneider, James W

    2013-07-08

    Hybridization analysis of short DNA and RNA targets presents many challenges for detection. The commonly employed sandwich hybridization approach cannot be implemented for these short targets due to insufficient probe-target binding strengths for unmodified DNA probes. Here, we present a method capable of rapid and stable sandwich hybridization detection for 22 nucleotide DNA and RNA targets. Stable hybridization is achieved using an n-alkylated, polyethylene glycol γ-carbon modified peptide nucleic acid (γPNA) amphiphile. The γPNA's exceptionally high affinity enables stable hybridization of a second DNA-based probe to the remaining bases of the short target. Upon hybridization of both probes, an electrophoretic mobility shift is measured via interaction of the n-alkane modification on the γPNA with capillary electrophoresis running buffer containing nonionic surfactant micelles. We find that sandwich hybridization of both probes is stable under multiple binding configurations and demonstrate single base mismatch discrimination. The binding strength of both probes is also stabilized via coaxial stacking on adjacent hybridization to targets. We conclude with a discussion on the implementation of the proposed sandwich hybridization assay as a high-throughput microRNA detection method.

  8. A reliable and sensitive bead-based fluorescence assay for identification of nucleic acid sequences

    NASA Astrophysics Data System (ADS)

    Klamp, Tobias; Yahiatène, Idir; Lampe, André; Schüttpelz, Mark; Sauer, Markus

    2011-03-01

    The sensitive and rapid detection of pathogenic DNA is of tremendous importance in the field of diagnostics. We demonstrate the ability of detecting and quantifying single- and double-stranded pathogenic DNA with picomolar sensitivity in a bead-based fluorescence assay. Selecting appropriate capturing and detection sequences enables rapid (2 h) and reliable DNA quantification. We show that synthetic sequences of S. pneumoniae and M. luteus can be quantified in very small sample volumes (20 μL) across a linear detection range over four orders of magnitude from 1 nM to 1 pM, using a miniaturized wide-field fluorescence microscope without amplification steps. The method offers single molecule detection sensitivity without using complex setups and thus volunteers as simple, robust, and reliable method for the sensitive detection of DNA and RNA sequences.

  9. Current methods for fluorescence-based universal sequence-dependent detection of nucleic acids in homogenous assays and clinical applications.

    PubMed

    Faltin, Bernd; Zengerle, Roland; von Stetten, Felix

    2013-11-01

    Specific and sensitive nucleic acid (NA) testing in research and clinical diagnostics is usually performed by use of labeled oligonucleotide probes. However, the use of target-specific fluorogenic probes increases the cost of analysis. Therefore, universal sequence-dependent (USD) NA detection methods have been developed to facilitate cost-effective target detection using standardized reagents. We provide a comprehensive review of the current methods for fluorescence-based USD NA detection. Initially, we focus on the emergence of these methods as a means to overcome the shortcomings of common NA detection methods, such as hydrolysis probes and molecular beacons. Thereafter, we provide a critical evaluation of the individual detection methods. These methods include (a) target amplification with bipartite primers introducing a universal detection tag to the amplicon (UniPrimer PCR, universal fluorescence energy transfer probe PCR, attached universal duplex probe PCR, and universal strand displacement amplification) or combined with bipartite probes comprising a universal detection region (mediator probe PCR, universal strand displacement amplification, universal quenching probe PCR) and (b) amplification-independent assays employing either a universal variant of the invader assay or universal NA hybridization sensors. We discuss differences between the methods and review clinical applications. The current methods for USD NA testing are cost-effective and flexible and have concordant analytical performance in comparison with common probe-based techniques. They can detect any target sequence by the simple use of a label-free, low-cost primer or probe combined with a universal fluorogenic reporter. The methods differ in the number of target specificities, capability of multiplexing, and incubation requirements (isothermal/thermocycling). Extensive clinical applications comprise detection of single-nucleotide polymorphisms, study of gene expression, in situ PCR, and

  10. 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

  11. Reactivity of Nucleic Acid Radicals.

    PubMed

    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.

  12. 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.

  13. Multiplexed Nucleic Acid Hybridization Assays Using Single-FRET-Pair Distance-Tuning.

    PubMed

    Qiu, Xue; Guo, Jiajia; Jin, Zongwen; Petreto, Alexandra; Medintz, Igor L; Hildebrandt, Niko

    2017-07-01

    Multiplexed photoluminescence (PL) detection plays an important role in chemical and biological sensing. Here, it is shown that time-gated (TG) detection of a single terbium-donor-based Förster resonance energy transfer (FRET) pair can be used to selectively quantify low nanomolar concentrations of multiple DNAs or microRNAs in a single sample. This study demonstrates the applicability of single-TG-FRET-pair multiplexing for molecular (Tb-to-dye) and nanoparticle (Tb-to-quantum-dot) biosensing. Both systems use acceptor-sensitization and donor-quenching for quantifying biomolecular recognition and modification of the donor-acceptor distance for tuning the PL decays. TG intensity detection provides extremely low background noise and a quick and simple one-step assay format. Single-TG-FRET-pair multiplexing can be combined with spectral and spatial resolution, paving the way for biosensing with unprecedented high-order multiplexing capabilities. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Enzyme-linked immunosorbent/chemiluminescence assays, recombinant immunoblot assays and nucleic acid tests in the diagnosis of HCV infection.

    PubMed

    Pondé, Robério Amorim de Almeida

    2013-08-01

    The diagnosis of hepatitis C virus (HCV) infection is defined according to the results obtained from screening assays, and confirmation made by supplemental tests, in order to exclude the possibility of false-positive and false-negative results and, therefore, a misdiagnosis. Identifying the patient's true clinical status is of crucial importance to direct an accurate course of therapy, but, often, the definition of this status is only possible after conjunctions and analysis of the results obtained from each methodology applied, considering the limitations of each assay. In this manuscript, it is discussed briefly the possible results obtained from the three methods most commonly applied in routine laboratory and their contribution in the diagnosis of HCV infection.

  15. Functional nucleic acid probes and uses thereof

    DOEpatents

    Nilsen-Hamilton, Marit

    2006-10-03

    The present invention provides functional nucleic acid probes, and methods of using functional nucleic acid probes, for binding a target to carry out a desired function. The probes have at least one functional nucleic acid, at least one regulating nucleic acid, and at least one attenuator. The functional nucleic acid is maintained in an inactive state by the attenuator and activated by the regulating nucleic acid only in the presence of a regulating nucleic acid target. In its activated state the functional nucleic acid can bind to its target to carry out a desired function, such as generating a signal, cleaving a nucleic acid, or catalyzing a reaction.

  16. A Simpler Nucleic Acid

    NASA Technical Reports Server (NTRS)

    Orgel, Leslie

    2000-01-01

    It has been supposed that for a nucleic acid analog to pair with RNA it must, like RNA, have a backbone with at least a sixatom repeat; a shorter backbone presumably would not stretch far enough to bind RNA properly. The Eschenmoser group has shown, however, that this first impression is incorrect.As they report in their new paper, Eschenmoser and co-workers ( I ) have now synthesized a substantial number of these polymers, which are called (L)-a-threofuranosyl oligonucleotides or TNAs. They are composed of bases linked to a threose sugar-phosphate backbone, with phosphodiester bonds connecting the nucleotides. The investigators discovered that pairs of complementary TNAs do indeed form stable Watson-Crick double helices and, perhaps more importantly, that TNAs form stable double helices with complementary RNAs and DNAs.

  17. A Simpler Nucleic Acid

    NASA Technical Reports Server (NTRS)

    Orgel, Leslie

    2000-01-01

    It has been supposed that for a nucleic acid analog to pair with RNA it must, like RNA, have a backbone with at least a sixatom repeat; a shorter backbone presumably would not stretch far enough to bind RNA properly. The Eschenmoser group has shown, however, that this first impression is incorrect.As they report in their new paper, Eschenmoser and co-workers ( I ) have now synthesized a substantial number of these polymers, which are called (L)-a-threofuranosyl oligonucleotides or TNAs. They are composed of bases linked to a threose sugar-phosphate backbone, with phosphodiester bonds connecting the nucleotides. The investigators discovered that pairs of complementary TNAs do indeed form stable Watson-Crick double helices and, perhaps more importantly, that TNAs form stable double helices with complementary RNAs and DNAs.

  18. Sample preparation: a challenge in the development of point-of-care nucleic acid-based assays for resource-limited settings.

    PubMed

    Dineva, Magda Anastassova; MahiLum-Tapay, Lourdes; Lee, Helen

    2007-12-01

    Currently available nucleic acid testing (NAT)-based assays are complex and time-consuming, and they require expensive instrumentation and dedicated laboratory spaces for sample preparation as well as for amplification and detection of the nucleic acid target. Reagents required for these tests are also expensive and must be transported and stored refrigerated or frozen. These characteristics have limited the use of such assays for point-of-care (POC) testing, especially in resource-poor settings. Efforts to develop simple and rapid NAT-based assays have focused predominantly on the amplification and detection steps, with sample preparation and nucleic acid extraction remaining the bottleneck in the development of NAT systems suitable for POC applications or resource-limited settings. A review of NAT platforms and technologies currently under development and validation for rapid field testing revealed that, in addition to requiring expensive and complex instrumentation, many of these systems also require off-line sample preparation and reagent handling. In their current format, they are therefore not appropriate for POC testing in resource-limited settings. We evaluated several commercially available technologies and procedures for the isolation of nucleic acid with the extraction of HIV-1 RNA from human plasma as a model system. Our results indicate that solid-phase extraction with silica or glass in the presence of a chaotropic salt provides the highest extraction efficiency. However, none of the existing methods and technologies is readily adaptable to a POC system. The integration of sample preparation procedures well suited to NAT-based assays in resource-limited settings therefore remains a challenge.

  19. Development and Evaluation of Novel Real-Time Reverse Transcription-PCR Assays with Locked Nucleic Acid Probes Targeting Leader Sequences of Human-Pathogenic Coronaviruses.

    PubMed

    Chan, Jasper Fuk-Woo; Choi, Garnet Kwan-Yue; Tsang, Alan Ka-Lun; Tee, Kah-Meng; Lam, Ho-Yin; Yip, Cyril Chik-Yan; To, Kelvin Kai-Wang; Cheng, Vincent Chi-Chung; Yeung, Man-Lung; Lau, Susanna Kar-Pui; Woo, Patrick Chiu-Yat; Chan, Kwok-Hung; Tang, Bone Siu-Fai; Yuen, Kwok-Yung

    2015-08-01

    Based on findings in small RNA-sequencing (Seq) data analysis, we developed highly sensitive and specific real-time reverse transcription (RT)-PCR assays with locked nucleic acid probes targeting the abundantly expressed leader sequences of Middle East respiratory syndrome coronavirus (MERS-CoV) and other human coronaviruses. Analytical and clinical evaluations showed their noninferiority to a commercial multiplex PCR test for the detection of these coronaviruses.

  20. Resonance Energy Transfer-Based Nucleic Acid Hybridization Assays on Paper-Based Platforms Using Emissive Nanoparticles as Donors.

    PubMed

    Doughan, Samer; Noor, M Omair; Han, Yi; Krull, Ulrich J

    2017-01-01

    Quantum dots (QDs) and upconverting nanoparticles (UCNPs) are luminescent nanoparticles (NPs) commonly used in bioassays and biosensors as resonance energy transfer (RET) donors. The narrow and tunable emissions of both QDs and UCNPs make them versatile RET donors that can be paired with a wide range of acceptors. Ratiometric signal processing that compares donor and acceptor emission in RET-based transduction offers improved precision, as it accounts for fluctuations in the absolute photoluminescence (PL) intensities of the donor and acceptor that can result from experimental and instrumental variations. Immobilizing NPs on a solid support avoids problems such as those that can arise with their aggregation in solution, and allows for facile layer-by-layer assembly of the interfacial chemistry. Paper is an attractive solid support for the development of point-of-care diagnostic assays given its ubiquity, low-cost, and intrinsic fluid transport by capillary action. Integration of nanomaterials with paper-based analytical devices (PADs) provides avenues to augment the analytical performance of PADs, given the unique optoelectronic properties of nanomaterials. Herein, we describe methodology for the development of PADs using QDs and UCNPs as RET donors for optical transduction of nucleic acid hybridization. Immobilization of green-emitting QDs (gQDs) on imidazole functionalized cellulose paper is described for use as RET donors with Cy3 molecular dye as acceptors for the detection of SMN1 gene fragment. We also describe the covalent immobilization of blue-emitting UCNPs on aldehyde modified cellulose paper for use as RET donors with orange-emitting QDs (oQDs) as acceptors for the detection of HPRT1 gene fragment. The data described herein is acquired using an epifluorescence microscope, and can also be collected using technology such as a typical electronic camera.

  1. Development of an Interaction Assay between Single-Stranded Nucleic Acids Trapped with Silica Particles and Fluorescent Compounds

    PubMed Central

    Isoda, T.; Maeda, R.

    2012-01-01

    Biopolymers are easily denatured by heating, a change in pH or chemical substances when they are immobilized on a substrate. To prevent denaturation of biopolymers, we developed a method to trap a polynucleotide on a substrate by hydrogen bonding using silica particles with surfaces modified by aminoalkyl chains ([A-AM silane]/SiO2). [A-AM silane]/SiO2 was synthesized by silane coupling reaction of N-2-(aminoethyl)-3-aminopropyltrimethoxysilane (A-AM silane) with SiO2 particles with a diameter of 5 μm at 100 °C for 20 min. The surface chemical structure of [A-AM silane]/SiO2 was characterized by Fourier transform infrared spectroscopy and molecular orbital calculations. The surface of the silica particles was modified with A-AM silane and primary amine groups were formed. [A-AM silane]/SiO2 was trapped with single-stranded nucleic acids [(Poly-X; X = A (adenine), G (guanine) and C (cytosine)] in PBS solution at 37 °C for 1 h. The single-stranded nucleic acids were trapped on the surface of the [A-AM silane]/SiO2 by hydrogen bonding to form conjugated materials. The resulting complexes were further conjugated by derivatives of acridine orange (AO) as fluorescent labels under the same conditions to form [AO:Poly-X:A-AM silane]/SiO2 complexes. Changes in the fluorescence intensity of these complexes originating from interactions between the single-stranded nucleic acid and aromatic compounds were also evaluated. The change in intensity displayed the order [AO: Poly-G: A-AM silane]/SiO2 > [AO:Poly-A:A-AM silane]/SiO2 >> [AO:Poly-C:A-AM silane]/SiO2. This suggests that the single-stranded nucleic acids conjugated with aminoalkyl chains on the surfaces of SiO2 particles and the change in fluorescence intensity reflected the molecular interaction between AO and the nucleic-acid base in a polynucleotide. PMID:24955635

  2. 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.

  3. Specific detection of DNA and RNA targets using a novel isothermal nucleic acid amplification assay based on the formation of a three-way junction structure.

    PubMed

    Wharam, S D; Marsh, P; Lloyd, J S; Ray, T D; Mock, G A; Assenberg, R; McPhee, J E; Brown, P; Weston, A; Cardy, D L

    2001-06-01

    The formation of DNA three-way junction (3WJ) structures has been utilised to develop a novel isothermal nucleic acid amplification assay (SMART) for the detection of specific DNA or RNA targets. The assay consists of two oligonucleotide probes that hybridise to a specific target sequence and, only then, to each other forming a 3WJ structure. One probe (template for the RNA signal) contains a non-functional single-stranded T7 RNA polymerase promoter sequence. This promoter sequence is made double-stranded (hence functional) by DNA polymerase, allowing T7 RNA polymerase to generate a target-dependent RNA signal which is measured by an enzyme-linked oligosorbent assay (ELOSA). The sequence of the RNA signal is always the same, regardless of the original target sequence. The SMART assay was successfully tested in model systems with several single-stranded synthetic targets, both DNA and RNA. The assay could also detect specific target sequences in both genomic DNA and total RNA from Escherichia coli. It was also possible to generate signal from E.coli samples without prior extraction of nucleic acid, showing that for some targets, sample purification may not be required. The assay is simple to perform and easily adaptable to different targets.

  4. 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.

  5. Comparison of the Simplexa™ Flu A/B & RSV kit (nucleic acid extraction-dependent assay) and the Prodessa ProFlu+™ assay for detecting influenza and respiratory syncytial viruses.

    PubMed

    Selvaraju, Suresh B; Bambach, Adrienne V; Leber, Amy L; Patru, Maria-Magdalena; Patel, Anami; Menegus, Marilyn A

    2014-09-01

    The relative performance of 2 widely used reverse transcription polymerase chain reaction (RT-PCR) assays, the Focus diagnostics Simplexa™ Flu A/B & RSV kit (nucleic acid extraction-dependent assay) and the Prodessa Proflu+™ assay, was evaluated using 735 prospectively and retrospectively collected nasopharyngeal swab specimens. Overall, the assays showed positive and negative agreements of 100% and 99.7% for influenza A, 98.1% and 99.9% for influenza B, and 99.3% and 99.5% for respiratory syncytial virus. The relative analytical sensitivity of the 2 assays was also similar.

  6. 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.

  7. Polymer-Nucleic Acid Interactions.

    PubMed

    Shen, Zhuang-Lin; Xia, Yi-Qi; Yang, Qiu-Song; Tian, Wen-de; Chen, Kang; Ma, Yu-Qiang

    2017-04-01

    Gene therapy is an important therapeutic strategy in the treatment of a wide range of genetic disorders. Polymers forming stable complexes with nucleic acids (NAs) are non-viral gene carriers. The self-assembly of polymers and nucleic acids is typically a complex process that involves many types of interaction at different scales. Electrostatic interaction, hydrophobic interaction, and hydrogen bonds are three important and prevalent interactions in the polymer/nucleic acid system. Electrostatic interactions and hydrogen bonds are the main driving forces for the condensation of nucleic acids, while hydrophobic interactions play a significant role in the cellular uptake and endosomal escape of polymer-nucleic acid complexes. To design high-efficiency polymer candidates for the DNA and siRNA delivery, it is necessary to have a detailed understanding of the interactions between them in solution. In this chapter, we survey the roles of the three important interactions between polymers and nucleic acids during the formation of polyplexes and summarize recent understandings of the linear polyelectrolyte-NA interactions and dendrimer-NA interactions. We also review recent progress optimizing the gene delivery system by tuning these interactions.

  8. Development of a novel nonradiometric assay for nucleic acid binding to TDP-43 suitable for high-throughput screening using AlphaScreen technology.

    PubMed

    Cassel, Joel A; Blass, Benjamin E; Reitz, Allen B; Pawlyk, Aaron C

    2010-10-01

    TAR DNA binding protein 43 (TDP-43) is a nucleic acid binding protein that is associated with the pathology of cystic fibrosis and neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar dementia. We have developed a robust, quantitative, nonradiometric high-throughput assay measuring oligonucleotide binding to TDP-43 using AlphaScreen technology. Biotinylated single-stranded TAR DNA (bt-TAR-32) and 6 TG repeats (bt-TG6) bound with high affinity to TDP-43, with K(D) values of 0.75 nM and 0.63 nM, respectively. Both oligonucleotides exhibited slow dissociation rates, with half-lives of 750 min for bt-TAR-32 and 150 min for bt-TG6. The affinities of unlabeled oligonucleotides, as determined by displacement of either bt-TAR-32 or bt-TG6, were consistent with previous reports of nucleic acid interactions with TDP-43, where increasing TG or UG repeats yield greater affinity. A diversity library of 7360 compounds was screened for inhibition of TDP-43 binding to bt-TAR-32, and a series of compounds was discovered with nascent SAR and IC(50) values ranging from 100 nM to 10 µM. These compounds may prove to be useful biochemical tools to elucidate the function of TDP-43 and may lead to novel therapeutics for indications where the TDP-43 nucleic acid interaction is causal to the associated pathology.

  9. Acidic domains around nucleic acids.

    PubMed Central

    Lamm, G; Pack, G R

    1990-01-01

    The hydrogen ion concentration in the vicinity of DNA was mapped out within the Poisson-Boltzmann approximation. Experimental conditions were modeled by assuming Na-DNA to be solvated in a buffer solution containing 45 mM Tris and 3 mM Mg cations at pH 7.5. Three regions of high H+ concentration (greater than 10 microM) are predicted: one throughout the minor groove of DNA and two localized in the major groove near N7 of guanine and C5 of cytosine for a G.C base pair. These acidic domains correlate well with the observed covalent binding sites of benzo[a]pyrene epoxide (N2 of guanine) and of aflatoxin B1 epoxide (N7 of guanine), chemical carcinogens that presumably undergo acid catalysis to form highly reactive carbocations that ultimately bind to DNA. It is suggested that these regions of high H+ concentration may also be of concern in understanding interactions involving proteins and noncarcinogenic molecules with or near nucleic acids. PMID:2123348

  10. 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.

  11. Infection with a plasmid-free variant Chlamydia related to Chlamydia trachomatis identified by using multiple assays for nucleic acid detection.

    PubMed Central

    An, Q; Radcliffe, G; Vassallo, R; Buxton, D; O'Brien, W J; Pelletier, D A; Weisburg, W G; Klinger, J D; Olive, D M

    1992-01-01

    Clinical samples in transport media from 40 patients exhibiting pathologies potentially caused by Chlamydia trachomatis infection were analyzed for chlamydial nucleic acid, and the results were compared with those of culture. Chlamydial culture was performed by a shell vial centrifugation method with HeLa 229 host cells. Polymerase chain reaction (PCR) assays were used to detect either regions on a 7.5-kb plasmid characteristic of C. trachomatis (plasmid-PCR) or a segment of the 16S rRNA genes (rRNA-PCR). All PCR results were confirmed by hybridization with probes for the specific amplified products in either a Southern or a dot blot format. An RNase protection (RNP) assay was used to detect genus-specific chlamydial 16S rRNA directly from the clinical samples. The PCR assays detected C. trachomatis but not other bacteria, including Chlamydia spp. C. trachomatis was isolated from six samples which were positive by the rDNA-PCR and plasmid-PCR assays. Five of the culture-positive specimens were positive by the RNP assay. Twenty-two samples were negative by all criteria. Surprisingly, nine samples were positive by rRNA-PCR and RNP assays only. Nucleic acid sequencing of the rRNA-PCR-amplified products indicated a close relationship between the variants and C. trachomatis. The data may indicate an unrecognized process in C. trachomatis infection or that these patients were infected by a variant strain of C. trachomatis which lacks the C. trachomatis-specific plasmid. Images PMID:1280642

  12. Nucleic acid based molecular devices.

    PubMed

    Krishnan, Yamuna; Simmel, Friedrich C

    2011-03-28

    In biology, nucleic acids are carriers of molecular information: DNA's base sequence stores and imparts genetic instructions, while RNA's sequence plays the role of a messenger and a regulator of gene expression. As biopolymers, nucleic acids also have exciting physicochemical properties, which can be rationally influenced by the base sequence in myriad ways. Consequently, in recent years nucleic acids have also become important building blocks for bottom-up nanotechnology: as molecules for the self-assembly of molecular nanostructures and also as a material for building machinelike nanodevices. In this Review we will cover the most important developments in this growing field of nucleic acid nanodevices. We also provide an overview of the biochemical and biophysical background of this field and the major "historical" influences that shaped its development. Particular emphasis is laid on DNA molecular motors, molecular robotics, molecular information processing, and applications of nucleic acid nanodevices in biology. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis.

    PubMed

    Zhao, Yong; Li, Guilian; Sun, Chongyun; Li, Chao; Wang, Xiaochen; Liu, Haican; Zhang, Pingping; Zhao, Xiuqin; Wang, Xinrui; Jiang, Yi; Yang, Ruifu; Wan, Kanglin; Zhou, Lei

    2015-01-01

    Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA) probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP) resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a "probe dropout" manner (quantification cycle = 0); thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE)/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100%) were correctly detected through the assay. Of these isolates, 88/88 (100%) were determined as RFP susceptible and 52/54 (96.3%) were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories.

  14. Ultra-high-throughput, automated nucleic acid detection of human immunodeficiency virus (HIV) for infant infection diagnosis using the Gen-Probe Aptima HIV-1 screening assay.

    PubMed

    Stevens, Wendy S; Noble, Lara; Berrie, Leigh; Sarang, Somaya; Scott, Lesley E

    2009-08-01

    The early diagnosis of human immunodeficiency virus (HIV) infection in infants is critical to ensure the initiation of treatment before significant immunological compromise. Each year an estimated 300,000 HIV-exposed infants in South Africa require access to tests for the diagnosis of HIV infection. Currently, testing is performed at several facilities by using PCR amplification of HIV DNA at 6 weeks of age by the use of dried blood spots (DBSs) and whole blood (WB). The Gen-Probe Aptima HIV type 1 (HIV-1) screening assay (the Aptima assay) is a qualitative nucleic acid test based on transcription-mediated amplification (TMA), a technology routinely used in blood banks in South Africa. The performance characteristics of Gen-Probe's TMA technology compared well to those of the Roche Amplicor HIV-1 DNA (version 1.5) assay. The sensitivity of the assay with WB and DBS samples was 100%, and the specificities were 99.4% and 99.5% for DBSs and WB, respectively. The detection of HIV by the Aptima assay at greater levels of dilution in samples negative by the comparator assay indicates an improvement in sensitivity by the use of the TMA technology. The ability to process 1,900 samples in a 24-h period on the Tigris instrument makes the Aptima assay an attractive option for high-volume, centralized laboratories.

  15. [Circulating nucleic acids and infertility].

    PubMed

    Scalici, E; Mullet, T; Ferrières Hoa, A; Gala, A; Loup, V; Anahory, T; Belloc, S; Hamamah, S

    2015-09-01

    Circulating nucleic acids (cell-free DNA and microRNAs) have for particularity to be easily detectable in the biological fluids of the body. Therefore, they constitute biomarkers of interest in female and male infertility care. Indeed, in female, they can be used to detect ovarian reserve disorders (polycystic ovary syndrome and low functional ovarian reserve) as well as to assess follicular microenvironment quality. Moreover, in men, their expression levels can vary in case of spermatogenesis abnormalities. Finally, circulating nucleic acids have also the ability to predict successfully the quality of in vitro embryo development. Their multiple contributions during assisted reproductive technology (ART) make of them biomarkers of interest, for the development of new diagnostic and/or prognostic tests, applied to our specialty. Circulating nucleic acids would so offer the possibility of personalized medical care for infertile couples in ART.

  16. Anions in Nucleic Acid Crystallography.

    PubMed

    D'Ascenzo, Luigi; Auffinger, Pascal

    2016-01-01

    Nucleic acid crystallization buffers contain a large variety of chemicals fitting specific needs. Among them, anions are often solely considered for pH-regulating purposes and as cationic co-salts while their ability to directly bind to nucleic acid structures is rarely taken into account. Here we review current knowledge related to the use of anions in crystallization buffers along with data on their biological prevalence. Chloride ions are frequently identified in crystal structures but display low cytosolic concentrations. Hence, they are thought to be distant from nucleic acid structures in the cell. Sulfate ions are also frequently identified in crystal structures but their localization in the cell remains elusive. Nevertheless, the characterization of the binding properties of these ions is essential for better interpreting the solvent structure in crystals and consequently, avoiding mislabeling of electron densities. Furthermore, understanding the binding properties of these anions should help to get clues related to their potential effects in crowded cellular environments.

  17. A microfluidic liquid phase nucleic acid purification chip to selectively isolate DNA or RNA from low copy/single bacterial cells in minute sample volume followed by direct on-chip quantitative PCR assay.

    PubMed

    Zhang, Rui; Gong, Hai-Qing; Zeng, Xudong; Lou, ChaoPing; Sze, ChunChau

    2013-02-05

    Purification of nucleic acids from a low quantity of bacterial cells in minute volume is important in many clinical and biological applications. We developed a novel microfluidic liquid phase nucleic acid purification chip to selectively isolate DNA or RNA from bacterial cells in the range of 5000 down to a single cell in the sample volume of 1 μl or 125 nl, which can be directly put through on-chip quantitative PCR assay. The aqueous phase bacterial lysate was isolated in an array of microwells, after which an immiscible organic (phenol-chloroform) phase was introduced in a headspace channel connecting the microwell array. Continuous flow of the organic phase increases the interfacial contact with the aqueous phase to achieve purification of target nucleic acid through phase partitioning. Significantly enhanced nucleic acid recovery yield, up to 10 fold higher, was achieved using the chip-based liquid phase nucleic acid purification technique compared to that obtained by the conventional column-based solid phase nucleic acid extraction method. One step vacuum-driven microfluidics allowed an on-chip quantitative PCR assay to be carried out in the same microwells within which bacterial nucleic acids were isolated, avoiding sample loss during liquid transfer. Using this nucleic acid purification device set in a two-dimensional (2D) array format of 900 microwells, it was demonstrated for the first time that high-throughput extraction of RNA couple with direct on-chip PCR analysis from single bacterial cells could be achieved. Our microfluidic platform offered a simple and effective solution for nucleic acid preparation, which can be integrated for automated bacterial pathogen detection and high throughput transcriptional profiling.

  18. High speed 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

    2011-05-17

    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. Each type of labeled nucleotide comprises an acceptor fluorophore attached to a phosphate portion of the nucleotide such that the fluorophore is removed upon incorporation into a growing strand. Fluorescent signal is emitted via fluorescent resonance energy transfer between the donor fluorophore and the acceptor fluorophore as each nucleotide is incorporated into the growing strand. The sequence is deduced by identifying which base is being incorporated into the growing strand.

  19. A nanodiagnostic colorimetric assay for 18S rRNA of Leishmania pathogens using nucleic acid sequence-based amplification and gold nanorods.

    PubMed

    Niazi, Alireza; Jorjani, Oghol-Niaz; Nikbakht, Hassan; Gill, Pooria

    2013-12-01

    We describe here a nanodiagnostic colorimetric assay for 18S rRNA of Leishmania pathogens that uses nucleic acid sequence-based amplification (NASBA) and gold nanorods (GNRs). NASBA primers targeting 18S rRNA were used for amplification of RNA in an isothermal process. The electrostatic interactions between the phosphate groups of the RNA amplicons and the cetyl trimethylammonium bromide layer on the GNRs resulting in their aggregation. This phenomenon changes the color of the GNR solution from red to purple. Our data showed 100% sensitivity and 80% specificity with the colorimetric assay compared with results using reverse transcription polymerase chain reaction. The nanodiagnostic method we describe simplifies the detection of NASBA amplicons without the need for gel electrophoresis.

  20. Relative analytical sensitivity of donor nucleic acid amplification technology screening and diagnostic real-time polymerase chain reaction assays for detection of Zika virus RNA.

    PubMed

    Stone, Mars; Lanteri, Marion C; Bakkour, Sonia; Deng, Xutao; Galel, Susan A; Linnen, Jeffrey M; Muñoz-Jordán, Jorge L; Lanciotti, Robert S; Rios, Maria; Gallian, Pierre; Musso, Didier; Levi, José E; Sabino, Ester C; Coffey, Lark L; Busch, Michael P

    2017-03-01

    Zika virus (ZIKV) has spread rapidly in the Pacific and throughout the Americas and is associated with severe congenital and adult neurologic outcomes. Nucleic acid amplification technology (NAT) assays were developed for diagnostic applications and for blood donor screening on high-throughput NAT systems. We distributed blinded panels to compare the analytical performance of blood screening relative to diagnostic NAT assays. A 25-member, coded panel (11 half-log dilutions of a 2013 French Polynesia ZIKV isolate and 2015 Brazilian donor plasma implicated in transfusion transmission, and 3 negative controls) was sent to 11 laboratories that performed 17 assays with 2 to 12 replicates per panel member. Results were analyzed for the percentage reactivity at each dilution and by probit analysis to estimate the 50% and 95% limits of detection (LOD50 and LOD95 , respectively). Donor-screening NAT assays that process approximately 500 µL of plasma into amplification reactions were comparable in sensitivity (LOD50 and LOD95 , 2.5 and 15-18 copies/mL) and were approximately 10-fold to 100-fold more sensitive than research laboratory-developed and diagnostic reverse transcriptase-polymerase chain reaction tests that process from 10 to 30 µL of plasma per amplification. Increasing sample input volume assayed with the Centers for Disease Control and Prevention reverse transcriptase-polymerase chain reaction assays increased the LODs by 10-fold to 30-fold. Blood donor-screening ZIKV NAT assays demonstrate similar excellent sensitivities to assays currently used for screening for transfusion-transmitted viruses and are substantially more sensitive than most other laboratory-developed and diagnostic ZIKV reverse transcriptase-polymerase chain reaction assays. Enhancing sensitivities of laboratory-developed and diagnostic assays may be achievable by increasing sample input. © 2017 AABB.

  1. 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.

  2. Advantages of one step nucleic acid amplification (OSNA) whole node assay in sentinel lymph node (SLN) analysis in breast cancer.

    PubMed

    Santaballa, Ana; De La Cueva, Helena; Salvador, Carmen; García-Martínez, Ana M; Guarín, María J; Lorente, David; Palomar, Laura; Aznar, Ismael; Dobón, Fernando; Bello, Pilar

    2013-01-01

    The purpose of this study is to present our first results of sentinel node analysis (SLN) by one step nucleic acid amplification (OSNA) in routine clinical practice in our centre and compare them with the results of classic histopathological analysis in a historical cohort from our same institution. 407 patients (total study population) with early breast cancer and no clinical nodal involvement underwent SLN biopsy in our institution. The SLN was analysed by OSNA in 164 biopsies. OSNA results were compared with the conventional histopathology study of 244 patients who had undergone SLN biopsy previously. The characteristics of the patients in both groups were evaluated and a comparison was made of the rate of metastases detected by both methods and of the surgical procedures needed in each group. We also investigated the state of non-sentinel lymph nodes if micrometastases where found in SLN. SLN biopsy result was considered as positive in 45 patients (28%) in the OSNA group and in 58 in the historical group (24%). There was no difference in the rate of macrometastases (16,5% for OSNA, 20% for HE) but we found differences in the rate of micrometastases (11% for OSNA and 3,6% for HE p = 0.0007). Axillary lymphadenectomy (ALND) was performed in 43/45 cases in the OSNA group and in 51/58 of the historical group. In all patients diagnosed by OSNA, ALND was performed during the initial surgical procedure. In the historical cohort ALND was performed during the initial surgical procedure in 41 patients and in a second surgical procedure in 10 patients. Patients from both groups with micrometastases in the SLN had no metastases in other nodes when the ALND was performed. OSNA analysis allows the detection of SLN metastases as precisely as conventional pathology with an increased detection of micrometastases. The OSNA method can reduce the need of a deferred lymphadenectomy.

  3. Nucleic acid amplification using microfluidic systems.

    PubMed

    Chang, Chen-Min; Chang, Wen-Hsin; Wang, Chih-Hung; Wang, Jung-Hao; Mai, John D; Lee, Gwo-Bin

    2013-04-07

    In the post-human-genome-project era, the development of molecular diagnostic techniques has advanced the frontiers of biomedical research. Nucleic-acid-based technology (NAT) plays an especially important role in molecular diagnosis. However, most research and clinical protocols still rely on the manual analysis of individual samples by skilled technicians which is a time-consuming and labor-intensive process. Recently, with advances in microfluidic designs, integrated micro total-analysis-systems have emerged to overcome the limitations of traditional detection assays. These microfluidic systems have the capability to rapidly perform experiments in parallel and with a high-throughput which allows a NAT analysis to be completed in a few hours or even a few minutes. These features have a significant beneficial influence on many aspects of traditional biological or biochemical research and this new technology is promising for improving molecular diagnosis. Thus, in the foreseeable future, microfluidic systems developed for molecular diagnosis using NAT will become an important tool in clinical diagnosis. One of the critical issues for NAT is nucleic acid amplification. In this review article, recent advances in nucleic acid amplification techniques using microfluidic systems will be reviewed. Different approaches for fast amplification of nucleic acids for molecular diagnosis will be highlighted.

  4. A paper-based resonance energy transfer nucleic acid hybridization assay using upconversion nanoparticles as donors and quantum dots as acceptors.

    PubMed

    Doughan, Samer; Uddayasankar, Uvaraj; Krull, Ulrich J

    2015-06-09

    Monodisperse aqueous upconverting nanoparticles (UCNPs) were covalently immobilized on aldehyde modified cellulose paper via reduction amination to develop a luminescence resonance energy transfer (LRET)-based nucleic acid hybridization assay. This first account of covalent immobilization of UCNPs on paper for a bioassay reports an optically responsive method that is sensitive, reproducible and robust. The immobilized UCNPs were decorated with oligonucleotide probes to capture HPRT1 housekeeping gene fragments, which in turn brought reporter conjugated quantum dots (QDs) in close proximity to the UCNPs for LRET. This sandwich assay could detect unlabeled oligonucleotide target, and had a limit of detection of 13 fmol and a dynamic range spanning nearly 3 orders of magnitude. The use of QDs, which are excellent LRET acceptors, demonstrated improved sensitivity, limit of detection, dynamic range and selectivity compared to similar assays that have used molecular fluorophores as acceptors. The selectivity of the assay was attributed to the decoration of the QDs with polyethylene glycol to eliminate non-specific adsorption. The kinetics of hybridization were determined to be diffusion limited and full signal development occurred within 3 min.

  5. A Paper-Based Sandwich Format Hybridization Assay for Unlabeled Nucleic Acid Detection Using Upconversion Nanoparticles as Energy Donors in Luminescence Resonance Energy Transfer

    PubMed Central

    Zhou, Feng; Noor, M. Omair; Krull, Ulrich J.

    2015-01-01

    Bioassays based on cellulose paper substrates are gaining increasing popularity for the development of field portable and low-cost diagnostic applications. Herein, we report a paper-based nucleic acid hybridization assay using immobilized upconversion nanoparticles (UCNPs) as donors in luminescence resonance energy transfer (LRET). UCNPs with intense green emission served as donors with Cy3 dye as the acceptor. The avidin functionalized UCNPs were immobilized on cellulose paper and subsequently bioconjugated to biotinylated oligonucleotide probes. Introduction of unlabeled oligonucleotide targets resulted in a formation of probe-target duplexes. A subsequent hybridization of Cy3 labeled reporter with the remaining single stranded portion of target brought the Cy3 dye in close proximity to the UCNPs to trigger a LRET-sensitized emission from the acceptor dye. The hybridization assays provided a limit of detection (LOD) of 146.0 fmol and exhibited selectivity for one base pair mismatch discrimination. The assay was functional even in undiluted serum samples. This work embodies important progress in developing DNA hybridization assays on paper. Detection of unlabeled targets is achieved using UCNPs as LRET donors, with minimization of background signal from paper substrates owing to the implementation of low energy near-infrared (NIR) excitation. PMID:28347081

  6. Yellow lupine cyclophilin interacts with nucleic acids.

    PubMed

    Nuc, Katarzyna; Leśniewicz, Krzysztof; Nuc, Przemysław; Słomski, Ryszard

    2008-01-01

    To investigate properties of yellow lupine cytosolic cyclophilin, an expression vector pET15CYP was constructed. The CyP cDNA (GenBank accession no.Y16088) reveals an open reading frame of 172 amino acids with the conserved tryptophan residue at position 128 and an insertion of seven amino acids spanning positions 48-54. Yellow lupine cyclophilin, purified after expression in E. coli cells, exhibits peptidyl-prolyl cis/trans isomerase activity when assayed with a synthetic oligopeptide. We have demonstrated that the recombinant cyclophilin is able to interact with nucleic acids, both single and double stranded DNA fragments as well as RNA.

  7. A sensitive SERS assay for detecting proteins and nucleic acids using a triple-helix molecular switch for cascade signal amplification.

    PubMed

    Ye, Sujuan; Wu, Yanying; Zhang, Wen; Li, Na; Tang, Bo

    2014-08-25

    A novel surface-enhanced Raman scattering (SERS) detection system is developed for proteins and nucleic acids based on a triple-helix molecular switch for multiple cycle signal amplification, achieving high sensitivity, universality, rapid analysis, and high selectivity.

  8. 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.

  9. 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.

  10. 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.

  11. Nucleic acid based logical systems.

    PubMed

    Han, Da; Kang, Huaizhi; Zhang, Tao; Wu, Cuichen; Zhou, Cuisong; You, Mingxu; Chen, Zhuo; Zhang, Xiaobing; Tan, Weihong

    2014-05-12

    Researchers increasingly visualize a significant role for artificial biochemical logical systems in biological engineering, much like digital logic circuits in electrical engineering. Those logical systems could be utilized as a type of servomechanism to control nanodevices in vitro, monitor chemical reactions in situ, or regulate gene expression in vivo. Nucleic acids (NA), as carriers of genetic information with well-regulated and predictable structures, are promising materials for the design and engineering of biochemical circuits. A number of logical devices based on nucleic acids (NA) have been designed to handle various processes for technological or biotechnological purposes. This article focuses on the most recent and important developments in NA-based logical devices and their evolution from in vitro, through cellular, even towards in vivo biological applications.

  12. Immunological detection of nucleic acids and antibodies to nucleic acids and nuclear antigens by counterimmunoelectrophoresis

    PubMed Central

    Schur, P. H.; De Angelis, Diane; Jackson, Jean M.

