Nucleic acid sequence detection using multiplexed oligonucleotide PCR

DOEpatents

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

2006-12-26

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

Development of a Digital Microarray with Interferometric Reflectance Imaging

NASA Astrophysics Data System (ADS)

Sevenler, Derin

This dissertation describes a new type of molecular assay for nucleic acids and proteins. We call this technique a digital microarray since it is conceptually similar to conventional fluorescence microarrays, yet it performs enumerative ('digital') counting of the number captured molecules. Digital microarrays are approximately 10,000-fold more sensitive than fluorescence microarrays, yet maintain all of the strengths of the platform including low cost and high multiplexing (i.e., many different tests on the same sample simultaneously). Digital microarrays use gold nanorods to label the captured target molecules. Each gold nanorod on the array is individually detected based on its light scattering, with an interferometric microscopy technique called SP-IRIS. Our optimized high-throughput version of SP-IRIS is able to scan a typical array of 500 spots in less than 10 minutes. Digital DNA microarrays may have utility in applications where sequencing is prohibitively expensive or slow. As an example, we describe a digital microarray assay for gene expression markers of bacterial drug resistance.

Label-free direct surface-enhanced Raman scattering (SERS) of nucleic acids (Conference Presentation)

NASA Astrophysics Data System (ADS)

Guerrini, Luca; Morla-Folch, Judit; Gisbert-Quilis, Patricia; Xie, Hainan; Alvarez-Puebla, Ramon

2016-03-01

Recently, plasmonic-based biosensing has experienced an unprecedented level of attention, with a particular focus on the nucleic acid detection, offering efficient solutions to engineer simple, fast, highly sensitive sensing platforms while overcoming important limitations of PCR and microarray techniques. In the broad field of plasmonics, surface-enhanced Raman scattering (SERS) spectroscopy has arisen as a powerful analytical tool for detection and structural characterization of biomolecules. Today applications of SERS to nucleic acid analysis largely rely on indirect strategies, which have been demonstrated very effective for pure sensing purposes but completely dismiss the exquisite structural information provided by the direct acquisition of the biomolecular vibrational fingerprint. Contrarily, direct label-free SERS of nucleic acid shows an outstanding potential in terms of chemical-specific information which, however, remained largely unexpressed mainly because of the inherent poor spectral reproducibility and/or limited sensitivity. To address these limitations, we developed a fast and affordable high-throughput screening direct SERS method for gaining detailed genomic information on nucleic acids (DNA and RNA) and for the characterization and quantitative recognition of DNA interactions with exogenous agents. The simple strategy relies on the electrostatic adhesion of DNA/RNA onto positively-charged silver colloids that promotes the nanoparticle aggregation into stable clusters yielding intense and reproducible SERS spectra at picogram level (i.e. the analysis can be performed without the necessity of amplification steps thus providing realistic direct information of the nucleic acid in its native state). We anticipate this method to gain a vast impact and set of applications in different fields, including medical diagnostics, genomic screening, drug discovery, forensic science and even molecular electronics.

Evaluation of a Field-Portable DNA Microarray Platform and Nucleic Acid Amplification Strategies for the Detection of Arboviruses, Arthropods, and Bloodmeals

PubMed Central

Grubaugh, Nathan D.; Petz, Lawrence N.; Melanson, Vanessa R.; McMenamy, Scott S.; Turell, Michael J.; Long, Lewis S.; Pisarcik, Sarah E.; Kengluecha, Ampornpan; Jaichapor, Boonsong; O'Guinn, Monica L.; Lee, John S.

2013-01-01

Highly multiplexed assays, such as microarrays, can benefit arbovirus surveillance by allowing researchers to screen for hundreds of targets at once. We evaluated amplification strategies and the practicality of a portable DNA microarray platform to analyze virus-infected mosquitoes. The prototype microarray design used here targeted the non-structural protein 5, ribosomal RNA, and cytochrome b genes for the detection of flaviviruses, mosquitoes, and bloodmeals, respectively. We identified 13 of 14 flaviviruses from virus inoculated mosquitoes and cultured cells. Additionally, we differentiated between four mosquito genera and eight whole blood samples. The microarray platform was field evaluated in Thailand and successfully identified flaviviruses (Culex flavivirus, dengue-3, and Japanese encephalitis viruses), differentiated between mosquito genera (Aedes, Armigeres, Culex, and Mansonia), and detected mammalian bloodmeals (human and dog). We showed that the microarray platform and amplification strategies described here can be used to discern specific information on a wide variety of viruses and their vectors. PMID:23249687

Optimization of an AMBER Force Field for the Artificial Nucleic Acid, LNA, and Benchmarking with NMR of L(CAAU)