    1974-01-01

    A counterimmunoelectrophoresis (CIEP) technique has been developed for the rapid, simple, specific detection of nucleic acids as antigens, or for the detection of precipitating antibodies to nucleic acids or nuclear antigens. The majority of precipitins could be detected within 1 hr. As little as 0·0015 μg of antigen per ml (e.g. poly A: poly U) could be detected. Specificity of rabbit antisera to nucleic acids was demonstrated by selective reactions using a panel of polynucleotides. 1091 patient sera were examined for precipitins to DNA, single-stranded DNA, nucleoprotein and calf thymus nucleoprotein. Precipitins to DNA were found in 42% of systemic lupus erythematosus sera, 9% of rheumatoid arthritis sera and 4% of juvenile rheumatoid arthritis sera. Results with the CIEP method showed equal sensitivity as results obtained by complement fixation or binding assays, but were more sensitive than double diffusion in agar (Ouchterlony). PMID:4549570

  13. Nucleic acid detection using MNAzymes.

    PubMed

    Gerasimova, Yulia V; Kolpashchikov, Dmitry M

    2010-02-26

    Deoxyribozymes are promising biotechnological tools. In a recent JACS article, Mokany et al. reported on the design of multi-component deoxyribozyme (MNAzyme) sensors based on 10-23 and 8-17 DNA enzymes. The sensors can detect down to 5 pM of a specific nucleic acid. The versatility of MNAzyme platform allows the design of catalytic cascades for signal amplification. This work is a step forward to PCR-free molecular diagnostics. Copyright 2010 Elsevier Ltd. All rights reserved.

  14. On-chip multiplexed solid-phase nucleic acid hybridization assay using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer.

    PubMed

    Noor, M Omair; Tavares, Anthony J; Krull, Ulrich J

    2013-07-25

    A microfluidic based solid-phase assay for the multiplexed detection of nucleic acid hybridization using quantum dot (QD) mediated fluorescence resonance energy transfer (FRET) is described herein. The glass surface of hybrid glass-polydimethylsiloxane (PDMS) microfluidic channels was chemically modified to assemble the biorecognition interface. Multiplexing was demonstrated using a detection system that was comprised of two colors of immobilized semi-conductor QDs and two different oligonucleotide probe sequences. Green-emitting and red-emitting QDs were paired with Cy3 and Alexa Fluor 647 (A647) labeled oligonucleotides, respectively. The QDs served as energy donors for the transduction of dye labeled oligonucleotide targets. The in-channel assembly of the biorecognition interface and the subsequent introduction of oligonucleotide targets was accomplished within minutes using a combination of electroosmotic flow and electrophoretic force. The concurrent quantification of femtomole quantities of two target sequences was possible by measuring the spatial coverage of FRET sensitized emission along the length of the channel. In previous reports, multiplexed QD-FRET hybridization assays that employed a ratiometric method for quantification had challenges associated with lower analytical sensitivity arising from both donor and acceptor dilution that resulted in reduced energy transfer pathways as compared to single-color hybridization assays. Herein, a spatial method for quantification that is based on in-channel QD-FRET profiles provided higher analytical sensitivity in the multiplexed assay format as compared to single-color hybridization assays. The selectivity of the multiplexed hybridization assays was demonstrated by discrimination between a fully-complementary sequence and a 3 base pair sequence at a contrast ratio of 8 to 1.

  15. Paper-based solid-phase multiplexed nucleic acid hybridization assay with tunable dynamic range using immobilized quantum dots as donors in fluorescence resonance energy transfer.

    PubMed

    Noor, M Omair; Krull, Ulrich J

    2013-08-06

    A multiplexed solid-phase nucleic acid hybridization assay on a paper-based platform is presented using multicolor immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). The surface of paper was modified with imidazole groups to immobilize two types of QD-probe oligonucleotide conjugates that were assembled in solution. Green-emitting QDs (gQDs) and red-emitting QDs (rQDs) served as donors with Cy3 and Alexa Fluor 647 (A647) acceptors. The gQD/Cy3 FRET pair served as an internal standard, while the rQD/A647 FRET pair served as a detection channel, combining the control and analytical test zones in one physical location. Hybridization of dye-labeled oligonucleotide targets provided the proximity for FRET sensitized emission from the acceptor dyes, which served as an analytical signal. Hybridization assays in the multicolor format provided a limit of detection of 90 fmol and an upper limit of dynamic range of 3.5 pmol. The use of an array of detection zones was designed to provide improved analytical figures of merit compared to that which could be achieved on one type of array design in terms of relative concentration of multicolor QDs. The hybridization assays showed excellent resistance to nonspecific adsorption of oligonucleotides. Selectivity of the two-plex hybridization assay was demonstrated by single nucleotide polymorphism (SNP) detection at a contrast ratio of 50:1. Additionally, it is shown that the use of preformed QD-probe oligonucleotide conjugates and consideration of the relative number density of the two types of QD-probe conjugates in the two-color assay format is advantageous to maximize assay sensitivity and the upper limit of dynamic range.

  16. Nucleic acid delivery with microbubbles and ultrasound

    PubMed Central

    Rychak, Joshua J.; Klibanov, Alexander L.

    2014-01-01

    Nucleic acid-based therapy is a growing field of drug delivery research. Although ultrasound has been suggested to enhance transfection decades ago, it took a combination of ultrasound with nucleic acid carrier systems (microbubbles, liposomes, polyplexes, viral carriers) to achieve reasonable nucleic acid delivery efficacy. Microbubbles serve as foci for local deposition of ultrasound energy near the target cell, and greatly enhance sonoporation. Major advantage of this approach is in the minimal transfection in the non-insonated non-target tissues. Microbubbles can be simply co-administered with the nucleic acid carrier or can be modified to carry nucleic acid themselves. Liposomes with embedded gas or gas precursor particles can also be used to carry nucleic acid, release and deliver it by the ultrasound trigger. Successful testing in a wide variety of animal models (myocardium, solid tumors, skeletal muscle, pancreas) proves the potential usefulness of this technique for nucleic acid drug delivery. PMID:24486388

  17. Allele Specific Locked Nucleic Acid Quantitative PCR (ASLNAqPCR): An Accurate and Cost-Effective Assay to Diagnose and Quantify KRAS and BRAF Mutation

    PubMed Central

    Morandi, Luca; de Biase, Dario; Visani, Michela; Cesari, Valentina; De Maglio, Giovanna; Pizzolitto, Stefano; Pession, Annalisa; Tallini, Giovanni

    2012-01-01

    The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes. PMID:22558339

  18. Toward an on-chip multiplexed nucleic acid hybridization assay using immobilized quantum dot-oligonucleotide conjugates and fluorescence resonance energy transfer

    NASA Astrophysics Data System (ADS)

    Tavares, Anthony J.; Noor, M. Omair; Algar, W. Russ; Vannoy, Charles H.; Chen, Lu; Krull, Ulrich J.

    2011-03-01

    Semiconductor quantum dots (QD) are a class of NP with photophysical properties that are ideally suited for optical multiplexing and use as donors in fluorescence resonance energy transfer (FRET). A new strategy is presented for the development of multiplexed DNA hybridization assays using immobilized QDs in a microfluidic system. Green- or red-emitting QDs were immobilized via self-assembly with a multidentate-thiol-derivatized glass slide, and subsequently conjugated with amine-terminated probe oligonucleotides using carbodiimide activation. Immobilized QD-probe conjugates were then passivated with adsorbed non-complementary oligonucleotides to achieve selectivity in microfluidic assays. Target nucleic acid sequences hybridized with QD-probe conjugates and were labeled with Cy3 or Alexa Fluor 647 as acceptor dyes for the QD donors, where FRET-sensitized dye emission provided a signal for the detection of picomolar quantities of target. The simultaneous immobilization of green- and red-emitting QDs at different ratios within a microfluidic channel was demonstrated as a step toward multiplexed assays.

  19. Interfacial Chemistry and the Design of Solid-Phase Nucleic Acid Hybridization Assays Using Immobilized Quantum Dots as Donors in Fluorescence Resonance Energy Transfer

    PubMed Central

    Algar, W. Russ; Krull, Ulrich J.

    2011-01-01

    The use of quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET) offer several advantages for the development of multiplexed solid-phase QD-FRET nucleic acid hybridization assays. Designs for multiplexing have been demonstrated, but important challenges remain in the optimization of these systems. In this work, we identify several strategies based on the design of interfacial chemistry for improving sensitivity, obtaining lower limits of detection (LOD) and enabling the regeneration and reuse of solid-phase QD-FRET hybridization assays. FRET-sensitized emission from acceptor dyes associated with hybridization events at immobilized QD donors provides the analytical signal in these assays. The minimization of active sensing area reduces background from QD donor PL and allows the resolution of smaller amounts of acceptor emission, thus lowering the LOD. The association of multiple acceptor dyes with each hybridization event can enhance FRET efficiency, thereby improving sensitivity. Many previous studies have used interfacial protein layers to generate selectivity; however, transient destabilization of these layers is shown to prevent efficient regeneration. To this end, we report a protein-free interfacial chemistry and demonstrate the specific detection of as little as 2 pmol of target, as well as an improved capacity for regeneration. PMID:22163951

  20. Interfacial chemistry and the design of solid-phase nucleic acid hybridization assays using immobilized quantum dots as donors in fluorescence resonance energy transfer.

    PubMed

    Algar, W Russ; Krull, Ulrich J

    2011-01-01

    The use of quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET) offer several advantages for the development of multiplexed solid-phase QD-FRET nucleic acid hybridization assays. Designs for multiplexing have been demonstrated, but important challenges remain in the optimization of these systems. In this work, we identify several strategies based on the design of interfacial chemistry for improving sensitivity, obtaining lower limits of detection (LOD) and enabling the regeneration and reuse of solid-phase QD-FRET hybridization assays. FRET-sensitized emission from acceptor dyes associated with hybridization events at immobilized QD donors provides the analytical signal in these assays. The minimization of active sensing area reduces background from QD donor PL and allows the resolution of smaller amounts of acceptor emission, thus lowering the LOD. The association of multiple acceptor dyes with each hybridization event can enhance FRET efficiency, thereby improving sensitivity. Many previous studies have used interfacial protein layers to generate selectivity; however, transient destabilization of these layers is shown to prevent efficient regeneration. To this end, we report a protein-free interfacial chemistry and demonstrate the specific detection of as little as 2 pmol of target, as well as an improved capacity for regeneration.

  1. Rapid hybridization of nucleic acids using isotachophoresis

    PubMed Central

    Bercovici, Moran; Han, Crystal M.; Liao, Joseph C.; Santiago, Juan G.

    2012-01-01

    We use isotachophoresis (ITP) to control and increase the rate of nucleic acid hybridization reactions in free solution. We present a new physical model, validation experiments, and demonstrations of this assay. We studied the coupled physicochemical processes of preconcentration, mixing, and chemical reaction kinetics under ITP. Our experimentally validated model enables a closed form solution for ITP-aided reaction kinetics, and reveals a new characteristic time scale which correctly predicts order 10,000-fold speed-up of chemical reaction rate for order 100 pM reactants, and greater enhancement at lower concentrations. At 500 pM concentration, we measured a reaction time which is 14,000-fold lower than that predicted for standard second-order hybridization. The model and method are generally applicable to acceleration of reactions involving nucleic acids, and may be applicable to a wide range of reactions involving ionic reactants. PMID:22733732

  2. Rapid detection of equine influenza virus H3N8 subtype by insulated isothermal RT-PCR (iiRT-PCR) assay using the POCKIT™ Nucleic Acid Analyzer.

    PubMed

    Balasuriya, Udeni B R; Lee, Pei-Yu Alison; Tiwari, Ashish; Skillman, Ashley; Nam, Bora; Chambers, Thomas M; Tsai, Yun-Long; Ma, Li-Juan; Yang, Pai-Chun; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas

    2014-10-01

    horses. Therefore, the EIV H3N8 subtype specific iiRT-PCR assay along with the portable POCKIT™ Nucleic Acid Analyzer provides a highly reliable, sensitive and specific on-site detection system of both equine and canine influenza viruses.

  3. Translation of a laboratory-validated equine herpesvirus-1 specific real-time PCR assay into an insulated isothermal polymerase chain reaction (iiPCR) assay for point-of-need diagnosis using POCKIT™ nucleic acid analyzer.

    PubMed

    Balasuriya, Udeni B R; Lee, Pei-Yu Alison; Tsai, Yun-Long; Tsai, Chuan-Fu; Shen, Yu-Han; Chang, Hsiao-Fen Grace; Skillman, Ashley; Wang, Hwa-Tang Thomas; Pronost, Stéphane; Zhang, Yan

    2017-03-01

    Equine herpesvirus myeloencephalopathy (EHM), a major problem for the equine industry in the United States, is caused by equine herpesvirus-1 (EHV-1). In addition, EHV-1 is associated with upper respiratory disease, abortion, and chorioretinal lesions in horses. Here we describe the development and evaluation of an inexpensive, user-friendly insulated isothermal PCR (iiPCR) method targeting open reading 30 (ORF30) to detect both neuropathogenic and non-neuropathogenic strains on the field-deployable POCKIT™ device for point-of-need detection of EHV-1. The analytical sensitivity of the EHV-1 iiPCR assay was 13 genome equivalents per reaction. The assay did not cross react with ten non-target equine viral pathogens. Performance of the EHV-1 iiPCR assay was compared to two previously described real-time PCR (qPCR) assays in two laboratories by using 104 archived clinical samples. All 53 qPCR-positive and 46 of the 51 qPCR-negative samples tested positive and negative, respectively, by the iiPCR. The agreement between the two assays was 95.19% (confidence interval 90.48-99.90%) with a kappa value of 0.90. In conclusion, the newly developed EHV-1 iiPCR assay is robust to provide specificity and sensitivity comparable to qPCR assays for the detection of EHV-1 nucleic acid in clinical specimens.

  4. Treatment of cytomegalovirus infection and disease pre- and post-quantitative nucleic acid test standardization: does use of a more sensitive assay lead to longer treatment duration?

    PubMed

    Dioverti, M Veronica; Lahr, Brian; Razonable, Raymund R

    2016-02-01

    Quantitative cytomegalovirus (CMV) nucleic acid testing (NAT) has been standardized using the World Health Organization (WHO) international calibration standard. A new FDA-approved WHO-calibrated assay (CA) was found to be more sensitive than a laboratory-developed test (LDT). We hypothesized that monitoring therapeutic response using a more sensitive assay may lead to longer antiviral therapy in solid organ and hematopoietic stem cell transplant patients with CMV infection. We reviewed transplant patients with CMV disease retrospectively, and divided them into two groups: those diagnosed and managed based on LDT and those managed using WHO-CA. Compared to patients monitored by LDT, the time to reach an undetectable viral load was significantly longer in the group monitored by the WHO-CA. However, a trend toward shorter duration of antiviral treatment was observed (median, 34 vs. 41 d; p = 0.058), with earlier discontinuation of induction antiviral therapy upon reaching undetectable viral load using WHO-CA (11 vs. 18 d; p = 002). We concluded that despite using a more sensitive CMV NAT, the total duration of antiviral treatment was not significantly prolonged in transplant patients with CMV infection and disease. Relapse rates did not differ between the two groups. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Establishment of the 1st World Health Organization International Standard for Plasmodium falciparum DNA for nucleic acid amplification technique (NAT)-based assays

    PubMed Central

    Padley, David J; Heath, Alan B; Sutherland, Colin; Chiodini, Peter L; Baylis, Sally A

    2008-01-01

    Background In order to harmonize results for the detection and quantification of Plasmodium falciparum DNA by nucleic acid amplification technique (NAT)-based assays, a World Health Organization (WHO) collaborative study was performed, evaluating a series of candidate standard preparations. Methods Fourteen laboratories from 10 different countries participated in the collaborative study. Four candidate preparations based upon blood samples parasitaemic for P. falciparum were evaluated in the study. Sample AA was lyophilized, whilst samples BB, CC and DD were liquid/frozen preparations. The candidate standards were tested by each laboratory at a range of dilutions in four independent assays, using both qualitative and quantitative NAT-based assays. The results were collated and analysed statistically. Results Twenty sets of data were returned from the participating laboratories and used to determine the mean P. falciparum DNA content for each sample. The mean log10 "equivalents"/ml were 8.51 for sample AA, 8.45 for sample BB, 8.35 for sample CC, and 5.51 for sample DD. The freeze-dried preparation AA, was examined by accelerated thermal degradation studies and found to be highly stable. Conclusion On the basis of the collaborative study, the freeze-dried material, AA (NIBSC code No. 04/176) was established as the 1st WHO International Standard for P. falciparum DNA NAT-based assays and has been assigned a potency of 109 International Units (IU) per ml. Each vial contains 5 × 108 IU, equivalent to 0.5 ml of material after reconstitution. PMID:18652656

  6. A Locked Nucleic Acid (LNA)-Based Real-Time PCR Assay for the Rapid Detection of Multiple Bacterial Antibiotic Resistance Genes Directly from Positive Blood Culture

    PubMed Central

    Zhu, Lingxiang; Shen, Dingxia; Zhou, Qiming; Li, Zexia; Fang, Xiangdong; Li, Quan-Zhen

    2015-01-01

    Bacterial strains resistant to various antibiotic drugs are frequently encountered in clinical infections, and the rapid identification of drug-resistant strains is highly essential for clinical treatment. We developed a locked nucleic acid (LNA)-based quantitative real-time PCR (LNA-qPCR) method for the rapid detection of 13 antibiotic resistance genes and successfully used it to distinguish drug-resistant bacterial strains from positive blood culture samples. A sequence-specific primer-probe set was designed, and the specificity of the assays was assessed using 27 ATCC bacterial strains and 77 negative blood culture samples. No cross-reaction was identified among bacterial strains and in negative samples, indicating 100% specificity. The sensitivity of the assays was determined by spiking each bacterial strain into negative blood samples, and the detection limit was 1–10 colony forming units (CFU) per reaction. The LNA-qPCR assays were first applied to 72 clinical bacterial isolates for the identification of known drug resistance genes, and the results were verified by the direct sequencing of PCR products. Finally, the LNA-qPCR assays were used for the detection in 47 positive blood culture samples, 19 of which (40.4%) were positive for antibiotic resistance genes, showing 91.5% consistency with phenotypic susceptibility results. In conclusion, LNA-qPCR is a reliable method for the rapid detection of bacterial antibiotic resistance genes and can be used as a supplement to phenotypic susceptibility testing for the early detection of antimicrobial resistance to allow the selection of appropriate antimicrobial treatment and to prevent the spread of resistant isolates. PMID:25775001

  7. A locked nucleic acid (LNA)-based real-time PCR assay for the rapid detection of multiple bacterial antibiotic resistance genes directly from positive blood culture.

    PubMed

    Zhu, Lingxiang; Shen, Dingxia; Zhou, Qiming; Li, Zexia; Fang, Xiangdong; Li, Quan-Zhen

    2015-01-01

    Bacterial strains resistant to various antibiotic drugs are frequently encountered in clinical infections, and the rapid identification of drug-resistant strains is highly essential for clinical treatment. We developed a locked nucleic acid (LNA)-based quantitative real-time PCR (LNA-qPCR) method for the rapid detection of 13 antibiotic resistance genes and successfully used it to distinguish drug-resistant bacterial strains from positive blood culture samples. A sequence-specific primer-probe set was designed, and the specificity of the assays was assessed using 27 ATCC bacterial strains and 77 negative blood culture samples. No cross-reaction was identified among bacterial strains and in negative samples, indicating 100% specificity. The sensitivity of the assays was determined by spiking each bacterial strain into negative blood samples, and the detection limit was 1-10 colony forming units (CFU) per reaction. The LNA-qPCR assays were first applied to 72 clinical bacterial isolates for the identification of known drug resistance genes, and the results were verified by the direct sequencing of PCR products. Finally, the LNA-qPCR assays were used for the detection in 47 positive blood culture samples, 19 of which (40.4%) were positive for antibiotic resistance genes, showing 91.5% consistency with phenotypic susceptibility results. In conclusion, LNA-qPCR is a reliable method for the rapid detection of bacterial antibiotic resistance genes and can be used as a supplement to phenotypic susceptibility testing for the early detection of antimicrobial resistance to allow the selection of appropriate antimicrobial treatment and to prevent the spread of resistant isolates.

  8. Double stranded nucleic acid biochips

    DOEpatents

    Chernov, Boris; Golova, Julia

    2006-05-23

    This invention describes a new method of constructing double-stranded DNA (dsDNA) microarrays based on the use of pre-synthesized or natural DNA duplexes without a stem-loop structure. The complementary oligonucleotide chains are bonded together by a novel connector that includes a linker for immobilization on a matrix. A non-enzymatic method for synthesizing double-stranded nucleic acids with this novel connector enables the construction of inexpensive and robust dsDNA/dsRNA microarrays. DNA-DNA and DNA-protein interactions are investigated using the microarrays.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. Multiplexed microfluidic approach for nucleic acid enrichment

    SciTech Connect

    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.

  14. Magnetic nanoparticles for high-sensitivity detection on nucleic acids via superconducting-quantum-interference-device-based immunomagnetic reduction assay

    NASA Astrophysics Data System (ADS)

    Yang, S. Y.; Chieh, J. J.; Wang, W. C.; Yu, C. Y.; Hing, N. S.; Horng, H. E.; Hong, Chin-Yih; Yang, H. C.; Chang, C. F.; Lin, H. Y.

    2011-03-01

    In this work, we investigate the feasibility of detecting quantitatively DNA molecules utilizing the technology named after the immunomagnetic reduction (IMR) assay. Magnetic nanoparticles dispersed in a phosphate buffer saline solution were bio-functionalized with probing single-strand DNA. A superconducting quantum interference device (SQUID) ac magnetosusceptometer was employed to detect IMR signals related to the concentration of the target DNA. The results reveal that use of IMR assay had merits such as a high convenience level, e.g. wash-free processes and high sensitivity, down to pM, for DNA detection.

  15. Do nucleic acids moonlight as molecular chaperones?

    PubMed

    Docter, Brianne E; Horowitz, Scott; Gray, Michael J; Jakob, Ursula; Bardwell, James C A

    2016-06-02

    Organisms use molecular chaperones to combat the unfolding and aggregation of proteins. While protein chaperones have been widely studied, here we demonstrate that DNA and RNA exhibit potent chaperone activity in vitro Nucleic acids suppress the aggregation of classic chaperone substrates up to 300-fold more effectively than the protein chaperone GroEL. Additionally, RNA cooperates with the DnaK chaperone system to refold purified luciferase. Our findings reveal a possible new role for nucleic acids within the cell: that nucleic acids directly participate in maintaining proteostasis by preventing protein aggregation. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. Indicator-based and indicator-free magnetic assays connected with disposable electrochemical nucleic acid sensor system.

    PubMed

    Karadeniz, Hakan; Erdem, Arzum; Kuralay, Filiz; Jelen, Frantisek

    2009-04-15

    An indicator-based and indicator-free magnetic assays connected with a disposable pencil graphite electrode (PGE) were successfully developed, and also compared for the electrochemical detection of DNA hybridization. The oxidation signals of echinomycin (ECHI) and electroactive DNA bases, guanine and adenine, respectively were monitored in the presence of DNA hybridization by using differential pulse voltammetry (DPV) technique. The biotinylated probe was immobilized onto the magnetic beads (magnetic particles, microspheres) and hybridization with its complementary target at the surface of particles within the medium was exhibited successfully using electrochemical sensor system. For the selectivity studies, the results represent that both indicator-based and indicator-free magnetic assays provide a better discrimination for DNA hybridization compared to duplex with one-base or more mismatches. The detection limits (S/N=3) of the magnetic assays based on indicator or indicator-free were found in nM concentration level of target using disposable sensor technology with good reproducibility. The characterization and advantages of both proposed magnetic assays connected with a disposable electrochemical sensor are also discussed and compared with those methods previously reported in the literature.

  17. Multiplexed detection of fungal nucleic acid signatures.

    PubMed

    Diaz, Mara R; Dunbar, Sherry A; Jacobson, James W

    2008-04-01

    Diagnoses of opportunistic mycotic infections constitute an increasing clinical problem. Conventional diagnostic tests are time consuming and lack specificity and sensitivity for accurate and timely prognoses. This unit provides a comprehensive description of a fungal detection method that combines nucleic acid signatures with flow cytometry. The multiplexed assay, which uses xMAP technology, consists of unique fluorescent microspheres covalently bound to species-specific fungal oligonucleotide probes. In the presence of the complementary target sequence, the probe hybridizes to its biotinylated target. Quantification of the reaction is based on the fluorescence signal of the reporter molecule that binds to the biotin moieties of the target. The assay can be expanded to include other microorganisms and has the capability to simultaneously test 100 different fungal probes per tube/well. The speed, flexibility in design, and high-throughput capability makes this assay an attractive diagnostic tool for fungal infections and other related maladies.

  18. [Viral safety of biologicals: evaluation of hepatitis C virus (HCV) nucleic acid amplification test (NAT) assay and development of concentration method of HCV for sensitive detection by NAT].

    PubMed

    Uchida, Eriko; Yamaguchi, Teruhide

    2010-02-01

    The most important issue for the safety of biological products and blood products derived from human sources is how to prevent transmission of infectious agents. The hepatitis C virus (HCV) is a major public health problem due to its high prevalence. HCV is mainly transmitted by exposure to blood and highly infectious during the early window period with extremely low viral loads. Therefore it is important to develop more sensitive detection methods for HCV. In the case of blood products, both serological test and nucleic acid amplification test (NAT) are required to detect HCV. Since NAT is highly sensitive, establishment of a new standard is required for validation of NAT assay. NAT guideline and establishment of the standard for HCV RNA and HCV genotype panel is introduced in this review. On the other hand, to enhance the sensitivity of virus detection by NAT, a novel viral concentration method using polyethyleneimine (PEI)-conjugated magnetic beads (PEI beads) was developed. PEI beads concentration method is applicable to a wide range of viruses including HCV. Studies using the national standard for HCV RNA, HCV genotype panel and seroconversion panel, suggest that virus concentration method using PEI-beads is useful for improvement of the sensitivity of HCV detection by NAT and applicable to donor screening for HCV.

  19. Unravelling the Bacterial Vaginosis-Associated Biofilm: A Multiplex Gardnerella vaginalis and Atopobium vaginae Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes

    PubMed Central

    Hardy, Liselotte; Jespers, Vicky; Dahchour, Nassira; Mwambarangwe, Lambert; Musengamana, Viateur; Vaneechoutte, Mario; Crucitti, Tania

    2015-01-01

    Bacterial vaginosis (BV), a condition defined by increased vaginal discharge without significant inflammation, is characterized by a change in the bacterial composition of the vagina. Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms. These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment. In this study, we examined the presence of Gardnerella vaginalis and Atopobium vaginae in vaginal biofilms using Peptide Nucleic Acid (PNA) probes targeting these bacteria. For this purpose, we developed three new PNA probes for A. vaginae. The most specific A. vaginae probe, AtoITM1, was selected and then used in an assay with two existing probes, Gard162 and BacUni-1, to evaluate multiplex FISH on clinical samples. Using quantitative polymerase chain reaction (qPCR) as the gold standard, we demonstrated a sensitivity of 66.7% (95% confidence interval: 54.5% - 77.1%) and a specificity of 89.4% (95% confidence interval: 76.1% - 96%) of the new AtoITM1 probe. FISH enabled us to show the presence of a polymicrobial biofilm in bacterial vaginosis, in which Atopobium vaginae is part of a Gardnerella vaginalis-dominated biofilm. We showed that the presence of this biofilm is associated with high bacterial loads of A. vaginae and G. vaginalis. PMID:26305575

  20. Unravelling the Bacterial Vaginosis-Associated Biofilm: A Multiplex Gardnerella vaginalis and Atopobium vaginae Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes.

    PubMed

    Hardy, Liselotte; Jespers, Vicky; Dahchour, Nassira; Mwambarangwe, Lambert; Musengamana, Viateur; Vaneechoutte, Mario; Crucitti, Tania

    2015-01-01

    Bacterial vaginosis (BV), a condition defined by increased vaginal discharge without significant inflammation, is characterized by a change in the bacterial composition of the vagina. Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms. These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment. In this study, we examined the presence of Gardnerella vaginalis and Atopobium vaginae in vaginal biofilms using Peptide Nucleic Acid (PNA) probes targeting these bacteria. For this purpose, we developed three new PNA probes for A. vaginae. The most specific A. vaginae probe, AtoITM1, was selected and then used in an assay with two existing probes, Gard162 and BacUni-1, to evaluate multiplex FISH on clinical samples. Using quantitative polymerase chain reaction (qPCR) as the gold standard, we demonstrated a sensitivity of 66.7% (95% confidence interval: 54.5% - 77.1%) and a specificity of 89.4% (95% confidence interval: 76.1% - 96%) of the new AtoITM1 probe. FISH enabled us to show the presence of a polymicrobial biofilm in bacterial vaginosis, in which Atopobium vaginae is part of a Gardnerella vaginalis-dominated biofilm. We showed that the presence of this biofilm is associated with high bacterial loads of A. vaginae and G. vaginalis.

  1. Visual detection and differentiation of Classic Swine Fever Virus strains using nucleic acid sequence-based amplification (NASBA) and G-quadruplex DNAzyme assay

    PubMed Central

    Lu, Xiaolu; Shi, Xueyao; Wu, Gege; Wu, Tiantian; Qin, Rui; Wang, Yi

    2017-01-01

    The split G-quadruplex DNAzyme has emerged as a valuable tool for visual DNA detection. Here, we successfully integrated colorimetric split G-quadruplex DNAzyme assay with nucleic acid sequence-based amplification to generate a novel detection approach, allowing visual and rapid detection for the RNA of Shimen and HCLV strains of Classic Swine Fever Virus (CSFV). CSFV is a RNA virus that causes a highly contagious disease in domestic pigs and wild boar. With this method, we were able to detect as little as 10 copies/ml of CSF viral RNA within 3 h in serum samples taken from the field. No interference was encountered in the amplification and detection of Classic Swine Fever Virus in the presence of non-target RNA or DNA. Moreover, Shimen and HCLV strains of Classic Swine Fever Virus could be easily differentiated using the NASBA-DNAzyme system. These findings indicate the NASBA-DNAzyme system is a rapid and practical technique for detecting and discriminating CSFV strains and may be applied to the detection of other RNA viruses. PMID:28287135

  2. Molecular modeling of nucleic acid structure

    PubMed Central

    Galindo-Murillo, Rodrigo; Bergonzo, Christina

    2013-01-01

    This unit is the first in a series of four units covering the analysis of nucleic acid structure by molecular modeling. This unit provides an overview of computer simulation of nucleic acids. Topics include the static structure model, computational graphics and energy models, generation of an initial model, and characterization of the overall three-dimensional structure. PMID:18428873

  3. Building biologically active nucleic acid nanocomplexes.

    PubMed

    Smith, C I Edvard; Lundin, Karin E; Simonson, Oscar E; Moreno, Pedro M D; Svahn, Mathias G; Wenska, Malgorzata; Strömberg, Roger

    2008-01-01

    The Bioplex technology allows the hybridization of functional entities to various forms of nucleic acids by the use of synthetic nucleic acid analogs. Such supramolecular assemblies can be made in a predetermined fashion and can confer new properties. The Zorro technology is based on a novel construct generated to simultaneously bind to both DNA strands. Such compounds may have gene silencing activity.

  4. Nucleic acids as therapeutic agents.

    PubMed

    Alvarez-Salas, Luis M

    2008-01-01

    Therapeutic nucleic acids (TNAs) and its precursors are applied to treat several pathologies and infections. TNA-based therapy has different rationales and mechanisms and can be classified into three main groups: 1) Therapeutic nucleotides and nucleosides; 2) Therapeutic oligonucleotides; and 3) Therapeutic polynucleotides. This review will focus in those TNAs that have reached clinical trials with anticancer and antiviral protocols, the two most common applications of TNAs. Although therapeutic nucleotides and nucleosides that interfere with nucleic acid metabolism and DNA polymerization have been successfully used as anticancer and antiviral drugs, they often produce toxic secondary effects related to dosage and continuous use. The use of oligonucleotides such as ribozyme and antisense oligodeoxynucleotides (AS-ODNs) showed promise as therapeutic moieties but faced several issues such as nuclease sensitivity, off-target effects and efficient delivery. Nevertheless, immunostimulatory oligodeoxynucleotides and AS-ODNs represent the most successful group of therapeutic oligonucleotides in the clinic. A newer group of therapeutic oligonucleotides, the aptamers, is rapidly advancing towards early detection and treatment alternatives the have reached the commercial interest. Despite the very high in vitro efficiency of small interfering RNAs (siRNAs) they present issues with intracellular target accessibility, specificity and delivery. DNA vaccines showed great promise, but they resulted in very poor responses in the clinic and further development is uncertain. Despite their many issues, the exquisite specificity and versatility of therapeutic oligonucleotides attracts a great deal of research and resources that will certainly convert them in the TNA of choice for treating cancer and viral diseases in the near future.

  5. A new gravity-driven microfluidic-based electrochemical assay coupled to magnetic beads for nucleic acid detection.

    PubMed

    Laschi, Serena; Miranda-Castro, Rebeca; González-Fernández, Eva; Palchetti, Ilaria; Reymond, Frédéric; Rossier, Joël S; Marrazza, Giovanna

    2010-11-01

    In this work, the characterisation and the optimisation of hybridisation assays based on a novel, rapid and sensitive micro-analytical, gravity-driven, flow device is reported. This device combines a special chip containing eight polymer microchannels, with a portable, computer-controlled instrument. The device is used as a platform for affinity experiments using oligonucleotide-modified paramagnetic particles. In our approach, both hybridisation and labelling events are performed on streptavidin-coated paramagnetic microparticles functionalized with a biotinylated capture probe. Modified particles, introduced in the microchannel inlet of the chip, accumulate near the electrode surface by virtue of a magnetic holder. After hybridisation with the complementary sequence, the hybrid is labelled with an alkaline phosphatase conjugate. The electrochemical substrate for alkaline phosphatase revelation is p-aminophenyl phosphate. Solutions and reagents are sequentially passed through the microchannels, until enzyme substrate is added for in situ signal detection. Upon readout, the magnet array is flipped away, beads are removed by addition of regeneration buffer, and the so-regenerated chip is ready for further analysis. This protocol has been applied to the analytical detection of specific DNA sequences of Legionella pneumophila, with an RSD=8.5% and a detection limit of 0.33 nM.

  6. Recent progress in nucleic acids isotachophoresis.

    PubMed

    Datinská, Vladimíra; Voráčová, Ivona; Schlecht, Ulrich; Berka, Jan; Foret, František

    2017-10-05

    Progress achieved between 2014-2017 in the extraction and sample preparation of nucleic acid by isotachophoresis is reviewed in this paper. The isolation and purification of nucleic acids is very often compromised by a complex matrix such as blood and other bodily fluids, samples from the scene of crime, fossil samples, etc. While most of the common nucleic acids isolation techniques are based on extraction with inherent limitations with regard to quantitative results, isotachophoretic focusing is a quantitative process with a theoretically unlimited concentration factor. Since isotachophoresis belongs to less traditional approaches of nucleic acids purification, we present not only the latest developments in the application of isotachophoresis for the nucleic acids concentration but also a brief description of the principles of this method. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  7. Characterization and Multicentric Validation of a Common Standard for Toxoplasma gondii Detection Using Nucleic Acid Amplification Assays

    PubMed Central

    Varlet-Marie, Emmanuelle; Sterkers, Yvon; Brenier-Pinchart, Marie-Pierre; Cassaing, Sophie; Dalle, Frédéric; Delhaes, Laurence; Filisetti, Denis; Pelloux, Hervé; Touafek, Fériel; Yera, Hélène

    2014-01-01

    The molecular diagnosis of toxoplasmosis essentially relies upon laboratory-developed methods and suffers from lack of standardization, hence the large diversity of performances between laboratories. Moreover, quantifications of parasitic loads differ among centers, a fact which prevents the possible prediction of the severity of this disease as a function of parasitic loads. The objectives of this multicentric study performed in eight proficient laboratories of the Molecular Biology Pole of the French National Reference Center for Toxoplasmosis (NRC-T) were (i) to assess the suitability of a lyophilized preparation of Toxoplasma gondii as a common standard for use in this PCR-based molecular diagnosis and (ii) to make this standard available to the community. High-quality written procedures were used for the production and qualification of this standard. Three independent batches of this standard, containing concentrations ranging from 104 to 0.01 T. gondii genome equivalents per PCR, were first assessed: the linear dynamic range was ≥6 log, the intra-assay coefficients of variation (CV) from a sample containing 10 T. gondii organisms per PCR were 0.3% to 0.42%, and the interassay CV over a 2-week period was 0.76% to 1.47%. A further assessment in eight diagnostic centers showed that the standard is stable, robust, and reliable. These lyophilized standards can easily be produced at a larger scale when needed and can be made widely available at the national level. To our knowledge, this is the first quality control assessment of a common standard which is usable both for self-evaluation in laboratories and for accurate quantification of parasitic loads in T. gondii prenatal infections. PMID:25187637

  8. Characterization and multicentric validation of a common standard for Toxoplasma gondii detection using nucleic acid amplification assays.

    PubMed

    Varlet-Marie, Emmanuelle; Sterkers, Yvon; Brenier-Pinchart, Marie-Pierre; Cassaing, Sophie; Dalle, Frédéric; Delhaes, Laurence; Filisetti, Denis; Pelloux, Hervé; Touafek, Fériel; Yera, Hélène; Bastien, Patrick

    2014-11-01

    The molecular diagnosis of toxoplasmosis essentially relies upon laboratory-developed methods and suffers from lack of standardization, hence the large diversity of performances between laboratories. Moreover, quantifications of parasitic loads differ among centers, a fact which prevents the possible prediction of the severity of this disease as a function of parasitic loads. The objectives of this multicentric study performed in eight proficient laboratories of the Molecular Biology Pole of the French National Reference Center for Toxoplasmosis (NRC-T) were (i) to assess the suitability of a lyophilized preparation of Toxoplasma gondii as a common standard for use in this PCR-based molecular diagnosis and (ii) to make this standard available to the community. High-quality written procedures were used for the production and qualification of this standard. Three independent batches of this standard, containing concentrations ranging from 10(4) to 0.01 T. gondii genome equivalents per PCR, were first assessed: the linear dynamic range was ≥ 6 log, the intra-assay coefficients of variation (CV) from a sample containing 10 T. gondii organisms per PCR were 0.3% to 0.42%, and the interassay CV over a 2-week period was 0.76% to 1.47%. A further assessment in eight diagnostic centers showed that the standard is stable, robust, and reliable. These lyophilized standards can easily be produced at a larger scale when needed and can be made widely available at the national level. To our knowledge, this is the first quality control assessment of a common standard which is usable both for self-evaluation in laboratories and for accurate quantification of parasitic loads in T. gondii prenatal infections.

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

    NASA Technical Reports Server (NTRS)

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

  10. Real-time nucleic acid sequence-based amplification (NASBA) assay targeting MIC1 for detection of Cryptosporidium parvum and Cryptosporidium hominis oocysts.

    PubMed

    Hønsvall, Birgitte K; Robertson, Lucy J

    2017-01-01

    Both Cryptosporidium parvum and Cryptosporidium hominis are often associated with cryptosporidiosis in humans, but whereas humans are the main host for C. hominis, C. parvum is zoonotic and able to infect a variety of species. The oocyst transmission stages of both species of parasites are morphologically identical and molecular techniques, usually polymerase chain reaction (PCR), are required to distinguish between oocysts detected by standard methods in environmental samples, such as water. In this study, we developed two primer sets for real-time nucleic acid sequence-based amplification (NASBA), targeting the MIC1 transcript in C. parvum (CpMIC1) and C. hominis (ChMIC1). Using these primer sets, we were not only able to detect low numbers of C. parvum and C. hominis oocysts (down to 5 oocysts in 10 μl, and down to 1 oocyst using diluted RNA samples), but also distinguish between them. One of the primer sets targeted an exon only occurring in CpMIC1, thereby providing a tool for distinguishing C. parvum from other Cryptosporidium species. Although mRNA has been suggested as a tool for assessing viability of Cryptosporidium oocysts, as it is short-lived and may have high transcription, this NASBA assay detected MIC1 mRNA in inactivated oocysts. RNA within the oocysts seems to be protected from degradation, even when the oocysts have been killed by heating or freeze-thawing. Thus, our approach detects both viable and non-viable oocysts, and RNA does not seem to be a suitable marker for assessing oocyst viability.

  11. An ultrasensitive photoelectrochemical nucleic acid biosensor

    PubMed Central

    Gao, Zhiqiang; Tansil, Natalia C.

    2005-01-01

    A simple and ultrasensitive procedure for non-labeling detection of nucleic acids is described in this study. It is based on the photoelectrochemical detection of target nucleic acids by forming a nucleic acid/photoreporter adduct layer on an ITO electrode. The target nucleic acids were hybridized with immobilized oligonucleotide capture probes on the ITO electrode. A subsequent binding of a photoreporter—a photoactive threading bis-intercalator consisting of two N,N′-bis(3-propyl-imidazole)-1,4,5,8-naphthalene diimides (PIND) linked by a Ru(bpy)22+ (bpy = 2,2′-bipyridine) complex (PIND–Ru–PIND)—allowed for photoelectrochemical detection of the target nucleic acids. The extremely low dissociation rate of the adduct and the highly reversible photoelectrochemical response under visible light illumination (490 nm) make it possible to conduct nucleic acid detection, with a sensitivity enhancement of four orders of magnitude over voltammetry. These results demonstrate for the first time the potential of photoelectrochemical biosensors for PCR-free ultrasensitive detection of nucleic acids. PMID:16061935

  12. 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.

  13. Dendrimers as Nanovectors for Nucleic Acid Delivery

    NASA Astrophysics Data System (ADS)

    Liu, Xiaoxuan; Wang, Qi; Peng, Ling

    2013-09-01

    Nucleic acid based gene therapy holds great promise in the treatment of various diseases. However, the success of both DNA- and siRNAbased gene therapies depends critically on safe and efficient nucleic acid delivery systems. Owing to their well-defined structure and multivalent cooperativity, dendrimers have attracted particular attention as ideal nanocarriers for nucleic acid delivery. The present chapter highlights the current status of dendrimers as non-viral nanovectors for both DNA and siRNA delivery, focusing on the different dendrimers investigated for their delivery efficiency with respect to structural alterations in the view to developing safe and efficient nanovectors for gene therapy application.

  14. Ribozyme-Spherical Nucleic Acids

    PubMed Central

    Hao, Liangliang; Kouri, Fotini M.; Briley, William E.; Stegh, Alexander H.; Mirkin, Chad A.

    2015-01-01

    Ribozymes are highly structured RNA sequences that can be tailored to recognize and cleave specific stretches of mRNA. Their current therapeutic efficacy remains low due to their large size and structural instability compared to shorter therapeutically relevant RNA such as small interfering RNA (siRNA) and microRNA (miRNA). Herein, a synthetic strategy that makes use of the spherical nucleic acid (SNA) architecture to stabilize ribozymes and transfect them into live cells is reported. The properties of this novel ribozyme SNA are characterized in the context of the targeted knockdown of O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein involved in chemotherapeutic resistance of solid tumors, foremost glioblastoma multiforme (GBM). Data showing the direct cleavage of full-length MGMT mRNA, knockdown of MGMT protein, and increased sensitization of GBM cells to therapy-mediated apoptosis, independent of transfection agents, provide compelling evidence for the promising properties of this new chemical architecture. PMID:26271335

  15. Nucleic acids, proteins, and chirality

    NASA Technical Reports Server (NTRS)

    Usher, D. A.; Profy, A. T.; Walstrum, S. A.; Needels, M. C.; Bulack, S. C.; Lo, K. M.

    1984-01-01

    The present investigation is concerned with experimental results related, in one case, to the chirality of nucleotides, and, in another case, to the possibility of a link between the chirality of nucleic acids, and that of peptides. It has been found that aminoacylation of the 'internal' hydroxyl group of a dinucleoside monophosphate can occur stereoselectively. However, this reaction has not yet been made a part of a working peptide synthesis scheme. The formation and cleavage of oligonucleotides is considered. In the event of the formation of a helical complex between the oligonucleotide and the polymer, 1-prime,5-prime-bonds in the oligomer are found to become more resistant towards cleavage. The conditions required for peptide bond formation are examined, taking into account the known structures of RNA and possible mechanisms for prebiotic peptide bond formation. The possibility is considered that the 2-prime,5-prime-internucleotide linkage could have played an important part in the early days of biological peptide synthesis.

  16. 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.

  17. Methods for analyzing nucleic acid sequences

    DOEpatents

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

    2011-05-17

    The present invention is directed to a method of sequencing a target nucleic acid. The method provides a complex comprising a polymerase enzyme, a target nucleic acid molecule, and a primer, wherein the complex is immobilized on a support Fluorescent label is attached to a terminal phosphate group of the nucleotide or nucleotide analog. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The time duration of the signal from labeled nucleotides or nucleotide analogs that become incorporated is distinguished from freely diffusing labels by a longer retention in the observation volume for the nucleotides or nucleotide analogs that become incorporated than for the freely diffusing labels.

  18. NMR studies of nucleic acid dynamics

    NASA Astrophysics Data System (ADS)

    Al-Hashimi, Hashim M.

    2013-12-01

    Nucleic acid structures have to satisfy two diametrically opposite requirements; on one hand they have to adopt well-defined 3D structures that can be specifically recognized by proteins; on the other hand, their structures must be sufficiently flexible to undergo very large conformational changes that are required during key biochemical processes, including replication, transcription, and translation. How do nucleic acids introduce flexibility into their 3D structure without losing biological specificity? Here, I describe the development and application of NMR spectroscopic techniques in my laboratory for characterizing the dynamic properties of nucleic acids that tightly integrate a broad set of NMR measurements, including residual dipolar couplings, spin relaxation, and relaxation dispersion with sample engineering and computational approaches. This approach allowed us to obtain fundamental new insights into directional flexibility in nucleic acids that enable their structures to change in a very specific functional manner.

  19. Fast, facile and ethidium bromide-free assay based on the use of adsorption indicator for the estimation of polyethylenimine to nucleic acid ratio of complete polyplex assembly for gene delivery.

    PubMed

    Plianwong, Samarwadee; Opanasopit, Praneet; Ngawhirunpat, Tanasait; Rojanarata, Theerasak

    2013-10-15

    A new method was developed for the estimation of polyethylenimine (PEI) to nucleic acid ratio at which the polyplex was completely formed. The assay relied on the attraction of dichlorofluoresceinate dye to adsorb on self-assembling particles as counterions, as induced by the surface charge of the polyplex which became positive once PEI associated equivalently with nucleic acid. This phenomenon resulted in the appearance of pink colored pellets of the polyplex after centrifugation. By the other means, sodium hydroxide solution might be added to free the adsorbed dye into the solution, producing conspicuous green fluorescence under UV light (366 nm). The assay was well applied to the polyplex formulations of PEI and plasmid DNA or siRNA with satisfactory detectability and gave the results in agreement with those from gel retardation method and zeta potential analysis. Importantly, the proposed method required no sophisticated instruments, time-consuming gel electrophoresis, carcinogenic ethidium bromide as well as costly dyes and the analysis could be accomplished within less than 10 min. Hence, it was a fast, facile, cost-effective and safe-for-operator alternative method, suited for the investigation of the optimal PEI to nucleic acid ratio for gene delivery.

  20. Perfect Strangers: Inorganic Photochemistry and Nucleic Acids

    NASA Astrophysics Data System (ADS)

    Carter, Pamela J.; Ciftan, Suzanne A.; Sistare, Mark F.; Holden Thorp, H.

    1997-06-01

    The applications of inorganic photochemistry to nucleic acid chemistry are discussed. A brief review of nucleic acid structure is given. Methods for probing DNA using emissive inorganic complexes are discussed. Photoreactions that damage DNA by hydrogen atom transfer from sugar or electron abstraction from guanine are presented. The method of photochemical footprinting using a diplatinum photocatalyst is described. The final section discusses advances in combinatorial selection experiments that increase the urgency for rapid screening methods such as those derived from inorganic photochemistry.

  1. 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).

  2. In vitro evolution of nucleic acids

    NASA Technical Reports Server (NTRS)

    Joyce, G. F.; Miller, S. L. (Principal Investigator)

    1994-01-01

    The author reviews recent published reports of in vitro selection and evolution of nucleic acids. These nucleic acids will bind to a target ligand or catalyze a specific chemical reaction. The terms aptamers and systematic evolution of ligands by exponential enrichment (SELEX) are explained. The review focuses on protein binders, small molecule binders, and ribozymes obtained by directed evolution. The reference list identifies articles of special or outstanding interest.

  3. Rapid nuclear import of short nucleic acids.

    PubMed

    Kitagawa, Mai; Okamoto, Akimitsu

    2016-10-01

    Exogenous short-chain nucleic acids undergo rapid import into the nucleus. Fluorescence-labeled dT1-13 DNA microinjected into the cytoplasm domain of a HeLa cell was rapidly imported into the nucleus domain within 1min. This is much more rapid than what has been observed for intracellular diffusion of small molecules. In contrast, import of longer nucleic acids with a length of over 30nt into the nucleus was suppressed.

  4. Peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) assay for specific detection of Mycobacterium immunogenum and DNA-FISH assay for analysis of pseudomonads in metalworking fluids and sputum.

    PubMed

    Selvaraju, Suresh B; Kapoor, Renuka; Yadav, Jagjit S

    2008-01-01

    Specific and rapid detection and quantification of mycobacteria in contaminated metalworking fluid (MWF) are problematic due to complexity of the matrix and heavy background co-occurring microflora. Furthermore, cross-reactivity among neighboring species of Mycobacterium makes species differentiation difficult for this genus. Here, we report for the first time a species-specific peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) method for Mycobacterium immunogenum, a non-tuberculous Mycobacterium species prevalent in MWF and implicated in occupational lung disease hypersensitivity pneumonitis and pseudo-outbreaks. A novel species-specific 14-bp PNA probe was designed for M. immunogenum based on its 16S rRNA gene sequence and was validated for specificity, by testing against a panel of other phylogenetically closely related rapidly growing mycobacteria and representative species of gram-positive, gram-negative, and acid fast organisms. In addition, a DNA-FISH protocol was optimized for co-detection of Pseudomonas, the most predominantly co-occurring genus in contaminated MWF. Reliable quantification for both the test organisms was achieved at or above a cell density of 10(3)cellsml(-1), a recognized minimum limit for microscopic quantification. The mycobacterial PNA-FISH assay was successfully adapted to human sputum demonstrating its potential for clinical diagnostic applications in addition to industrial MWF monitoring, to assess MWF-associated exposures and pseudo-outbreaks.

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

    SciTech Connect

    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.

  6. Recent Advances in Developing Small Molecules Targeting Nucleic Acid

    PubMed Central

    Wang, Maolin; Yu, Yuanyuan; Liang, Chao; Lu, Aiping; Zhang, Ge

    2016-01-01

    Nucleic acids participate in a large number of biological processes. However, current approaches for small molecules targeting protein are incompatible with nucleic acids. On the other hand, the lack of crystallization of nucleic acid is the limiting factor for nucleic acid drug design. Because of the improvements in crystallization in recent years, a great many structures of nucleic acids have been reported, providing basic information for nucleic acid drug discovery. This review focuses on the discovery and development of small molecules targeting nucleic acids. PMID:27248995

  7. Continuously tunable nucleic acid hybridization probes.

    PubMed

    Wu, Lucia R; Wang, Juexiao Sherry; Fang, John Z; Evans, Emily R; Pinto, Alessandro; Pekker, Irena; Boykin, Richard; Ngouenet, Celine; Webster, Philippa J; Beechem, Joseph; Zhang, David Yu

    2015-12-01

    In silico-designed nucleic acid probes and primers often do not achieve favorable specificity and sensitivity tradeoffs on the first try, and iterative empirical sequence-based optimization is needed, particularly in multiplexed assays. We present a novel, on-the-fly method of tuning probe affinity and selectivity by adjusting the stoichiometry of auxiliary species, which allows for independent and decoupled adjustment of the hybridization yield for different probes in multiplexed assays. Using this method, we achieved near-continuous tuning of probe effective free energy. To demonstrate our approach, we enforced uniform capture efficiency of 31 DNA molecules (GC content, 0-100%), maximized the signal difference for 11 pairs of single-nucleotide variants and performed tunable hybrid capture of mRNA from total RNA. Using the Nanostring nCounter platform, we applied stoichiometric tuning to simultaneously adjust yields for a 24-plex assay, and we show multiplexed quantitation of RNA sequences and variants from formalin-fixed, paraffin-embedded samples.

  8. Continuously Tunable Nucleic Acid Hybridization Probes

    PubMed Central

    Wu, Lucia R.; Wang, J. Sherry; Fang, John Z.; Reiser, Emily; Pinto, Alessandro; Pekker, Irena; Boykin, Richard; Ngouenet, Celine; Webster, Philippa J.; Beechem, Joseph; Zhang, David Yu

    2015-01-01

    In silico designed nucleic acid probes and primers often fail to achieve favorable specificity and sensitivity tradeoffs on the first try, and iterative empirical sequence-based optimization is needed, particularly in multiplexed assays. Here, we present a novel, on-the-fly method of tuning probe affinity and selectivity via the stoichiometry of auxiliary species, allowing independent and decoupled adjustment of hybridization yield for different probes in multiplexed assays. Using this method, we achieve near-continuous tuning of probe effective free energy (0.03 kcal·mol−1 granularity). As applications, we enforced uniform capture efficiency of 31 DNA molecules (GC content 0% – 100%), maximized signal difference for 11 pairs of single nucleotide variants, and performed tunable hybrid-capture of mRNA from total RNA. Using the Nanostring nCounter platform, we applied stoichiometric tuning to simultaneously adjust yields for a 24-plex assay, and we show multiplexed quantitation of RNA sequences and variants from formalin-fixed, paraffin-embedded samples (FFPE). PMID:26480474

  9. Modeling Electrical Transport through Nucleic Acids

    NASA Astrophysics Data System (ADS)

    Qi, Jianqing

    Nucleic acids play a vital role in many biological systems and activities. In recent years, engineers and scientists have been interested in studying their electrical properties. The motivation for these studies stems from the following facts: (1) the bases, which form the building blocks of nucleic acids, have unique ionization potentials. Further, nucleic acids are one of the few nanomaterials that can be reproducibly manufactured with a high degree of accuracy (though admittedly their placement at desired locations remains a challenge). As a result, designed strands with specific sequences may offer unique device properties; (2) electrical methods offer potential for sequencing nucleic acids based on a single molecule; (3) electrical methods for disease detection based on the current flowing through nucleic acids are beginning to be demonstrated. While experiments in the above mentioned areas is promising, a deeper understanding of the electrical current flow through the nucleic acids needs to be developed. The modeling of current flowing in these molecules is complex because: (1) they are based on atomic scale contacts between nucleic acids and metal, which cannot be reproducibly built; (2) the conductivity of nucleic acids is easily influenced by the environment, which is constantly changing; and (3) the nucleic acids by themselves are floppy. This thesis focuses on the modeling of electrical transport through nucleic acids that are connected to two metal electrodes at nanoscale. We first develop a decoherent transport model for the double-stranded helix based on the Landauer-Buttiker framework. This model is rationalized by comparison with an experiment that measured the conductance of four different DNA strands. The developed model is then used to study the: (1) potential to make barriers and wells for quantum transport using specifically engineered sequences; (2) change in the electrical properties of a specific DNA strand with and without methylation; (3

  10. Nucleic acid hybridization-an alternative tool in diagnostic microbiology.

    PubMed

    Pettersson, U; Hyypiä, T

    1985-09-01

    The use of radioimmunoossays (RIAs) and enzyme-linked immunosorbent assays (ELISAs) has revolutionized diagnostic microbiology. Their high specificity and sensitivity make them versatile, they are simple to carry out either for direct detection of microorganisms in specimens or for serological diagnosis, and they can easily and reliably be standardized. Monoclonal antibodies have further improved these immunoassays. However, the development of simple and highly sensitive detection methods for nucleic acids has nevertheless promoted an interest also in diagnostic methods based on nucleic acid hybridization. Here Ulf Pettersson and Timo Hyypiä discuss methods which are likely to become a useful complement to the immunoassays in the near future.

  11. 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.

  12. 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.

  13. 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.

  14. A label-free signal amplification assay for DNA detection based on exonuclease III and nucleic acid dye SYBR Green I.

    PubMed

    Zheng, Aihua; Luo, Ming; Xiang, Dongshan; Xiang, Xia; Ji, Xinghu; He, Zhike

    2013-09-30

    We have developed a new fluorescence method for specific single-stranded DNA sequences with exonuclease III (Exo III) and nucleic acid dye SYBR Green I. It is demonstrated by a reverse transcription oligonucleotide sequence (target DNA, 27 bases) of RNA fragment of human immunodeficiency virus (HIV) as a model system. In the absence of the target DNA, the hairpin-probe is in the stem-closed structure, the fluorescence of SYBR Green I is very strong. In the presence of the target DNA, the hairpin-probe hybridizes with the target DNA to form double-stranded structure with a blunt 3'-terminus. Thus, in the presence of Exo III, only the 3'-terminus of probe is subjected to digestion. Exo III catalyzes the stepwise removal of mononucleotides from this terminus, releasing the target DNA. The released target DNA then hybridizes with another probe, whence the cycle starts anew. The signal of SYBR Green I decreases greatly. This system provides a detection limit of 160 pM, which is comparable to the existing signal amplification methods that utilized Exo III as a signal amplification nuclease. Due to the unique property of Exo III, this method shows excellent detection selectivity for single-base discrimination. More importantly, superiors to other methods based on Exo III, these probes have the advantages of easier to design, synthesize, purify and thus are much cheaper and more applicable. This new approach could be widely applied to sensitive and selective nucleic acids detection.

  15. Plasmonics-based SERS nanobiosensor for homogeneous nucleic acid detection.

    PubMed

    Wang, Hsin-Neng; Fales, Andrew M; Vo-Dinh, Tuan

    2015-05-01

    Developing a simple and efficient nucleic acid detection technology is essential for clinical diagnostics. Here, we describe a new conceptually simple and selective "turn on" plasmonics-based nanobiosensor, which integrates non-enzymatic DNA strand-displacement hybridization for specific nucleic acid target identification with surface-enhanced Raman scattering (SERS) detection. This SERS nanobiosensor is a target label-free, and rapid nanoparticle-based biosensing system using a homogeneous assay format that offers a simple and efficient tool for nucleic acid diagnostics. Our results showed that the nanobiosensor provided a limit of detection of ~0.1nM (200amol) in the current bioassay system, and exhibited high specificity for single nucleotide mismatch discrimination. Surface-enhanced Raman scattering (SERS) is a sensitive technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces. The enhancement means that the technique may even detect single molecules. In this article, the authors describe a simple and efficient nucleic acid detection technology using SERS, with "OFF-to-ON" signal switch upon nucleic acid target identification and capture, which provides high sensitivity and specificity for single nucleotide mismatch discrimination. This new technology will be most welcomed in clinical diagnostics. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Applications of peptide nucleic acids (PNAs) and locked nucleic acids (LNAs) in biosensor development.

    PubMed

    Briones, Carlos; Moreno, Miguel

    2012-04-01

    Nucleic acid biosensors have a growing number of applications in genetics and biomedicine. This contribution is a critical review of the current state of the art concerning the use of nucleic acid analogues, in particular peptide nucleic acids (PNA) and locked nucleic acids (LNA), for the development of high-performance affinity biosensors. Both PNA and LNA have outstanding affinity for natural nucleic acids, and the destabilizing effect of base mismatches in PNA- or LNA-containing heterodimers is much higher than in double-stranded DNA or RNA. Therefore, PNA- and LNA-based biosensors have unprecedented sensitivity and specificity, with special applicability in DNA genotyping. Herein, the most relevant PNA- and LNA-based biosensors are presented, and their advantages and their current limitations are discussed. Some of the reviewed technology, while promising, still needs to bridge the gap between experimental status and the harder reality of biotechnological or biomedical applications.

  17. Nucleic acid visualization with UCSF Chimera

    PubMed Central

    Couch, Gregory S.; Hendrix, Donna K.; Ferrin, Thomas E.

    2006-01-01

    With the increase in the number of large, 3D, high-resolution nucleic acid structures, particularly of the 30S and 50S ribosomal subunits and the intact bacterial ribosome, advancements in the visualization of nucleic acid structural features are essential. Large molecular structures are complicated and detailed, and one goal of visualization software is to allow the user to simplify the display of some features and accent others. We describe an extension to the UCSF Chimera molecular visualization system for the purpose of displaying and highlighting nucleic acid characteristics, including a new representation of sugar pucker, several options for abstraction of base geometries that emphasize stacking and base pairing, and an adaptation of the ribbon backbone to accommodate the nucleic acid backbone. Molecules are displayed and manipulated interactively, allowing the user to change the representations as desired for small molecules, proteins and nucleic acids. This software is available as part of the UCSF Chimera molecular visualization system and thus is integrated with a suite of existing tools for molecular graphics. PMID:16478715

  18. The graphene/nucleic acid nanobiointerface.

    PubMed

    Tang, Longhua; Wang, Ying; Li, Jinghong

    2015-10-07

    The combination of nanomaterials with biomolecules yields functional nanostructured biointerfaces with synergistic properties and functions. Owing to a unique combination of its crystallographic and electronic structure, graphene and its derivatives exhibit several superior and typical properties, and has emerged as an attractive candidate for the fabrication of novel nanobiointerfaces with different kinds of unique applications. As is known, nucleic acids are stable and can easily handle modification, and can recognize a wide range of targets with high selectivity, specificity, and affinity. The integration of nucleic acids with graphene-based materials has been substantially advanced over the past few years, achieving amazing properties and functions, thereby exhibiting attractive potential applications in biosensing, diagnostics, drug screening and biomedicine. Herein, this review addresses the recent progress on the design and fabrication of graphene/nucleic acid nanostructured biointerfaces, and the fundamental understanding of their interfacial properties, as well as the various nanobiotechnological applications. To begin with, we summarize the basic features of the graphene and nucleic acid-based nanobiointerface, especially the interfacial interaction mechanism and the resulting biological effects. Then, the fabrication and characterization methodology of graphene and nucleic acid-based nanobiointerfaces are discussed. Next, particular emphasis is directed towards the exploration of their biosensing and biomedical applications, including small molecule detection, protein and DNA sensing/sequencing, as well as gene delivery and therapy. Finally, some significant prospects, further opportunities and challenges in this emerging field are also suggested.

  19. Peptide nucleic acid probes with charged photocleavable mass markers

    PubMed Central

    Ball, Rachel J; Green, Philip S; Gale, Nittaya; Langley, G John

    2010-01-01

    Halogen-labelled peptide organic acid (HPOA) monomers have been synthesised and incorporated into sequence-specific peptide nucleic acid (PNA) probes. Three different types of probe have been prepared; the unmodified PNA probe, the PNA probe with a mass marker, and the PNA probe with photocleavable mass marker. All three types of probe have been used in model studies to develop a mass spectrometry-based hybridisation assay for detection of point mutations in DNA. PMID:21687524

  20. Sodium and Potassium Interactions with Nucleic Acids.

    PubMed

    Auffinger, Pascal; D'Ascenzo, Luigi; Ennifar, Eric

    2016-01-01

    Metal ions are essential cofactors for the structure and functions of nucleic acids. Yet, the early discovery in the 70s of the crucial role of Mg(2+) in stabilizing tRNA structures has occulted for a long time the importance of monovalent cations. Renewed interest in these ions was brought in the late 90s by the discovery of specific potassium metal ions in the core of a group I intron. Their importance in nucleic acid folding and catalytic activity is now well established. However, detection of K(+) and Na(+) ions is notoriously problematic and the question about their specificity is recurrent. Here we review the different methods that can be used to detect K(+) and Na(+) ions in nucleic acid structures such as X-ray crystallography, nuclear magnetic resonance or molecular dynamics simulations. We also discuss specific versus non-specific binding to different structures through various examples.

  1. Novel biochip platform for nucleic acid analysis.

    PubMed

    Pernagallo, Salvatore; Ventimiglia, Giorgio; Cavalluzzo, Claudia; Alessi, Enrico; Ilyine, Hugh; Bradley, Mark; Diaz-Mochon, Juan J

    2012-01-01

    This manuscript describes the use of a novel biochip platform for the rapid analysis/identification of nucleic acids, including DNA and microRNAs, with very high specificity. This approach combines a unique dynamic chemistry approach for nucleic acid testing and analysis developed by DestiNA Genomics with the STMicroelectronics In-Check platform, which comprises two microfluidic optimized and independent PCR reaction chambers, and a sequential microarray area for nucleic acid capture and identification by fluorescence. With its compact bench-top "footprint" requiring only a single technician to operate, the biochip system promises to transform and expand routine clinical diagnostic testing and screening for genetic diseases, cancers, drug toxicology and heart disease, as well as employment in the emerging companion diagnostics market.

  2. 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.

  3. Imaging Functional Nucleic Acid Delivery to Skin.

    PubMed

    Kaspar, Roger L; Hickerson, Robyn P; González-González, Emilio; Flores, Manuel A; Speaker, Tycho P; Rogers, Faye A; Milstone, Leonard M; Contag, Christopher H

    2016-01-01

    Monogenic skin diseases arise from well-defined single gene mutations, and in some cases a single point mutation. As the target cells are superficial, these diseases are ideally suited for treatment by nucleic acid-based therapies as well as monitoring through a variety of noninvasive imaging technologies. Despite the accessibility of the skin, there remain formidable barriers for functional delivery of nucleic acids to the target cells within the dermis and epidermis. These barriers include the stratum corneum and the layered structure of the skin, as well as more locally, the cellular, endosomal and nuclear membranes. A wide range of technologies for traversing these barriers has been described and moderate success has been reported for several approaches. The lessons learned from these studies include the need for combinations of approaches to facilitate nucleic acid delivery across these skin barriers and then functional delivery across the cellular and nuclear membranes for expression (e.g., reporter genes, DNA oligonucleotides or shRNA) or into the cytoplasm for regulation (e.g., siRNA, miRNA, antisense oligos). The tools for topical delivery that have been evaluated include chemical, physical and electrical methods, and the development and testing of each of these approaches has been greatly enabled by imaging tools. These techniques allow delivery and real time monitoring of reporter genes, therapeutic nucleic acids and also triplex nucleic acids for gene editing. Optical imaging is comprised of a number of modalities based on properties of light-tissue interaction (e.g., scattering, autofluorescence, and reflectance), the interaction of light with specific molecules (e.g., absorbtion, fluorescence), or enzymatic reactions that produce light (bioluminescence). Optical imaging technologies operate over a range of scales from macroscopic to microscopic and if necessary, nanoscopic, and thus can be used to assess nucleic acid delivery to organs, regions, cells

  4. The world of catalytic nucleic acids

    NASA Astrophysics Data System (ADS)

    Jaeger, Luc

    2001-08-01

    The finding by Tom Cech and Sydney Altman that RNA can be both informational and catalytic is the pillar on which the RNA world hypothesis stands. This hypothesis relies on the premise that RNA can catalyze its own replication, can synthesize proteins and can chemically sustain a primitive metabolism. This paper presents an overview of the catalytic potential of nucleic acids that have been uncovered the past 10 years by in vitro evolution techniques. Besides the fact that new catalytic nucleic acids uphold the prevalence of RNA at an early stage of life, they can have interesting applications in therapeutics, biotechnology and nanotechnology.

  5. Improved DNA hybridization parameters by Twisted Intercalating Nucleic Acid (TINA).

    PubMed

    Schneider, Uffe Vest

    2012-01-01

    This thesis establishes oligonucleotide design rules and applications of a novel group of DNA stabilizing molecules collectively called Twisted Intercalating Nucleic Acid - TINA. Three peer-reviewed publications form the basis for the thesis. One publication describes an improved and rapid method for determination of DNA melting points and two publications describe the effects of positioning TINA molecules in parallel triplex helix and antiparallel duplex helix forming DNA structures. The third publication establishes that TINA molecules containing oligonucleotides improve an antiparallel duplex hybridization based capture assay's analytical sensitivity compared to conventionel DNA oligonucleotides. Clinical microbiology is traditionally based on pathogenic microorganisms' culture and serological tests. The introduction of DNA target amplification methods like PCR has improved the analytical sensitivity and total turn around time involved in clinical diagnostics of infections. Due to the relatively weak hybridization between the two strands of double stranded DNA, a number of nucleic acid stabilizing molecules have been developed to improve the sensitivity of DNA based diagnostics through superior binding properties. A short introduction is given to Watson-Crick and Hoogsteen based DNA binding and the derived DNA structures. A number of other nucleic acid stabilizing molecules are described. The stabilizing effect of TINA molecules on different DNA structures is discussed and considered in relation to other nucleic acid stabilizing molecules and in relation to future use of TINA containing oligonucleotides in clinical diagnostics and therapy. In conclusion, design of TINA modified oligonucleotides for antiparallel duplex helixes and parallel triplex helixes follows simple purpose dependent rules. TINA molecules are well suited for improving multiplex PCR assays and can be used as part of novel technologies. Future research should test whether combinations of TINA

  6. 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.

  7. Design and Evaluation of a Human Immunodeficiency Virus Type 1 RNA Assay Using Nucleic Acid Sequence-Based Amplification Technology Able To Quantify Both Group M and O Viruses by Using the Long Terminal Repeat as Target

    PubMed Central

    de Baar, Michel P.; van der Schoot, Audrey M.; Goudsmit, Jaap; Jacobs, Femke; Ehren, Ron; van der Horn, Karin H. M.; Oudshoorn, Peter; de Wolf, Frank; de Ronde, Anthony

    1999-01-01

    Currently available human immunodeficiency virus type 1 (HIV-1) RNA quantification assays can detect most viruses of the group M subtypes, but a substantial number are missed or not quantified reliably. Viruses of HIV-1 group O cannot be detected by any commercially available assay. We developed and evaluated a quantitative assay based on nucleic acid sequence-based amplification (NASBA) technology, with primers and probes located in the conserved long terminal repeat (LTR) region of the HIV-1 genome. In 68 of 72 serum samples from individuals infected with HIV-1 subtypes A to H of group M, viruses could be detected and quantified. In serum samples from two patients infected with HIV-1 group O viruses, these viruses as well could be detected and quantified. In contrast, the currently used gag-based assay underestimated the presence of subtype A viruses and could not detect subtype G and group O viruses. The discrepancy between the results of the two assays may be explained by the number of mismatches found within and among the probe and primer regions of the subtype isolates. These data indicate that LTR-based assays, including the NASBA format chosen here, are better suited to monitoring HIV-1 therapy than are gag-based assays in an era in which multiple HIV-1 subtypes and groups are spreading worldwide. PMID:10325329

  8. Optical Melting Measurements of Nucleic Acid Thermodynamics

    PubMed Central

    Turner, Douglas H.

    2014-01-01

    Optical melting experiments provide measurements of thermodynamic parameters for nucleic acids. These thermodynamic parameters are widely used in RNA structure prediction programs and DNA primer design software. This review briefly summarizes the theory and underlying assumptions of the method and provides practical details for instrument calibration, experimental design, and data interpretation. PMID:20946778

  9. Thermodynamic Analysis of Nylon Nucleic Acids

    PubMed Central

    Liu, Yu; Wang, Risheng; Ding, Liang; Sha, Ruojie; Lukeman, Philip S.

    2010-01-01

    The stability and structure of nylon nucleic acid duplexes with complementary DNA and RNA strands was examined. Thermal denaturing studies of a series of oligonucleotides containing nylon nucleic acids (1 to 5 amide linkages) revealed that the amide linkage enhanced significantly the binding affinity of nylon nucleic acids towards both complementary DNA (up to 26°C increase in the thermal transition temperature (Tm) for 5 linkages) and RNA (around 15°C increase in Tm for 5 linkages) when compared with non-amide linked precursor strands. For both DNA and RNA complements, increasing derivatization decreased the melting temperatures of uncoupled molecules relative to unmodified strands; by contrast, increasing lengths of coupled copolymer raised Tm from less to slightly greater than Tm of unmodified strands. Thermodynamic data extracted from melting curves and CD spectra of nylon nucleic acid duplexes were consistent with loss of stability due to incorporation of pendent groups on the 2′ position of ribose, and recovery of stability upon linkage of the side chains. PMID:18543259

  10. Nucleic acid-coupled colorimetric analyte detectors

    DOEpatents

    Charych, Deborah H.; Jonas, Ulrich

    2001-01-01

    The present invention relates to methods and compositions for the direct detection of analytes and membrane conformational changes through the detection of color changes in biopolymeric materials. In particular, the present invention provide for the direct colorimetric detection of analytes using nucleic acid ligands at surfaces of polydiacetylene liposomes and related molecular layer systems.

  11. Advances in nucleic acid-based detection methods.

    PubMed Central

    Wolcott, M J

    1992-01-01

    Laboratory techniques based on nucleic acid methods have increased in popularity over the last decade with clinical microbiologists and other laboratory scientists who are concerned with the diagnosis of infectious agents. This increase in popularity is a result primarily of advances made in nucleic acid amplification and detection techniques. Polymerase chain reaction, the original nucleic acid amplification technique, changed the way many people viewed and used nucleic acid techniques in clinical settings. After the potential of polymerase chain reaction became apparent, other methods of nucleic acid amplification and detection were developed. These alternative nucleic acid amplification methods may become serious contenders for application to routine laboratory analyses. This review presents some background information on nucleic acid analyses that might be used in clinical and anatomical laboratories and describes some recent advances in the amplification and detection of nucleic acids. PMID:1423216

  12. Nanoconstructions Based on Spatially Ordered Nucleic Acid Molecules

    NASA Astrophysics Data System (ADS)

    Yevdokimov, Yu. M.

    Different strategies for the design of nanoconstructions whose building blocks are both linear molecules of double-stranded nucleic acids and nucleic acid molecules fixed in the spatial structure of particles of liquid-crystalline dispersions are described.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. Development of a peptide nucleic acid polymerase chain reaction clamping assay for semiquantitative evaluation of genetically modified organism content in food.

    PubMed

    Peano, C; Lesignoli, F; Gulli, M; Corradini, R; Samson, M C; Marchelli, R; Marmiroli, N

    2005-09-15

    In the present study a peptide nucleic acid (PNA)-mediated polymerase chain reaction (PCR) clamping method was developed and applied to the detection of genetically modified organisms (GMO), to test PCR products for band identity and to obtain a semiquantitative evaluation of GMO content. The minimal concentration of PNA necessary to block the PCR was determined by comparing PCRs containing a constant amount of DNA in the presence of increasing concentration of target-specific PNA. The lowest PNA concentration at which specific inhibition took place, by the inhibition of primer extension and/or steric hindrance, was the most efficient condition. Optimization of PCR clamping by PNA was observed by testing five different PNAs with a minimum of 13 bp to a maximum of 15 bp, designed on the target sequence of Roundup Ready soybean. The results obtained on the DNA extracted from Roundup Ready soybean standard flour were verified also on DNA extracted from standard flours of maize GA21, Bt176, Bt11, and MON810. A correlation between the PNA concentration necessary for inducing PCR clamping and the percentage of the GMO target sequence in the sample was found.

  18. Dimethyl adipimidate/Thin film Sample processing (DTS); A simple, low-cost, and versatile nucleic acid extraction assay for downstream analysis.

    PubMed

    Shin, Yong; Lim, Swee Yin; Lee, Tae Yoon; Park, Mi Kyoung

    2015-09-15

    Sample processing, especially that involving nucleic acid extraction, is a prerequisite step for the isolation of high quantities of relatively pure DNA for downstream analyses in many life science and biomedical engineering studies. However, existing methods still have major problems, including labor-intensive time-consuming methods and high costs, as well as requirements for a centrifuge and the complex fabrication of filters and membranes. Here, we first report a versatile Dimethyl adipimidate/Thin film based Sample processing (DTS) procedure without the limitations of existing methods. This procedure is useful for the extraction of DNA from a variety of sources, including 6 eukaryotic cells, 6 bacteria cells, and 2 body fluids in a single step. Specifically, the DTS procedure does not require a centrifuge and has improved time efficiency (30 min), affordability, and sensitivity in downstream analysis. We validated the DTS procedure for the extraction of DNA from human body fluids, as well as confirmed that the quality and quantity of the extracted DNA were sufficient to allow robust detection of genetic and epigenetic biomarkers in downstream analysis.

  19. Dimethyl adipimidate/Thin film Sample processing (DTS); A simple, low-cost, and versatile nucleic acid extraction assay for downstream analysis

    PubMed Central

    Shin, Yong; Lim, Swee Yin; Lee, Tae Yoon; Park, Mi Kyoung

    2015-01-01

    Sample processing, especially that involving nucleic acid extraction, is a prerequisite step for the isolation of high quantities of relatively pure DNA for downstream analyses in many life science and biomedical engineering studies. However, existing methods still have major problems, including labor-intensive time-consuming methods and high costs, as well as requirements for a centrifuge and the complex fabrication of filters and membranes. Here, we first report a versatile Dimethyl adipimidate/Thin film based Sample processing (DTS) procedure without the limitations of existing methods. This procedure is useful for the extraction of DNA from a variety of sources, including 6 eukaryotic cells, 6 bacteria cells, and 2 body fluids in a single step. Specifically, the DTS procedure does not require a centrifuge and has improved time efficiency (30 min), affordability, and sensitivity in downstream analysis. We validated the DTS procedure for the extraction of DNA from human body fluids, as well as confirmed that the quality and quantity of the extracted DNA were sufficient to allow robust detection of genetic and epigenetic biomarkers in downstream analysis. PMID:26370251

  20. Total tumor load assessed by one-step nucleic acid amplification assay as an intraoperative predictor for non-sentinel lymph node metastasis in breast cancer.

    PubMed

    Nabais, Celso; Figueiredo, Joana; Lopes, Paulina; Martins, Manuela; Araújo, António

    2017-04-01

    This study aimed to determine the relationship between CK19 mRNA copy number in sentinel lymph nodes (SLN) assessed by one-step nucleic acid amplification (OSNA) technique, and non-sentinel lymph nodes (NSLN) metastization in invasive breast cancer. A model using total tumor load (TTL) obtained by OSNA technique was also constructed to evaluate its predictability. We conducted an observational retrospective study including 598 patients with clinically T1-T3 and node negative invasive breast cancer. Of the 88 patients with positive SLN, 58 patients fulfill the inclusion criteria. In the analyzed group 25.86% had at least one positive NSLN in axillary lymph node dissection. Univariate analysis showed that tumor size, TTL and number of SLN macrometastases were predictive factors for NSLN metastases. In multivariate analysis just the TTL was predictive for positive NSLN (OR 2.67; 95% CI 1.06-6.70; P = 0.036). The ROC curve for the model using TTL alone was obtained and an AUC of 0.805 (95% CI 0.69-0.92) was achieved. For TTL >1.9 × 10(5) copies/μL we got 73.3% sensitivity, 74.4% specificity and 88.9% negative predictive value to predict NSLN metastases. When using OSNA technique to evaluate SLN, NSLN metastases can be predicted intraoperatively. This prediction tool could help in decision for axillary lymph node dissection. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Discovery of the unique self-assembly behavior of terminal suckers-contained dsDNA onto GNP and novel "light-up" colorimetric assay of nucleic acids.

    PubMed

    Qiu, Liping; Shen, Zhifa; Wu, Zai-Sheng; Shen, Guo-Li; Yu, Ruqin

    2015-02-15

    Noble metal nanoparticles are currently of great interest because of their unique optical properties and potential applications in disease diagnostics and cancer treatment. In the present work, a discovery was reported that dsDNA with terminal thiols at its two ends could lie easily flat onto the gold nanoparticle (GNP) surface rather than cross linked different GNPs, indicating an unique self-assembly behavior of newly-designed molecules on GNPs. This could intensively stabilize gold nanoparticles against aggregation even at a high salt concentration. On the basis of this discovery, a novel light-up colorimetric sensing strategy was developed for the detection of p53 gene by combining with the cyclical nucleic acid strand-displacement polymerization (CNDP). For the described colorimetric system, GNPs require no any surface functionalization, and target recognition reaction and CNDP amplification could be conducted under the optimized conditions to achieve a high efficiency. The high detection sensitivity and desirable selectivity are achieved, and the potential practical application was demonstrated. Besides, this sensing system can function in a wide range of salts, making it a suitable platform to cooperate with many biological processes. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.

    PubMed

    Thomas, Marshall P; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron J; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

    2014-06-01

    Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. In this study, we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein targets, whereas adding RNA to recombinant RNA binding protein substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Preincubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G. During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps, which bind NE and cathepsin G. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and neutrophil extracellular traps in a DNA-dependent manner. Thus, high-affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation.

  3. Leukocyte Protease Binding to Nucleic Acids Promotes Nuclear Localization and Cleavage of Nucleic Acid Binding Proteins

    PubMed Central

    Thomas, Marshall P.; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

    2014-01-01

    Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. Here we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein (RBP) targets, while adding RNA to recombinant RBP substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Pre-incubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G (CATG). During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps (NETs), which bind NE and CATG. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and NETs in a DNA-dependent manner. Thus, high affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation. PMID:24771851

  4. Method for nucleic acid isolation using supercritical fluids

    SciTech Connect

    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.

  5. 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.

  6. Cytoplasmic nucleic acid sensors in antiviral immunity.

    PubMed

    Ranjan, Priya; Bowzard, J Bradford; Schwerzmann, Joy W; Jeisy-Scott, Victoria; Fujita, Takashi; Sambhara, Suryaprakash

    2009-08-01

    The innate immune system uses pattern recognition receptors (PRRs) to sense invading microbes and initiate a rapid protective response. PRRs bind and are activated by structural motifs, such as nucleic acids or bacterial and fungal cell wall components, collectively known as pathogen-associated molecular patterns. PRRs that recognize pathogen-derived nucleic acids are present in vesicular compartments and in the cytosol of most cell types. Here, we review recent studies of these cytosolic sensors, focusing on the nature of the ligands for DNA-dependent activator of interferon (DAI)-regulatory factors, absent in melanoma 2 (AIM2), and the retinoic acid-inducible gene I-like helicase (RLH) family of receptors, the basis of ligand recognition and the signaling pathways triggered by the activation of these receptors. An increased understanding of these molecular aspects of innate immunity will guide the development of novel antiviral therapeutics.