PubMed Central

2013-01-01

Locked Nucleic Acids (LNAs) are RNA analogues with an O2′-C4′ methylene bridge which locks the sugar into a C3′-endo conformation. This enhances hybridization to DNA and RNA, making LNAs useful in microarrays and potential therapeutics. Here, the LNA, L(CAAU), provides a simplified benchmark for testing the ability of molecular dynamics (MD) to approximate nucleic acid properties. LNA χ torsions and partial charges were parametrized to create AMBER parm99_LNA. The revisions were tested by comparing MD predictions with AMBER parm99 and parm99_LNA against a 200 ms NOESY NMR spectrum of L(CAAU). NMR indicates an A-Form equilibrium ensemble. In 3000 ns simulations starting with an A-form structure, parm99_LNA and parm99 provide 66% and 35% agreement, respectively, with NMR NOE volumes and 3J-couplings. In simulations of L(CAAU) starting with all χ torsions in a syn conformation, only parm99_LNA is able to repair the structure. This implies methods for parametrizing force fields for nucleic acid mimics can reasonably approximate key interactions and that parm99_LNA will improve reliability of MD studies for systems with LNA. A method for approximating χ population distribution on the basis of base to sugar NOEs is also introduced. PMID:24377321

Construction of a novel peptide nucleic acid piezoelectric gene sensor microarray detection system.

PubMed

Chen, Ming; Liu, Minghua; Yu, Lili; Cai, Guoru; Chen, Qinghai; Wu, Rong; Wang, Feng; Zhang, Bo; Jiang, Tianlun; Fu, Welling

2005-08-01

A novel 2 x 5 clamped style piezoelectric gene sensor microarray has been successfully constructed. Every crystal unit of the fabricated gene sensor can oscillate independently without interfering with each other. The bis-peptide nucleic acid (bis-PNA) probe, which can combine with target DNA or RNA sequences more effectively and specifically than a DNA probe, was designed and immobilized on the surface of the gene sensor microarray to substitute the conventional DNA probe for direct detection of the hepatitis B virus (HBV) genomic DNA. Detection conditions were then explored and optimized. Results showed that PBS buffer of pH 6.8, an ion concentration of 20 mmol/liter, and a probe concentration of 1.5 micromol/liter were optimal for the detection system. Under such optimized experimental conditions, the specificity of bis-PNA was proved much higher than that of DNA probe. The relationship between quantity of target and decrease of frequency showed a typical saturation curve when concentrations of target HBV DNA varied from 10 pg/liter to 100 microg/liter, and 10 microg/liter was the watershed, with a statistic linear regression equation of I gC = -2.7455 + 0.0691 deltaF and the correlating coefficient of 0.9923. Fortunately, this is exactly the most ordinary variant range of the HBV virus concentration in clinical hepatitis samples. So, a good technical platform is successfully constructed and it will be applied to detect HBV quantitatively in clinical samples.

Serological and Genetic Evidence for Altered Complement System Functionality in Systemic Lupus Erythematosus: Findings of the GAPAID Consortium.

PubMed

Prechl, József; Papp, Krisztián; Hérincs, Zoltán; Péterfy, Hajna; Lóránd, Veronika; Szittner, Zoltán; Estonba, Andone; Rovero, Paolo; Paolini, Ilaria; Del Amo, Jokin; Uribarri, Maria; Alcaro, Maria Claudia; Ruiz-Larrañaga, Otsanda; Migliorini, Paola; Czirják, László

2016-01-01

Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption we examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n = 211), with other systemic autoimmune diseases (n = 65) and non-autoimmune control subjects (n = 149). Standard clinical and laboratory data were collected and serum complement levels were determined. The genotype of SNP rs1143679 in the ITGAM gene was also determined. Ex vivo formation of immune complexes, with respect to IgM, IgG, complement C4 and C3 binding, was examined using a functional immunoassay on autoantigen microarray comprising nucleic acids, proteins and lipids. Complement consumption of nucleic acids increased upon binding of IgM and IgG even when serum complement levels were decreased due to consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, complement deposition on tested protein and lipid autoantigens showed positive correlation with C4 levels. Genetic analysis revealed that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) had lower levels of dsDNA specific IgM among SLE patients. Both the non-synonymous variant rs1143679 and the high ratio of nucleic acid specific IgG/IgM were associated with multiple organ involvement. In summary, secondary complement deficiency in SLE does not impair opsonization of nucleic-acid-containing autoantigens but does affect other antigens and potentially other complement dependent processes. Dysfunction of the receptor recognizing complement opsonized immune complexes promotes the development of class-switched autoantibodies targeting nucleic acids.

A multi-model approach to nucleic acid-based drug development.