  7. Handheld advanced nucleic acid analyzer

    NASA Astrophysics Data System (ADS)

    Benett, William J.; Richards, James B.; Stratton, Paul; Hadley, Dean R.; Bodtker, Brian H.; Nasarabadi, Shanavaz L.; Milanovich, Fred P.; Mariella, Raymond P., Jr.; Koopman, Ronald P.; Belgrader, Philip

    2000-12-01

    There is a growing need for portable, lightweight, battery operated instruments capable of detecting and identifying bio-warfare and bio-terrorism agents in the field. To address this need, we have developed a handheld PCR instrument. LLNLs advanced thermal cycling technology and expertise with portable, field tested biological instrumentation, combined with the development of real-time, fluorescence based PCR assays, has enabled the development of a very portable, versatile, power efficient PCR instrument with a simplified operating system designed for use by first responders. The heart of the instrument is the sample module, which incorporates the advanced silicon thermal cycler developed at LLNL.

  8. Diagnostic applications of nucleic acid circuits.

    PubMed

    Jung, Cheulhee; Ellington, Andrew D

    2014-06-17

    CONSPECTUS: While the field of DNA computing and molecular programming was engendered in large measure as a curiosity-driven exercise, it has taken on increasing importance for analytical applications. This is in large measure because of the modularity of DNA circuitry, which can serve as a programmable intermediate between inputs and outputs. These qualities may make nucleic acid circuits useful for making decisions relevant to diagnostic applications. This is especially true given that nucleic acid circuits can potentially directly interact with and be triggered by diagnostic nucleic acids and other analytes. Chemists are, by and large, unaware of many of these advances, and this Account provides a means of touching on what might seem to be an arcane field. We begin by explaining nucleic acid amplification reactions that can lead to signal amplification, such as catalytic hairpin assembly (CHA) and the hybridization chain reaction (HCR). In these circuits, a single-stranded input acts on kinetically trapped substrates via exposed toeholds and strand exchange reactions, refolding the substrates and allowing them to interact with one another. As multiple duplexes (CHA) or concatemers of increasing length (HCR) are generated, there are opportunities to couple these outputs to different analytical modalities, including transduction to fluorescent, electrochemical, and colorimetric signals. Because both amplification and transduction are at their root dependent on the programmability of Waston-Crick base pairing, nucleic acid circuits can be much more readily tuned and adapted to new applications than can many other biomolecular amplifiers. As an example, robust methods for real-time monitoring of isothermal amplification reactions have been developed recently. Beyond amplification, nucleic acid circuits can include logic gates and thresholding components that allow them to be used for analysis and decision making. Scalable and complex DNA circuits (seesaw gates

  9. Comparison of Levels of Human Immunodeficiency Virus Type 1 RNA in Plasma as Measured by the NucliSens Nucleic Acid Sequence-Based Amplification and Quantiplex Branched-DNA Assays

    PubMed Central

    Ginocchio, C. C.; Tetali, S.; Washburn, D.; Zhang, F.; Kaplan, M. H.

    1999-01-01

    This study compared levels of human immunodeficiency virus type 1 RNA in plasma as measured by the Quantiplex branched-DNA and NucliSens nucleic acid sequence-based amplification assays. RNA was detectable in 118 of 184 samples (64.13%) by the Quantiplex assay and in 171 of 184 samples (92.94%) by the NucliSens assay. Regression analysis indicated that a linear relationship existed between the two sets of values (P < 0.0001), although the Quantiplex and NucliSens values were significantly different (P < 0.001), with the NucliSens values being approximately 0.323 log higher. Spearman correlation analysis indicated that the overall changes in patient viral load patterns were highly correlative between the two assays: r = 0.912, P < 0.0001. The lower limits of sensitivity were determined to be approximately 100 copies/ml and 1,200 to 1,400 copies/ml for the NucliSens and Quantiplex assays, respectively. PMID:10074556

  10. Comparison of levels of human immunodeficiency virus type 1 RNA in plasma as measured by the NucliSens nucleic acid sequence-based amplification and Quantiplex branched-DNA assays.

    PubMed

    Ginocchio, C C; Tetali, S; Washburn, D; Zhang, F; Kaplan, M H

    1999-04-01

    This study compared levels of human immunodeficiency virus type 1 RNA in plasma as measured by the Quantiplex branched-DNA and NucliSens nucleic acid sequence-based amplification assays. RNA was detectable in 118 of 184 samples (64.13%) by the Quantiplex assay and in 171 of 184 samples (92.94%) by the NucliSens assay. Regression analysis indicated that a linear relationship existed between the two sets of values (P < 0.0001), although the Quantiplex and NucliSens values were significantly different (P < 0.001), with the NucliSens values being approximately 0.323 log higher. Spearman correlation analysis indicated that the overall changes in patient viral load patterns were highly correlative between the two assays: r = 0.912, P < 0.0001. The lower limits of sensitivity were determined to be approximately 100 copies/ml and 1,200 to 1,400 copies/ml for the NucliSens and Quantiplex assays, respectively.

  11. 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.

  12. Quick chip assay using locked nucleic acid modified epithelial cell adhesion molecule and nucleolin aptamers for the capture of circulating tumor cells

    PubMed Central

    Maremanda, Nihal G.; Roy, Kislay; Kanwar, Rupinder K.; Shyamsundar, Vidyarani; Ramshankar, Vijayalakshmi; Krishnamurthy, Arvind; Krishnakumar, Subramanian; Kanwar, Jagat R.

    2015-01-01

    The role of circulating tumor cells (CTCs) in disease diagnosis, prognosis, monitoring of the therapeutic efficacy, and clinical decision making is immense and has attracted tremendous focus in the last decade. We designed and fabricated simple, flat channel microfluidic devices polydimethylsiloxane (PDMS based) functionalized with locked nucleic acid (LNA) modified aptamers (targeting epithelial cell adhesion molecule (EpCAM) and nucleolin expression) for quick and efficient capture of CTCs and cancer cells. With optimized flow rates (10 μl/min), it was revealed that the aptamer modified devices offered reusability for up to six times while retaining optimal capture efficiency (>90%) and specificity. High capture sensitivity (92%) and specificity (100%) was observed in whole blood samples spiked with Caco-2 cells (10–100 cells/ml). Analysis of blood samples obtained from 25 head and neck cancer patients on the EpCAM LNA aptamer functionalized chip revealed that an average count of 5 ± 3 CTCs/ml of blood were captured from 22/25 samples (88%). EpCAM intracellular domain (EpICD) immunohistochemistry on 9 oral squamous cell carcinomas showed the EpICD positivity in the tumor cells, confirming the EpCAM expression in CTCs from head and neck cancers. These microfluidic devices also maintained viability for in vitro culture and characterization. Use of LNA modified aptamers provided added benefits in terms of cost effectiveness due to increased reusability and sustainability of the devices. Our results present a robust, quick, and efficient CTC capture platform with the use of simple PDMS based devices that are easy to fabricate at low cost and have an immense potential in cancer diagnosis, prognosis, and therapeutic planning. PMID:26487896

  13. 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

  14. 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

  15. Counting the ions surrounding nucleic acids

    PubMed Central

    2017-01-01

    Abstract Nucleic acids are strongly negatively charged, and thus electrostatic interactions—screened by ions in solution—play an important role in governing their ability to fold and participate in biomolecular interactions. The negative charge creates a region, known as the ion atmosphere, in which cation and anion concentrations are perturbed from their bulk values. Ion counting experiments quantify the ion atmosphere by measuring the preferential ion interaction coefficient: the net total number of excess ions above, or below, the number expected due to the bulk concentration. The results of such studies provide important constraints on theories, which typically predict the full three-dimensional distribution of the screening cloud. This article reviews the state of nucleic acid ion counting measurements and critically analyzes their ability to test both analytical and simulation-based models. PMID:28034959

  16. Enzyme-free nucleic acid logic circuits.

    PubMed

    Seelig, Georg; Soloveichik, David; Zhang, David Yu; Winfree, Erik

    2006-12-08

    Biological organisms perform complex information processing and control tasks using sophisticated biochemical circuits, yet the engineering of such circuits remains ineffective compared with that of electronic circuits. To systematically create complex yet reliable circuits, electrical engineers use digital logic, wherein gates and subcircuits are composed modularly and signal restoration prevents signal degradation. We report the design and experimental implementation of DNA-based digital logic circuits. We demonstrate AND, OR, and NOT gates, signal restoration, amplification, feedback, and cascading. Gate design and circuit construction is modular. The gates use single-stranded nucleic acids as inputs and outputs, and the mechanism relies exclusively on sequence recognition and strand displacement. Biological nucleic acids such as microRNAs can serve as inputs, suggesting applications in biotechnology and bioengineering.

  17. Digital Microfluidics for Nucleic Acid Amplification.

    PubMed

    Coelho, Beatriz; Veigas, Bruno; Fortunato, Elvira; Martins, Rodrigo; Águas, Hugo; Igreja, Rui; Baptista, Pedro V

    2017-06-25

    Digital Microfluidics (DMF) has emerged as a disruptive methodology for the control and manipulation of low volume droplets. In DMF, each droplet acts as a single reactor, which allows for extensive multiparallelization of biological and chemical reactions at a much smaller scale. DMF devices open entirely new and promising pathways for multiplex analysis and reaction occurring in a miniaturized format, thus allowing for healthcare decentralization from major laboratories to point-of-care with accurate, robust and inexpensive molecular diagnostics. Here, we shall focus on DMF platforms specifically designed for nucleic acid amplification, which is key for molecular diagnostics of several diseases and conditions, from pathogen identification to cancer mutations detection. Particular attention will be given to the device architecture, materials and nucleic acid amplification applications in validated settings.

  18. Digital Microfluidics for Nucleic Acid Amplification

    PubMed Central

    Veigas, Bruno; Fortunato, Elvira; Martins, Rodrigo; Águas, Hugo; Igreja, Rui; Baptista, Pedro V.

    2017-01-01

    Digital Microfluidics (DMF) has emerged as a disruptive methodology for the control and manipulation of low volume droplets. In DMF, each droplet acts as a single reactor, which allows for extensive multiparallelization of biological and chemical reactions at a much smaller scale. DMF devices open entirely new and promising pathways for multiplex analysis and reaction occurring in a miniaturized format, thus allowing for healthcare decentralization from major laboratories to point-of-care with accurate, robust and inexpensive molecular diagnostics. Here, we shall focus on DMF platforms specifically designed for nucleic acid amplification, which is key for molecular diagnostics of several diseases and conditions, from pathogen identification to cancer mutations detection. Particular attention will be given to the device architecture, materials and nucleic acid amplification applications in validated settings. PMID:28672827

  19. Nucleic Acid Templated Reactions for Chemical Biology.

    PubMed

    Di Pisa, Margherita; Seitz, Oliver

    2017-06-21

    Nucleic acid directed bioorthogonal reactions offer the fascinating opportunity to unveil and redirect a plethora of intracellular mechanisms. Nano- to picomolar amounts of specific RNA molecules serve as templates and catalyze the selective formation of molecules that 1) exert biological effects, or 2) provide measurable signals for RNA detection. Turnover of reactants on the template is a valuable asset when concentrations of RNA templates are low. The idea is to use RNA-templated reactions to fully control the biodistribution of drugs and to push the detection limits of DNA or RNA analytes to extraordinary sensitivities. Herein we review recent and instructive examples of conditional synthesis or release of compounds for in cellulo protein interference and intracellular nucleic acid imaging. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  20. Nucleic acid compositions and the encoding proteins

    DOEpatents

    Preston, III, James F.; Chow, Virginia; Nong, Guang; Rice, John D.; St. John, Franz J.

    2014-09-02

    The subject invention provides at least one nucleic acid sequence encoding an aldouronate-utilization regulon isolated from Paenibacillus sp. strain JDR-2, a bacterium which efficiently utilizes xylan and metabolizes aldouronates (methylglucuronoxylosaccharides). The subject invention also provides a means for providing a coordinately regulated process in which xylan depolymerization and product assimilation are coupled in Paenibacillus sp. strain JDR-2 to provide a favorable system for the conversion of lignocellulosic biomass to biobased products. Additionally, the nucleic acid sequences encoding the aldouronate-utilization regulon can be used to transform other bacteria to form organisms capable of producing a desired product (e.g., ethanol, 1-butanol, acetoin, 2,3-butanediol, 1,3-propanediol, succinate, lactate, acetate, malate or alanine) from lignocellulosic biomass.

  1. Nucleic acids, compositions and uses thereof

    DOEpatents

    Preston, III, James F.; Chow, Virginia [Gainesville, FL; Nong, Guang [Gainesville, FL; Rice, John D [Gainesville, FL; John, Franz J [Baltimore, MD

    2012-02-21

    The subject invention provides at least one nucleic acid sequence encoding an aldouronate-utilization regulon isolated from Paenibacillus sp. strain JDR-2, a bacterium which efficiently utilizes xylan and metabolizes aldouronates (methylglucuronoxylosaccharides). The subject invention also provides a means for providing a coordinately regulated process in which xylan depolymerization and product assimilation are coupled in Paenibacillus sp. strain JDR-2 to provide a favorable system for the conversion of lignocellulosic biomass to biobased products. Additionally, the nucleic acid sequences encoding the aldouronate-utilization regulon can be used to transform other bacteria to form organisms capable of producing a desired product (e.g., ethanol, 1-butanol, acetoin, 2,3-butanediol, 1,3-propanediol, succinate, lactate, acetate, malate or alanine) from lignocellulosic biomass.

  2. Nanotechnology for delivery of peptide nucleic acids (PNAs).

    PubMed

    Gupta, Anisha; Bahal, Raman; Gupta, Meera; Glazer, Peter M; Saltzman, W Mark

    2016-10-28

    Over the past three decades, peptide nucleic acids have been employed in numerous chemical and biological applications. Peptide nucleic acids possess enormous potential because of their superior biophysical properties, compared to other oligonucleotide chemistries. However, for therapeutic applications, intracellular delivery of peptide nucleic acids remains a challenge. In this review, we summarize the progress that has been made in delivering peptide nucleic acids to intracellular targets. In addition, we emphasize recent nanoparticle-based strategies for efficient delivery of conventional and chemically-modified peptides nucleic acids.

  3. Spectrofluorimetric study of the binding of codeine to nucleic acids

    NASA Astrophysics Data System (ADS)

    Wang, Feng; Huang, Wei; Su, Liang; Dong, Zijia; Zhang, Shuai

    2009-06-01

    The characteristics of the interaction between codeine (CD) and nucleic acids were studied by ultraviolet-visible spectra and fluorescent spectra. It shows that there is a powerful ability in nucleic acids to quench the fluorescence intensity of codeine. The fluorescence quenching data were analyzed according to Stern-Volmer equation and Förster's nonradiative energy transfer mechanism. Thus the binding constant and the thermodynamic parameters between codeine and nucleic acids were obtained. The results show that codeine interacts with nucleic acids in a mode of groove binding and -OCH 3 of the codeine molecular combines with the groove of nucleic acids through hydrogen bond or van der Waals force.

  4. Orthogonal Synthesis of Xeno Nucleic Acids.

    PubMed

    Fiers, Guillaume; Chouikhi, Dalila; Oswald, Laurence; Al Ouahabi, Abdelaziz; Chan-Seng, Delphine; Charles, Laurence; Lutz, Jean-François

    2016-12-12

    Sequence-defined peptide triazole nucleic acids (PTzNA) were synthesized by means of a solid-phase orthogonal "AB+CD" iterative strategy. In this approach, AB and CD building blocks containing carboxylic acid (A), azide (B), alkyne (C), and primary amine (D) functions are assembled together by successive copper-catalyzed azide-alkyne cycloaddition (CuAAC) and acid-amine coupling steps. Different PTzNA genetic sequences were prepared using a library of eight building blocks (i.e., four AB and four CD building blocks).

  5. Comparison of peptide nucleic acid fluorescence in situ hybridization assays with culture-based matrix-assisted laser desorption/ionization-time of flight mass spectrometry for the identification of bacteria and yeasts from blood cultures and cerebrospinal fluid cultures.

    PubMed

    Calderaro, A; Martinelli, M; Motta, F; Larini, S; Arcangeletti, M C; Medici, M C; Chezzi, C; De Conto, F

    2014-08-01

    Peptide nucleic acid fluorescence in situ hybridization (PNA FISH) is a molecular diagnostic tool for the rapid detection of pathogens directly from liquid media. The aim of this study was to prospectively evaluate PNA FISH assays in comparison with culture-based matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) identification, as a reference method, for both blood and cerebrospinal fluid (CSF) cultures, during a 1-year investigation. On the basis of the Gram stain microscopy results, four different PNA FISH commercially available assays were used ('Staphylococcus aureus/CNS', 'Enterococcus faecalis/OE', 'GNR Traffic Light' and 'Yeasts Traffic Light' PNA FISH assays, AdvanDx). The four PNA FISH assays were applied to 956 positive blood cultures (921 for bacteria and 35 for yeasts) and 11 CSF cultures. Among the 921 blood samples positive for bacteria, PNA FISH gave concordant results with MALDI-TOF MS in 908/921 (98.64%) samples, showing an agreement of 99.4% in the case of monomicrobial infections. As regards yeasts, the PNA FISH assay showed a 100% agreement with the result obtained by MALDI-TOF MS. When PNA FISH assays were tested on the 11 CSF cultures, the results agreed with the reference method in all cases (100%). PNA FISH assays provided species identification at least one work-day before the MALDI-TOF MS culture-based identification. PNA FISH assays showed an excellent efficacy in the prompt identification of main pathogens, yielding a significant reduction in reporting time and leading to more appropriate patient management and therapy in cases of sepsis and severe infections. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

  6. Optimizing the specificity of nucleic acid hybridization

    NASA Astrophysics Data System (ADS)

    Zhang, David Yu; Chen, Sherry Xi; Yin, Peng

    2012-03-01

    The specific hybridization of complementary sequences is an essential property of nucleic acids, enabling diverse biological and biotechnological reactions and functions. However, the specificity of nucleic acid hybridization is compromised for long strands, except near the melting temperature. Here, we analytically derived the thermodynamic properties of a hybridization probe that would enable near-optimal single-base discrimination and perform robustly across diverse temperature, salt and concentration conditions. We rationally designed ‘toehold exchange’ probes that approximate these properties, and comprehensively tested them against five different DNA targets and 55 spurious analogues with energetically representative single-base changes (replacements, deletions and insertions). These probes produced discrimination factors between 3 and 100+ (median, 26). Without retuning, our probes function robustly from 10 °C to 37 °C, from 1 mM Mg2+ to 47 mM Mg2+, and with nucleic acid concentrations from 1 nM to 5 µM. Experiments with RNA also showed effective single-base change discrimination.

  7. Optimizing the specificity of nucleic acid hybridization.

    PubMed

    Zhang, David Yu; Chen, Sherry Xi; Yin, Peng

    2012-01-22

    The specific hybridization of complementary sequences is an essential property of nucleic acids, enabling diverse biological and biotechnological reactions and functions. However, the specificity of nucleic acid hybridization is compromised for long strands, except near the melting temperature. Here, we analytically derived the thermodynamic properties of a hybridization probe that would enable near-optimal single-base discrimination and perform robustly across diverse temperature, salt and concentration conditions. We rationally designed 'toehold exchange' probes that approximate these properties, and comprehensively tested them against five different DNA targets and 55 spurious analogues with energetically representative single-base changes (replacements, deletions and insertions). These probes produced discrimination factors between 3 and 100+ (median, 26). Without retuning, our probes function robustly from 10 °C to 37 °C, from 1 mM Mg(2+) to 47 mM Mg(2+), and with nucleic acid concentrations from 1 nM to 5 µM. Experiments with RNA also showed effective single-base change discrimination.

  8. Carbohydrate Polymers for Nonviral Nucleic Acid Delivery

    PubMed Central

    Sizovs, Antons; McLendon, Patrick M.; Srinivasachari, Sathya

    2014-01-01

    Carbohydrates have been investigated and developed as delivery vehicles for shuttling nucleic acids into cells. In this review, we present the state of the art in carbohydrate-based polymeric vehicles for nucleic acid delivery, with the focus on the recent successes in preclinical models, both in vitro and in vivo. Polymeric scaffolds based on the natural polysaccharides chitosan, hyaluronan, pullulan, dextran, and schizophyllan each have unique properties and potential for modification, and these results are discussed with the focus on facile synthetic routes and favorable performance in biological systems. Many of these carbohydrates have been used to develop alternative types of biomaterials for nucleic acid delivery to typical polyplexes, and these novel materials are discussed. Also presented are polymeric vehicles that incorporate copolymerized carbohydrates into polymer backbones based on polyethylenimine and polylysine and their effect on transfection and biocompatibility. Unique scaffolds, such as clusters and polymers based on cyclodextrin (CD), are also discussed, with the focus on recent successes in vivo and in the clinic. These results are presented with the emphasis on the role of carbohydrate and charge on transfection. Use of carbohydrates as molecular recognition ligands for cell-type specific delivery is also briefly reviewed. We contend that carbohydrates have contributed significantly to progress in the field of non-viral DNA delivery, and these new discoveries are impactful for developing new vehicles and materials for treatment of human disease. PMID:21504102

  9. Optimizing the specificity of nucleic acid hybridization

    PubMed Central

    Zhang, David Yu; Chen, Sherry Xi; Yin, Peng

    2014-01-01

    The specific hybridization of complementary sequences is an essential property of nucleic acids, enabling diverse biological and biotechnological reactions and functions. However, the specificity of nucleic acid hybridization is compromised for long strands, except near the melting temperature. Here, we analytically derived the thermodynamic properties of a hybridization probe that would enable near-optimal single-base discrimination and perform robustly across diverse temperature, salt and concentration conditions. We rationally designed ‘toehold exchange’ probes that approximate these properties, and comprehensively tested them against five different DNA targets and 55 spurious analogues with energetically representative single-base changes (replacements, deletions and insertions). These probes produced discrimination factors between 3 and 100+ (median, 26). Without retuning, our probes function robustly from 10 °C to 37 °C, from 1 mM Mg2+ to 47 mM Mg2+, and with nucleic acid concentrations from 1 nM to 5 μM. Experiments with RNA also showed effective single-base change discrimination. PMID:22354435

  10. 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.

  11. 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.

  12. 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.

  13. Control of stability and structure of nucleic acids using cosolutes.

    PubMed

    Tateishi-Karimta, Hisae; Sugimoto, Naoki

    2014-05-15

    The stabilities, structures, and functions of nucleic acids are responsive to surrounding conditions. Living cells contain biomolecules, including nucleic acids, proteins, polysaccharides, and other soluble and insoluble low-molecular weight components, that occupy a significant fraction of the cellular volume (up to 40%), resulting in a highly crowded intracellular environment. We have proven that conditions that mimic features of this intra-cellular environment alter the physical properties affect the stability, structure, and function of nucleic acids. The ability to control structure of nucleic acids by mimicking intra-cellular conditions will be useful in nanotechnology applications of nucleic acids. This paper describes methods that can be used to analyze quantitatively the intra-cellular environment effects caused by cosolutes on nucleic acid structures and to regulate properties of nucleic acids using cosolutes.

  14. Zip nucleic acids are potent hydrolysis probes for quantitative PCR

    PubMed Central

    Paris, Clément; Moreau, Valérie; Deglane, Gaëlle; Voirin, Emilie; Erbacher, Patrick; Lenne-Samuel, Nathalie

    2010-01-01

    Zip nucleic acids (ZNAs) are oligonucleotides conjugated with cationic spermine units that increase affinity for their target. ZNAs were recently shown to enable specific and sensitive reactions when used as primers for polymerase chain reaction (PCR) and reverse-transcription. Here, we report their use as quantitative PCR hydrolysis probes. Ultraviolet duplex melting data demonstrate that attachment of cationic residues to the 3′ end of an oligonucleotide does not alter its ability to discriminate nucleotides nor the destabilization pattern relative to mismatch location in the oligonucleotide sequence. The stability increase provided by the cationic charges allows the use of short dual-labeled probes that significantly improve single-nucleotide polymorphism genotyping. Longer ZNA probes were shown to display reduced background fluorescence, therefore, generating greater sensitivity and signal level as compared to standard probes. ZNA probes thus provide broad flexibility in assay design and also represent an effective alternative to minor groove binder- and locked nucleic-acid-containing probes. PMID:20071749

  15. NanoDrop microvolume quantitation of nucleic acids.

    PubMed

    Desjardins, Philippe; Conklin, Deborah

    2010-11-22

    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

  16. Identification of Nucleic Acid High Affinity Binding Sequences of Proteins by SELEX.

    PubMed

    Bouvet, Philippe

    2015-01-01

    A technique is described for the identification of nucleic acid sequences bound with high affinity by proteins or by other molecules suitable for a partitioning assay. Here, a histidine-tagged protein is allowed to interact with a pool of nucleic acids and the protein-nucleic acid complexes formed are retained on a Ni-NTA matrix. Nucleic acids with a low level of recognition by the protein are washed away. The pool of recovered nucleic acids is amplified by the polymerase chain reaction and is submitted to further rounds of selection. Each round of selection increases the proportion of sequences that are avidly bound by the protein of interest. The cloning and sequencing of these sequences finally completes their identification.

  17. Identification of nucleic acid high-affinity binding sequences of proteins by SELEX.

    PubMed

    Bouvet, Philippe

    2009-01-01

    A technique is described for the identification of nucleic acid sequences bound with high affinity by proteins or by other molecules suitable for a partitioning assay. Here, a histidine-tagged protein is allowed to interact with a pool of nucleic acids and the protein-nucleic acid complexes formed are retained on a Ni-NTA matrix. Nucleic acids with a low level of recognition by the protein are washed away. The pool of recovered nucleic acids is amplified by the polymerase chain reaction and is submitted to further rounds of selection. Each round of selection increases the proportion of sequences that are avidly bound by the protein of interest. The cloning and sequencing of these sequences finally completes their identification.

  18. 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.

  19. 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.

  20. 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.

  1. Nucleic Acid Database: a Repository of Three-Dimensional Information about Nucleic Acids

    DOE Data Explorer

    Berman, H. M.; Olson, W. K.; Beveridge, D. L.; Westbrook, J.; Gelbin, A.; Demeny, T.; Hsieh, S. H.; Srinivasan, A. R.; Schneider, B.

    The Nucleic Acid Database (NDB) provides 3-D structural information about nucleic acids.  It is a relational database designed to facilitate the easy search for nucleic acid structures using any of the stored primary or derived structural features. Reports can then be created describing any properties of the selected structures and structures may be viewed in several different formats, including the mmCIF format, the NDB Atlas format, the NDB coordinate format, or the PDB coordinate format. Browsing structure images created directly from coordinates in the repository can also be done. More than 7000 structures have been released as of May 2014. This website also includes a number of specialized tools and interfaces. The NDB Project is funded by the National Institutes of Health and has been funded by the National Science Foundation and the Department of Energy in the past.

  2. Suitability of an automated nucleic acid extractor (easyMAG) for use with hepatitis C virus and human immunodeficiency virus type 1 nucleic acid amplification testing.

    PubMed

    Jarvis, L M; Mulligan, K; Dunsford, T H; McGowan, K; Petrik, J

    2011-02-01

    Serological screening assays have greatly reduced, but not eliminated, the risk of transmission of viral infections by transfusion of blood and blood products. In addition, the 1999 regulation of the European Agency for the Evaluation of Medicinal Products requiring all plasma for fractionation to have tested negative for hepatitis C virus (HCV) RNA (CPMP/BWP/390/97, 1998) led many blood transfusion services to introduce nucleic acid amplification technology (NAT) to screen blood donations for HCV, and in some services for human immunodeficiency virus (HIV) and hepatitis B virus (HBV). BioMérieux's second-generation system, the NucliSENS easyMAG, was evaluated as a suitable platform for the automated extraction of nucleic acids for use with the existing SNBTS NAT assays. Two nucleic acid extraction protocols were examined, either lysis on the easyMAG (on board) or a 30-min pre-incubation of the sample with lysis buffer at 37 °C (off board). Off board lysis was found to extract nucleic acid more efficiently for both HCV and HIV NAT assays although the improvement was more marked with HIV. The 95% limit of detections (LODs) were 10.11 IU/ml (on board) and 7.21 IU/ml (off board) for HCV and 55.11 IU/ml (on board) and 34.13 (off board) for HIV. Using the more sensitive off board lysis, nucleic acid extraction specificity, robustness and reliability of the easyMAG were examined and over 10,000 Scottish blood donations (in 107 pools of 95 donations) were tested for HCV and HIV in parallel with the existing assay. The results indicate that the easyMAG is a suitable and flexible nucleic acid extraction system, providing high quality nucleic acids and a rapid response alternative to commercial, fully automated, approved blood screening platforms. © 2010 Elsevier B.V. All rights reserved.

  3. A Pan-Dengue Virus Reverse Transcription-Insulated Isothermal PCR Assay Intended for Point-of-Need Diagnosis of Dengue Virus Infection by Use of the POCKIT Nucleic Acid Analyzer

    PubMed Central

    Rajapakse, R. P. V. Jayanthe; Kularatne, Senanayake A. M.; Lee, Pei-Yu Alison; Ku, Keun Bon; Nam, Sangwoo; Chou, Pin-Hsing; Tsai, Yun-Long; Liu, Yu-Lun; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas

    2016-01-01

    Dengue virus (DENV) infection is considered a major public health problem in developing tropical countries where the virus is endemic and continues to cause major disease outbreaks every year. Here, we describe the development of a novel, inexpensive, and user-friendly diagnostic assay based on a reverse transcription-insulated isothermal PCR (RT-iiPCR) method for the detection of all four serotypes of DENV in clinical samples. The diagnostic performance of the newly established pan-DENV RT-iiPCR assay targeting a conserved 3′ untranslated region of the viral genome was evaluated. The limit of detection with a 95% confidence was estimated to be 10 copies of in vitro-transcribed (IVT) RNA. Sensitivity analysis using RNA prepared from 10-fold serial dilutions of tissue culture fluid containing DENVs suggested that the RT-iiPCR assay was comparable to the multiplex real-time quantitative RT-PCR (qRT-PCR) assay for DENV-1, -3, and -4 detection but 10-fold less sensitive for DENV-2 detection. Subsequently, plasma collected from patients suspected of dengue virus infection (n = 220) and individuals not suspected of dengue virus infection (n = 45) were tested by the RT-iiPCR and compared to original test results using a DENV NS1 antigen rapid test and the qRT-PCR. The diagnostic agreement of the pan-DENV RT-iiPCR, NS1 antigen rapid test, and qRT-PCR tests was 93.9%, 84.5%, and 97.4%, respectively, compared to the composite reference results. This new RT-iiPCR assay along with the portable POCKIT nucleic acid analyzer could provide a highly reliable, sensitive, and specific point-of-need diagnostic assay for the diagnosis of DENV in clinics and hospitals in developing countries. PMID:27030492

  4. Nucleic acid extraction from Agrobacterium strains.

    PubMed

    Wise, Arlene A; Liu, Zhenying; Binns, Andrew N

    2006-01-01

    Agrobacterium is routinely used as a tool for moving genetic constructs into plant cells. The successful use of Agrobacterium as a tool for the genetic engineering of plant cells often requires the manipulation and analysis of nucleic acids present in recombinant Agrobacterium strains. Here we present dependable methods for the isolation of genomic (total) DNA, mega-plasmid DNA, shuttle or binary plasmid DNA, and RNA. In addition, we provide a simple method for the electronic transfer of shuttle plasmids from Agrobacterium to E. coli for use when their low copy number in Agrobacterium impedes plasmid isolation from that strain.

  5. Nucleic acid amplification using modular branched primers

    DOEpatents

    Ulanovsky, Levy

    2001-01-01

    Methods and compositions expand the options for making primers for use in amplifying nucleic acid segments. The invention eliminates the step of custom synthesis of primers for Polymerase Chain Reactions (PCR). Instead of being custom-synthesized, a primer is replaced by a combination of several oligonucleotide modules selected from a pre-synthesized library. A modular combination of just a few oligonucleotides essentially mimics the performance of a conventional, custom-made primer by matching the sequence of the priming site in the template. Each oligonucleotide module has a segment that matches one of the stretches within the priming site.

  6. Kinetics of ligand binding to nucleic acids.

    PubMed

    Arakelyan, V B; Babayan, S Y; Tairyan, V I; Arakelyan, A V; Parsadanyan, M A; Vardevanyan, P O

    2006-02-01

    Ligand binding to nucleic acids (NA) is considered as a stationary Markov process. It is shown that the probabilistic description of ligand-NA binding allows one to describe not only the kinetics of the change of number of bound ligands at arbitrary fillings but also to calculate stationary values of the number of bound ligands and its dispersion. The general analysis of absorption isotherms and kinetics of ligand binding to NA make it possible to determine of rate constants of ligand-NA complex formation and dissociation.

  7. Nucleic acid probes in diagnostic medicine

    NASA Technical Reports Server (NTRS)

    Oberry, Phillip A.

    1991-01-01

    The need for improved diagnostic procedures is outlined and variations in probe technology are briefly reviewed. A discussion of the application of probe technology to the diagnosis of disease in animals and humans is presented. A comparison of probe versus nonprobe diagnostics and isotopic versus nonisotopic probes is made and the current state of sequence amplification is described. The current market status of nucleic acid probes is reviewed with respect to their diagnostic application in human and veterinary medicine. Representative product examples are described and information on probes being developed that offer promise as future products is discussed.

  8. Nucleic acid amplification using modular branched primers

    DOEpatents

    Ulanovsky, Levy; Raja, Mugasimangalam C.

    2001-01-01

    Methods and compositions expand the options for making primers for use in amplifying nucleic acid segments. The invention eliminates the step of custom synthesis of primers for Polymerase Chain Reactions (PCR). Instead of being custom-synthesized, a primer is replaced by a combination of several oligonucleotide modules selected from a pre-synthesized library. A modular combination of just a few oligonucleotides essentially mimics the performance of a conventional, custom-made primer by matching the sequence of the priming site in the template. Each oligonucleotide module has a segment that matches one of the stretches within the priming site.

  9. Electrocatalysis in proteins, nucleic acids and carbohydrates.

    PubMed

    Paleček, Emil; Bartošík, Martin; Ostatná, Veronika; Trefulka, Mojmír

    2012-02-01

    The ability of proteins to catalyze hydrogen evolution has been known for more than 80 years, but the poorly developed d.c. polarographic "pre-sodium wave" was of little analytical use. Recently, we have shown that by using constant current chronopotentiometric stripping analysis, proteins produce a well-developed peak H at hanging mercury drop and solid amalgam electrodes. Peak H sensitively reflects changes in protein structures due to protein denaturation, single amino acid exchange, etc. at the picomole level. Unmodified DNA and RNA do not yield such a peak, but they produce electrocatalytic voltammetric signals after modification with osmium tetroxide complexes with nitrogen ligands [Os(VIII)L], binding covalently to pyrimidine bases in nucleic acids. Recently, it has been shown that six-valent [Os(VI)L] complexes bind to 1,2-diols in polysaccharides and oligosaccharides, producing voltammetric responses similar to those of DNA-Os(VIII)L adducts. Electrocatalytic peaks produced by Os-modified nucleic acids, proteins (reaction with tryptophan residues) and carbohydrates are due to the catalytic hydrogen evolution, allowing determination of oligomers at the picomolar level.

  10. 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.

  11. 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

  12. Designer nucleic acids to probe and program the cell.

    PubMed

    Krishnan, Yamuna; Bathe, Mark

    2012-12-01

    Recent advances in nucleic acid sequencing, structural, and computational technologies have resulted in dramatic progress in our understanding of nucleic acid structure and function in the cell. This knowledge, together with the predictable base-pairing of nucleic acids and powerful synthesis and expression capabilities now offers the unique ability to program nucleic acids to form precise 3D architectures with diverse applications in synthetic and cell biology. The unique modularity of structural motifs that include aptamers, DNAzymes, and ribozymes, together with their well-defined construction rules, enables the synthesis of functional higher-order nucleic acid complexes from these subcomponents. As we illustrate here, these highly programmable, smart complexes are increasingly enabling researchers to probe and program the cell in a sophisticated manner that moves well beyond the use of nucleic acids for conventional genetic manipulation alone.

  13. Geranyl diphosphate synthase molecules, and nucleic acid molecules encoding same

    DOEpatents

    Croteau, Rodney Bruce; Burke, Charles Cullen

    2008-06-24

    In one aspect, the present invention provides isolated nucleic acid molecules that each encode a geranyl diphosphate synthase protein, wherein each isolated nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:1 under conditions of 5.times.SSC at 45.degree. C. for one hour. The present invention also provides isolated geranyl diphosphate synthase proteins, and methods for altering the level of expression of geranyl diphosphate synthase protein in a host cell.

  14. SnapShot: Nucleic acid immune sensors, part 2.

    PubMed

    Hornung, Veit

    2014-12-18

    The innate immune system has evolved sensors that can detect specific molecular fingerprints of non-self RNA or DNA. At the same time, some receptors respond to nucleic acids of both exogenous and endogenous origin, yet they are spatially segregated from endogenous nucleic acids. This SnapShot schematizes families and individual members of nucleic acid sensors with a focus on their ligands and the signaling pathways they employ.

  15. [Kinetics of ligand binding to nucleic acids at random fillings].

    PubMed

    Arakelian, V B; Babaian, S Iu; Tairian, V I; Arakelian, A V; Parsadanian, M A; Vardevanian, P O

    2006-01-01

    Ligand binding with nucleic acids is described in frames of the theory of random processes. It is shown that the probabilistic description of binding of a ligand to nucleic acid allows one to describe not only the kinetics of changes in the number of bound ligands at arbitrary fillings but also to calculate stationary values of the number of bound ligands and its dispersion. A general analysis of absorption isotherms and the kinetics of ligand binding with nucleic acids allows one to determine the rate constants of formation and decomposition of the ligand-nucleic acid complex. A comparison of the results obtained with the case of low fillings is conducted.

  16. CRC handbook of chromatography: Nucleic acids and related compounds

    SciTech Connect

    Krstulovic, A.M.

    1987-01-01

    This book's contents include: Structure Elucidation of Nucleic Acid Components; Fundamentals of HPLC; Analysis of Nucleic Acids and Oligonucleotides; Extraction of Nucleic Acids from Tissues; Gel Filtration Chromatography of RNAs and DNS Fragments; Separation of tRNAs and Oligonucleotides by Mixed Mode Chromatography; Anion-Exchange and Reversed-Phase HPLC of Synthetic Oligonucleotides; Nucleic Acid Components in Biological Fluids; RPLC Separation of RNA and DNA Hydrolysates; Nucleotides in Tissue Extracts; and Determination of Adenine Nucleotides and Creatine Phosphate in Various Mammalian Tissues.

  17. NALDB: nucleic acid ligand database for small molecules targeting nucleic acid.

    PubMed

    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.

  18. 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

  19. 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.

  20. 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.

  1. EGVII endoglucanase and nucleic acids encoding the same

    SciTech Connect

    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

    SciTech Connect

    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.

  3. EGVII endoglucanase and nucleic acids encoding the same

    DOEpatents

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

    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.

  4. EGVI endoglucanase and nucleic acids encoding the same

    DOEpatents

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

    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.

  5. 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.

  6. EGVII endoglucanase and nucleic acids encoding the same

    DOEpatents

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

    2008-11-11

    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.

  7. 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.

  8. Novel application of Locked Nucleic Acid chemistry for a TaqMan assay for measuring diverse Human Immunodeficiency Virus type 1 subtypes

    PubMed Central

    Li, Peilin; Ruel, Theodore; Fujimoto, Katsuya; Hatano, Hiroyu; Yukl, Steven; Eller, Leigh Anne; Liegler, Teri; Kamya, Moses; Gassasira, Anne; Dorsey, Grant; Rosenthal, Phil; Havlir, Diane; Wong, Joseph K.

    2010-01-01

    Summary There remains a need for sensitive and cost-effective assays to monitor therapy in human immunodeficiency virus type-1 (HIV-1) infection. However, the genetic diversity of HIV poses difficulties for traditional real-time PCR assays that require long oligonucleotides probes. LNA™ probes may be useful in overcoming these limits to traditional probe design. A new application of LNA™ chemistry in a Taqman assay applicable to a wide range of HIV-1 subtypes is described. This assay, based on a 13-mer LNA™ probe that matches the majority of HIV-1 sequences in the Los Alamos database, exhibited a wide dynamic range (101 to 107 copies of HIV DNA), high sensitivity (limit of detection of 1 copy of HIV DNA in 105 cells), and broad applicability to a range of HIV-1 subtypes (including A, B, C, D, F, H, B/C, and A/E CRFs). Using the LNA™ probe assay, HIV-1 DNA was detected in all dried blood spots (DBS) from treatment naïve HIV-1 positive Ugandan children, and HIV DNA levels significantly correlated with viral RNA levels in plasma (r=0.765, p<0.0001). This approach to Taqman probe design should be explored further for use in diagnosis and monitoring of HIV in resource-limited settings, especially where several subtypes co-circulate. PMID:20863855

  9. 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.