PubMed

Gautherot, Isabelle; Sodoyer, Regís

2004-01-01

With the advent of functional genomics and the shift of interest towards sequence-based therapeutics, the past decades have witnessed intense research efforts on nucleic acid-mediated gene regulation technologies. Today, RNA interference is emerging as a groundbreaking discovery, holding promise for development of genetic modulators of unprecedented potency. Twenty-five years after the discovery of antisense RNA and ribozymes, gene control therapeutics are still facing developmental difficulties, with only one US FDA-approved antisense drug currently available in the clinic. Limited predictability of target site selection models is recognized as one major stumbling block that is shared by all of the so-called complementary technologies, slowing the progress towards a commercial product. Currently employed in vitro systems for target site selection include RNAse H-based mapping, antisense oligonucleotide microarrays, and functional screening approaches using libraries of catalysts with randomized target-binding arms to identify optimal ribozyme/DNAzyme cleavage sites. Individually, each strategy has its drawbacks from a drug development perspective. Utilization of message-modulating sequences as therapeutic agents requires that their action on a given target transcript meets criteria of potency and selectivity in the natural physiological environment. In addition to sequence-dependent characteristics, other factors will influence annealing reactions and duplex stability, as well as nucleic acid-mediated catalysis. Parallel consideration of physiological selection systems thus appears essential for screening for nucleic acid compounds proposed for therapeutic applications. Cellular message-targeting studies face issues relating to efficient nucleic acid delivery and appropriate analysis of response. For reliability and simplicity, prokaryotic systems can provide a rapid and cost-effective means of studying message targeting under pseudo-cellular conditions, but such approaches also have limitations. To streamline nucleic acid drug discovery, we propose a multi-model strategy integrating high-throughput-adapted bacterial screening, followed by reporter-based and/or natural cellular models and potentially also in vitro assays for characterization of the most promising candidate sequences, before final in vivo testing.

Label-Free Potentiometry for Detecting DNA Hybridization Using Peptide Nucleic Acid and DNA Probes

PubMed Central

Goda, Tatsuro; Singi, Ankit Balram; Maeda, Yasuhiro; Matsumoto, Akira; Torimura, Masaki; Aoki, Hiroshi; Miyahara, Yuji

2013-01-01

Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry. PMID:23435052

Cell and tissue microarray technologies for protein and nucleic acid expression profiling.

PubMed

Cardano, Marina; Diaferia, Giuseppe R; Falavigna, Maurizio; Spinelli, Chiara C; Sessa, Fausto; DeBlasio, Pasquale; Biunno, Ida

2013-02-01

Tissue microarray (TMA) and cell microarray (CMA) are two powerful techniques that allow for the immunophenotypical characterization of hundreds of samples simultaneously. In particular, the CMA approach is particularly useful for immunophenotyping new stem cell lines (e.g., cardiac, neural, mesenchymal) using conventional markers, as well as for testing the specificity and the efficacy of newly developed antibodies. We propose the use of a tissue arrayer not only to perform protein expression profiling by immunohistochemistry but also to carry out molecular genetics studies. In fact, starting with several tissues or cell lines, it is possible to obtain the complete signature of each sample, describing the protein, mRNA and microRNA expression, and DNA mutations, or eventually to analyze the epigenetic processes that control protein regulation. Here we show the results obtained using the Galileo CK4500 TMA platform.

Sequence specificity of single-stranded DNA-binding proteins: a novel DNA microarray approach

PubMed Central

Morgan, Hugh P.; Estibeiro, Peter; Wear, Martin A.; Max, Klaas E.A.; Heinemann, Udo; Cubeddu, Liza; Gallagher, Maurice P.; Sadler, Peter J.; Walkinshaw, Malcolm D.

2007-01-01

We have developed a novel DNA microarray-based approach for identification of the sequence-specificity of single-stranded nucleic-acid-binding proteins (SNABPs). For verification, we have shown that the major cold shock protein (CspB) from Bacillus subtilis binds with high affinity to pyrimidine-rich sequences, with a binding preference for the consensus sequence, 5′-GTCTTTG/T-3′. The sequence was modelled onto the known structure of CspB and a cytosine-binding pocket was identified, which explains the strong preference for a cytosine base at position 3. This microarray method offers a rapid high-throughput approach for determining the specificity and strength of ss DNA–protein interactions. Further screening of this newly emerging family of transcription factors will help provide an insight into their cellular function. PMID:17488853

DNA stable-isotope probing (DNA-SIP).