  10. Enzyme-assisted target recycling (EATR) for nucleic acid detection.

    PubMed

    Gerasimova, Yulia V; Kolpashchikov, Dmitry M

    2014-09-07

    Fast, reliable and sensitive methods for nucleic acid detection are of growing practical interest with respect to molecular diagnostics of cancer, infectious and genetic diseases. Currently, PCR-based and other target amplification strategies are most extensively used in practice. At the same time, such assays have limitations that can be overcome by alternative approaches. There is a recent explosion in the design of methods that amplify the signal produced by a nucleic acid target, without changing its copy number. This review aims at systematization and critical analysis of the enzyme-assisted target recycling (EATR) signal amplification technique. The approach uses nucleases to recognize and cleave the probe-target complex. Cleavage reactions produce a detectable signal. The advantages of such techniques are potentially low sensitivity to contamination and lack of the requirement of a thermal cycler. Nucleases used for EATR include sequence-dependent restriction or nicking endonucleases or sequence independent exonuclease III, lambda exonuclease, RNase H, RNase HII, AP endonuclease, duplex-specific nuclease, DNase I, or T7 exonuclease. EATR-based assays are potentially useful for point-of-care diagnostics, single nucleotide polymorphisms genotyping and microRNA analysis. Specificity, limit of detection and the potential impact of EATR strategies on molecular diagnostics are discussed.

  11. The 'helix clamp' in HIV-1 reverse transcriptase: a new nucleic acid binding motif common in nucleic acid polymerases.

    PubMed Central

    Hermann, T; Meier, T; Götte, M; Heumann, H

    1994-01-01

    Amino acid sequences homologous to 259KLVGKL (X)16KLLR284 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) are conserved in several nucleotide polymerizing enzymes. This amino acid motif has been identified in the crystal structure model as an element of the enzyme's nucleic acid binding apparatus. It is part of the helix-turn-helix structure, alpha H-turn-alpha I, within the 'thumb' region of HIV-1 RT. The motif grasps the complexed nucleic acid at one side. Molecular modeling studies on HIV-1 RT in complex with a nucleic acid fragment suggest that the motif has binding function in the p66 subunit as well as in the p51 subunit, acting as a kind of 'helix clamp'. Given its wide distribution within the nucleic acid polymerases, the helix clamp motif is assumed to be a structure of general significance for nucleic acid binding. Images PMID:7527138

  12. Nucleic Acid Aptamers for Living Cell Analysis

    NASA Astrophysics Data System (ADS)

    Xiong, Xiangling; Lv, Yifan; Chen, Tao; Zhang, Xiaobing; Wang, Kemin; Tan, Weihong

    2014-06-01

    Cells as the building blocks of life determine the basic functions and properties of a living organism. Understanding the structure and components of a cell aids in the elucidation of its biological functions. Moreover, knowledge of the similarities and differences between diseased and healthy cells is essential to understanding pathological mechanisms, identifying diagnostic markers, and designing therapeutic molecules. However, monitoring the structures and activities of a living cell remains a challenging task in bioanalytical and life science research. To meet the requirements of this task, aptamers, as “chemical antibodies,” have become increasingly powerful tools for cellular analysis. This article reviews recent advances in the development of nucleic acid aptamers in the areas of cell membrane analysis, cell detection and isolation, real-time monitoring of cell secretion, and intracellular delivery and analysis with living cell models. Limitations of aptamers and possible solutions are also discussed.

  13. Towards biomedical applications for nucleic acid nanodevices.

    PubMed

    Simmel, Friedrich C

    2007-12-01

    DNA and RNA can be used to construct artificial nanodevices with strong potential for future biomedical applications. DNA nanodevices can function as biosensors, which detect and report the presence of proteins and naturally occurring nucleic acids, such as mRNA or microRNAs. Complex sensors can be realized by supporting DNA devices with DNA-based information processing. Artificial DNA-based reaction networks can be created that amplify molecular signals or evaluate logical functions to report the simultaneous presence of several disease-related molecules. Other applications for DNA nanodevices are found in controlled release and drug delivery. DNA can be used to build nanocontainers for drugs or switchable hydrogels, which can trap and release compounds. For in vivo applications of DNA nanodevices, techniques for efficient packaging and delivery have been developed and the first examples of intracellular RNA-based nanodevices have already been demonstrated.

  14. 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.

  15. 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.

  16. 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…

  17. Detecting Microbial Nucleic Acids within Nematode Bodies: A Photo Essay

    USDA-ARS?s Scientific Manuscript database

    We developed a taxa-specific, fluorescence in situ hybridization (FISH) technique to localize microbial nucleic acids within nematode bodies. This technique involves hybridization of a nucleic acid probe to target microbial sequences. Hybridization is detected microscopically, as the probes have f...

  18. 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…

  19. Fluorescence upconversion microbarcodes for multiplexed biological detection: nucleic acid encoding.

    PubMed

    Zhang, Fan; Shi, Qihui; Zhang, Yichi; Shi, Yifeng; Ding, Kunlun; Zhao, Dongyuan; Stucky, Galen D

    2011-09-01

    Fluoride rare-earth-doped upconversion microbarcodes have been successfully developed for multiplexed signaling and nucleic-acid encoding. This kind of novel barcode material can be used for rapid and sensitive analysis of nucleic acids and antigens, which would have many potential applications in clinical, food, and environment detection.

  20. 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.

  1. 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.

  2. Ferrocene-containing nucleic acids. Synthesis and electrochemical properties

    NASA Astrophysics Data System (ADS)

    Zatsepin, Timofei S.; Andreev, Sergei Yu; Hianik, T.; Oretskaya, Tat'yana S.

    2003-06-01

    The published data on the synthesis of ferrocene-containing nucleic acid fragments are generalised. The known methods are systematised and their advantages and disadvantages are discussed. The use of nucleoside, nucleotide and oligonucleotide conjugates with ferrocene in the design of systems for electrochemical detection of nucleic acids is considered.

  3. 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.

  4. Nucleic Acid--Based Nanodevices in Biological Imaging

    PubMed Central

    Chakraborty, Kasturi; Veetil, Aneesh T.

    2017-01-01

    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. PMID:27294440

  5. Antibiotic treatment algorithm development based on a microarray nucleic acid assay for rapid bacterial identification and resistance determination from positive blood cultures.

    PubMed

    Rödel, Jürgen; Karrasch, Matthias; Edel, Birgit; Stoll, Sylvia; Bohnert, Jürgen; Löffler, Bettina; Saupe, Angela; Pfister, Wolfgang

    2016-03-01

    Rapid diagnosis of bloodstream infections remains a challenge for the early targeting of an antibiotic therapy in sepsis patients. In recent studies, the reliability of the Nanosphere Verigene Gram-positive and Gram-negative blood culture (BC-GP and BC-GN) assays for the rapid identification of bacteria and resistance genes directly from positive BCs has been demonstrated. In this work, we have developed a model to define treatment recommendations by combining Verigene test results with knowledge on local antibiotic resistance patterns of bacterial pathogens. The data of 275 positive BCs were analyzed. Two hundred sixty-three isolates (95.6%) were included in the Verigene assay panels, and 257 isolates (93.5%) were correctly identified. The agreement of the detection of resistance genes with subsequent phenotypic susceptibility testing was 100%. The hospital antibiogram was used to develop a treatment algorithm on the basis of Verigene results that may contribute to a faster patient management.

  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. Comparison of the acetylation of proteins and nucleic acids

    PubMed Central

    Paik, Woon Ki; Kim, Sangduk

    1970-01-01

    The possibility of acetylation of nucleic acids was examined. Although protein is actively acetylated with [1-14C]acetic acid in rat liver systems in vivo and in vitro and in a frog liver system in vivo, nucleic acids are not acetylated under these conditions; nucleic acids purified from these sources are without radioactivity. Requirements for acetylation in vitro of protein in rat liver are different from those in frog liver; GSH has no effect in the rat liver system and is inhibitory in the frog liver system. Among various acetylated proteins, proteins insoluble in 0.1m-sulphuric acid have the highest radioactivity. PMID:5435491

  8. Fluorescent complexes of nucleic acids/8-hydroxyquinoline/lanthanum(III) and the fluorometry of nucleic acids

    SciTech Connect

    Cheng Zhi Huang; Ke An Li; Shen Yang Tong

    1996-07-01

    The ternary fluorescent complexes of nucleic acids/8-hydroxyquinoline/lanthanum (III) were studied. Nucleic acids in the study involve natured and thermally denatured calf thymus DNA, fish sperm DNA and yeast RNA. In the range of PH 8.0-8.4 (controlled by NH{sub 3}-NH{sub 4}Cl buffer) ternary fluorescent complexes are formed which emit at 485.0 nm for calf thymus DNA and at 480.0 nm for fish sperm DNA when excited at 265.0 nm. Based on the fluorescence reactions sensitive fluorometric methods for nucleic acids were proposed. Using optimal conditions, the calibration curves were linear in the range of 0.4 --3.6 {mu}g{sup .}ml{sup -1} for calf thymus DNA, 0.4 -- 4.0 {mu}g{sup .}ml{sup -1} for fish sperm DNA and 0.4 --4.0{mu}g{sup .}ml{sup -1} for yeast RNA, respectively. Five synthetic samples were determined with satisfaction.

  9. Gamma Peptide Nucleic Acids: As Orthogonal Nucleic Acid Recognition Codes for Organizing Molecular Self-Assembly.

    PubMed

    Sacui, Iulia; Hsieh, Wei-Che; Manna, Arunava; Sahu, Bichismita; Ly, Danith H

    2015-07-08

    Nucleic acids are an attractive platform for organizing molecular self-assembly because of their specific nucleobase interactions and defined length scale. Routinely employed in the organization and assembly of materials in vitro, however, they have rarely been exploited in vivo, due to the concerns for enzymatic degradation and cross-hybridization with the host's genetic materials. Herein we report the development of a tight-binding, orthogonal, synthetically versatile, and informationally interfaced nucleic acid platform for programming molecular interactions, with implications for in vivo molecular assembly and computing. The system consists of three molecular entities: the right-handed and left-handed conformers and a nonhelical domain. The first two are orthogonal to each other in recognition, while the third is capable of binding to both, providing a means for interfacing the two conformers as well as the natural nucleic acid biopolymers (i.e., DNA and RNA). The three molecular entities are prepared from the same monomeric chemical scaffold, with the exception of the stereochemistry or lack thereof at the γ-backbone that determines if the corresponding oligo adopts a right-handed or left-handed helix, or a nonhelical motif. These conformers hybridize to each other with exquisite affinity, sequence selectivity, and level of orthogonality. Recognition modules as short as five nucleotides in length are capable of organizing molecular assembly.

  10. Characteristics of Cell-Penetrating Peptide/Nucleic Acid Nanoparticles.

    PubMed

    Margus, Helerin; Arukuusk, Piret; Langel, Ülo; Pooga, Margus

    2016-01-04

    Nucleic acids are highly promising candidates for the treatment of various genetic diseases. However, due to the large size and negative charge, nucleic acids are not efficiently taken up by cells, and thus, their clinical potential remains limited so far. Therefore, various delivery vehicles have been designed to assist the cellular uptake of nucleic acids. Among these, cell-penetrating peptides (CPPs) have gained increasing popularity as efficient and nontoxic delivery vectors. CPPs can be coupled to nucleic acids either by covalent or noncovalent association. Noncovalent coupling, which is based on the formation of nanoparticle-like nanocomplexes (NP), has received much attention in recent years, and the number of studies employing the strategy is explosively increasing due to the high therapeutic potential. However, the properties of CPP/nucleic acid NPs have not been characterized in sufficient detail yet. We performed a comprehensive analysis of the size and morphology of nucleic acid nanoparticles with novel transfection peptides, PepFects (PFs) and NickFects (NFs), using negative staining transmission electron microscopy (TEM). In addition, we examined whether the attachment of fluorescence or (nano)gold label to nucleic acid affects the nanocomplex formation or its morphology. We demonstrated that transportan-10-based new generation CPPs from PF and NF families condense nucleic acids to NPs of homogeneous size and shape. The size and shape of assembled nanoparticles depend on the type of the complexed nucleic acid and the sequence of the used peptide, whereas the label on the nucleic acid does not influence the gross characteristics of formed NPs.

  11. Performance of self-collected penile-meatal swabs compared to clinician-collected urethral swabs for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and Mycoplasma genitalium by nucleic acid amplification assays.

    PubMed

    Dize, Laura; Barnes, Perry; Barnes, Mathilda; Hsieh, Yu-Hsiang; Marsiglia, Vincent; Duncan, Della; Hardick, Justin; Gaydos, Charlotte A

    2016-10-01

    Men were enrolled in a study to assess the performance and acceptability of self-collected penile meatal swabs as compared to clinician-collected urethral swabs for sexually transmitted infections (STIs). We expected penile-meatal swabs to perform favorably to urethral swabs for Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), and Mycoplasma genitalium (MG) detection by nucleic acid amplification assays (NAATs). Of 203 swab pairs tested; for CT, penile-meatal swab sensitivity was 96.8% and specificity was 98.8%. NG sensitivity and specificity were 100% and 98.9%, respectively. For TV, sensitivity was 85.0% and specificity was 96.7%. For MG sensitivity and specificity were 79.3% and 99.4%, respectively. No significant statistical differences between sample type accuracy (CT: P=0.625; NG: P=0.248; TV: P=0.344; and MG: P=0.070) existed. Most men, 90.1%, reported self-collection of penile-meatal swabs as "Very Easy" or "Easy". Self-collected penile-meatal swabs appeared acceptable for NAAT STI detection and an acceptable collection method by men.

  12. A Simple, Low-Cost Platform for Real-Time Isothermal Nucleic Acid Amplification.

    PubMed

    Craw, Pascal; Mackay, Ruth E; Naveenathayalan, Angel; Hudson, Chris; Branavan, Manoharanehru; Sadiq, S Tariq; Balachandran, Wamadeva

    2015-09-16

    Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays have resulted in a range of solutions for nucleic acid amplification tests suited for point of care and field use. However, miniaturisation of instrumentation for such assays has not seen such rapid advances and fluorescence based assays still depend on complex, bulky and expensive optics such as fluorescence microscopes, photomultiplier tubes and sensitive lens assemblies. In this work we demonstrate a robust, low cost platform for isothermal nucleic acid amplification on a microfluidic device. Using easily obtainable materials and commercial off-the-shelf components, we show real time fluorescence detection using a low cost photodiode and operational amplifier without need for lenses. Temperature regulation on the device is achieved using a heater fabricated with standard printed circuit board fabrication methods. These facile construction methods allow fabrications at a cost compatible with widespread deployment to resource poor settings.

  13. A Simple, Low-Cost Platform for Real-Time Isothermal Nucleic Acid Amplification

    PubMed Central

    Craw, Pascal; Mackay, Ruth E.; Naveenathayalan, Angel; Hudson, Chris; Branavan, Manoharanehru; Sadiq, S. Tariq; Balachandran, Wamadeva

    2015-01-01

    Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays have resulted in a range of solutions for nucleic acid amplification tests suited for point of care and field use. However, miniaturisation of instrumentation for such assays has not seen such rapid advances and fluorescence based assays still depend on complex, bulky and expensive optics such as fluorescence microscopes, photomultiplier tubes and sensitive lens assemblies. In this work we demonstrate a robust, low cost platform for isothermal nucleic acid amplification on a microfluidic device. Using easily obtainable materials and commercial off-the-shelf components, we show real time fluorescence detection using a low cost photodiode and operational amplifier without need for lenses. Temperature regulation on the device is achieved using a heater fabricated with standard printed circuit board fabrication methods. These facile construction methods allow fabrications at a cost compatible with widespread deployment to resource poor settings. PMID:26389913

  14. 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.

  15. A Nucleic Acid Probe and Method for the Rapid Detection of Typhoid Fever Bacteria.

    DTIC Science & Technology

    This invention relates to a nucleic acid probe and method for the rapid detection of typhoid fever bacteria by use of a nucleic acid hybridization...a nucleic acid hybridization reaction with a clinical specimen containing typhoid fever bacteria.

  16. Sensitivity of the Quidel Sofia Fluorescent Immunoassay Compared With 2 Nucleic Acid Assays and Viral Culture to Detect Pandemic Influenza A(H1N1)pdm09.

    PubMed

    Arbefeville, Sophie S; Fickle, Ann R; Ferrieri, Patricia

    2015-01-01

    To confirm a diagnosis of influenza at the point of care, healthcare professionals may rely on rapid influenza diagnostic tests (RIDTs). RIDTs have low to moderate sensitivity compared with viral culture or real-time reverse-transcription polymerase chain reaction (rRT-PCR). With the resurgence of the influenza A (Flu A; subtype H1N1) pandemic 2009 (pdm09) strain in the years 2013 and 2014, we evaluated the accuracy of the United State Food and Drug Administration (FDA)-approved Sofia Influenza A+B Fluorescent Immunoassay to detect epidemic Flu A(H1N1)pdm09 in specimens from the upper-respiratory tract. During a 3-month period, we collected 40 specimens that tested positive via PCR and/or culture for Flu A of the H1N1 pdm09 subtype. Of the 40 specimens, 27 tested positive (67.5%) via Sofia assay for Flu A. Of the 13 specimens with a negative result via Sofia testing, 4 had coinfection, as detected by the GenMark Diagnostics eSensor Respiratory Viral Panel. This sensitivity of the RIDT Sofia assay to detect Flu A(H1N1) pdm09 was comparable to previously reported sensitivities ranging from 10% to 75% for older RIDTs.

  17. 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

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

    SciTech Connect

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

    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. Insights gained from these studies are described and future directions of this field are also discussed.

  19. 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

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

    PubMed

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

    2014-12-10

    In this review, 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. Insights gained from these studies are described and future directions of this field are also discussed.

  1. Scaffolding along nucleic acid duplexes using 2'-amino-locked nucleic acids.

    PubMed

    Astakhova, I Kira; Wengel, Jesper

    2014-06-17

    CONSPECTUS: Incorporation of chemically modified nucleotide scaffolds into nucleic acids to form assemblies rich in function is an innovative area with great promise for nanotechnology and biomedical and material science applications. The intrinsic biorecognition potential of nucleic acids combined with advanced properties of the locked nucleic acids (LNAs) provide opportunities to develop new nanomaterials and devices like sensors, aptamers, and machines. In this Account, we describe recent research on preparation and investigation of the properties of LNA/DNA hybrids containing functionalized 2'-amino-LNA nucleotides. By application of different chemical reactions, modification of 2'-amino-LNA scaffolds can be efficiently performed in high yields and with various tags, postsynthetically or during the automated oligonucleotide synthesis. The choice of a synthetic method for scaffolding along 2'-amino-LNA mainly depends on the chemical nature of the modification, its price, its availability, and applications of the product. One of the most useful applications of the product LNA/DNA scaffolds containing 2'-amino-LNA is to detect complementary DNA and RNA targets. Examples of these applications include sensing of clinically important single-nucleotide polymorphisms (SNPs) and imaging of nucleic acids in vitro, in cell culture, and in vivo. According to our studies, 2'-amino-LNA scaffolds are efficient within diagnostic probes for DNA and RNA targets and as therapeutics, whereas both 2'-amino- and isomeric 2'-α-l-amino-LNA scaffolds have promising properties for stabilization and detection of DNA nanostructures. Attachment of fluorescent groups to the 2'-amino group results in very high fluorescent quantum yields of the duplexes and remarkable sensitivity of the fluorescence signal to target binding. Notably, fluorescent LNA/DNA probes bind nucleic acid targets with advantages of high affinity and specificity. Thus, molecular motion of nanodevices and programmable

  2. Computational Approaches to Nucleic Acid Origami.

    PubMed

    Jabbari, Hosna; Aminpour, Maral; Montemagno, Carlo

    2015-10-12

    Recent advances in experimental DNA origami have dramatically expanded the horizon of DNA nanotechnology. Complex 3D suprastructures have been designed and developed using DNA origami with applications in biomaterial science, nanomedicine, nanorobotics, and molecular computation. Ribonucleic acid (RNA) origami has recently been realized as a new approach. Similar to DNA, RNA molecules can be designed to form complex 3D structures through complementary base pairings. RNA origami structures are, however, more compact and more thermodynamically stable due to RNA's non-canonical base pairing and tertiary interactions. With all these advantages, the development of RNA origami lags behind DNA origami by a large gap. Furthermore, although computational methods have proven to be effective in designing DNA and RNA origami structures and in their evaluation, advances in computational nucleic acid origami is even more limited. In this paper, we review major milestones in experimental and computational DNA and RNA origami and present current challenges in these fields. We believe collaboration between experimental nanotechnologists and computer scientists are critical for advancing these new research paradigms.

  3. Metal ion-catalyzed nucleic acid alkylation and fragmentation.

    PubMed

    Browne, Kenneth A

    2002-07-10

    occurred even without incorporation of phosphorothioate moieties into the RNA and DNA target molecules. In fact, LDC conditions were found in which RNA could be fragmented into its component monomers, allowing simultaneous sequencing from both the 5'- and the 3'-termini by mass spectrometry. The results can be explained by alkylation of the (thio)phosphodiester linkages to form less hydrolytically stable (thio)phosphotriesters, which then decompose into 2',3'-cyclic phosphate (or 2'-phosphate) and 5'-hydroxyl terminal products. Analysis of fragmentation and alkylation products of Mycobacterium tuberculosis (Mtb) ribosomal RNA (rRNA) transcripts by polyacrylamide gel electrophoresis was consistent with the model studies. Building upon these results, I found that products from Mtb rRNA amplification products were processed with fluorescent reporters and metal ions in a single reaction milieu for analysis on an Affymetrix GeneChip. Mild conditions were discovered which balanced the need for aggressive alkylation and the need for controlled fragmentation, advantageously yielding GeneChip results with greater than 98% of the nucleotides reported correctly relative to reference sequences, results sufficient for accurately identifying Mtb from other Mycobacterium species. Thus, LDC is a new, straightforward, and rapid aqueous chemistry that is based on metal ion-catalyzed alkylation and alkylation-catalyzed fragmentation of nucleic acids for analysis on microarrays or other hybridization assays and that, possibly, has utility in similar processing of other appropriately functionalized biomolecules.

  4. Entrapment of nucleic acids in liposomes.

    PubMed

    Monnard, P A; Oberholzer, T; Luisi, P

    1997-10-02

    The entrapment efficiency of three main methods used in the literature for the encapsulation of nucleic acids in liposomes were studied using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes. In particular the reverse phase method, the dehydration/rehydration method, and the freeze/thawing method were compared to each other under standardised conditions, i.e. using in every case the same concentration of guest molecules (DNA, tRNA and ATP as low molecular weight analogue) and equally extruded liposomes. The percentage of entrapment strictly referred to the material localized inside the liposomes, i.e. particular care was devoted to ruling out the contribution of the nucleic acid material bound to the outer surface of the liposomes: this was eliminated by extensive enzymatic digestion prior to column chromatography. Depending on the conditions used, the percentage of the entrapped material varied between 10 and 54% of the initial amount. Further, the encapsulation efficiency was markedly affected by the salt concentration, by the size of liposomes, but to a lower degree by the molecular weight of the guest molecules. In general, we observed that the freeze/thawing encapsulation procedure was the most efficient one. In a second part of the work the freeze/thawing method was applied to encapsulate DNA (369 bp and 3368 bp, respectively) using liposomes obtained from POPC mixed with 1-10% charged cosurfactant, i.e. phosphatidylserine (PS) or didodecyldimethylammonium bromide (DDAB), respectively. Whereas PS had no significant effect, the entrapment efficiency went up to 60% in POPC/DDAB (97.5:2.5) liposomes. The large entrapment efficiency of DNA permits spectroscopic investigations of the DNA encapsulated in the water pool of the liposomes. UV absorption and circular dichroism spectra were practically the same as in water, indicating no appreciable perturbation of the electronic transitions or of the conformation of the entrapped biopolymer. This was

  5. 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

  6. Cell-free fetal nucleic acids in amniotic fluid

    PubMed Central

    Hui, L.; Bianchi, D.W.

    2011-01-01

    BACKGROUND Research into cell-free fetal (cff) nucleic acids has primarily focused on maternal plasma; however, cff DNA and RNA are also detectable in other body fluids such as amniotic fluid (AF). In AF, cff DNA is present in much greater concentrations than in maternal plasma and represents a pure fetal sample uncontaminated by maternal- and trophoblast-derived nucleic acids. The aim of this review was to summarize the current knowledge on cff nucleic acids in AF and to outline future research directions. METHODS MEDLINE and PREMEDLINE were searched up to August 2010 for original investigations of cell-free RNA or DNA in AF. Sixteen studies were included in the review. RESULTS AF cff DNA represents a physiologically separate pool from cff DNA in maternal plasma. The placenta is not a major source of nucleic acids in AF. It is feasible to isolate cff nucleic acids from small volumes of discarded AF supernatant in sufficient quality and quantity to perform microarray studies and downstream applications such as pathway analysis. This ‘discovery-driven approach’ has resulted in new information on the pathogenesis of Down syndrome and polyhydramnios. There is otherwise a paucity of information relating to the basic biology and clinical applications of cff nucleic acids in AF. CONCLUSIONS AF supernatant is a valuable and widely available but under-utilized biological resource. Further studies of cff nucleic acids in AF may lead to new insights into human fetal development and ultimately new approaches to antenatal treatment of human disease. PMID:20923874

  7. Nucleic acid amplification of HIV-1 integrase sequence subtypes CRF01_AE and B for development of HIV anti-integrase drug resistance genotyping assay

    NASA Astrophysics Data System (ADS)

    Adlar, F. R.; Bela, B.

    2017-08-01

    To anticipate the potential use of anti-integrase drugs in Indonesia for treatment of HIV-1 infection, the development of a drug resistance genotyping assay for anti-integrase is crucial in identifying the genetic drug resistance profile of Indonesian HIV-1 strains. This experiment aimed to amplify a target region in the integrase gene of Indonesian HIV-1 subtypes CRF01_AE and B that contain genetic mutations known to confer resistance to anti-integrase drug. Eleven archived plasma samples from individuals living with HIV-1 were obtained from the Virology and Cancer Pathobiology Research Center for Health Service (VCPRC FKUI-RSCM) laboratory. One of the plasma samples contained HIV-1 subtype B, and the remaining plasma samples contained subtype CRF01_AE. The target regions for all samples were amplified through RT-PCR, with an annealing temperature of 55 °C, using the primer pair AE_POL 4086F and AE_POL 5232R that were designed by VCPRC FKUI-RSCM. The results of this experiment show that 18.2% (2/11) of the samples were successfully amplified using the one-step RT-PCR. While the primer pair was effective in amplifying the target region in the integrase gene sequence for subtype B (100%; 1/1), it had a low efficacy (10%, 1/10) for subtype CRF01_AE. In conclusion, the primer pair can be used to amplify the target region in Indonesian HIV-1 strain subtypes CRF01_AE and B. However, optimization of the PCR condition and an increased number of samples would help to determine an accurate representation of the efficacy of the primer pair.

  8. Ligation with nucleic acid sequence-based amplification.

    PubMed

    Ong, Carmichael; Tai, Warren; Sarma, Aartik; Opal, Steven M; Artenstein, Andrew W; Tripathi, Anubhav

    2012-01-01

    This work presents a novel method for detecting nucleic acid targets using a ligation step along with an isothermal, exponential amplification step. We use an engineered ssDNA with two variable regions on the ends, allowing us to design the probe for optimal reaction kinetics and primer binding. This two-part probe is ligated by T4 DNA Ligase only when both parts bind adjacently to the target. The assay demonstrates that the expected 72-nt RNA product appears only when the synthetic target, T4 ligase, and both probe fragments are present during the ligation step. An extraneous 38-nt RNA product also appears due to linear amplification of unligated probe (P3), but its presence does not cause a false-positive result. In addition, 40 mmol/L KCl in the final amplification mix was found to be optimal. It was also found that increasing P5 in excess of P3 helped with ligation and reduced the extraneous 38-nt RNA product. The assay was also tested with a single nucleotide polymorphism target, changing one base at the ligation site. The assay was able to yield a negative signal despite only a single-base change. Finally, using P3 and P5 with longer binding sites results in increased overall sensitivity of the reaction, showing that increasing ligation efficiency can improve the assay overall. We believe that this method can be used effectively for a number of diagnostic assays.

  9. Nucleic acid sequence detection using multiplexed oligonucleotide PCR

    DOEpatents

    Nolan, John P.; White, P. Scott

    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.

  10. A novel nucleic acid analogue shows strong angiogenic activity

    SciTech Connect

    Tsukamoto, Ikuko; Sakakibara, Norikazu; Maruyama, Tokumi; Igarashi, Junsuke; Kosaka, Hiroaki; Kubota, Yasuo; Tokuda, Masaaki; Ashino, Hiromi; Hattori, Kenichi; Tanaka, Shinji; Kawata, Mitsuhiro; Konishi, Ryoji

    2010-09-03

    Research highlights: {yields} A novel nucleic acid analogue (2Cl-C.OXT-A, m.w. 284) showed angiogenic potency. {yields} It stimulated the tube formation, proliferation and migration of HUVEC in vitro. {yields} 2Cl-C.OXT-A induced the activation of ERK1/2 and MEK in HUVEC. {yields} Angiogenic potency in vivo was confirmed in CAM assay and rabbit cornea assay. {yields} A synthesized small angiogenic agent would have great clinical therapeutic value. -- Abstract: A novel nucleic acid analogue (2Cl-C.OXT-A) significantly stimulated tube formation of human umbilical endothelial cells (HUVEC). Its maximum potency at 100 {mu}M was stronger than that of vascular endothelial growth factor (VEGF), a positive control. At this concentration, 2Cl-C.OXT-A moderately stimulated proliferation as well as migration of HUVEC. To gain mechanistic insights how 2Cl-C.OXT-A promotes angiogenic responses in HUVEC, we performed immunoblot analyses using phospho-specific antibodies as probes. 2Cl-C.OXT-A induced robust phosphorylation/activation of MAP kinase ERK1/2 and an upstream MAP kinase kinase MEK. Conversely, a MEK inhibitor PD98059 abolished ERK1/2 activation and tube formation both enhanced by 2Cl-C.OXT-A. In contrast, MAP kinase responses elicited by 2Cl-C.OXT-A were not inhibited by SU5416, a specific inhibitor of VEGF receptor tyrosine kinase. Collectively these results suggest that 2Cl-C.OXT-A-induces angiogenic responses in HUVEC mediated by a MAP kinase cascade comprising MEK and ERK1/2, but independently of VEGF receptor tyrosine kinase. In vivo assay using chicken chorioallantoic membrane (CAM) and rabbit cornea also suggested the angiogenic potency of 2Cl-C.OXT-A.

  11. ATR-IR spectroscopy as applied to nucleic acid films

    NASA Astrophysics Data System (ADS)

    Stepanyugin, Andriy V.; Samijlenko, Svitlana P.; Martynenko, Olena I.; Hovorun, Dmytro M.

    2005-07-01

    For the first time the ATR technique was applied to obtain IR absorption spectra of DNA and RNA dry films. There was worked out procedure of the nucleic acid removal from germanium plate, which obviously was a main obstacle to application of ATR-IR spectroscopy to nucleic acids. This technique of IR spectroscopy was applied to confirmation of RNA tropism of aurin tricarboxylic acid observed by molecular biological methods.

  12. 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.

  13. 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.

  14. Lateral flow biosensors for the detection of nucleic acid.

    PubMed

    Zeng, Lingwen; Lie, Puchang; Fang, Zhiyuan; Xiao, Zhuo

    2013-01-01

    The detection of nucleic acid is of central importance for the diagnosis of genetic diseases, infectious agents, and biowarfare agents. Traditional strategies and technologies for nucleic acid detection are time-consuming and labor-intensive. Recently, isothermal strand-displacement reaction-based lateral flow biosensors have attracted a great deal of research interest because they are sensitive, simple, fast, and easy to use. Here, we describe a lateral flow biosensor based on isothermal strand-displacement polymerase reaction and gold nanoparticles for the visual detection of nucleic acid.

  15. Properties of nucleic acid staining dyes used in gel electrophoresis.

    PubMed

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

    2015-03-01

    Nucleic acid staining dyes are used for detecting nucleic acids in electrophoresis gels. Historically, the most common dye used for gel staining is ethidium bromide, however due to its toxicity and mutagenicity other dyes that are safer to the user and the environment are preferred. This Short Communication details the properties of dyes now available and their sensitivity for detection of DNA and their ability to permeate the cell membrane. It was found that GelRed™ was the most sensitive and safest dye to use with UV light excitation, and both GelGreen™ and Diamond™ Nucleic Acid Dye were sensitive and the safer dyes using blue light excitation.

  16. In vitro selection of functional nucleic acids

    NASA Technical Reports Server (NTRS)

    Wilson, D. S.; Szostak, J. W.

    1999-01-01

    In vitro selection allows rare functional RNA or DNA molecules to be isolated from pools of over 10(15) different sequences. This approach has been used to identify RNA and DNA ligands for numerous small molecules, and recent three-dimensional structure solutions have revealed the basis for ligand recognition in several cases. By selecting high-affinity and -specificity nucleic acid ligands for proteins, promising new therapeutic and diagnostic reagents have been identified. Selection experiments have also been carried out to identify ribozymes that catalyze a variety of chemical transformations, including RNA cleavage, ligation, and synthesis, as well as alkylation and acyl-transfer reactions and N-glycosidic and peptide bond formation. The existence of such RNA enzymes supports the notion that ribozymes could have directed a primitive metabolism before the evolution of protein synthesis. New in vitro protein selection techniques should allow for a direct comparison of the frequency of ligand binding and catalytic structures in pools of random sequence polynucleotides versus polypeptides.

  17. In vitro selection of functional nucleic acids

    NASA Technical Reports Server (NTRS)

    Wilson, D. S.; Szostak, J. W.

    1999-01-01

    In vitro selection allows rare functional RNA or DNA molecules to be isolated from pools of over 10(15) different sequences. This approach has been used to identify RNA and DNA ligands for numerous small molecules, and recent three-dimensional structure solutions have revealed the basis for ligand recognition in several cases. By selecting high-affinity and -specificity nucleic acid ligands for proteins, promising new therapeutic and diagnostic reagents have been identified. Selection experiments have also been carried out to identify ribozymes that catalyze a variety of chemical transformations, including RNA cleavage, ligation, and synthesis, as well as alkylation and acyl-transfer reactions and N-glycosidic and peptide bond formation. The existence of such RNA enzymes supports the notion that ribozymes could have directed a primitive metabolism before the evolution of protein synthesis. New in vitro protein selection techniques should allow for a direct comparison of the frequency of ligand binding and catalytic structures in pools of random sequence polynucleotides versus polypeptides.

  18. Fluorescent hybridization probes for nucleic acid detection.

    PubMed

    Guo, Jia; Ju, Jingyue; Turro, Nicholas J

    2012-04-01

    Due to their high sensitivity and selectivity, minimum interference with living biological systems, and ease of design and synthesis, fluorescent hybridization probes have been widely used to detect nucleic acids both in vivo and in vitro. Molecular beacons (MBs) and binary probes (BPs) are two very important hybridization probes that are designed based on well-established photophysical principles. These probes have shown particular applicability in a variety of studies, such as mRNA tracking, single nucleotide polymorphism (SNP) detection, polymerase chain reaction (PCR) monitoring, and microorganism identification. Molecular beacons are hairpin oligonucleotide probes that present distinctive fluorescent signatures in the presence and absence of their target. Binary probes consist of two fluorescently labeled oligonucleotide strands that can hybridize to adjacent regions of their target and generate distinctive fluorescence signals. These probes have been extensively studied and modified for different applications by modulating their structures or using various combinations of fluorophores, excimer-forming molecules, and metal complexes. This review describes the applicability and advantages of various hybridization probes that utilize novel and creative design to enhance their target detection sensitivity and specificity.

  19. Selenium Derivatization of Nucleic Acids for Crystallography

    SciTech Connect

    Jiang,J.; Sheng, J.; Carrasco, N.; Huang, Z.

    2007-01-01

    The high-resolution structure of the DNA (5'-GTGTACA-C-3') with the selenium derivatization at the 2'-position of T2 was determined via MAD and SAD phasing. The selenium-derivatized structure (1.28 {angstrom} resolution) with the 2'-Se modification in the minor groove is isomorphorous to the native structure (2.0 {angstrom}). To directly compare with the conventional bromine derivatization, we incorporated bromine into the 5-postion of T4, determined the bromine-derivatized DNA structure at 1.5 {angstrom} resolution, and found that the local backbone torsion angles and solvent hydration patterns were altered in the structure with the Br incorporation in the major groove. Furthermore, while the native and Br-derivatized DNAs needed over a week to form reasonable-size crystals, we observed that the Se-derivatized DNAs grew crystals overnight with high-diffraction quality, suggesting that the Se derivatization facilitated the crystal formation. In addition, the Se-derivatized DNA sequences crystallized under a broader range of buffer conditions, and generally had a faster crystal growth rate. Our experimental results indicate that the selenium derivatization of DNAs may facilitate the determination of nucleic acid X-ray crystal structures in phasing and high-quality crystal growth. In addition, our results suggest that the Se derivatization can be an alternative to the conventional Br derivatization.

  20. NAPP: the Nucleic Acid Phylogenetic Profile Database.

    PubMed

    Ott, Alban; Idali, Anouar; Marchais, Antonin; Gautheret, Daniel

    2012-01-01

    Nucleic acid phylogenetic profiling (NAPP) classifies coding and non-coding sequences in a genome according to their pattern of conservation across other genomes. This procedure efficiently distinguishes clusters of functional non-coding elements in bacteria, particularly small RNAs and cis-regulatory RNAs, from other conserved sequences. In contrast to other non-coding RNA detection pipelines, NAPP does not require the presence of conserved RNA secondary structure and therefore is likely to identify previously undetected RNA genes or elements. Furthermore, as NAPP clusters contain both coding and non-coding sequences with similar occurrence profiles, they can be analyzed under a functional perspective. We recently improved the NAPP pipeline and applied it to a collection of 949 bacterial and 68 archaeal species. The database and web interface available at http://napp.u-psud.fr/ enable detailed analysis of NAPP clusters enriched in non-coding RNAs, graphical display of phylogenetic profiles, visualization of predicted RNAs in their genome context and extraction of predicted RNAs for use with genome browsers or other software.

  1. NAPP: the Nucleic Acid Phylogenetic Profile Database

    PubMed Central

    Ott, Alban; Idali, Anouar; Marchais, Antonin; Gautheret, Daniel

    2012-01-01

    Nucleic acid phylogenetic profiling (NAPP) classifies coding and non-coding sequences in a genome according to their pattern of conservation across other genomes. This procedure efficiently distinguishes clusters of functional non-coding elements in bacteria, particularly small RNAs and cis-regulatory RNAs, from other conserved sequences. In contrast to other non-coding RNA detection pipelines, NAPP does not require the presence of conserved RNA secondary structure and therefore is likely to identify previously undetected RNA genes or elements. Furthermore, as NAPP clusters contain both coding and non-coding sequences with similar occurrence profiles, they can be analyzed under a functional perspective. We recently improved the NAPP pipeline and applied it to a collection of 949 bacterial and 68 archaeal species. The database and web interface available at http://napp.u-psud.fr/ enable detailed analysis of NAPP clusters enriched in non-coding RNAs, graphical display of phylogenetic profiles, visualization of predicted RNAs in their genome context and extraction of predicted RNAs for use with genome browsers or other software. PMID:21984475

  2. Nucleic Acid Amplification Testing for Neisseria gonorrhoeae

    PubMed Central

    Whiley, David M.; Tapsall, John W.; Sloots, Theo P.

    2006-01-01

    Nucleic acid amplification tests (NAATs) for the detection of Neisseria gonorrhoeae became available in the early 1990s. Although offering several advantages over traditional detection methods, N. gonorrhoeae NAATs do have some limitations. These include cost, risk of carryover contamination, inhibition, and inability to provide antibiotic resistance data. In addition, there are sequence-related limitations that are unique to N. gonorrhoeae NAATs. In particular, false-positive results are a major consideration. These primarily stem from the frequent horizontal genetic exchange occurring within the Neisseria genus, leading to commensal Neisseria species acquiring N. gonorrhoeae genes. Furthermore, some N. gonorrhoeae subtypes may lack specific sequences targeted by a particular NAAT. Therefore, NAAT false-negative results because of sequence variation may occur in some gonococcal populations. Overall, the N. gonorrhoeae species continues to present a considerable challenge for molecular diagnostics. The need to evaluate N. gonorrhoeae NAATs before their use in any new patient population and to educate physicians on the limitations of these tests is emphasized in this review. PMID:16436629

  3. BeadCons: detection of nucleic acid sequences by flow cytometry.

    PubMed

    Horejsh, Douglas; Martini, Federico; Capobianchi, Maria Rosaria

    2005-11-01

    Molecular beacons are single-stranded nucleic acid structures with a terminal fluorophore and a distal, terminal quencher. These molecules are typically used in real-time PCR assays, but have also been conjugated with solid matrices. This unit describes protocols related to molecular beacon-conjugated beads (BeadCons), whose specific hybridization with complementary target sequences can be resolved by cytometry. Assay sensitivity is achieved through the concentration of fluorescence signal on discrete particles. By using molecular beacons with different fluorophores and microspheres of different sizes, it is possible to construct a fluid array system with each bead corresponding to a specific target nucleic acid. Methods are presented for the design, construction, and use of BeadCons for the specific, multiplexed detection of unlabeled nucleic acids in solution. The use of bead-based detection methods will likely lead to the design of new multiplex molecular diagnostic tools.

  4. Digital MDA for enumeration of total nucleic acid contamination

    PubMed Central

    Blainey, Paul C.; Quake, Stephen R.

    2011-01-01

    Multiple displacement amplification (MDA) is an isothermal, sequence-independent method for the amplification of high molecular weight DNA that is driven by ϕ29 DNA polymerase (DNAP). Here we report digital MDA (dMDA), an ultrasensitive method for quantifying nucleic acid fragments of unknown sequence. We use the new assay to show that our custom ϕ29 DNAP preparation is free of contamination at the limit of detection of the dMDA assay (1 contaminating molecule per assay microliter). Contamination in commercially available preparations is also investigated. The results of the dMDA assay provide strong evidence that the so-called ‘template-independent’ MDA background can be attributed to high-molecular weight contaminants and is not primer-derived in the commercial kits tested. dMDA is orders of magnitude more sensitive than PCR-based techniques for detection of microbial genomic DNA fragments and opens up new possibilities for the ultrasensitive quantification of DNA fragments in a wide variety of application areas using MDA chemistry and off-the-shelf hardware developed for digital PCR. PMID:21071419

  5. SAXS studies of ion-nucleic acid interactions.

    PubMed

    Pollack, Lois

    2011-01-01

    Positively charged ions, atoms, or molecules compensate the high negative charge of the nucleic acid backbone. Their presence is critical to the biological function of DNA and RNA. This review focuses on experimental studies probing (a) interactions between small ions and nucleic acids and (b) ion-mediated interactions between nucleic acid duplexes. Experimental results on these simple model systems can be compared with specific theoretical models to validate their predictions. Small angle X-ray scattering (SAXS) provides unique insight into these interactions. Anomalous SAXS reports the spatial correlations of condensed (e.g., locally concentrated) counterions to individual DNA or RNA duplexes. SAXS very effectively reports interactions between nucleic acid helices, which range from strongly repulsive to strongly attractive depending on the ionic species present. The sign and strength of interparticle interactions are easily deduced from dramatic changes in the scattering profiles of interacting duplexes.

  6. Nucleic Acid Conjugated Nanomaterials for Enhanced Molecular Recognition

    PubMed Central

    Wang, Hao; Yang, Ronghua; Yang, Liu; Tan, Weihong

    2009-01-01

    Nucleic acids, whether designed or selected in vitro, play important roles in biosensing, medical diagnostics and therapy. Specifically, the conjugation of functional nucleic acid-based probe molecules and nanomaterials has resulted in an unprecedented improvement in the field of molecular recognition. With their unique physical and chemical properties, nanomaterials facilitate the sensing process and amplify the signal of recognition events. Thus, the coupling of nucleic acids with various nanomaterials opens up a promising future for molecular recognition. The literature offers a broad spectrum of recent advances in biosensing by employing different nano-platforms with designed nucleic acids, especially gold nanoparticles, carbon nanotubes, silica nanoparticles and quantum dots. The advantages of these novel combinations are discussed from the perspective of molecular recognition in chemistry, biology and medicine, along with the problems confronting future applications. PMID:19658387

  7. 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).

  8. 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.

  9. 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.

  10. Isolated menthone reductase and nucleic acid molecules encoding same

    DOEpatents

    Croteau, Rodney B; Davis, Edward M; Ringer, Kerry L

    2013-04-23

    The present invention provides isolated menthone reductase proteins, isolated nucleic acid molecules encoding menthone reductase proteins, methods for expressing and isolating menthone reductase proteins, and transgenic plants expressing elevated levels of menthone reductase protein.

  11. Chemical processes induced by OH attack on nucleic acids

    NASA Astrophysics Data System (ADS)

    Kuwabara, Mikinori

    Recent studies concerning the chemical processes in nucleic acids starting with OH attack to produce free radicals and ending with the formation of stable products were reviewed. Using nucleosides, nucleotides and homopolynucleotides as model compounds, and DNA itself, free radicals produced by OH attack on nucleic acids have been mainly studied by a method combining ESR, spin trapping and high-performance liquid chromatography. For identification of final products in both base and sugar moieties of nucleic acids, mass and NMR spectroscopies combined with gas chromatography or high-performance liquid chromatography are usually employed. Kinetic measurements of structural alterations in the polynucleotides and DNA after OH attack have been made by a method combining electron-pulse irradiation and laser-light scattering. From these studies, the chemical reaction processes from the generation of free radicals in nucleic acids by OH attack, through the formation of unstable intermediates, to the formation of final products can be described.

  12. Nucleic Acid Duplexes Incorporating a Dissociable Covalent Base Pair

    NASA Astrophysics Data System (ADS)

    Gao, Kui; Orgel, Leslie E.

    1999-12-01

    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.

  13. Changes of nucleic acids of wheat seedlings under spaceflight conditions

    NASA Technical Reports Server (NTRS)

    Sytnyk, K. M.; Musatenko, L. I.

    1983-01-01

    The effects of space flight on the growth of wheat seedlings and their nucleic acid content were studied. It was shown that both space and ground seedlings have almost the same appearance, dry weight and nucleic acid content in the root, coleoptile and leaves. The only difference found is in the RNA and DNA content, which is twice as much in the ground seedling apices as in the space-grown seedlings.

  14. Nucleic Acid Delivery for Endothelial Dysfunction in Cardiovascular Diseases

    PubMed Central

    Deshpande, Dipti; Janero, David R.; Segura-Ibarra, Victor; Blanco, Elvin; Amiji, Mansoor M.

    2016-01-01

    Endothelial dysfunction has been implicated in the pathophysiology of multiple cardiovascular diseases and involves components of both innate and acquired immune mechanisms. Identifying signature patterns and targets associated with endothelial dysfunction can help in the development of novel nanotherapeutic platforms for treatment of vascular diseases. This review discusses nucleic acid-based regulation of endothelial function and the different nucleic acid-based nanotherapeutic approaches designed to target endothelial dysfunction in cardiovascular disorders. PMID:27826366

  15. Molecular beacons: nucleic acid hybridization and emerging applications.

    PubMed

    Antony, T; Subramaniam, V

    2001-12-01

    Molecular beacons (MBs) are a novel class of nucleic acid probes that become fluorescent when bound to a complementary sequence. Because of this characteristic, coupled with the sequence specificity of nucleic acid hybridization and the sensitivity of fluorescence techniques, MBs are very useful probes for a variety of applications requiring the detection of DNA or RNA. We survey various applications of MBs, including the monitoring of DNA triplex formation, and describe recent developments in MB design that enhance their sensitivity.

  16. 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.

  17. Digital PCR analysis of circulating nucleic acids.

    PubMed

    Hudecova, Irena

    2015-10-01

    Detection of plasma circulating nucleic acids (CNAs) requires the use of extremely sensitive and precise methods. The commonly used quantitative real-time polymerase chain reaction (PCR) poses certain technical limitations in relation to the precise measurement of CNAs whereas the costs of massively parallel sequencing are still relatively high. Digital PCR (dPCR) now represents an affordable and powerful single molecule counting strategy to detect minute amounts of genetic material with performance surpassing many quantitative methods. Microfluidic (chip) and emulsion (droplet)-based technologies have already been integrated into platforms offering hundreds to millions of nanoliter- or even picoliter-scale reaction partitions. The compelling observations reported in the field of cancer research, prenatal testing, transplantation medicine and virology support translation of this technology into routine use. Extremely sensitive plasma detection of rare mutations originating from tumor or placental cells among a large background of homologous sequences facilitates unraveling of the early stages of cancer or the detection of fetal mutations. Digital measurement of quantitative changes in plasma CNAs associated with cancer or graft rejection provides valuable information on the monitoring of disease burden or the recipient's immune response and subsequent therapy treatment. Furthermore, careful quantitative assessment of the viral load offers great value for effective monitoring of antiviral therapy for immunosuppressed or transplant patients. The present review describes the inherent features of dPCR that make it exceptionally robust in precise and sensitive quantification of CNAs. Moreover, I provide an insight into the types of potential clinical applications that have been developed by researchers to date.

  18. 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

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

    PubMed Central

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

    2016-01-01

    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. PMID:27528673

  20. 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.

  1. Bioanalytical applications of isothermal nucleic acid amplification techniques.

    PubMed

    Deng, Huimin; Gao, Zhiqiang

    2015-01-01

    The most popular in vitro nucleic acid amplification techniques like polymerase chain reaction (PCR) including real-time PCR are costly and require thermocycling, rendering them unsuitable for uses at point-of-care. Highly efficient in vitro nucleic acid amplification techniques using simple, portable and low-cost instruments are crucial in disease diagnosis, mutation detection and biodefense. Toward this goal, isothermal amplification techniques that represent a group of attractive in vitro nucleic acid amplification techniques for bioanalysis have been developed. Unlike PCR where polymerases are easily deactivated by thermally labile constituents in a sample, some of the isothermal nucleic acid amplification techniques, such as helicase-dependent amplification and nucleic acid sequence-based amplification, enable the detection of bioanalytes with much simplified protocols and with minimal sample preparations since the entire amplification processes are performed isothermally. This review focuses on the isothermal nucleic acid amplification techniques and their applications in bioanalytical chemistry. Starting off from their amplification mechanisms and significant properties, the adoption of isothermal amplification techniques in bioanalytical chemistry and their future perspectives are discussed. Representative examples illustrating the performance and advantages of each isothermal amplification technique are discussed along with some discussion on the advantages and disadvantages of each technique.

  2. Sensors of Infection: Viral Nucleic Acid PRRs in Fish

    PubMed Central

    Poynter, Sarah; Lisser, Graeme; Monjo, Andrea; DeWitte-Orr, Stephanie

    2015-01-01

    Viruses produce nucleic acids during their replication, either during genomic replication or transcription. These nucleic acids are present in the cytoplasm or endosome of an infected cell, or in the extracellular space to be sensed by neighboring cells during lytic infections. Cells have mechanisms of sensing virus-generated nucleic acids; these nucleic acids act as flags to the cell, indicating an infection requiring defense mechanisms. The viral nucleic acids are called pathogen-associated molecular patterns (PAMPs) and the sensors that bind them are called pattern recognition receptors (PRRs). This review article focuses on the most recent findings regarding nucleic acids PRRs in fish, including: Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), cytoplasmic DNA sensors (CDSs) and class A scavenger receptors (SR-As). It also discusses what is currently known of the downstream signaling molecules for each PRR family and the resulting antiviral response, either type I interferons (IFNs) or pro-inflammatory cytokine production. The review highlights what is known but also defines what still requires elucidation in this economically important animal. Understanding innate immune systems to virus infections will aid in the development of better antiviral therapies and vaccines for the future. PMID:26184332

  3. 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.

  4. 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.

  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. 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.

  7. Diagnostics based on nucleic acid sequence variant profiling: PCR, hybridization, and NGS approaches.

    PubMed

    Khodakov, Dmitriy; Wang, Chunyan; Zhang, David Yu

    2016-10-01

    Nucleic acid sequence variations have been implicated in many diseases, and reliable detection and quantitation of DNA/RNA biomarkers can inform effective therapeutic action, enabling precision medicine. Nucleic acid analysis technologies being translated into the clinic can broadly be classified into hybridization, PCR, and sequencing, as well as their combinations. Here we review the molecular mechanisms of popular commercial assays, and their progress in translation into in vitro diagnostics. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  8. CKC: isolation of nucleic acids from a diversity of plants using CTAB and silica columns.

    PubMed

    Henderson, David C; Hammond, John

    2013-02-01

    To assay for viruses in plant samples, we required a method for nucleic acid isolation that is rapid, simple, and applicable to the widest possible variety of plants. A protocol for isolation of total nucleic acid (TNA) was developed by combining common CTAB methods with silica spin columns. We report data on TNA purity and RNA quality from over 30 plant genera representing 25 families. Measurements showed that RNA is of high quality, and one-step RT-PCR was successfully performed on all samples. The protocol can be completed in less than 2 h.

  9. Microbial Nucleic Acid Sensing in Oral and Systemic Diseases.

    PubMed

    Crump, K E; Sahingur, S E

    2016-01-01

    One challenge in studying chronic infectious and inflammatory disorders is understanding how host pattern recognition receptors (PRRs), specifically toll-like receptors (TLRs), sense and respond to pathogen- or damage-associated molecular patterns, their communication with each other and different components of the immune system, and their role in propagating inflammatory stages of disease. The discovery of innate immune activation through nucleic acid recognition by intracellular PRRs such as endosomal TLRs (TLR3, TLR7, TLR8, and TLR9) and cytoplasmic proteins (absent in melanoma 2 and DNA-dependent activator of interferon regulatory factor) opened a new paradigm: Nucleic acid sensing is now implicated in multiple immune and inflammatory conditions (e.g., atherosclerosis, cancer), viral (e.g., human papillomavirus, herpes virus) and bacterial (e.g., Helicobacter pylori, pneumonia) diseases, and autoimmune disorders (e.g., systemic lupus erythematosus, rheumatoid arthritis). Clinical investigations reveal the overexpression of specific nucleic acid sensors in diseased tissues. In vivo animal models show enhanced disease progression associated with receptor activation. The involvement of nucleic acid sensors in various systemic conditions is further supported by studies reporting receptor knockout mice being either protected from or prone to disease. TLR9-mediated inflammation is also implicated in periodontal diseases. Considering that persistent inflammation in the oral cavity is associated with systemic diseases and that oral microbial DNA is isolated at distal sites, nucleic acid sensing may potentially be a link between oral and systemic diseases. In this review, we discuss recent advances in how intracellular PRRs respond to microbial nucleic acids and emerging views on the role of nucleic acid sensors in various systemic diseases. We also highlight new information on the role of intracellular PRRs in the pathogenesis of oral diseases including periodontitis

  10. 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

  11. 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

  12. [Method for automated extraction and purification of nucleic acids and its implementation in microfluidic system].

    PubMed

    Mamaev, D D; Khodakov, D A; Dement'eva, E I; Filatov, I V; Iurasov, D A; Cherepanov, A I; Vasiliskov, V A; Smoldovskaia, O V; Zimenkov, D V; Griadunov, D A; Mikhaĭlovich, V M; Zasedatelev, A S

    2011-01-01

    A method and a microfluidic device for automated extraction and purification of nucleic acids from biological samples have been developed. The method involves disruption of bacterial cells and/or viral particles by combining enzymatic and chemical lysis procedures followed by solid-phase sorbent extraction and purification of nucleic acids. The procedure is carried out in an automated mode in a microfluidic module isolated from the outside environment, which minimizes contact of the researcher with potentially infectious samples and, consequently, decreases the risk of laboratory-acquired infections. The module includes reservoirs with lyophilized components for lysis and washing buffers; a microcolumn with a solid-phase sorbent; reservoirs containing water, ethanol, and water-ethanol buffer solutions for dissolving freeze-dried buffer components, rinsing the microcolumn, and eluting of nucleic acids; and microchannels and valves needed for directing fluids inside the module. The microfluidic module is placed into the control unit that delivers pressure, heats, mixes reagents, and flows solutions within the microfluidic module. The microfluidic system performs extraction and purification of nucleic acids with high efficiency in 40 min, and nucleic acids extracted can be directly used in PCR reaction and microarray assays.

  13. Nucleic acid binding and other biomedical properties of artificial oligolysines

    PubMed Central

    Roviello, Giovanni N; Vicidomini, Caterina; Costanzo, Vincenzo; Roviello, Valentina

    2016-01-01

    In the present study, we report the interaction of an artificial oligolysine (referred to as AOL) realized in our laboratory with targets of biomedical importance. These included polyinosinic acid (poly rI) and its complex with polycytidylic acid (poly I:C), RNAs with well-known interferon-inducing ability, and double-stranded (ds) DNA. The ability of the peptide to bind both single-stranded poly rI and ds poly I:C RNAs emerged from our circular dichroism (CD) and ultraviolet (UV) studies. In addition, we found that AOL forms complexes with dsDNA, as shown by spectroscopic binding assays and UV thermal denaturation experiments. These findings are encouraging for the possible use of AOL in biomedicine for nucleic acid targeting and oligonucleotide condensation, with the latter being a key step preceding their clinical application. Moreover, we tested the ability of AOL to bind to proteins, using serum albumin as a model protein. We demonstrated the oligolysine–protein binding by CD experiments which suggested that AOL, positively charged under physiological conditions, binds to the protein regions rich in anionic residues. Finally, the morphology characterization of the solid oligolysine, performed by scanning electron microscopy, showed different crystal forms including cubic-shaped crystals confirming the high purity of AOL. PMID:28115843

  14. Ability of two commercially available assays (Abbott RealTime HIV-1 and Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 Version 2.0) to quantify low HIV-1 RNA Levels (<1,000 copies/milliliter): comparison with clinical samples and NIBSC working reagent for nucleic acid testing assays.

    PubMed

    Amendola, Alessandra; Marsella, Patrizia; Bloisi, Maria; Forbici, Federica; Angeletti, Claudio; Capobianchi, Maria R

    2014-06-01

    Concordance between molecular assays may be suboptimal at low HIV-1 viremia levels (<1,000 copies/ml); therefore, it may be difficult to define and compare virologic endpoints for successful and failed therapy. We compared two commercial assays (the Abbott RealTime HIV-1 and the Roche Cobas AmpliPrep/TaqMan HIV-1 version 2.0) for their ability to detect and quantify low viral loads. A comparison was performed using 167 residual clinical samples (with values ranging from "not detected" to 1,000 copies/ml, as measured by the Abbott assay) and the National Institute and Biological Standards and Control (NIBSC) HIV-1 RNA working reagent 1 for nucleic acid amplification techniques (NAT) assays (serially diluted to a range from 1 to 1,000 copies/ml). Quantitative results were compared using Lin's concordance correlation coefficient and a Bland-Altman plot. Concordance with the qualitative results was measured by Cohen's kappa statistic. With clinical samples, the degree of interassay concordance of the qualitative results at a 40-copies/ml HIV-1 RNA threshold was substantial (κ = 0.762); the correlation among the quantified samples was suboptimal (concordance correlation coefficient, 0.728; P < 0.0001); the mean difference of the values between the Roche and Abbott assays was 0.193 log10 copies/ml. Using the HIV-1 RNA working reagent 1 for NAT assays, the results provided by the Roche assay were, on average, 3 times higher than expected, while the Abbott assay showed high accuracy. The Roche assay was highly sensitive, being able to detect a level as low as 3.5 copies/ml HIV-1 RNA with 95% probability. The performance characteristics of each molecular assay should be taken into account when HIV-1 RNA threshold values for "virologic suppression," "virologic failure," "persistent low viral loads," etc., are defined and indicated in the support of clinical decisions. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  15. Nucleic acid modifications in regulation of gene expression

    PubMed Central

    Chen, Kai; Zhao, Boxuan Simen; He, Chuan

    2016-01-01

    Nucleic acids carry a wide range of different chemical modifications. In contrast to previous views that these modifications are static and only play fine-tuning functions, recent research advances paint a much more dynamic picture. Nucleic acids carry diverse modifications and employ these chemical marks to exert essential or critical influences in a variety of cellular processes in eukaryotic organisms. This review covers several nucleic acid modifications that play important regulatory roles in biological systems, especially in regulation of gene expression: 5-methylcytosine (5mC) and its oxidative derivatives, and N6 -methyladenine (6mA) in DNA; N6 -methyladenosine (m6A), pseudouridine (), and 5-methylcytosine (m5C) in messenger RNA and long non-coding RNA. Modifications in other non-coding RNAs, such as tRNA, miRNA, and snRNA, are also briefly summarized. We provide brief historical perspective of the field, and highlight recent progress in identifying diverse nucleic acid modifications and exploring their functions in different organisms. Overall, we believe that work in this field will yield additional layers of both chemical and biological complexity as we continue to uncover functional consequences of known nucleic acid modifications and discover new ones. PMID:26933737

  16. Plant viral and bacteriophage delivery of nucleic acid therapeutics.

    PubMed

    Lam, Patricia; Steinmetz, Nicole F

    2017-07-18

    Nucleic acid therapeutics have emerged as a powerful method for treatment of many diseases. However, the challenge lies in safe and efficient delivery of nucleic acids to their target site, as they need to cross various extracellular and intracellular barriers. Mammalian viruses have initially been favored for delivery of nucleic acid therapeutics, but safety concerns regarding their immunogenicity and potential of integration have fueled the search for alternative delivery strategies. For example, chemistry and bioengineering have led to advances in the use of nonviral vectors composed of lipids and other polymers; nevertheless, the synthetic systems often do not match the efficiency achieved using the biological systems. More recently, researchers have turned toward the development of plant viruses and bacteriophages and virus-like particles as an alternative or complementary approach. These systems unite the properties of both the viral and nonviral systems and as such are a new exciting avenue toward nucleic acid delivery. This review highlights the benefits of plant viral and bacteriophage delivery of nucleic acids and provides a summary of the current progress in research in this field. For further resources related to this article, please visit the WIREs website. © 2017 Wiley Periodicals, Inc.

  17. Nucleic Acid Modifications in Regulation of Gene Expression.

    PubMed

    Chen, Kai; Zhao, Boxuan Simen; He, Chuan

    2016-01-21

    Nucleic acids carry a wide range of different chemical modifications. In contrast to previous views that these modifications are static and only play fine-tuning functions, recent research advances paint a much more dynamic picture. Nucleic acids carry diverse modifications and employ these chemical marks to exert essential or critical influences in a variety of cellular processes in eukaryotic organisms. This review covers several nucleic acid modifications that play important regulatory roles in biological systems, especially in regulation of gene expression: 5-methylcytosine (5mC) and its oxidative derivatives, and N(6)-methyladenine (6mA) in DNA; N(6)-methyladenosine (m(6)A), pseudouridine (Ψ), and 5-methylcytidine (m(5)C) in mRNA and long non-coding RNA. Modifications in other non-coding RNAs, such as tRNA, miRNA, and snRNA, are also briefly summarized. We provide brief historical perspective of the field, and highlight recent progress in identifying diverse nucleic acid modifications and exploring their functions in different organisms. Overall, we believe that work in this field will yield additional layers of both chemical and biological complexity as we continue to uncover functional consequences of known nucleic acid modifications and discover new ones. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Nucleic acid detection and quantification in the developing world.

    PubMed

    Huggett, Jim; Green, Clare; Zumla, Alimuddin

    2009-04-01

    Techniques using nucleic acid amplification have not had the same amount of impact on research and clinical diagnosis in the developing world as that observed in the West. This is unsurprising when the costs and infrastructure required to perform nucleic acid amplification are considered. Despite this, nucleic acid amplification is being increasingly used in both research and diagnosis in countries such as Zambia and Tanzania. Scientific research in the developing world is made possible through the support and development of the necessary laboratory infrastructure and the establishment of special transport for the reagents and samples. This has enabled world-leading country-relevant research to be performed by local scientists on subjects ranging from rapid diagnosis of infectious diseases to measuring the RNA gene expression in an immune response. Concomitantly, the challenge presented by the need for tests that are more appropriate for a resource-poor setting has led to a number of newer methodologies for nucleic acid detection, which can be tailored to be performed in the field without the need for training in molecular biology. As nucleic acid amplification techniques become both simpler and cheaper, their impact is likely to play an increasingly crucial role in research and diagnosis in the developing world.

  19. 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

  20. Carbon nanotubes and nucleic acids: tools and targets

    NASA Astrophysics Data System (ADS)

    Onoa, Bibiana; Zheng, Ming; Dresselhaus, Mildred S.; Diner, Bruce A.

    2006-05-01

    Nucleic acids, with their intrinsic structural properties as well as their high specificity, are playing an important role in the rapid development of nano-technologies. In turn, these new technologies and their efficient performance enable fast and precise methods for detection of nucleic acids, improving the diagnosis of diseases and identification of pathogens. We discuss the use of nucleic acids to disperse and sort single walled carbon nanotubes (SWNTs), and carbon nanotube-based field effect transistors (CNT-FETs) to electrically detect specific nucleic acid sequences. Both DNA and RNA are efficient agents for dispersion and separation of SWNTs by diameter and chirality. Fractions enriched in a narrow band gap distribution of DNA:SWNT hybrids do not alter the electronic performance of field effect transistors. A CNT-FET fulfills the requirements for a nanosensing device that can greatly exceed the existing technologies. Electrical detection of specific nucleic acid sequence could potentially overcome the current limitations of optical detection, by increasing sensitivity and speed, while reducing sample manipulation, size, and cost.

  1. 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.

  2. Efficacy of Nucleic Acid Probes for Detection of Poliovirus in Water Disinfected by Chlorine, Chlorine Dioxide, Ozone, and UV Radiation

    PubMed Central

    Moore, Norman J.; Margolin, Aaron B.

    1994-01-01

    MilliQ water was inoculated with poliovirus type 1 strain LSc-1 and was treated with disinfectants, including chlorine, chlorine dioxide, ozone, and UV light. No relationship between probes and plaque assays were seen, demonstrating that viral nucleic acids were not destroyed. These findings suggest that nucleic acid probes cannot distinguish between infectious and noninfectious viruses and cannot be used in the evaluation of treated waters. PMID:16349448

  3. Rapid genotyping using pyrene−perylene locked nucleic acid complexes

    PubMed Central

    Kumar, T. Santhosh; Myznikova, Anna; Samokhina, Evgeniya; Astakhova, Irina Kira

    2013-01-01

    We have developed an assay for single strand DNA and RNA detection which is based on novel pyrene−perylene FRET pairs attached to short LNA/DNA probes. The assay is based on ratiometric emission upon binding of target DNA/RNA by three combinations of fluorescent LNA/DNA reporter strands. Specific geometry of the pyrene fluorophore attached to the 2′-amino group of 2′-amino-LNA in position 4 allows for the first time to efficiently utilize dipole−dipole orientation parameter for sensing of single-nucleotide polymorphisms (SNPs) in nucleic acid targets by FRET. Using novel probes, SNP detection is achieved with advantages of large Stokes shift (115 nm), high fluorescence quantum yields and low limit of target detection values (< 5 nM). Rapid and accurate genotyping of highly polymorphic HIV Pol cDNA and RNA fragments performed herein proves the possibility for broad application of the novel pyrene−perylene FRET pairs, e.g., in imaging and clinical diagnostics. PMID:24044052

  4. 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.

  5. Challenges and Perspectives in Nucleic Acid Enzyme Engineering.

    PubMed

    Balke, Darko; Hieronymus, Robert; Müller, Sabine

    2017-08-04

    Engineering of nucleic acids has been a goal in research for many years. Since the discovery of catalytic nucleic acids (ribozymes and DNAzymes), this field has attracted even more attention. One reason for the increased interest is that a large number of ribozymes have been engineered that catalyze a broad range of reactions of relevance to the origin of life. Another reason is that the structures of ribozymes or DNAzymes have been modulated such that activity is dependent on allosteric regulation by an external cofactor. Such constructs have great potential for application as biosensors in medicinal or environmental diagnostics, and as molecular tools for control of cellular processes. In addition to the development of nucleic acid enzymes by in vitro selection, rational design is a powerful strategy for the engineering of ribozymes or DNAzymes with tailored features. The structures and mechanisms of a large number of nucleic acid catalysts are now well understood. Therefore, specific design of their functional properties by structural modulation is a good option for the development of custom-made molecular tools. For rational design, several parameters have to be considered, and a number of tools are available to help/guide sequence design. Here, we discuss sequence, structural and functional design using the example of hairpin ribozyme variants to highlight the challenges and opportunities of rational nucleic enzyme engineering. Graphical Abstract.

  6. Hybrids of Nucleic Acids and Carbon Nanotubes for Nanobiotechnology

    PubMed Central

    Umemura, Kazuo

    2015-01-01

    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. PMID:28347014

  7. Geometric properties of nucleic acids with potential for autobuilding

    PubMed Central

    Gruene, Tim; Sheldrick, George M.

    2011-01-01

    Medium- to high-resolution X-ray structures of DNA and RNA molecules were investigated to find geometric properties useful for automated model building in crystallographic electron-density maps. We describe a simple method, starting from a list of electron-density ‘blobs’, for identifying backbone phosphates and nucleic acid bases based on properties of the local electron-density distribution. This knowledge should be useful for the automated building of nucleic acid models into electron-density maps. We show that the distances and angles involving C1′ and the P atoms, using the pseudo-torsion angles and that describe the …P—C1′—P—C1′… chain, provide a promising basis for building the nucleic acid polymer. These quantities show reasonably narrow distributions with asymmetry that should allow the direction of the phosphate backbone to be established. PMID:21173468

  8. Beyond DNA origami: the unfolding prospects of nucleic acid nanotechnology.

    PubMed

    Michelotti, Nicole; Johnson-Buck, Alexander; Manzo, Anthony J; Walter, Nils G

    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 two-dimensional and three-dimensional 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.

  9. Innate immune sensing of nucleic acids from pathogens.

    PubMed

    Oliveira, Sergio C

    2014-12-01

    The innate immune system is important as the first line of defense to sense invading pathogens. Nucleic acids represent critical pathogen signatures that trigger a host proinflammatory immune response. Much progress has been made in understanding how DNA and RNA trigger host defense countermeasures, however, several aspects of how cytosolic nucleic acids are sensed remain unclear. This special issue reviews how the host innate immune system senses nucleic acids from Brucella abortus, Mycobacterium sp and Legionella pneumophila, viral DNA, the role of STING in DNA sensing and inflammatory diseases and the mechanism of viral RNA recognition by the small interfering RNA pathway in Drosophila melanogaster. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  10. 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.

  11. Vibrational Raman optical activity of proteins, nucleic acids, and viruses.

    PubMed

    Blanch, Ewan W; Hecht, Lutz; Barron, Laurence D

    2003-02-01

    Due to its sensitivity to chirality, Raman optical activity (ROA), which may be measured as a small difference in vibrational Raman scattering from chiral molecules in right- and left-circularly polarized incident light, is a powerful probe of biomolecular structure in solution. Protein ROA spectra provide information on the secondary and tertiary structures of the polypeptide backbone, hydration, side-chain conformation, and structural elements present in denatured states. Nucleic acid ROA spectra yield information on the sugar ring conformation, the base stacking arrangement, and the mutual orientation of the sugar and base rings around the C-N glycosidic linkage. ROA is able to simultaneously probe the structures of both the protein and the nucleic acid components of intact viruses. This article gives a brief account of the theory and measurement of ROA and presents the ROA spectra of a selection of proteins, nucleic acids, and viruses which illustrate the applications of ROA spectroscopy in biomolecular research.

  12. Soni-removal of nucleic acids from inclusion bodies.

    PubMed

    Neerathilingam, Muniasamy; Mysore, Sumukh; Gandham, Sai Hari A

    2014-05-23

    Inclusion bodies (IBs) are commonly formed in Escherichia coli due to over expression of recombinant proteins in non-native state. Isolation, denaturation and refolding of these IBs is generally performed to obtain functional protein. However, during this process IBs tend to form non-specific interactions with sheared nucleic acids from the genome, thus getting carried over into downstream processes. This may hinder the refolding of IBs into their native state. To circumvent this, we demonstrate a methodology termed soni-removal which involves disruption of nucleic acid-inclusion body interaction using sonication; followed by solvent based separation. As opposed to conventional techniques that use enzymes and column-based separations, soni-removal is a cost effective alternative for complete elimination of buried and/or strongly bound short nucleic acid contaminants from IBs. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Pyrophosphate-condensing activity linked to nucleic acid synthesis.

    PubMed Central

    Volloch, V Z; Rits, S; Tumerman, L

    1979-01-01

    In some preparations of DNA dependent RNA polymerase a new enzymatic activity has been found which catalyzes the condensation of two pyrophosphate molecules, liberated in the process of RNA synthesis, to one molecule of orthophosphate and one molecule of Mg (or Mn) - chelate complex with trimetaphosphate. This activity can also cooperate with DNA-polymerase, on condition that both enzymes originate from the same cells. These results point to two general conclusions. First, energy is conserved in the overall process of nucleic acid synthesis and turnover, so that the process does not require an energy influx from the cell's general resources. Second, the synthesis of nucleic acids is catalyzed by a complex enzyme system which contains at least two separate enzymes, one responsible for nucleic acid polymerization and the other for energy conservation via pyrophosphate condensation. Images PMID:88040

  14. A Polyaniline-based Sensor of Nucleic Acids.

    PubMed

    Sengupta, Partha Pratim; Gloria, Jared N; Parker, Marcus K; Flynt, Alex S

    2016-11-01

    Detection of nucleic acids is at the center of diagnostic technologies used in research and the clinic. Standard approaches used in these technologies rely on enzymatic modification that can introduce bias and artifacts. A critical element of next generation detection platforms will be direct molecular sensing, thereby avoiding a need for amplification or labels. Advanced nanomaterials may provide the suitable chemical modalities to realize label-free sensors. Conjugated polymers are ideal for biological sensing, possessing properties compatible with biomolecules and exhibit high sensitivity to localized environmental changes. In this article, a method is presented for detecting nucleic acids using the electroconductive polymer polyaniline. Simple DNA "probe" oligonucleotides complementary to target nucleic acids are attached electrostatically to the polymer, creating a sensor system that can differentiate single nucleotide differences in target molecules. Outside the specific and unbiased nature of this technology, it is highly cost effective.

  15. Importance of Nucleic Acid Recognition in Inflammation and Autoimmunity.

    PubMed

    Barrat, Franck J; Elkon, Keith B; Fitzgerald, Katherine A

    2016-01-01

    An important concept in immunology is the classification of immune responses as either innate or adaptive, based on whether the antigen receptors are encoded in the germline or generated somatically by gene rearrangement. The innate immune system is an ancient mode of immunity, and by being a first layer in our defense against infectious agents, it is essential for our ability to develop rapid and sustained responses to pathogens. We discuss the importance of nucleic acid recognition by the innate immune system to mounting an appropriate immune response to pathogens and also how inflammation driven by uncontrolled recognition of self-nucleic acids can lead to autoimmune diseases. We also summarize current efforts to either harness the immune system using agonists of nucleic acid-specific innate sensors or, on the contrary, by using inhibitors in autoimmune situations.

  16. Iron-sulphur clusters in nucleic acid processing enzymes.

    PubMed

    White, Malcolm F; Dillingham, Mark S

    2012-02-01

    Several unexpected reports of iron-sulphur clusters in nucleic acid binding proteins have recently appeared in the literature. Once thought to be relatively rare in these systems, iron-sulphur clusters are now known to be essential components of diverse nucleic acid processing machinery including glycosylases, primases, helicases, nucleases, transcription factors, RNA polymerases and RNA methyltransferases. In many cases, the function of the cluster is poorly understood and crystal structures of these iron-sulphur enzymes reveal little in common between them. In this article, we review the recent developments in the field and discuss to what extent there might exist common mechanistic roles for iron-sulphur clusters in nucleic acid enzymes. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. 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.

  18. Accurate ab initio prediction of NMR chemical shifts of nucleic acids and nucleic acids/protein complexes

    PubMed Central

    Victora, Andrea; Möller, Heiko M.; Exner, Thomas E.

    2014-01-01

    NMR chemical shift predictions based on empirical methods are nowadays indispensable tools during resonance assignment and 3D structure calculation of proteins. However, owing to the very limited statistical data basis, such methods are still in their infancy in the field of nucleic acids, especially when non-canonical structures and nucleic acid complexes are considered. Here, we present an ab initio approach for predicting proton chemical shifts of arbitrary nucleic acid structures based on state-of-the-art fragment-based quantum chemical calculations. We tested our prediction method on a diverse set of nucleic acid structures including double-stranded DNA, hairpins, DNA/protein complexes and chemically-modified DNA. Overall, our quantum chemical calculations yield highly/very accurate predictions with mean absolute deviations of 0.3–0.6 ppm and correlation coefficients (r2) usually above 0.9. This will allow for identifying misassignments and validating 3D structures. Furthermore, our calculations reveal that chemical shifts of protons involved in hydrogen bonding are predicted significantly less accurately. This is in part caused by insufficient inclusion of solvation effects. However, it also points toward shortcomings of current force fields used for structure determination of nucleic acids. Our quantum chemical calculations could therefore provide input for force field optimization. PMID:25404135

  19. Nucleic acid structure analysis: Local, mathematically rigorous, comparable

    SciTech Connect

    Babcock, M.S.

    1993-01-01

    A more sophisticated mathematical treatments for analyzing nucleic acid coordinate data is presented. The methodology is both rigorous and comparable for parameterizing nucleic acids in terms of the local structural morphology of complementary and neighboring base pairs. Chapter 1 clearly defines the problems of nucleic acid structure parameterization by examining the consequences of the EMBO workshop guidelines published in 1989. Chapter 2 defines mathematics to rigorously and comparably calculate all of the parameters for nucleic acid structure from a local viewpoint. The mathematics satisfies all EMBO guidelines for local structural parameters. One of the main features making this program flexible is that any base pair relationship can be rigorously analyzed. This is because the meaning of zero for the complementary base parameters is clearly definable for any base pairing relationship. Chapter 3 analyses and explains why certain pairwise parameter correlations were observed between rotational and translational parameters. It was observed that the method of calculating the rotational parameters greatly affected the calculated translational parameters. As a result of our analysis, we determined the optimum location about which rotations should be performed in order to reduce and/or eliminate the correlations which are artifacts of the mathematics employed and do not reflect true structural properties of nucleic acids. Chapter 4 presents an analysis of the available nucleic acid X-ray crystallographic structural data, showing that the experimental base pairs do not generally have the ideal Watson-Crick structure. By utilizing a hybrid between helical and Cartesian parameterization methods, the relative distribution of the complementary base parameters was examined as a function of the nearest neighboring base pairs. The final chapter includes a review article explaining each of the available methods in plain English as well as giving the mathematics.

  20. 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

  1. 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.

  2. Towards Improved Accuracy of Bordetella pertussis Nucleic Acid Amplification Tests

    PubMed Central

    2012-01-01

    In many clinical microbiology laboratories, nucleic acid amplification tests such as PCR have become the routine methods for the diagnosis of pertussis. While PCR has greatly increased the ability of laboratories to detect Bordetella pertussis infections, it has also been associated with false-positive results that can, given the tendency of B. pertussis to cause outbreaks, result in unnecessary and costly control measures. The species specificity of Bordetella gene targets and their number of copies per genome greatly impact the performance characteristics of nucleic acid amplification tests for B. pertussis. It is crucial that laboratorians recognize these characteristics, to limit false-positive test results and prevent pseudo-outbreaks. PMID:22442315

  3. Biomimetic high density lipoprotein nanoparticles for nucleic acid delivery.

    PubMed

    McMahon, Kaylin M; Mutharasan, R Kannan; Tripathy, Sushant; Veliceasa, Dorina; Bobeica, Mariana; Shumaker, Dale K; Luthi, Andrea J; Helfand, Brian T; Ardehali, Hossein; Mirkin, Chad A; Volpert, Olga; Thaxton, C Shad

    2011-03-09

    We report a gold nanoparticle-templated high density lipoprotein (HDL AuNP) platform for gene therapy that combines lipid-based nucleic acid transfection strategies with HDL biomimicry. For proof-of-concept, HDL AuNPs are shown to adsorb antisense cholesterylated DNA. The conjugates are internalized by human cells, can be tracked within cells using transmission electron microscopy, and regulate target gene expression. Overall, the ability to directly image the AuNP core within cells, the chemical tailorability of the HDL AuNP platform, and the potential for cell-specific targeting afforded by HDL biomimicry make this platform appealing for nucleic acid delivery.

  4. Methods of introducing nucleic acids into cellular DNA

    DOEpatents

    Lajoie, Marc J.; Gregg, Christopher J.; Mosberg, Joshua A.; Church, George M.

    2017-06-27

    A method of introducing a nucleic acid sequence into a cell is provided where the cell has impaired or inhibited or disrupted DnaG primase activity or impaired or inhibited or disrupted DnaB helicase activity, or larger or increased gaps or distance between Okazaki fragments or lowered or reduced frequency of Okazaki fragment initiation, or the cell has increased single stranded DNA (ssDNA) on the lagging strand of the replication fork including transforming the cell through recombination with a nucleic acid oligomer.

  5. Nanopores and nucleic acids: prospects for ultrarapid sequencing

    NASA Technical Reports Server (NTRS)

    Deamer, D. W.; Akeson, M.

    2000-01-01

    DNA and RNA molecules can be detected as they are driven through a nanopore by an applied electric field at rates ranging from several hundred microseconds to a few milliseconds per molecule. The nanopore can rapidly discriminate between pyrimidine and purine segments along a single-stranded nucleic acid molecule. Nanopore detection and characterization of single molecules represents a new method for directly reading information encoded in linear polymers. If single-nucleotide resolution can be achieved, it is possible that nucleic acid sequences can be determined at rates exceeding a thousand bases per second.