PubMed

Dunford, Eric A; Neufeld, Josh D

2010-08-02

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations

PubMed Central

Law, Jodi Woan-Fei; Ab Mutalib, Nurul-Syakima; Chan, Kok-Gan; Lee, Learn-Han

2015-01-01

The incidence of foodborne diseases has increased over the years and resulted in major public health problem globally. Foodborne pathogens can be found in various foods and it is important to detect foodborne pathogens to provide safe food supply and to prevent foodborne diseases. The conventional methods used to detect foodborne pathogen are time consuming and laborious. Hence, a variety of methods have been developed for rapid detection of foodborne pathogens as it is required in many food analyses. Rapid detection methods can be categorized into nucleic acid-based, biosensor-based and immunological-based methods. This review emphasizes on the principles and application of recent rapid methods for the detection of foodborne bacterial pathogens. Detection methods included are simple polymerase chain reaction (PCR), multiplex PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP) and oligonucleotide DNA microarray which classified as nucleic acid-based methods; optical, electrochemical and mass-based biosensors which classified as biosensor-based methods; enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay which classified as immunological-based methods. In general, rapid detection methods are generally time-efficient, sensitive, specific and labor-saving. The developments of rapid detection methods are vital in prevention and treatment of foodborne diseases. PMID:25628612

Isolation of Microarray-Grade Total RNA, MicroRNA, and DNA from a Single PAXgene Blood RNA Tube

PubMed Central

Kruhøffer, Mogens; Dyrskjøt, Lars; Voss, Thorsten; Lindberg, Raija L.P.; Wyrich, Ralf; Thykjaer, Thomas; Orntoft, Torben F.

2007-01-01

We have developed a procedure for isolation of microRNA and genomic DNA in addition to total RNA from whole blood stabilized in PAXgene Blood RNA tubes. The procedure is based on automatic extraction on a BioRobot MDx and includes isolation of DNA from a fraction of the stabilized blood and recovery of small RNA species that are otherwise lost. The procedure presented here is suitable for large-scale experiments and is amenable to further automation. Procured total RNA and DNA was tested using Affymetrix Expression and single-nucleotide polymorphism GeneChips, respectively, and isolated microRNA was tested using spotted locked nucleic acid-based microarrays. We conclude that the yield and quality of total RNA, microRNA, and DNA from a single PAXgene blood RNA tube is sufficient for downstream microarray analysis. PMID:17690207

Cell and Tissue Microarray Technologies for Protein and Nucleic Acid Expression Profiling

PubMed Central

Cardano, Marina; Diaferia, Giuseppe R.; Falavigna, Maurizio; Spinelli, Chiara C.; Sessa, Fausto; DeBlasio, Pasquale

2013-01-01

Tissue microarray (TMA) and cell microarray (CMA) are two powerful techniques that allow for the immunophenotypical characterization of hundreds of samples simultaneously. In particular, the CMA approach is particularly useful for immunophenotyping new stem cell lines (e.g., cardiac, neural, mesenchymal) using conventional markers, as well as for testing the specificity and the efficacy of newly developed antibodies. We propose the use of a tissue arrayer not only to perform protein expression profiling by immunohistochemistry but also to carry out molecular genetics studies. In fact, starting with several tissues or cell lines, it is possible to obtain the complete signature of each sample, describing the protein, mRNA and microRNA expression, and DNA mutations, or eventually to analyze the epigenetic processes that control protein regulation. Here we show the results obtained using the Galileo CK4500 TMA platform. PMID:23172795

Systematic spatial bias in DNA microarray hybridization is caused by probe spot position-dependent variability in lateral diffusion.

PubMed

Steger, Doris; Berry, David; Haider, Susanne; Horn, Matthias; Wagner, Michael; Stocker, Roman; Loy, Alexander

2011-01-01

The hybridization of nucleic acid targets with surface-immobilized probes is a widely used assay for the parallel detection of multiple targets in medical and biological research. Despite its widespread application, DNA microarray technology still suffers from several biases and lack of reproducibility, stemming in part from an incomplete understanding of the processes governing surface hybridization. In particular, non-random spatial variations within individual microarray hybridizations are often observed, but the mechanisms underpinning this positional bias remain incompletely explained. This study identifies and rationalizes a systematic spatial bias in the intensity of surface hybridization, characterized by markedly increased signal intensity of spots located at the boundaries of the spotted areas of the microarray slide. Combining observations from a simplified single-probe block array format with predictions from a mathematical model, the mechanism responsible for this bias is found to be a position-dependent variation in lateral diffusion of target molecules. Numerical simulations reveal a strong influence of microarray well geometry on the spatial bias. Reciprocal adjustment of the size of the microarray hybridization chamber to the area of surface-bound probes is a simple and effective measure to minimize or eliminate the diffusion-based bias, resulting in increased uniformity and accuracy of quantitative DNA microarray hybridization.