  6. Nanopores and nucleic acids: prospects for ultrarapid sequencing

    NASA Technical Reports Server (NTRS)

    Deamer, D. W.; Akeson, M.

    2000-01-01

    DNA and RNA molecules can be detected as they are driven through a nanopore by an applied electric field at rates ranging from several hundred microseconds to a few milliseconds per molecule. The nanopore can rapidly discriminate between pyrimidine and purine segments along a single-stranded nucleic acid molecule. Nanopore detection and characterization of single molecules represents a new method for directly reading information encoded in linear polymers. If single-nucleotide resolution can be achieved, it is possible that nucleic acid sequences can be determined at rates exceeding a thousand bases per second.

  7. Biomimetic High Density Lipoprotein Nanoparticles For Nucleic Acid Delivery

    PubMed Central

    McMahon, Kaylin M.; Mutharasan, R. Kannan; Tripathy, Sushant; Veliceasa, Dorina; Bobeica, Mariana; Shumaker, Dale K.; Luthi, Andrea J.; Helfand, Brian T.; Ardehali, Hossein; Mirkin, Chad A.; Volpert, Olga; Thaxton, C. Shad

    2014-01-01

    We report a gold nanoparticle-templated high density lipoprotein (HDL AuNP) platform for gene therapy which combines lipid-based nucleic acid transfection strategies with HDL biomimicry. For proof-of-concept, HDL AuNPs are shown to adsorb antisense cholesterylated DNA. The conjugates are internalized by human cells, can be tracked within cells using transmission electron microscopy (TEM), and regulate target gene expression. Overall, the ability to directly image the AuNP core within cells, the chemical tailorability of the HDL AuNP platform, and the potential for cell-specific targeting afforded by HDL biomimicry make this platform appealing for nucleic acid delivery. PMID:21319839

  8. Functional nucleic acids in high throughput screening and drug discovery.

    PubMed

    Srivatsan, Seergazhi G; Famulok, Michael

    2007-09-01

    In vitro selection can be used to generate functional nucleic acids such as aptamers and ribozymes that can recognize a variety of molecules with high affinity and specificity. Most often these recognition events are associated with structural alterations that can be converted into detectable signals. Several signaling aptamers and ribozymes constructed by both design and selection have been successfully utilized as sensitive detection reagents. Here we summarize the development of different types of signaling nucleic acids, and approaches that have been implemented in the screening format.

  9. Nanopore-based sequencing and detection of nucleic acids.

    PubMed

    Ying, Yi-Lun; Zhang, Junji; Gao, Rui; Long, Yi-Tao

    2013-12-09

    Nanopore-based techniques, which mimic the functions of natural ion channels, have attracted increasing attention as unique methods for single-molecule detection. The technology allows the real-time, selective, high-throughput analysis of nucleic acids through both biological and solid-state nanopores. In this Minireview, the background and latest progress in nanopore-based sequencing and detection of nucleic acids are summarized, and light is shed on a novel platform for nanopore-based detection. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Nucleic acid aptamers: an emerging frontier in cancer therapy.

    PubMed

    Zhu, Guizhi; Ye, Mao; Donovan, Michael J; Song, Erqun; Zhao, Zilong; Tan, Weihong

    2012-11-04

    The last two decades have witnessed the development and application of nucleic acid aptamers in a variety of fields, including target analysis, disease therapy, and molecular and cellular engineering. The efficient and widely applicable aptamer selection, reproducible chemical synthesis and modification, generally impressive target binding selectivity and affinity, relatively rapid tissue penetration, low immunogenicity, and rapid systemic clearance make aptamers ideal recognition elements for use as therapeutics or for in vivo delivery of therapeutics. In this feature article, we discuss the development and biomedical application of nucleic acid aptamers, with emphasis on cancer cell aptamer isolation, targeted cancer therapy, oncology biomarker identification and drug discovery.

  11. Multifunctional combinatorial-designed nanoparticles for nucleic acid therapy

    NASA Astrophysics Data System (ADS)

    Amiji, Mansoor M.

    2016-05-01

    Recent advances in biomedical sciences, especially in the field of human genetics, is increasingly considered to facilitate a new frontier in development of novel disease-modifying therapeutics. One of major challenges in the development of nucleic acid therapeutics is efficient and specific delivery of the molecules to the target tissue and cell upon systemic administration. In this report, I discuss our strategy to develop combinatorial-designed multifunctional nanoparticle assemblies based on natural biocompatible and biodegradable polymers for nucleic acid delivery in: (1) overcoming tumor drug resistance and (2) genetic modulation of macrophage functional phenotype from M1 to M2 in treatment of inflammatory diseases.

  12. Spherical Nucleic Acids as Intracellular Agents for Nucleic Acid Based Therapeutics

    NASA Astrophysics Data System (ADS)

    Hao, Liangliang

    Recent functional discoveries on the noncoding sequences of human genome and transcriptome could lead to revolutionary treatment modalities because the noncoding RNAs (ncRNAs) can be applied as therapeutic agents to manipulate disease-causing genes. To date few nucleic acid-based therapeutics have been translated into the clinic due to challenges in the delivery of the oligonucleotide agents in an effective, cell specific, and non-toxic fashion. Unmodified oligonucleotide agents are destroyed rapidly in biological fluids by enzymatic degradation and have difficulty crossing the plasma membrane without the aid of transfection reagents, which often cause inflammatory, cytotoxic, or immunogenic side effects. Spherical nucleic acids (SNAs), nanoparticles consisting of densely organized and highly oriented oligonucleotides, pose one possible solution to circumventing these problems in both the antisense and RNA interference (RNAi) pathways. The unique three dimensional architecture of SNAs protects the bioactive oligonucleotides from unspecific degradation during delivery and supports their targeting of class A scavenger receptors and endocytosis via a lipid-raft-dependent, caveolae-mediated pathway. Owing to their unique structure, SNAs are able to cross cell membranes and regulate target genes expression as a single entity, without triggering the cellular innate immune response. Herein, my thesis has focused on understanding the interactions between SNAs and cellular components and developing SNA-based nanostructures to improve therapeutic capabilities. Specifically, I developed a novel SNA-based, nanoscale agent for delivery of therapeutic oligonucleotides to manipulate microRNAs (miRNAs), the endogenous post-transcriptional gene regulators. I investigated the role of SNAs involving miRNAs in anti-cancer or anti-inflammation responses in cells and in in vivo murine disease models via systemic injection. Furthermore, I explored using different strategies to construct

  13. 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.

  14. 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

  15. Optimization of a Nucleic Acid-Based Reporter System To Detect Mycobacterium tuberculosis Antibiotic Sensitivity

    PubMed Central

    Lemmon, Margaret; Rotter, Stephanie; Lees, Jonathan; Einck, Leo; Nacy, Carol A.

    2014-01-01

    We previously reported the development of a prototype antibiotic sensitivity assay to detect drug-resistant Mycobacterium tuberculosis using infection by mycobacteriophage to create a novel nucleic acid transcript, a surrogate marker of mycobacterial viability, detected by reverse transcriptase PCR (M. C. Mulvey et al., mBio 3:e00312-11, 2012). This assay detects antibiotic resistance to all drugs, even drugs for which the resistance mechanism is unknown or complex: it is a phenotypic readout using nucleic acid detection. In this report, we describe development and characteristics of an optimized reporter system that directed expression of the RNA cyclase ribozyme, which generated circular RNA through an intramolecular splicing reaction and led to accumulation of a new nucleic acid sequence in phage-infected bacteria. These modifications simplified the assay, increased the limit of detection from 104 to <102 M. tuberculosis cells, and correctly identified the susceptibility profile of M. tuberculosis strains exposed for 16 h to either first-line or second-line antitubercular drugs. In addition to phenotypic drug resistance or susceptibility, the assay reported streptomycin MICs and clearly detected 10% drug-resistant cells in an otherwise drug-susceptible population. PMID:25367910

  16. Submicrometer-Sized Thermometer Particles Exploiting Selective Nucleic Acid Stability.

    PubMed

    Puddu, Michela; Mikutis, Gediminas; Stark, Wendelin J; Grass, Robert N

    2016-01-27

    Encapsulated nucleic acid selective damage quantification by real-time polymerase chain reaction is used as sensing mechanism to build a novel class of submicrometer size thermometer. Thanks to the high thermal and chemical stability, and the capability of storing the read accumulated thermal history, the sensor overcomes some of current limitations in small scale thermometry.

  17. Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

    DOEpatents

    Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

    2010-06-22

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

  18. Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

    DOEpatents

    Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

    2012-10-16

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

  19. Watson-Crick hydrogen bonding of unlocked nucleic acids.

    PubMed

    Langkjær, Niels; Wengel, Jesper; Pasternak, Anna

    2015-11-15

    We herein describe the synthesis of two new unlocked nucleic acid building blocks containing hypoxanthine and 2,6-diaminopurine as nucleobase moieties and their incorporation into oligonucleotides. The modified oligonucleotides were used to examine the thermodynamic properties of UNA against unmodified oligonucleotides and the resulting thermodynamic data support that the hydrogen bonding face of UNA is Watson-Crick like.

  20. Cell cycle nucleic acids, polypeptides and uses thereof

    DOEpatents

    Gordon-Kamm, William J.; Lowe, Keith S.; Larkins, Brian A.; Dilkes, Brian R.; Sun, Yuejin

    2007-08-14

    The invention provides isolated nucleic acids and their encoded proteins that are involved in cell cycle regulation. The invention further provides recombinant expression cassettes, host cells, transgenic plants, and antibody compositions. The present invention provides methods and compositions relating to altering cell cycle protein content, cell cycle progression, cell number and/or composition of plants.

  1. Nucleic acid separations using superficially porous silica particles.

    PubMed

    Close, Elizabeth D; Nwokeoji, Alison O; Milton, Dafydd; Cook, Ken; Hindocha, Darsha M; Hook, Elliot C; Wood, Helen; Dickman, Mark J

    2016-04-01

    Ion pair reverse-phase liquid chromatography has been widely employed for nucleic acid separations. A wide range of alternative stationary phases have been utilised in conjunction with ion pair reverse-phase chromatography, including totally porous particles, non-porous particles, macroporous particles and monolithic stationary phases. In this study we have utilised superficially porous silica particles in conjunction with ion pair reverse-phase liquid chromatography for the analysis of nucleic acids. We have investigated a range of different pore-sizes and phases for the analysis of a diverse range of nucleic acids including oligonucleotides, oligoribonucleotides, phosphorothioate oligonucleotides and high molecular weight dsDNA and RNA. The pore size of the superficially porous silica particles was shown to significantly affect the resolution of the nucleic acids. Optimum separations of small oligonucleotides such as those generated in RNase mapping experiments were obtained with 80Å pore sizes and can readily be interfaced with mass spectrometry analysis. Improved resolution of larger oligonucleotides (>19mers) was observed with pore sizes of 150Å. The optimum resolution for larger dsDNA/RNA molecules was achieved using superficially porous silica particles with pore sizes of 400Å. Furthermore, we have utilised 150Å pore size solid-core particles to separate typical impurities of a fully phosphorothioated oligonucleotide, which are often generated in the synthesis of this important class of therapeutic oligonucleotide.

  2. Mouse Vk gene classification by nucleic acid sequence similarity.

    PubMed

    Strohal, R; Helmberg, A; Kroemer, G; Kofler, R

    1989-01-01

    Analyses of immunoglobulin (Ig) variable (V) region gene usage in the immune response, estimates of V gene germline complexity, and other nucleic acid hybridization-based studies depend on the extent to which such genes are related (i.e., sequence similarity) and their organization in gene families. While mouse Igh heavy chain V region (VH) gene families are relatively well-established, a corresponding systematic classification of Igk light chain V region (Vk) genes has not been reported. The present analysis, in the course of which we reviewed the known extent of the Vk germline gene repertoire and Vk gene usage in a variety of responses to foreign and self antigens, provides a classification of mouse Vk genes in gene families composed of members with greater than 80% overall nucleic acid sequence similarity. This classification differed in several aspects from that of VH genes: only some Vk gene families were as clearly separated (by greater than 25% sequence dissimilarity) as typical VH gene families; most Vk gene families were closely related and, in several instances, members from different families were very similar (greater than 80%) over large sequence portions; frequently, classification by nucleic acid sequence similarity diverged from existing classifications based on amino-terminal protein sequence similarity. Our data have implications for Vk gene analyses by nucleic acid hybridization and describe potentially important differences in sequence organization between VH and Vk genes.

  3. Computer analysis and structure prediction of nucleic acids and proteins.

    PubMed Central

    Kanehisa, M; Klein, P; Greif, P; DeLisi, C

    1984-01-01

    We have developed an integrated computer system for analysis of nucleic acid and protein sequences, which consists of sequence and structure databases, a relational database, and software for structural analysis. The system is potentially applicable to a number of problems in structural biology including predictive classification of the function and location of oncogene products. PMID:6546426

  4. Circulating nucleic acids: An analysis of their occurrence in malignancies

    PubMed Central

    Suraj, Shankar; Dhar, Chirag; Srivastava, Sweta

    2017-01-01

    Through a regulated or fortuitous phenomenon, small portions of cell nucleic acids are thrown into circulation. Since the discovery of these circulating nucleic acids (CNAs) in 1948, numerous studies have been published to elucidate their clinical implications in multifarious diseases. Scientists have now discovered disease-specific genetic aberrations, such as mutations, microsatellite alterations, epigenetic modulations (including aberrant methylation), as well as viral DNA/RNA from nucleic acids in plasma and serum. CNAs have become increasingly popular due to their potential for use as a liquid biopsy, which is a tool for non-invasive diagnosis and monitoring of diseases, such as cancer, stroke, trauma, myocardial infarction, autoimmune disorders, and pregnancy-associated complications. While the diagnostic potential of CNAs has been investigated extensively, there is a paucity of understanding of their pathophysiological functions. Are these CNAs part of the cell's regular framework of functioning? Or do they act as molecular players in disease initiation and progression? The aim of this review is to investigate the origins and functions of the circulating cell-free nucleic acids in the plasma and serum of patients with various malignancies, and propose areas of study, which may elucidate the novel underlying mechanisms that are functioning during cancer initiation/progression. PMID:28123700

  5. Potentiometric sensing of nucleic acids using chemically modified nanopores.

    PubMed

    Makra, István; Brajnovits, Alexandra; Jágerszki, Gyula; Fürjes, Péter; Gyurcsányi, Róbert E

    2017-01-05

    Unlike the overwhelming majority of nanopore sensors that are based on the measurement of a transpore ionic current, here we introduce a potentiometric sensing scheme and demonstrate its application for the selective detection of nucleic acids. The sensing concept uses the charge inversion that occurs in the sensing zone of a nanopore upon binding of negatively charged microRNA strands to positively charged peptide-nucleic acid (PNA) modified nanopores. The initial anionic permselectivity of PNA-modified nanopores is thus gradually changed to cationic permselectivity, which can be detected simply by measuring the nanoporous membrane potential. A quantitative theoretical treatment of the potentiometric microRNA response is provided based on the Nernst-Planck/Poisson model for the nanopore system assuming first order kinetics for the nucleic acid hybridization. An excellent correlation between the theoretical and experimental results was observed, which revealed that the binding process is focused at the nanopore entrance with contributions from both in pore and out of pore sections of the nanoporous membrane. The theoretical treatment is able to give clear guidelines for further optimization of potentiometric nanopore-based nucleic acid sensors by predicting the effect of the most important experimental parameters on the potential response.

  6. Mosaic protein and nucleic acid vaccines against hepatitis C virus

    DOEpatents

    Yusim, Karina; Korber, Bette T. M.; Kuiken, Carla L.; Fischer, William M.

    2013-06-11

    The invention relates to immunogenic compositions useful as HCV vaccines. Provided are HCV mosaic polypeptide and nucleic acid compositions which provide higher levels of T-cell epitope coverage while minimizing the occurrence of unnatural and rare epitopes compared to natural HCV polypeptides and consensus HCV sequences.

  7. Circulating nucleic acids: An analysis of their occurrence in malignancies.

    PubMed

    Suraj, Shankar; Dhar, Chirag; Srivastava, Sweta

    2017-01-01

    Through a regulated or fortuitous phenomenon, small portions of cell nucleic acids are thrown into circulation. Since the discovery of these circulating nucleic acids (CNAs) in 1948, numerous studies have been published to elucidate their clinical implications in multifarious diseases. Scientists have now discovered disease-specific genetic aberrations, such as mutations, microsatellite alterations, epigenetic modulations (including aberrant methylation), as well as viral DNA/RNA from nucleic acids in plasma and serum. CNAs have become increasingly popular due to their potential for use as a liquid biopsy, which is a tool for non-invasive diagnosis and monitoring of diseases, such as cancer, stroke, trauma, myocardial infarction, autoimmune disorders, and pregnancy-associated complications. While the diagnostic potential of CNAs has been investigated extensively, there is a paucity of understanding of their pathophysiological functions. Are these CNAs part of the cell's regular framework of functioning? Or do they act as molecular players in disease initiation and progression? The aim of this review is to investigate the origins and functions of the circulating cell-free nucleic acids in the plasma and serum of patients with various malignancies, and propose areas of study, which may elucidate the novel underlying mechanisms that are functioning during cancer initiation/progression.

  8. Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

    SciTech Connect

    Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

    2016-08-09

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

  9. Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

    SciTech Connect

    Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

    2014-09-30

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

  10. A DNA origami nanorobot controlled by nucleic acid hybridization.

    PubMed

    Torelli, Emanuela; Marini, Monica; Palmano, Sabrina; Piantanida, Luca; Polano, Cesare; Scarpellini, Alice; Lazzarino, Marco; Firrao, Giuseppe

    2014-07-23

    A prototype for a DNA origami nanorobot is designed, produced, and tested. The cylindrical nanorobot (diameter of 14 nm and length of 48 nm) with a switchable flap, is able to respond to an external stimulus and reacts by a physical switch from a disarmed to an armed configuration able to deliver a cellular compatible message. In the tested design the robot weapon is a nucleic acid fully contained in the inner of the tube and linked to a single point of the internal face of the flap. Upon actuation the nanorobot moves the flap extracting the nucleic acid that assembles into a hemin/G-quadruplex horseradish peroxidase mimicking DNAzyme catalyzing a colorimetric reaction or chemiluminescence generation. The actuation switch is triggered by an external nucleic acid (target) that interacts with a complementary nucleic acid that is beard externally by the nanorobot (probe). Hybridization of probe and target produces a localized structural change that results in flap opening. The flap movement is studied on a two-dimensional prototype origami using Förster resonance energy transfer and is shown to be triggered by a variety of targets, including natural RNAs. The nanorobot has potential for in vivo biosensing and intelligent delivery of biological activators.

  11. Caged molecular beacons: controlling nucleic acid hybridization with light.

    PubMed

    Wang, Chunming; Zhu, Zhi; Song, Yanling; Lin, Hui; Yang, Chaoyong James; Tan, Weihong

    2011-05-28

    We have constructed a novel class of light-activatable caged molecular beacons (cMBs) that are caged by locking two stems with a photo-labile biomolecular interaction or covalent bond. With the cMBs, the nucleic acid hybridization process can be easily controlled with light, which offers the possibility for a high spatiotemporal resolution study of intracellular mRNAs.

  12. Nucleic acid amplification: Alternative methods of polymerase chain reaction.

    PubMed

    Fakruddin, Md; Mannan, Khanjada Shahnewaj Bin; Chowdhury, Abhijit; Mazumdar, Reaz Mohammad; Hossain, Md Nur; Islam, Sumaiya; Chowdhury, Md Alimuddin

    2013-10-01

    Nucleic acid amplification is a valuable molecular tool not only in basic research but also in application oriented fields, such as clinical medicine development, infectious diseases diagnosis, gene cloning and industrial quality control. A comperehensive review of the literature on the principles, applications, challenges and prospects of different alternative methods of polymerase chain reaction (PCR) was performed. PCR was the first nucleic acid amplification method. With the advancement of research, a no of alternative nucleic acid amplification methods has been developed such as loop mediated isothermal amplification, nucleic acid sequence based amplification, strand displacement amplification, multiple displacement amplification. Most of the alternative methods are isothermal obviating the need for thermal cyclers. Though principles of most of the alternate methods are relatively complex than that of PCR, they offer better applicability and sensitivity in cases where PCR has limitations. Most of the alternate methods still have to prove themselves through extensive validation studies and are not available in commercial form; they pose the potentiality to be used as replacements of PCR. Continuous research is going on in different parts of the world to make these methods viable technically and economically.

  13. [Photochemistry and UV Spectroscopy of Proteins and Nucleic Acids].

    PubMed

    Wierzchowski, Kazimierz Lech

    2015-01-01

    The article presents a short history of David Shugar studies in the field of photochemistry and UV spectroscopy of proteins and nucleic acids, carried out since the late 1940s. to the beginning of the 1970s. of the 20th century, with some references to the state of related research in those days.

  14. Nucleic acid amplification: Alternative methods of polymerase chain reaction

    PubMed Central

    Fakruddin, Md; Mannan, Khanjada Shahnewaj Bin; Chowdhury, Abhijit; Mazumdar, Reaz Mohammad; Hossain, Md. Nur; Islam, Sumaiya; Chowdhury, Md. Alimuddin

    2013-01-01

    Nucleic acid amplification is a valuable molecular tool not only in basic research but also in application oriented fields, such as clinical medicine development, infectious diseases diagnosis, gene cloning and industrial quality control. A comperehensive review of the literature on the principles, applications, challenges and prospects of different alternative methods of polymerase chain reaction (PCR) was performed. PCR was the first nucleic acid amplification method. With the advancement of research, a no of alternative nucleic acid amplification methods has been developed such as loop mediated isothermal amplification, nucleic acid sequence based amplification, strand displacement amplification, multiple displacement amplification. Most of the alternative methods are isothermal obviating the need for thermal cyclers. Though principles of most of the alternate methods are relatively complex than that of PCR, they offer better applicability and sensitivity in cases where PCR has limitations. Most of the alternate methods still have to prove themselves through extensive validation studies and are not available in commercial form; they pose the potentiality to be used as replacements of PCR. Continuous research is going on in different parts of the world to make these methods viable technically and economically. PMID:24302831

  15. Predicting nucleic acid binding interfaces from structural models of proteins

    PubMed Central

    Dror, Iris; Shazman, Shula; Mukherjee, Srayanta; Zhang, Yang; Glaser, Fabian; Mandel-Gutfreund, Yael

    2011-01-01

    The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared to patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. PMID:22086767

  16. Predicting nucleic acid binding interfaces from structural models of proteins.

    PubMed

    Dror, Iris; Shazman, Shula; Mukherjee, Srayanta; Zhang, Yang; Glaser, Fabian; Mandel-Gutfreund, Yael

    2012-02-01

    The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However, the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three-dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared with patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. Copyright © 2011 Wiley Periodicals, Inc.

  17. Progress in nucleic acid research and molecular biology

    SciTech Connect

    Cohn, W.E. . Biology Div.); Moldave, K. )

    1989-01-01

    This book contain the proceedings on progress in nucleic acid research and molecular biology. Topics covered include: polynucleotide-protein cross-links induced by ultraviolet light and their use for structural investigation of nucleoproteins; left-handed Z-DNA and genetic recombination, and structure and function of signal recognition particle RNA.

  18. Structure, stability and behaviour of nucleic acids in ionic liquids

    PubMed Central

    Tateishi-Karimata, Hisae; Sugimoto, Naoki

    2014-01-01

    Nucleic acids have become a powerful tool in nanotechnology because of their conformational polymorphism. However, lack of a medium in which nucleic acid structures exhibit long-term stability has been a bottleneck. Ionic liquids (ILs) are potential solvents in the nanotechnology field. Hydrated ILs, such as choline dihydrogen phosphate (choline dhp) and deep eutectic solvent (DES) prepared from choline chloride and urea, are ‘green’ solvents that ensure long-term stability of biomolecules. An understanding of the behaviour of nucleic acids in hydrated ILs is necessary for developing DNA materials. We here review current knowledge about the structures and stabilities of nucleic acids in choline dhp and DES. Interestingly, in choline dhp, A–T base pairs are more stable than G–C base pairs, the reverse of the situation in buffered NaCl solution. Moreover, DNA triplex formation is markedly stabilized in hydrated ILs compared with aqueous solution. In choline dhp, the stability of Hoogsteen base pairs is comparable to that of Watson–Crick base pairs. Moreover, the parallel form of the G-quadruplex is stabilized in DES compared with aqueous solution. The behaviours of various DNA molecules in ILs detailed here should be useful for designing oligonucleotides for the development of nanomaterials and nanodevices. PMID:25013178

  19. AquaLite, a bioluminescent label for immunoassay and nucleic acid detection: quantitative analyses at the attomol level

    NASA Astrophysics Data System (ADS)

    Smith, David F.; Stults, Nancy L.

    1996-04-01

    AquaLiteR is a direct, bioluminescent label capable of detecting attomol levels of analyte in clinical immunoassays and assays for the quantitative measurement of nucleic acids. Bioluminescent immunoassays (BIAs) require no radioisotopes and avoid complex fluorescent measurements and many of the variables of indirect enzyme immunoassays (EIAs). AquaLite, a recombinant form of the photoprotein aequorin from a bioluminescent jellyfish, is coupled directly to antibodies to prepare bioluminescent conjugates for assay development. When the AquaLite-antibody complex is exposed to a solution containing calcium ions, a flash of blue light ((lambda) max equals 469 nm) is generated. The light signal is measured in commercially available luminometers that simultaneously inject a calcium solution and detect subattomol photoprotein levies in either test tubes or microtiter plates. Immunometric or 'sandwich' type assays are available for the quantitative measurement of human endocrine hormones and nucleic acids. The AquaLite TSH assay can detect 1 attomol of thyroid stimulating hormone (TSH) in 0.2 mL of human serum and is a useful clinical tool for diagnosing hyperthyroid patients. AquaLite-based nucleic acid detection permits quantifying attomol levels of specific nucleic acid markers and represents possible solution to the difficult problem of quantifying the targets of nucleic acid amplification methods.

  20. Practical strategies for the evaluation of high-affinity protein/nucleic acid interactions

    PubMed Central

    Altschuler, Sarah E.; Lewis, Karen A.; Wuttke, Deborah S.

    2014-01-01

    The quantitative evaluation of binding interactions between proteins and nucleic acids is highly sensitive to a variety of experimental conditions. Optimization of these conditions is critical for obtaining high quality, reproducible data, particularly in the context of very high affinity interactions. Here, we discuss the practical considerations involved in optimizing the apparent binding constant of an interaction as measured by two common quantitative assays, electrophoretic mobility shift assay and double-filter binding when measuring extremely tight protein/nucleic acid interactions with sub-nanomolar binding affinities. We include specific examples from two telomere end-binding protein systems, Schizo -saccharomyces pombe Pot1 and Saccharomyces cerevisiae Cdc13, to demonstrate potential experimental pitfalls and some useful strategies for optimization. PMID:25197549

  1. The use of solid supports to generate nucleic acid carriers.

    PubMed

    Unciti-Broceta, Asier; Díaz-Mochón, Juan José; Sánchez-Martín, Rosario M; Bradley, Mark

    2012-07-17

    Nucleic acids are the foundation stone of all cellular processes. Consequently, the use of DNA or RNA to treat genetic and acquired disorders (so called gene therapy) offers enormous potential benefits. The restitution of defective genes or the suppression of malignant genes could target a range of diseases, including cancers, inherited diseases (cystic fibrosis, muscular dystrophy, etc.), and viral infections. However, this strategy has a major barrier: the size and charge of nucleic acids largely restricts their transit into eukaryotic cells. Potential strategies to solve this problem include the use of a variety of natural and synthetic nucleic acid carriers. Driven by the aim and ambition of translating this promising therapeutic approach into the clinic, researchers have been actively developing advanced delivery systems for nucleic acids for more than 20 years. A decade ago we began our investigations of solid-phase techniques to construct families of novel nucleic acid carriers for transfection. We envisaged that the solid-phase synthesis of polycationic dendrimers and derivatized polyamimes would offer distinct advantages over solution phase techniques. Notably in solid phase synthesis we could take advantage of mass action and streamlined purification procedures, while simplifying the handling of compounds with high polarities and plurality of functional groups. Parallel synthesis methods would also allow rapid access to libraries of compounds with improved purities and yields over comparable solution methodologies and facilitate the development of structure activity relationships. We also twisted the concept of the solid-phase support on its head: we devised miniaturized solid supports that provided an innovative cell delivery vehicle in their own right, carrying covalently conjugated cargos (biomolecules) into cells. In this Account, we summarize the main outcomes of this series of chemically related projects.

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

    DOEpatents

    Nasarabadi, Shanavaz [Livermore, CA

    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 reaction 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.

  3. Mesoporous Silica Nanoparticles as Carriers for Intracellular Delivery of Nucleic Acids and Subsequent Therapeutic Applications.

    PubMed

    Cha, Wenzhang; Fan, Rengen; Miao, Yufeng; Zhou, Yong; Qin, Chenglin; Shan, Xiangxiang; Wan, Xinqiang; Li, Jinbo

    2017-05-11

    Nucleic acids, including DNA, microRNA (miRNA), small interfering RNA (siRNA), and antisense oligonucleotide (ASO), are powerful gene regulators, which have been demonstrated as promising drug candidates for therapeutic treatments. Nevertheless, poor cellular membrane permeability and serum stability have greatly hindered the applications of nucleic acids in biomedicine. To address these issues, associate carriers that can encapsulate and protect nucleic acids are urgently required. Mesoporous silica nanoparticles (MSNs or MSNPs), which are nanomaterials with excellent biocompatibility, large surface area for functionalization, and tunable pore size for encapsulating different cargos, are emerging as novel and ideal biomaterials for different biomedical applications. In this review paper, we focus on the applications of MSNs in nucleic acid delivery and nucleic acid-guided therapeutic treatments. General strategies for the preparation of nucleic acid-MSN complexes will be firstly introduced, followed by a summary of recent applications of MSNs in nucleic acid delivery and nucleic acid-guided therapeutics.

  4. Variation-tolerant capture and multiplex detection of nucleic acids: application to detection of microbes.

    PubMed

    Ohrmalm, Christina; Eriksson, Ronnie; Jobs, Magnus; Simonson, Magnus; Strømme, Maria; Bondeson, Kåre; Herrmann, Björn; Melhus, Asa; Blomberg, Jonas

    2012-10-01

    In contrast to ordinary PCRs, which have a limited multiplex capacity and often return false-negative results due to target variation or inhibition, our new detection strategy, VOCMA (variation-tolerant capture multiplex assay), allows variation-tolerant, target-specific capture and detection of many nucleic acids in one test. Here we demonstrate the use of a single-tube, dual-step amplification strategy that overcomes the usual limitations of PCR multiplexing, allowing at least a 22-plex format with retained sensitivity. Variation tolerance was achieved using long primers and probes designed to withstand variation at known sites and a judicious mix of degeneration and universal bases. We tested VOCMA in situations where enrichment from a large sample volume with high sensitivity and multiplexity is important (sepsis; streptococci, enterococci, and staphylococci, several enterobacteria, candida, and the most important antibiotic resistance genes) and where variation tolerance and high multiplexity is important (gastroenteritis; astrovirus, adenovirus, rotavirus, norovirus genogroups I and II, and sapovirus, as well as enteroviruses, which are not associated with gastroenteritis). Detection sensitivities of 10 to 1,000 copies per reaction were achieved for many targets. VOCMA is a highly multiplex, variation-tolerant, general purpose nucleic acid detection concept. It is a specific and sensitive method for simultaneous detection of nucleic acids from viruses, bacteria, fungi, and protozoa, as well as host nucleic acid, in the same test. It can be run on an ordinary PCR and a Luminex machine and is suitable for both clinical diagnoses and microbial surveillance.

  5. Highly Stable and Sensitive Nucleic Acid Amplification and Cell-Phone-Based Readout.

    PubMed

    Kong, Janay E; Wei, Qingshan; Tseng, Derek; Zhang, Jingzi; Pan, Eric; Lewinski, Michael; Garner, Omai B; Ozcan, Aydogan; Di Carlo, Dino

    2017-03-02

    Key challenges with point-of-care (POC) nucleic acid tests include achieving a low-cost, portable form factor, and stable readout, while also retaining the same robust standards of benchtop lab-based tests. We addressed two crucial aspects of this problem, identifying a chemical additive, hydroxynaphthol blue, that both stabilizes and significantly enhances intercalator-based fluorescence readout of nucleic acid concentration, and developing a cost-effective fiber-optic bundle-based fluorescence microplate reader integrated onto a mobile phone. Using loop-mediated isothermal amplification on lambda DNA we achieve a 69-fold increase in signal above background, 20-fold higher than the gold standard, yielding an overall limit of detection of 25 copies/μL within an hour using our mobile-phone-based platform. Critical for a point-of-care system, we achieve a >60% increase in fluorescence stability as a function of temperature and time, obviating the need for manual baseline correction or secondary calibration dyes. This field-portable and cost-effective mobile-phone-based nucleic acid amplification and readout platform is broadly applicable to other real-time nucleic acid amplification tests by similarly modulating intercalating dye performance and is compatible with any fluorescence-based assay that can be run in a 96-well microplate format, making it especially valuable for POC and resource-limited settings.

  6. Visual detection of nucleic acids based on Mie scattering and the magnetophoretic effect.

    PubMed

    Zhao, Zichen; Chen, Shan; Ho, John Kin Lim; Chieng, Ching-Chang; Chen, Ting-Hsuan

    2015-12-07

    Visual detection of nucleic acid biomarkers is a simple and convenient approach to point-of-care applications. However, issues of sensitivity and the handling of complex bio-fluids have posed challenges. Here we report on a visual method detecting nucleic acids using Mie scattering of polystyrene microparticles and the magnetophoretic effect. Magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) were surface-functionalised with oligonucleotide probes, which can hybridise with target oligonucleotides in juxtaposition and lead to the formation of MMPs-targets-PMPs sandwich structures. Using an externally applied magnetic field, the magnetophoretic effect attracts the sandwich structure to the sidewall, which reduces the suspended PMPs and leads to a change in the light transmission via the Mie scattering. Based on the high extinction coefficient of the Mie scattering (∼3 orders of magnitude greater than that of the commonly used gold nanoparticles), our results showed the limit of detection to be 4 pM using a UV-Vis spectrometer or 10 pM by direct visual inspection. Meanwhile, we also demonstrated that this method is compatible with multiplex assays and detection in complex bio-fluids, such as whole blood or a pool of nucleic acids, without purification in advance. With a simplified operation procedure, low instrumentation requirement, high sensitivity and compatibility with complex bio-fluids, this method provides an ideal solution for visual detection of nucleic acids in resource-limited settings.

  7. 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.

  8. Complex formation with nucleic acids and aptamers alters the antigenic properties of platelet factor 4

    PubMed Central

    Jaax, Miriam E.; Krauel, Krystin; Marschall, Thomas; Brandt, Sven; Gansler, Julia; Fürll, Birgitt; Appel, Bettina; Fischer, Silvia; Block, Stephan; Helm, Christiane A.; Müller, Sabine; Preissner, Klaus T.

    2013-01-01

    The tight electrostatic binding of the chemokine platelet factor 4 (PF4) to polyanions induces heparin-induced thrombocytopenia, a prothrombotic adverse drug reaction caused by immunoglobulin G directed against PF4/polyanion complexes. This study demonstrates that nucleic acids, including aptamers, also bind to PF4 and enhance PF4 binding to platelets. Systematic assessment of RNA and DNA constructs, as well as 4 aptamers of different lengths and secondary structures, revealed that increasing length and double-stranded segments of nucleic acids augment complex formation with PF4, while single nucleotides or single-stranded polyA or polyC constructs do not. Aptamers were shown by circular dichroism spectroscopy to induce structural changes in PF4 that resemble those induced by heparin. Moreover, heparin-induced anti-human–PF4/heparin antibodies cross-reacted with human PF4/nucleic acid and PF4/aptamer complexes, as shown by an enzyme immunoassay and a functional platelet activation assay. Finally, administration of PF4/44mer–DNA protein C aptamer complexes in mice induced anti–PF4/aptamer antibodies, which cross-reacted with murine PF4/heparin complexes. These data indicate that the formation of anti-PF4/heparin antibodies in postoperative patients may be augmented by PF4/nucleic acid complexes. Moreover, administration of therapeutic aptamers has the potential to induce anti-PF4/polyanion antibodies and a prothrombotic diathesis. PMID:23673861

  9. Cationic liposome–nucleic acid complexes for gene delivery and gene silencing

    PubMed Central

    Ewert, Kai K.; Majzoub, Ramsey N.; Leal, Cecília

    2014-01-01

    Cationic liposomes (CLs) are studied worldwide as carriers of DNA and short interfering RNA (siRNA) for gene delivery and gene silencing, and related clinical trials are ongoing. Optimization of transfection efficiency and silencing efficiency by cationic liposome carriers requires a comprehensive understanding of the structures of CL–nucleic acid complexes and the nature of their interactions with cell membranes as well as events leading to release of active nucleic acids within the cytoplasm. Synchrotron x-ray scattering has revealed that CL–nucleic acid complexes spontaneously assemble into distinct liquid crystalline phases including the lamellar, inverse hexagonal, hexagonal, and gyroid cubic phases, and fluorescence microscopy has revealed CL–DNA pathways and interactions with cells. The combining of custom synthesis with characterization techniques and gene expression and silencing assays has begun to unveil structure–function relations in vitro. As a recent example, this review will briefly describe experiments with surface-functionalized PEGylated CL–DNA nanoparticles. The functionalization, which is achieved through custom synthesis, is intended to address and overcome cell targeting and endosomal escape barriers to nucleic acid delivery faced by PEGylated nanoparticles designed for in vivo applications. PMID:25587216

  10. MagaZorb: a simple tool for rapid isolation of viral nucleic acids.

    PubMed

    Nargessi, Dokhi; Ou, Chin-Yih

    2010-04-15

    Effective isolation of nucleic acids from samples containing viral materials is an essential step for accurate diagnosis of viral infections. The necessity of this critical step before analytical identification and diagnosis of viral infections is paramount to screening programs and to identifying and monitoring epidemics and pandemics. With molecular assays rapidly evolving into routine practice, clinical laboratories face several challenges, including presence of small amounts of viral nucleic acids in abundant levels of genomic DNA and total RNA, processing of various sample types, and carry-over of polymerase chain reaction inhibitors, which could significantly affect polymerase chain reaction and microarray results. MagaZorb nucleic acid isolation technology overcomes these challenges and offers a simple and reliable method for isolation of high-quality and high-yield nucleic acids. Although the MagaZorb technology is readily adaptable to automated platforms, it is also well suited to laboratories in remote areas of resource-poor countries, because a simple magnet is the only device required to perform the procedure manually. Performance characteristics and clinical application of the MagaZorb technology are briefly described here.

  11. Automated nucleic acid isolation in viral molecular diagnostics: evaluation of the QIAsymphony SP.

    PubMed

    Parham, N J; Parmar, S A; Kumar, N; Aliyu, S; Curran, M D; Zhang, H

    2012-01-01

    The Qiagen QIAsymphony SP is a high-throughput (up to 96 samples per run), fully-automated nucleic acid isolation system. It was implemented in the authors' laboratory to cope with the high demand for pandemic H1N1 influenza testing in 2009. This study evaluated the QIAsymphony SP for viral nucleic acid isolation from quality control materials, pure cultures and various clinical specimens. The effect of varying sample volume on detection sensitivity was investigated using serial 10-fold dilutions of pure viral specimens and target nucleic acids were detected by real-time polymerase chain reaction (PCR) assays. Little variability in detection sensitivity was observed for all the viral targets tested, although variation in cycle threshold values was apparent in some cases. Importantly, pathogens were detectable over a broad concentration range and from diverse clinical specimens. Removal of PCR inhibitors was generally effective, as demonstrated by detection of viral nucleic acids and/or internal controls. The results demonstrate that the QIAsymphony SP is suitable for use in routine virology molecular diagnostics, and provides a high-throughput capacity, which is needed in peak seasons of infection or in centralised laboratories.

  12. Nucleic Acid Chaperone Activity of the ORF1 Protein from the Mouse LINE-1 Retrotransposon

    PubMed Central

    Martin, Sandra L.; Bushman, Frederic D.