Systematic Spatial Bias in DNA Microarray Hybridization Is Caused by Probe Spot Position-Dependent Variability in Lateral Diffusion

PubMed Central

Haider, Susanne; Horn, Matthias; Wagner, Michael; Stocker, Roman; Loy, Alexander

2011-01-01

Background The hybridization of nucleic acid targets with surface-immobilized probes is a widely used assay for the parallel detection of multiple targets in medical and biological research. Despite its widespread application, DNA microarray technology still suffers from several biases and lack of reproducibility, stemming in part from an incomplete understanding of the processes governing surface hybridization. In particular, non-random spatial variations within individual microarray hybridizations are often observed, but the mechanisms underpinning this positional bias remain incompletely explained. Methodology/Principal Findings This study identifies and rationalizes a systematic spatial bias in the intensity of surface hybridization, characterized by markedly increased signal intensity of spots located at the boundaries of the spotted areas of the microarray slide. Combining observations from a simplified single-probe block array format with predictions from a mathematical model, the mechanism responsible for this bias is found to be a position-dependent variation in lateral diffusion of target molecules. Numerical simulations reveal a strong influence of microarray well geometry on the spatial bias. Conclusions Reciprocal adjustment of the size of the microarray hybridization chamber to the area of surface-bound probes is a simple and effective measure to minimize or eliminate the diffusion-based bias, resulting in increased uniformity and accuracy of quantitative DNA microarray hybridization. PMID:21858215

SERS diagnostic platforms, methods and systems microarrays, biosensors and biochips

DOEpatents

Vo-Dinh, Tuan [Knoxville, TN

2007-09-11

A Raman integrated sensor system for the detection of targets including biotargets includes at least one sampling platform, at least one receptor probe disposed on the sampling platform, and an integrated circuit detector system communicably connected to the receptor. The sampling platform is preferably a Raman active surface-enhanced scattering (SERS) platform, wherein the Raman sensor is a SERS sensor. The receptors can include at least one protein receptor and at least one nucleic acid receptor.

Microelectroporation device for genomic screening

DOEpatents

Perroud, Thomas D.; Renzi, Ronald F.; Negrete, Oscar; Claudnic, Mark R.

2014-09-09

We have developed an microelectroporation device that combines microarrays of oligonucleotides, microfluidic channels, and electroporation for cell transfection and high-throughput screening applications (e.g. RNA interference screens). Microarrays allow the deposition of thousands of different oligonucleotides in microscopic spots. Microfluidic channels and microwells enable efficient loading of cells into the device and prevent cross-contamination between different oligonucleotides spots. Electroporation allows optimal transfection of nucleic acids into cells (especially hard-to-transfect cells such as primary cells) by minimizing cell death while maximizing transfection efficiency. This invention has the advantage of a higher throughput and lower cost, while preventing cross-contamination compared to conventional screening technologies. Moreover, this device does not require bulky robotic liquid handling equipment and is inherently safer given that it is a closed system.

Fluorescent labeling of NASBA amplified tmRNA molecules for microarray applications

PubMed Central

Scheler, Ott; Glynn, Barry; Parkel, Sven; Palta, Priit; Toome, Kadri; Kaplinski, Lauris; Remm, Maido; Maher, Majella; Kurg, Ants

2009-01-01

Background Here we present a novel promising microbial diagnostic method that combines the sensitivity of Nucleic Acid Sequence Based Amplification (NASBA) with the high information content of microarray technology for the detection of bacterial tmRNA molecules. The NASBA protocol was modified to include aminoallyl-UTP (aaUTP) molecules that were incorporated into nascent RNA during the NASBA reaction. Post-amplification labeling with fluorescent dye was carried out subsequently and tmRNA hybridization signal intensities were measured using microarray technology. Significant optimization of the labeled NASBA protocol was required to maintain the required sensitivity of the reactions. Results Two different aaUTP salts were evaluated and optimum final concentrations were identified for both. The final 2 mM concentration of aaUTP Li-salt in NASBA reaction resulted in highest microarray signals overall, being twice as high as the strongest signals with 1 mM aaUTP Na-salt. Conclusion We have successfully demonstrated efficient combination of NASBA amplification technology with microarray based hybridization detection. The method is applicative for many different areas of microbial diagnostics including environmental monitoring, bio threat detection, industrial process monitoring and clinical microbiology. PMID:19445684

See what you eat--broad GMO screening with microarrays.

PubMed

von Götz, Franz

2010-03-01

Despite the controversy of whether genetically modified organisms (GMOs) are beneficial or harmful for humans, animals, and/or ecosystems, the number of cultivated GMOs is increasing every year. Many countries and federations have implemented safety and surveillance systems for GMOs. Potent testing technologies need to be developed and implemented to monitor the increasing number of GMOs. First, these GMO tests need to be comprehensive, i.e., should detect all, or at least the most important, GMOs on the market. This type of GMO screening requires a high degree of parallel tests or multiplexing. To date, DNA microarrays have the highest number of multiplexing capabilities when nucleic acids are analyzed. This trend article focuses on the evolution of DNA microarrays for GMO testing. Over the last 7 years, combinations of multiplex PCR detection and microarray detection have been developed to qualitatively assess the presence of GMOs. One example is the commercially available DualChip GMO (Eppendorf, Germany; http://www.eppendorf-biochip.com), which is the only GMO screening system successfully validated in a multicenter study. With use of innovative amplification techniques, promising steps have recently been taken to make GMO detection with microarrays quantitative.

Effect of Moderate Hypothermia on Gene Expression by THP-1 Cells: A DNA Microarray Study

DTIC Science & Technology

2006-01-01

h was found to improve both survival and neurological outcomes in newborn infants suffering from hypoxic-ischemic encephalopathy (17). Al- though the...from adults and newborns . J Interferon Cytokine Res 20: 1049–1055, 2000. 7. Farrell RE Jr. Determination of nucleic acid concentration and purity. In...hypoxic-ischemic encephalopathy . N Engl J Med 353: 1574–1584, 2005. 18. Sonna LA, Cullivan ML, Sheldon HK, Pratt RE, and Lilly CM. Effect of hypoxia

A statistical model for investigating binding probabilities of DNA nucleotide sequences using microarrays.

PubMed

Lee, Mei-Ling Ting; Bulyk, Martha L; Whitmore, G A; Church, George M

2002-12-01

There is considerable scientific interest in knowing the probability that a site-specific transcription factor will bind to a given DNA sequence. Microarray methods provide an effective means for assessing the binding affinities of a large number of DNA sequences as demonstrated by Bulyk et al. (2001, Proceedings of the National Academy of Sciences, USA 98, 7158-7163) in their study of the DNA-binding specificities of Zif268 zinc fingers using microarray technology. In a follow-up investigation, Bulyk, Johnson, and Church (2002, Nucleic Acid Research 30, 1255-1261) studied the interdependence of nucleotides on the binding affinities of transcription proteins. Our article is motivated by this pair of studies. We present a general statistical methodology for analyzing microarray intensity measurements reflecting DNA-protein interactions. The log probability of a protein binding to a DNA sequence on an array is modeled using a linear ANOVA model. This model is convenient because it employs familiar statistical concepts and procedures and also because it is effective for investigating the probability structure of the binding mechanism.

High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays.

PubMed

Yu, Xiaobo; LaBaer, Joshua

2015-05-01

AMPylation (adenylylation) has been recognized as an important post-translational modification that is used by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes, and it is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method for identifying new substrates using protein microarrays, which can markedly expand the list of potential substrates. Here we describe procedures for detecting AMPylated and auto-AMPylated proteins in a sensitive, high-throughput and nonradioactive manner. The approach uses high-density protein microarrays fabricated using nucleic acid programmable protein array (NAPPA) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide-alkyne cycloaddition (CuAAC). The assay can be accomplished within 11 h.

Optimization of single-base-pair mismatch discrimination in oligonucleotide microarrays

NASA Technical Reports Server (NTRS)

Urakawa, Hidetoshi; El Fantroussi, Said; Smidt, Hauke; Smoot, James C.; Tribou, Erik H.; Kelly, John J.; Noble, Peter A.; Stahl, David A.

2003-01-01

The discrimination between perfect-match and single-base-pair-mismatched nucleic acid duplexes was investigated by using oligonucleotide DNA microarrays and nonequilibrium dissociation rates (melting profiles). DNA and RNA versions of two synthetic targets corresponding to the 16S rRNA sequences of Staphylococcus epidermidis (38 nucleotides) and Nitrosomonas eutropha (39 nucleotides) were hybridized to perfect-match probes (18-mer and 19-mer) and to a set of probes having all possible single-base-pair mismatches. The melting profiles of all probe-target duplexes were determined in parallel by using an imposed temperature step gradient. We derived an optimum wash temperature for each probe and target by using a simple formula to calculate a discrimination index for each temperature of the step gradient. This optimum corresponded to the output of an independent analysis using a customized neural network program. These results together provide an experimental and analytical framework for optimizing mismatch discrimination among all probes on a DNA microarray.

Modular nucleic acid assembled p/MHC microarrays for multiplexed sorting of antigen-specific T cells.