    2001-01-01

    Non-LTR retrotransposons such as L1 elements are major components of the mammalian genome, but their mechanism of replication is incompletely understood. Like retroviruses and LTR-containing retrotransposons, non-LTR retrotransposons replicate by reverse transcription of an RNA intermediate. The details of cDNA priming and integration, however, differ between these two classes. In retroviruses, the nucleocapsid (NC) protein has been shown to assist reverse transcription by acting as a “nucleic acid chaperone,” promoting the formation of the most stable duplexes between nucleic acid molecules. A protein-coding region with an NC-like sequence is present in most non-LTR retrotransposons, but no such sequence is evident in mammalian L1 elements or other members of its class. Here we investigated the ORF1 protein from mouse L1 and found that it does in fact display nucleic acid chaperone activities in vitro. L1 ORF1p (i) promoted annealing of complementary DNA strands, (ii) facilitated strand exchange to form the most stable hybrids in competitive displacement assays, and (iii) facilitated melting of an imperfect duplex but stabilized perfect duplexes. These findings suggest a role for L1 ORF1p in mediating nucleic acid strand transfer steps during L1 reverse transcription. PMID:11134335

  13. 77 FR 16126 - Microbiology Devices; Reclassification of Nucleic Acid-Based Systems for Mycobacterium tuberculosis

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-19

    ... Food and Drug Administration 21 CFR Part 866 Microbiology Devices; Reclassification of Nucleic Acid...: Proposed rule. SUMMARY: The Food and Drug Administration (FDA) is proposing to reclassify nucleic acid... effectiveness of the device for its intended use. II. Regulatory Background of the Device Nucleic acid-based in...

  14. 78 FR 36698 - Microbiology Devices; Reclassification of Nucleic Acid-Based Systems for Mycobacterium tuberculosis

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-19

    ... Nucleic Acid-Based Systems for Mycobacterium tuberculosis Complex in Respiratory Specimens AGENCY: Food...) is proposing to reclassify nucleic acid-based in vitro diagnostic devices for the detection of... Controls Guideline: Nucleic Acid-Based In Vitro Diagnostic Devices for the Detection of Mycobacterium...

  15. Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes

    PubMed Central

    Greenough, Lucia; Schermerhorn, Kelly M.; Mazzola, Laurie; Bybee, Joanna; Rivizzigno, Danielle; Cantin, Elizabeth; Slatko, Barton E.; Gardner, Andrew F.

    2016-01-01

    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. PMID:26365239

  16. 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.

  17. Nucleic acids encoding antifungal polypeptides and uses thereof

    DOEpatents

    Altier, Daniel J.; Ellanskaya, I. A.; Gilliam, Jacob T.; Hunter-Cevera, Jennie; Presnail, James K; Schepers, Eric; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser

    2010-11-02

    Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include an amino acid sequence, and variants and fragments thereof, for an antipathogenic polypeptide that was isolated from a fungal fermentation broth. Nucleic acid molecules that encode the antipathogenic polypeptides of the invention, and antipathogenic domains thereof, are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention are also disclosed.

  18. Isolated nucleic acids encoding antipathogenic polypeptides and uses thereof

    DOEpatents

    Altier, Daniel J.; Crane, Virginia C.; Ellanskaya, Irina; Ellanskaya, Natalia; Gilliam, Jacob T.; Hunter-Cevera, Jennie; Presnail, James K.; Schepers, Eric J.; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser

    2010-04-20

    Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include amino acid sequences, and variants and fragments thereof, for antipathogenic polypeptides that were isolated from fungal fermentation broths. Nucleic acids that encode the antipathogenic polypeptides are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention are also disclosed.

  19. Recent Developments in Peptide-Based Nucleic Acid Delivery

    PubMed Central

    Veldhoen, Sandra; Laufer, Sandra D.; Restle, Tobias

    2008-01-01

    Despite the fact that non-viral nucleic acid delivery systems are generally considered to be less efficient than viral vectors, they have gained much interest in recent years due to their superior safety profile compared to their viral counterpart. Among these synthetic vectors are cationic polymers, branched dendrimers, cationic liposomes and cell-penetrating peptides (CPPs). The latter represent an assortment of fairly unrelated sequences essentially characterised by a high content of basic amino acids and a length of 10–30 residues. CPPs are capable of mediating the cellular uptake of hydrophilic macromolecules like peptides and nucleic acids (e.g. siRNAs, aptamers and antisense-oligonucleotides), which are internalised by cells at a very low rate when applied alone. Up to now, numerous sequences have been reported to show cell-penetrating properties and many of them have been used to successfully transport a variety of different cargos into mammalian cells. In recent years, it has become apparent that endocytosis is a major route of internalisation even though the mechanisms underlying the cellular translocation of CPPs are poorly understood and still subject to controversial discussions. In this review, we will summarise the latest developments in peptide-based cellular delivery of nucleic acid cargos. We will discuss different mechanisms of entry, the intracellular fate of the cargo, correlation studies of uptake versus biological activity of the cargo as well as technical problems and pitfalls. PMID:19325804

  20. Bioreducible polyethylenimine nanoparticles for the efficient delivery of nucleic acids.

    PubMed

    Bansal, Ruby; Tayal, Shweta; Gupta, K C; Kumar, Pradeep

    2015-03-14

    Recently, non-viral vectors for nucleic acid delivery have received considerable attention. Among the various non-viral vectors, branched polyethylenimine (bPEI, 25 kDa) has been one of the most widely used carrier systems due to its high transfection efficiency, however, it imparts high cytotoxicity. In this study, we have crosslinked bPEI with a bioreducible linker, 3,3'-dithiodipropionic acid (DTPA), via electrostatic interactions to obtain DTPA crosslinked bPEI (DP) nanoparticles. The crosslinking significantly reduced the cytotoxicity of the nanoparticles. To arrive at the best formulation in terms of nucleic acid transfection, a series of DP nanoparticles were prepared by varying the percentage of crosslinking. The dual action of DTPA, i.e. partial blocking of the charge density as well as crosslinking to convert bPEI into its nanoparticles, did not alter the pDNA condensation ability of the so-formed nanoparticles, rather the strategy favoured the unpackaging of the complexes inside the cells improving the release of pDNA, which resulted in a higher transfection efficiency. All the formulations carried nucleic acids inside the cells and exhibited significantly higher transfection efficiencies than native bPEI and the commercial transfection reagent, Lipofectamine™. Sequential siRNA delivery displayed significant suppression in the target gene expression. All together, the evaluation of the delivery systems demonstrates that the newly synthesized DP NPs are quite promising as non-viral gene carriers.

  1. Multiplex nucleic acid amplification test for diagnosis of dengue fever, malaria, and leptospirosis.

    PubMed

    Waggoner, Jesse J; Abeynayake, Janaki; Balassiano, Ilana; Lefterova, Martina; Sahoo, Malaya K; Liu, Yuanyuan; Vital-Brazil, Juliana Magalhães; Gresh, Lionel; Balmaseda, Angel; Harris, Eva; Banaei, Niaz; Pinsky, Benjamin A

    2014-06-01

    Dengue, leptospirosis, and malaria are among the most common etiologies of systemic undifferentiated febrile illness (UFI) among travelers to the developing world, and these pathogens all have the potential to cause life-threatening illness in returned travelers. The current study describes the development of an internally controlled multiplex nucleic acid amplification test for the detection of dengue virus (DENV) and Leptospira and Plasmodium species, with a specific callout for Plasmodium falciparum (referred to as the UFI assay). During analytical evaluation, the UFI assay displayed a wide dynamic range and a sensitive limit of detection for each target, including all four DENV serotypes. In a clinical evaluation including 210 previously tested samples, the sensitivities of the UFI assay were 98% for DENV (58/59 samples detected) and 100% for Leptospira and malaria (65/65 and 20/20 samples, respectively). Malaria samples included all five Plasmodium species known to cause human disease. The specificity of the UFI assay was 100% when evaluated with a panel of 66 negative clinical samples. Furthermore, no amplification was observed when extracted nucleic acids from related pathogens were tested. Compared with whole-blood samples, the UFI assay remained positive for Plasmodium in 11 plasma samples from patients with malaria (parasitemia levels of 0.0037 to 3.4%). The syndrome-based design of the UFI assay, combined with the sensitivities of the component tests, represents a significant improvement over the individual diagnostic tests available for these pathogens.

  2. Multiplex Nucleic Acid Amplification Test for Diagnosis of Dengue Fever, Malaria, and Leptospirosis

    PubMed Central

    Waggoner, Jesse J.; Abeynayake, Janaki; Balassiano, Ilana; Lefterova, Martina; Sahoo, Malaya K.; Liu, Yuanyuan; Vital-Brazil, Juliana Magalhães; Gresh, Lionel; Balmaseda, Angel; Harris, Eva; Banaei, Niaz

    2014-01-01

    Dengue, leptospirosis, and malaria are among the most common etiologies of systemic undifferentiated febrile illness (UFI) among travelers to the developing world, and these pathogens all have the potential to cause life-threatening illness in returned travelers. The current study describes the development of an internally controlled multiplex nucleic acid amplification test for the detection of dengue virus (DENV) and Leptospira and Plasmodium species, with a specific callout for Plasmodium falciparum (referred to as the UFI assay). During analytical evaluation, the UFI assay displayed a wide dynamic range and a sensitive limit of detection for each target, including all four DENV serotypes. In a clinical evaluation including 210 previously tested samples, the sensitivities of the UFI assay were 98% for DENV (58/59 samples detected) and 100% for Leptospira and malaria (65/65 and 20/20 samples, respectively). Malaria samples included all five Plasmodium species known to cause human disease. The specificity of the UFI assay was 100% when evaluated with a panel of 66 negative clinical samples. Furthermore, no amplification was observed when extracted nucleic acids from related pathogens were tested. Compared with whole-blood samples, the UFI assay remained positive for Plasmodium in 11 plasma samples from patients with malaria (parasitemia levels of 0.0037 to 3.4%). The syndrome-based design of the UFI assay, combined with the sensitivities of the component tests, represents a significant improvement over the individual diagnostic tests available for these pathogens. PMID:24671788

  3. Physical chemistry of nucleic acids and their complexes.

    PubMed

    Ghirlando, Rodolfo; Felsenfeld, Gary

    2013-12-01

    Studies of the physical properties of nucleic acids began almost immediately following the discovery of the DNA structure. Early investigations focused on the stability and specificity of multi-strand polynucleotide complexes, then gradually on their interaction with other molecules, particularly proteins. As molecular and structural biology expanded to provide detailed information about biochemical mechanisms, physical studies eventually acquired the additional constraint that they should be relevant to functioning biological systems. We describe work in our laboratory that began with investigations of relatively simple questions about the role of electrostatic interactions in the stabilization of multi-strand nucleic acid structures, and evolved to studies of chromatin structure in vitro and within the nucleus. Copyright © 2013 Wiley Periodicals, Inc.

  4. Nucleic Acid Engineering: RNA Following the Trail of DNA.

    PubMed

    Kim, Hyejin; Park, Yongkuk; Kim, Jieun; Jeong, Jaepil; Han, Sangwoo; Lee, Jae Sung; Lee, Jong Bum

    2016-02-08

    The self-assembly feature of the naturally occurring biopolymer, DNA, has fascinated researchers in the fields of materials science and bioengineering. With the improved understanding of the chemical and structural nature of DNA, DNA-based constructs have been designed and fabricated from two-dimensional arbitrary shapes to reconfigurable three-dimensional nanodevices. Although DNA has been used successfully as a building block in a finely organized and controlled manner, its applications need to be explored. Hence, with the myriad of biological functions, RNA has recently attracted considerable attention to further the application of nucleic acid-based structures. This Review categorizes different approaches of engineering nucleic acid-based structures and introduces the concepts, principles, and applications of each technique, focusing on how DNA engineering is applied as a guide to RNA engineering.

  5. Reaction mechanisms of riboflavin triplet state with nucleic acid bases.

    PubMed

    Lin, Weizhen; Lu, Changyuan; Du, Fuqiang; Shao, Zhiyong; Han, Zhenhui; Tu, Tiecheng; Yao, Side; Lin, Nianyun

    2006-04-01

    ESR and laser flash photolysis studies have determined a reasonable order of reactivity of nucleotides with triplet riboflavin (3Rb*) for the first time. ESR detection of triplet state reactivity of Rb with nucleoside, polynucleotide and DNA has been obtained simultaneously. In addition, ESR spin elimination measurement of the reactivity of 3Rb* with nucleotides in good accord with laser flash photolysis determination of the corresponding rate constants offers a simple and reliable method to detect the reactivities of nucleic acids and its components with photoexcited flavins. Kinetic, ESR and thermodynamic studies have demonstrated that Rb should be a strong endogenous photosensitizer capable of oxidizing all nucleic acid bases, and preferentially two purine nucleotides with high rate constants.

  6. Microwave-Assisted Rapid Enzymatic Synthesis of Nucleic Acids.

    PubMed

    Hari Das, Rakha; Ahirwar, Rajesh; Kumar, Saroj; Nahar, Pradip

    2016-07-02

    Herein we report microwave-induced enhancement of the reactions catalyzed by Escherichia coli DNA polymerase I and avian myeloblastosis virus-reverse transcriptase. The reactions induced by microwaves result in a highly selective synthesis of nucleic acids in 10-50 seconds. In contrast, same reactions failed to give desired reaction products when carried out in the same time periods, but without microwave irradiation. Each of the reactions was carried out for different duration of microwave exposure time to find the optimum reaction time. The products produced by the respective enzyme upon microwave irradiation of the reaction mixtures were identical to that produced by the conventional procedures. As the microwave-assisted reactions are rapid, microwave could be a useful alternative to the conventional and time consuming procedures of enzymatic synthesis of nucleic acids.

  7. Selected Nucleic Acid Precursors in Studies of Aquatic Microbial Ecology

    PubMed Central

    Karl, David M.

    1982-01-01

    The use of radiolabeled nucleosides and nucleic acid bases to estimate the rates of RNA and DNA synthesis in naturally occurring microbial assemblages requires numerous assumptions, several of which are evaluated herein. Comparative time series analyses of the uptake and incorporation, labeling specificity, and extent of catabolism of [2-3H]adenine, [methyl-3H]thymidine, and [5-3H]uridine were performed with pure bacterial and algal cultures, as well as with environmental samples. [3H]thymidine yielded the most variable results, especially with regard to the extent of nonspecific macromolecular labeling. The pathways of [3H]thymidine and [3H]adenine metabolism were further evaluated by isotope dilution methods and by comparing incorporation patterns of thymidine labeled at different sites of the molecule. The advantages, uncertainties, and limitations of the use of radiolabeled nucleic acid precursors in studies of aquatic microbial ecology are discussed and a prospectus for future studies presented. PMID:16346114

  8. Nucleic acid aptamers: research tools in disease diagnostics and therapeutics.

    PubMed

    Santosh, Baby; Yadava, Pramod K

    2014-01-01

    Aptamers are short sequences of nucleic acid (DNA or RNA) or peptide molecules which adopt a conformation and bind cognate ligands with high affinity and specificity in a manner akin to antibody-antigen interactions. It has been globally acknowledged that aptamers promise a plethora of diagnostic and therapeutic applications. Although use of nucleic acid aptamers as targeted therapeutics or mediators of targeted drug delivery is a relatively new avenue of research, one aptamer-based drug "Macugen" is FDA approved and a series of aptamer-based drugs are in clinical pipelines. The present review discusses the aspects of design, unique properties, applications, and development of different aptamers to aid in cancer diagnosis, prevention, and/or treatment under defined conditions.

  9. Optimization of encoded hydrogel particles for nucleic acid quantification.

    PubMed

    Pregibon, Daniel C; Doyle, Patrick S

    2009-06-15

    The accurate quantification of nucleic acids is of utmost importance for clinical diagnostics, drug discovery, and basic science research. These applications require the concurrent measurement of multiple targets while demanding high-throughput analysis, high sensitivity, specificity between closely related targets, and a wide dynamic range. In attempt to create a technology that can simultaneously meet these demands, we recently developed a method of multiplexed analysis using encoded hydrogel particles. Here, we demonstrate tuning of hydrogel porosity with semi-interpenetrating networks of poly(ethylene glycol), develop a quantitative model to understand hybridization kinetics, and use the findings from these studies to enhance particle design for nucleic acid detection. With an optimized particle design and efficient fluorescent labeling scheme, we demonstrate subattomole sensitivity and single-nucleotide specificity for small RNA targets.

  10. Ultrasensitive nucleic acid sequence detection by single-molecule electrophoresis

    SciTech Connect

    Castro, A; Shera, E.B.

    1996-09-01

    This is the final report of a one-year laboratory-directed research and development project at Los Alamos National Laboratory. There has been considerable interest in the development of very sensitive clinical diagnostic techniques over the last few years. Many pathogenic agents are often present in extremely small concentrations in clinical samples, especially at the initial stages of infection, making their detection very difficult. This project sought to develop a new technique for the detection and accurate quantification of specific bacterial and viral nucleic acid sequences in clinical samples. The scheme involved the use of novel hybridization probes for the detection of nucleic acids combined with our recently developed technique of single-molecule electrophoresis. This project is directly relevant to the DOE`s Defense Programs strategic directions in the area of biological warfare counter-proliferation.

  11. A quantitative measure of chirality inside nucleic acid databank.

    PubMed

    Pietropaolo, Adriana; Parrinello, Michele

    2011-08-01

    We show the capability of a chirality index (Pietropaolo et al., Proteins 2008;70:667-677) to investigate nucleic acid structures because of its high sensitivity to helical conformations. By analyzing selected structures of DNA and RNA, we have found that sequences rich in cytosine and guanine have a tendency to left-handed chirality, in contrast to regions rich in adenine or thymine which show strong negative, right-handed, chirality values. We also analyze RNA structures, where specific loops and hairpin motifs are characterized by a well-defined chirality value. We find that in nucleosome the chirality is exalted, whereas in ribosome it is reduced. Our results illustrate the sensitivity of this descriptor for nucleic acid conformations.

  12. A nanoliter-scale nucleic acid processor with parallel architecture.

    PubMed

    Hong, Jong Wook; Studer, Vincent; Hang, Giao; Anderson, W French; Quake, Stephen R

    2004-04-01

    The purification of nucleic acids from microbial and mammalian cells is a crucial step in many biological and medical applications. We have developed microfluidic chips for automated nucleic acid purification from small numbers of bacterial or mammalian cells. All processes, such as cell isolation, cell lysis, DNA or mRNA purification, and recovery, were carried out on a single microfluidic chip in nanoliter volumes without any pre- or postsample treatment. Measurable amounts of mRNA were extracted in an automated fashion from as little as a single mammalian cell and recovered from the chip. These microfluidic chips are capable of processing different samples in parallel, thereby illustrating how highly parallel microfluidic architectures can be constructed to perform integrated batch-processing functionalities for biological and medical applications.

  13. Activating frataxin expression by repeat-targeted nucleic acids

    PubMed Central

    Li, Liande; Matsui, Masayuki; Corey, David R.

    2016-01-01

    Friedreich's ataxia is an incurable genetic disorder caused by a mutant expansion of the trinucleotide GAA within an intronic FXN RNA. This expansion leads to reduced expression of frataxin (FXN) protein and evidence suggests that transcriptional repression is caused by an R-loop that forms between the expanded repeat RNA and complementary genomic DNA. Synthetic agents that increase levels of FXN protein might alleviate the disease. We demonstrate that introducing anti-GAA duplex RNAs or single-stranded locked nucleic acids into patient-derived cells increases FXN protein expression to levels similar to analogous wild-type cells. Our data are significant because synthetic nucleic acids that target GAA repeats can be lead compounds for restoring curative FXN levels. More broadly, our results demonstrate that interfering with R-loop formation can trigger gene activation and reveal a new strategy for upregulating gene expression. PMID:26842135

  14. Nucleic Acid Aptamers: Research Tools in Disease Diagnostics and Therapeutics

    PubMed Central

    Yadava, Pramod K.

    2014-01-01

    Aptamers are short sequences of nucleic acid (DNA or RNA) or peptide molecules which adopt a conformation and bind cognate ligands with high affinity and specificity in a manner akin to antibody-antigen interactions. It has been globally acknowledged that aptamers promise a plethora of diagnostic and therapeutic applications. Although use of nucleic acid aptamers as targeted therapeutics or mediators of targeted drug delivery is a relatively new avenue of research, one aptamer-based drug “Macugen” is FDA approved and a series of aptamer-based drugs are in clinical pipelines. The present review discusses the aspects of design, unique properties, applications, and development of different aptamers to aid in cancer diagnosis, prevention, and/or treatment under defined conditions. PMID:25050359

  15. The oral microbiota: general overview, taxonomy, and nucleic acid techniques.

    PubMed

    Siqueira, José F; Rôças, Isabela N

    2010-01-01

    Application of nucleic acid technology to the analysis of the bacterial diversity in the oral cavity in conditions of health and disease has not only confirmed the findings from early culture studies but also significantly expanded the list of oral inhabitants and candidate pathogens associated with the major oral diseases. Over 800 bacterial distinct species-level taxa have been detected in the oral cavity and recent studies using high-throughput technology suggest that the breadth of bacterial diversity can be much larger. This chapter provides an overview of the diversity and taxonomy of oral bacteria. Emphasis is also given on nucleic acid technologies that have been widely used for the study of the oral microbiota.

  16. Nonviral Approaches for Neuronal Delivery of Nucleic Acids

    PubMed Central

    Bergen, Jamie M.; Park, In-Kyu; Horner, Philip J.

    2007-01-01

    The delivery of therapeutic nucleic acids to neurons has the potential to treat neurological disease and spinal cord injury. While select viral vectors have shown promise as gene carriers to neurons, their potential as therapeutic agents is limited by their toxicity and immunogenicity, their broad tropism, and the cost of large-scale formulation. Nonviral vectors are an attractive alternative in that they offer improved safety profiles compared to viruses, are less expensive to produce, and can be targeted to specific neuronal subpopulations. However, most nonviral vectors suffer from significantly lower transfection efficiencies than neurotropic viruses, severely limiting their utility in neuron-targeted delivery applications. To realize the potential of nonviral delivery technology in neurons, vectors must be designed to overcome a series of extra- and intracellular barriers. In this article, we describe the challenges preventing successful nonviral delivery of nucleic acids to neurons and review strategies aimed at overcoming these challenges. PMID:17932730

  17. Hands-free sample preparation platform for nucleic acid analysis.

    PubMed

    Baier, T; Hansen-Hagge, T E; Gransee, R; Crombé, A; Schmahl, S; Paulus, C; Drese, K S; Keegan, H; Martin, C; O'Leary, J J; Furuberg, L; Solli, L; Grønn, P; Falang, I M; Karlgård, A; Gulliksen, A; Karlsen, F

    2009-12-07

    A Lab-On-Chip system with an instrument is presented which is capable of performing total sample preparation and automated extraction of nucleic acid from human cell samples fixed in a methanol based solution. The target application is extraction of mRNA from cervical liquid based cytology specimens for detection of transformed HPV-infections. The device accepts 3 ml of sample and performs the extraction in a disposable polymer chip of credit card size. All necessary reagents for cell lysis, washing, and elution are stored on-chip and the extraction is performed in two filter stages; one for cell pre-concentration and the other for nucleic acid capture. Tests performed using cancer cell lines and cervical liquid based cytology specimens confirm the extraction of HPV-mRNA by the system.

  18. Advancing polymeric delivery systems amidst a nucleic acid therapy renaissance

    PubMed Central

    Burke, Paul A.; Pun, Suzie H.; Reineke, Theresa M.

    2013-01-01

    Nucleic acid therapeutics are attracting renewed interest due to recent clinical advances and product approvals. Most leading programs use chemical conjugates, or viral vectors in the case of gene therapy, while several use no delivery system at all. Polymer systems, which have been at the periphery of this renaissance, often involve greater molecular complexity than competing approaches, which must be justified by their advantages. Advanced analytical methods, along with biological tools for characterizing biotransformation and intracellular trafficking, are increasingly being applied to nucleic acid delivery systems including those based on polymers. These frontiers of investigation create the opportunity for an era where highly defined polymer compositions are optimized based on mechanistic insights in a way that has not been previously possible, offering the prospect of greater differentiation from alternatives. This will require integrated collaboration between polymer scientists and those from other disciplines. PMID:24683504

  19. 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.

  20. Developing nucleic acid-based electrical detection systems

    PubMed Central

    Gabig-Ciminska, Magdalena

    2006-01-01

    Development of nucleic acid-based detection systems is the main focus of many research groups and high technology companies. The enormous work done in this field is particularly due to the broad versatility and variety of these sensing devices. From optical to electrical systems, from label-dependent to label-free approaches, from single to multi-analyte and array formats, this wide range of possibilities makes the research field very diversified and competitive. New challenges and requirements for an ideal detector suitable for nucleic acid analysis include high sensitivity and high specificity protocol that can be completed in a relatively short time offering at the same time low detection limit. Moreover, systems that can be miniaturized and automated present a significant advantage over conventional technology, especially if detection is needed in the field. Electrical system technology for nucleic acid-based detection is an enabling mode for making miniaturized to micro- and nanometer scale bio-monitoring devices via the fusion of modern micro- and nanofabrication technology and molecular biotechnology. The electrical biosensors that rely on the conversion of the Watson-Crick base-pair recognition event into a useful electrical signal are advancing rapidly, and recently are receiving much attention as a valuable tool for microbial pathogen detection. Pathogens may pose a serious threat to humans, animal and plants, thus their detection and analysis is a significant element of public health. Although different conventional methods for detection of pathogenic microorganisms and their toxins exist and are currently being applied, improvements of molecular-based detection methodologies have changed these traditional detection techniques and introduced a new era of rapid, miniaturized and automated electrical chip detection technologies into pathogen identification sector. In this review some developments and current directions in nucleic acid-based electrical

  1. Nucleic acid oxidation: an early feature of Alzheimer's disease.

    PubMed

    Bradley-Whitman, Melissa A; Timmons, Michael D; Beckett, Tina L; Murphy, Michael P; Lynn, Bert C; Lovell, Mark A

    2014-01-01

    Studies of oxidative damage during the progression of Alzheimer's disease (AD) suggest its central role in disease pathogenesis. To investigate levels of nucleic acid oxidation in both early and late stages of AD, levels of multiple base adducts were quantified in nuclear and mitochondrial DNA from the superior and middle temporal gyri (SMTG), inferior parietal lobule (IPL), and cerebellum (CER) of age-matched normal control subjects, subjects with mild cognitive impairment, preclinical AD, late-stage AD, and non-AD neurological disorders (diseased control; DC) using gas chromatography/mass spectrometry. Median levels of multiple DNA adducts in nuclear and mitochondrial DNA were significantly (p ≤ 0.05) elevated in the SMTG, IPL, and CER in multiple stages of AD and in DC subjects. Elevated levels of fapyguanine and fapyadenine in mitochondrial DNA suggest a hypoxic environment early in the progression of AD and in DC subjects. Overall, these data suggest that oxidative damage is an early event not only in the pathogenesis of AD but is also present in neurodegenerative diseases in general. Levels of oxidized nucleic acids in nDNA and mtDNA were found to be significantly elevated in mild cognitive impairment (MCI), preclinical Alzheimer's disease (PCAD), late-stage AD (LAD), and a pooled diseased control group (DC) of frontotemporal dementia (FTD) and dementia with Lewy bodies (DLB) subjects compared to normal control (NC) subjects. Nucleic acid oxidation peaked early in disease progression and remained elevated. The study suggests nucleic acid oxidation is a general event in neurodegeneration.

  2. Method and apparatus for staining immobilized nucleic acids

    DOEpatents

    Ramsey, J. Michael; Foote, Robert S.; Jacobson, Stephen C.

    2000-01-01

    A method for staining immobilized nucleic acids includes the steps of affixing DNA probes to a solid substrate, moving target DNA material into proximity with the DNA probes, whereby the target DNA hybridized with specific ones of the DNA probes, and moving a fluorescent dye into proximity with the hybridized target DNA, whereby the fluorescent dye binds to the hybridized DNA to enable subsequent detection of fluorescence.

  3. Silicon dioxide thin film mediated single cell nucleic acid isolation.

    PubMed

    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.

  4. 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

  5. Histidine-lysine peptides as carriers of nucleic acids.

    PubMed

    Leng, Qixin; Goldgeier, Lisa; Zhu, Jingsong; Cambell, Patricia; Ambulos, Nicholas; Mixson, A James

    2007-03-01

    With their biodegradability and diversity of permutations, peptides have significant potential as carriers of nucleic acids. This review will focus on the sequence and branching patterns of peptide carriers composed primarily of histidines and lysines. While lysines within peptides are important for binding to the negatively charged phosphates, histidines are critical for endosomal lysis enabling nucleic acids to reach the cytosol. Histidine-lysine (HK) polymers by either covalent or ionic bonds with liposomes augment transfection compared to liposome carriers alone. More recently, we have examined peptides as sole carriers of nucleic acids because of their intrinsic advantages compared to the bipartite HK/liposome carriers. With a protocol change and addition of a histidine-rich tail, HK peptides as sole carriers were more effective than liposomes alone in several cell lines. While four-branched polymers with a primary repeating sequence pattern of -HHK- were more effective as carriers of plasmids, eight-branched polymers with a sequence pattern of -HHHK- were more effective as carriers of siRNA. Compared to polyethylenimine, HK carriers of siRNA and plasmids had reduced toxicity. When injected intravenously, HK polymers in complex with plasmids encoding antiangiogenic proteins significantly decreased tumor growth. Furthermore, modification of HK polymers with polyethylene glycol and vascular-specific ligands increased specificity of the polyplex to the tumor by more than 40-fold. Together with further development and insight on the structure of HK polyplexes, HK peptides may prove to be useful as carriers of different forms of nucleic acids both in vitro and in vivo.

  6. Application of living free radical polymerization for nucleic acid delivery.

    PubMed

    Chu, David S H; Schellinger, Joan G; Shi, Julie; Convertine, Anthony J; Stayton, Patrick S; Pun, Suzie H

    2012-07-17

    Therapeutic gene delivery can alter protein function either through the replacement of nonfunctional genes to restore cellular health or through RNA interference (RNAi) to mask mutated and harmful genes. Researchers have investigated a range of nucleic acid-based therapeutics as potential treatments for hereditary, acquired, and infectious diseases. Candidate drugs include plasmids that induce gene expression and small, interfering RNAs (siRNAs) that silence target genes. Because of their self-assembly with nucleic acids into virus-sized nanoparticles and high transfection efficiency in vitro, cationic polymers have been extensively studied for nucleic acid delivery applications, but toxicity and particle stability have limited the clinical applications of these systems. The advent of living free radical polymerization has improved the quality, control, and reproducibility of these synthesized materials. This process yields well-defined, narrowly disperse materials with designed architectures and molecular weights. As a result, researchers can study the effects of polymer architecture and molecular weight on transfection efficiency and cytotoxicity, which will improve the design of next-generation vectors. In this Account, we review findings from structure-function studies that have elucidated key design motifs necessary for the development of effective nucleic acid vectors. Researchers have used robust methods such as atom transfer radical polymerization (ATRP), reverse addition-fragmentation chain transfer polymerization (RAFT), and ring-opening metastasis polymerization (ROMP) to engineer materials that enhance extracellular stability and cellular specificity and decrease toxicity. In addition, we discuss polymers that are biodegradable, form supramolecular structures, target specific cells, or facilitate endosomal release. Finally, we describe promising materials with a range of in vivo applications from pulmonary gene delivery to DNA vaccines.

  7. Computational design of nucleic acid feedback control circuits.

    PubMed

    Yordanov, Boyan; Kim, Jongmin; Petersen, Rasmus L; Shudy, Angelina; Kulkarni, Vishwesh V; Phillips, Andrew

    2014-08-15

    The design of synthetic circuits for controlling molecular-scale processes is an important goal of synthetic biology, with potential applications in future in vitro and in vivo biotechnology. In this paper, we present a computational approach for designing feedback control circuits constructed from nucleic acids. Our approach relies on an existing methodology for expressing signal processing and control circuits as biomolecular reactions. We first extend the methodology so that circuits can be expressed using just two classes of reactions: catalysis and annihilation. We then propose implementations of these reactions in three distinct classes of nucleic acid circuits, which rely on DNA strand displacement, DNA enzyme and RNA enzyme mechanisms, respectively. We use these implementations to design a Proportional Integral controller, capable of regulating the output of a system according to a given reference signal, and discuss the trade-offs between the different approaches. As a proof of principle, we implement our methodology as an extension to a DNA strand displacement software tool, thus allowing a broad range of nucleic acid circuits to be designed and analyzed within a common modeling framework.

  8. Nucleic acid-binding specificity of human FUS protein

    PubMed Central

    Wang, Xueyin; Schwartz, Jacob C.; Cech, Thomas R.

    2015-01-01

    FUS, a nuclear RNA-binding protein, plays multiple roles in RNA processing. Five specific FUS-binding RNA sequence/structure motifs have been proposed, but their affinities for FUS have not been directly compared. Here we find that human FUS binds all these sequences with Kdapp values spanning a 10-fold range. Furthermore, some RNAs that do not contain any of these motifs bind FUS with similar affinity. FUS binds RNA in a length-dependent manner, consistent with a substantial non-specific component to binding. Finally, investigation of FUS binding to different nucleic acids shows that it binds single-stranded DNA with three-fold lower affinity than ssRNA of the same length and sequence, while binding to double-stranded nucleic acids is weaker. We conclude that FUS has quite general nucleic acid-binding activity, with the various proposed RNA motifs being neither necessary for FUS binding nor sufficient to explain its diverse binding partners. PMID:26150427

  9. Nucleic acid-metal ion interactions in the solid state.

    PubMed

    Aoki, Katsuyuki; Murayama, Kazutaka

    2012-01-01

    Metal ions play a key role in nucleic acid structure and activity. Elucidation of the rules that govern the binding of metal ions is therefore an essential step for better understanding of the nucleic acid functions. This review is as an update to a preceding one (Metal Ions Biol. Syst., 1996, 32, 91-134), in which we offered a general view of metal ion interactions with mono-, di-, tri-, and oligonucleotides in the solid state, based on their crystal structures reported before 1994. In this chapter, we survey all the crystal structures of metal ion complexes with nucleotides involving oligonucleotides reported after 1994 and we have tried to uncover new characteristic metal bonding patterns for mononucleotides and oligonucleotides with A-RNA and A/B/Z-DNA fragments that form duplexes. We do not cover quadruplexes, duplexes with metal-mediated base-pairs, tRNAs, rRNAs in ribosome, ribozymes, and nucleic acid-drug and -protein complexes. Factors that affect metal binding to mononucleotides and oligonucleotide duplexes are also dealt with.

  10. IR-UV photochemistry of protein-nucleic acid systems

    SciTech Connect

    Kozub, J.; Edwards, G.

    1995-12-31

    UV light has often been used to induce the formation of covalent bonds between DNA (or RNA) and tightly-bound protein molecules. However, the internal photoreactions of nucleic acids and proteins limit the yield and complicate the analysis of intermolecular crosslinks. In an ongoing search for improved reaction specificity or new photoreactions in these systems, we have employed UV photons from a Nd:YAG-pumped dye laser and mid-IR photons from the Vanderbilt FEL. Having crosslinked several protein-nucleic acid systems with nanosecond UV laser pulses, we are currently studying the effect of various IR wavelengths on a model system (gene 32 protein and poly[dT]). We have found that irradiation with sufficiently intense FEL macropulses creates an altered form of gene 32 protein which was not observed with UV-only irradiation. The electrophoretic nobility of the product is consistent with the formation of a specific protein-protein crosslink. No evidence of the non-specific protein damage typically induced by UV light is found. The yield of the new photoproduct is apparently enhanced by exposure to FEL macropulses which are synchronized with UV laser pulses. With ideal exposure parameters, the two-color reaction effectively competes with UV-only reactions. Experiments designed to determine the reaction mechanism and to demonstrate FEL-induced reactions in other protein-nucleic acid systems are currently underway.

  11. Nucleic Acid Sample Preparation using Spontaneous Biphasic Plug Flow

    PubMed Central

    Thomas, Peter C.; Strotman, Lindsay N.; Theberge, Ashleigh B.; Berthier, Erwin; O’Connell, Rachel; Loeb, Jennifer M.; Berry, Scott M.; Beebe, David J.

    2013-01-01

    Nucleic acid (NA) extraction and purification has become a common technique in both research and clinical laboratories. Current methods require repetitive wash steps with a pipette that is laborious and time consuming making it inefficient for clinical settings. We present here a simple technique that relies on spontaneous biphasic plug flow inside a capillary to achieve sample preparation. By filling the sample with oil, aqueous contaminants were displaced from the captured NA without pipetting wash buffers or use of external force and equipment. mRNA from mammalian cell culture was purified and PCR amplification showed similar threshold cycle values as those obtained from a commercially available kit. HIV viral like particles were spiked into serum and a 5-fold increase in viral RNA extraction yield was achieved compared to the conventional wash method. In addition, viral RNA was successfully purified from human whole blood, and a limit of detection of approximately 14 copies of RNA extracted per sample. The results demonstrate the utility of the current technique for nucleic acid purification for clinical purposes, and the overall approach provides a potential method to implement nucleic acid testing in low resource settings. PMID:23941230

  12. Nucleic acid sample preparation using spontaneous biphasic plug flow.

    PubMed

    Thomas, Peter C; Strotman, Lindsay N; Theberge, Ashleigh B; Berthier, Erwin; O'Connell, Rachel; Loeb, Jennifer M; Berry, Scott M; Beebe, David J

    2013-09-17

    Nucleic acid (NA) extraction and purification has become a common technique in both research and clinical laboratories. Current methods require repetitive wash steps with a pipet that are laborious and time-consuming, making the procedure inefficient for clinical settings. We present here a simple technique that relies on spontaneous biphasic plug flow inside a capillary to achieve sample preparation. By filling the sample with oil, aqueous contaminants were displaced from the captured NA without pipetting wash buffers or use of external force and equipment. mRNA from mammalian cell culture was purified, and polymerase chain reaction (PCR) amplification showed similar threshold cycle values as those obtained from a commercially available kit. Human immunodeficiency virus (HIV) viral-like particles were spiked into serum and a 5-fold increase in viral RNA extraction yield was achieved compared to the conventional wash method. In addition, viral RNA was successfully purified from human whole blood, and a limit of detection of approximately 14 copies of RNA extracted per sample was determined. The results demonstrate the utility of the current technique for nucleic acid purification for clinical purposes, and the overall approach provides a potential method to implement nucleic acid testing in low-resource settings.

  13. Nucleic acid-lipid membrane interactions studied by DSC.

    PubMed

    Giatrellis, Sarantis; Nounesis, George

    2011-01-01

    The interactions of nucleic acids with lipid membranes are of great importance for biological mechanisms as well as for biotechnological applications in gene delivery and drug carriers. The optimization of liposomal vectors for clinical use is absolutely dependent upon the formation mechanisms, the morphology, and the molecular organization of the lipoplexes, that is, the complexes of lipid membranes with DNA. Differential scanning calorimetry (DSC) has emerged as an efficient and relatively easy-to-operate experimental technique that can straightforwardly provide data related to the thermodynamics and the kinetics of the DNA-lipid complexation and especially to the lipid organization and phase transitions within the membrane. In this review, we summarize DSC studies considering nucleic acid-membrane systems, accentuating DSC capabilities, and data analysis. Published work involving cationic, anionic, and zwitterionic lipids as well as lipid mixtures interacting with RNA and DNA of different sizes and conformations are included. It is shown that despite limitations, issues such as DNA- or RNA-induced phase separation and microdomain lipid segregation, liposomal aggregation and fusion, alterations of the lipid long-range molecular order, as well as membrane-induced structural changes of the nucleic acids can be efficiently treated by systematic high-sensitivity DSC studies.

  14. A microarray-based method to perform nucleic acid selections.

    PubMed

    Aminova, Olga; Disney, Matthew D

    2010-01-01

    This method describes a microarray-based platform to perform nucleic acid selections. Chemical ligands to which a nucleic acid binder is desired are immobilized onto an agarose microarray surface; the array is then incubated with an RNA library. Bound RNA library members are harvested directly from the array surface via gel excision at the position on the array where a ligand was immobilized. The RNA is then amplified via RT-PCR, cloned, and sequenced. This method has the following advantages over traditional resin-based Systematic Evolution of Ligands by Exponential Enrichment (SELEX): (1) multiple selections can be completed in parallel on a single microarray surface; (2) kinetic biases in the selections are mitigated since all RNA binders are harvested from an array via gel excision; (3) the amount of chemical ligand needed to perform a selection is minimized; (4) selections do not require expensive resins or equipment; and (5) the matrix used for selections is inexpensive and easy to prepare. Although this protocol was demonstrated for RNA selections, it should be applicable for any nucleic acid selection.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. Nucleic acids encoding human trithorax protein

    DOEpatents

    Evans, Glen A.; Djabali, Malek; Selleri, Licia; Parry, Pauline

    2001-01-01

    In accordance with the present invention, there is provided an isolated peptide having the characteristics of human trithorax protein (as well as DNA encoding same, antisense DNA derived therefrom and antagonists therefor). The invention peptide is characterized by having a DNA binding domain comprising multiple zinc fingers and at least 40% amino acid identity with respect to the DNA binding domain of Drosophila trithorax protein and at least 70% conserved sequence with respect to the DNA binding domain of Drosophila trithorax protein, and wherein said peptide is encoded by a gene located at chromosome 11 of the human genome at q23. Also provided are methods for the treatment of subject(s) suffering from immunodeficiency, developmental abnormality, inherited disease, or cancer by administering to said subject a therapeutically effective amount of one of the above-described agents (i.e., peptide, antagonist therefor, DNA encoding said peptide or antisense DNA derived therefrom). Also provided is a method for the diagnosis, in a subject, of immunodeficiency, developmental abnormality, inherited disease, or cancer associated with disruption of chromosome 11 at q23.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27775006