PubMed

Kwong, Gabriel A; Radu, Caius G; Hwang, Kiwook; Shu, Chengyi J; Ma, Chao; Koya, Richard C; Comin-Anduix, Begonya; Hadrup, Sine Reker; Bailey, Ryan C; Witte, Owen N; Schumacher, Ton N; Ribas, Antoni; Heath, James R

2009-07-22

The human immune system consists of a large number of T cells capable of recognizing and responding to antigens derived from various sources. The development of peptide-major histocompatibility (p/MHC) tetrameric complexes has enabled the direct detection of these antigen-specific T cells. With the goal of increasing throughput and multiplexing of T cell detection, protein microarrays spotted with defined p/MHC complexes have been reported, but studies have been limited due to the inherent instability and reproducibility of arrays produced via conventional spotted methods. Herein, we report on a platform for the detection of antigen-specific T cells on glass substrates that offers significant advantages over existing surface-bound schemes. In this approach, called "Nucleic Acid Cell Sorting (NACS)", single-stranded DNA oligomers conjugated site-specifically to p/MHC tetramers are employed to immobilize p/MHC tetramers via hybridization to a complementary-printed substrate. Fully assembled p/MHC arrays are used to detect and enumerate T cells captured from cellular suspensions, including primary human T cells collected from cancer patients. NACS arrays outperform conventional spotted arrays assessed in key criteria such as repeatability and homogeneity. The versatility of employing DNA sequences for cell sorting is exploited to enable the programmed, selective release of target populations of immobilized T cells with restriction endonucleases for downstream analysis. Because of the performance, facile and modular assembly of p/MHC tetramer arrays, NACS holds promise as a versatile platform for multiplexed T cell detection.

Comparison of gene expression microarray data with count-based RNA measurements informs microarray interpretation.

PubMed

Richard, Arianne C; Lyons, Paul A; Peters, James E; Biasci, Daniele; Flint, Shaun M; Lee, James C; McKinney, Eoin F; Siegel, Richard M; Smith, Kenneth G C

2014-08-04

Although numerous investigations have compared gene expression microarray platforms, preprocessing methods and batch correction algorithms using constructed spike-in or dilution datasets, there remains a paucity of studies examining the properties of microarray data using diverse biological samples. Most microarray experiments seek to identify subtle differences between samples with variable background noise, a scenario poorly represented by constructed datasets. Thus, microarray users lack important information regarding the complexities introduced in real-world experimental settings. The recent development of a multiplexed, digital technology for nucleic acid measurement enables counting of individual RNA molecules without amplification and, for the first time, permits such a study. Using a set of human leukocyte subset RNA samples, we compared previously acquired microarray expression values with RNA molecule counts determined by the nCounter Analysis System (NanoString Technologies) in selected genes. We found that gene measurements across samples correlated well between the two platforms, particularly for high-variance genes, while genes deemed unexpressed by the nCounter generally had both low expression and low variance on the microarray. Confirming previous findings from spike-in and dilution datasets, this "gold-standard" comparison demonstrated signal compression that varied dramatically by expression level and, to a lesser extent, by dataset. Most importantly, examination of three different cell types revealed that noise levels differed across tissues. Microarray measurements generally correlate with relative RNA molecule counts within optimal ranges but suffer from expression-dependent accuracy bias and precision that varies across datasets. We urge microarray users to consider expression-level effects in signal interpretation and to evaluate noise properties in each dataset independently.

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

DOEpatents

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

1998-03-24

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

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

DOEpatents

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

1998-01-01

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

Evaluation of a Field-Portable DNA Microarray Platform and Nucleic Acid Amplification Strategies for the Detection of Arboviruses, Arthropods, and Bloodmeals

DTIC Science & Technology

2013-01-01

heparin was purchased from Innovative Research (Novi, MI). Goat and horse whole blood was provided by our Veterinary Medicine Division (USAMRIID, Fort...quinquefasciatus (10) BA House NA Human Culex Th9-0122 Ae. aegypti (1) BA House DENV-3 DNP Aedes Th9-0164 Cx. tritaeniorhynchus (24) LT Farm JEV NA...Culex Th9-0167 Ae. albopictus (1) LT Farm NA NA Aedes Th9-0175 Cx. tritaeniorhynchus (25) LT Farm JEV NA Culex Th9-0235 Cx. tritaeniorhynchus (25) BA

PNA-encoded chemical libraries.

PubMed

Zambaldo, Claudio; Barluenga, Sofia; Winssinger, Nicolas

2015-06-01

Peptide nucleic acid (PNA)-encoded chemical libraries along with DNA-encoded libraries have provided a powerful new paradigm for library synthesis and ligand discovery. PNA-encoding stands out for its compatibility with standard solid phase synthesis and the technology has been used to prepare libraries of peptides, heterocycles and glycoconjugates. Different screening formats have now been reported including selection-based and microarray-based methods that have yielded specific ligands against diverse target classes including membrane receptors, lectins and challenging targets such as Hsp70. Copyright © 2015 Elsevier Ltd. All rights reserved.

Systematic evaluation of RNA quality, microarray data reliability and pathway analysis in fresh, fresh frozen and formalin-fixed paraffin-embedded tissue samples.

PubMed

Wimmer, Isabella; Tröscher, Anna R; Brunner, Florian; Rubino, Stephen J; Bien, Christian G; Weiner, Howard L; Lassmann, Hans; Bauer, Jan

2018-04-20

Formalin-fixed paraffin-embedded (FFPE) tissues are valuable resources commonly used in pathology. However, formalin fixation modifies nucleic acids challenging the isolation of high-quality RNA for genetic profiling. Here, we assessed feasibility and reliability of microarray studies analysing transcriptome data from fresh, fresh-frozen (FF) and FFPE tissues. We show that reproducible microarray data can be generated from only 2 ng FFPE-derived RNA. For RNA quality assessment, fragment size distribution (DV200) and qPCR proved most suitable. During RNA isolation, extending tissue lysis time to 10 hours reduced high-molecular-weight species, while additional incubation at 70 °C markedly increased RNA yields. Since FF- and FFPE-derived microarrays constitute different data entities, we used indirect measures to investigate gene signal variation and relative gene expression. Whole-genome analyses revealed high concordance rates, while reviewing on single-genes basis showed higher data variation in FFPE than FF arrays. Using an experimental model, gene set enrichment analysis (GSEA) of FFPE-derived microarrays and fresh tissue-derived RNA-Seq datasets yielded similarly affected pathways confirming the applicability of FFPE tissue in global gene expression analysis. Our study provides a workflow comprising RNA isolation, quality assessment and microarray profiling using minimal RNA input, thus enabling hypothesis-generating pathway analyses from limited amounts of precious, pathologically significant FFPE tissues.

Short non-coding RNAs as bacteria species identifiers detected by surface plasmon resonance enhanced common path interferometry

NASA Astrophysics Data System (ADS)

Greef, Charles; Petropavlovskikh, Viatcheslav; Nilsen, Oyvind; Khattatov, Boris; Plam, Mikhail; Gardner, Patrick; Hall, John

2008-04-01

Small non-coding RNA sequences have recently been discovered as unique identifiers of certain bacterial species, raising the possibility that they can be used as highly specific Biowarfare Agent detection markers in automated field deployable integrated detection systems. Because they are present in high abundance they could allow genomic based bacterial species identification without the need for pre-assay amplification. Further, a direct detection method would obviate the need for chemical labeling, enabling a rapid, efficient, high sensitivity mechanism for bacterial detection. Surface Plasmon Resonance enhanced Common Path Interferometry (SPR-CPI) is a potentially market disruptive, high sensitivity dual technology that allows real-time direct multiplex measurement of biomolecule interactions, including small molecules, nucleic acids, proteins, and microbes. SPR-CPI measures differences in phase shift of reflected S and P polarized light under Total Internal Reflection (TIR) conditions at a surface, caused by changes in refractive index induced by biomolecular interactions within the evanescent field at the TIR interface. The measurement is performed on a microarray of discrete 2-dimensional areas functionalized with biomolecule capture reagents, allowing simultaneous measurement of up to 100 separate analytes. The optical beam encompasses the entire microarray, allowing a solid state detector system with no scanning requirement. Output consists of simultaneous voltage measurements proportional to the phase differences resulting from the refractive index changes from each microarray feature, and is automatically processed and displayed graphically or delivered to a decision making algorithm, enabling a fully automatic detection system capable of rapid detection and quantification of small nucleic acids at extremely sensitive levels. Proof-of-concept experiments on model systems and cell culture samples have demonstrated utility of the system, and efforts are in progress for full development and deployment of the device. The technology has broad applicability as a universal detection platform for BWA detection, medical diagnostics, and drug discovery research, and represents a new class of instrumentation as a rapid, high sensitivity, label-free methodology.

Final Scientific/Technical Report, DE-FG02-06ER64171, Integrated Nucleic Acid System for In-Field Monitoring of Microbial Community Dynamics and Metabolic Activity – Subproject to Co-PI Eric E. Roden

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

Eric E. Roden

2009-07-08

This report summarizes research conducted in conjunction with a project entitled “Integrated Nucleic Acid System for In-Field Monitoring of Microbial Community Dynamics and Metabolic Activity”, which was funded through the Integrative Studies Element of the former NABIR Program (now the Environmental Remediation Sciences Program) within the Office of Biological and Environmental Research. Dr. Darrell Chandler (originally at Argonne National Laboratory, now with Akonni Biosystems) was the overall PI/PD for the project. The overall project goals were to (1) apply a model iron-reducer and sulfate-reducer microarray and instrumentation systems to sediment and groundwater samples from the Scheibe et al. FRC Areamore » 2 field site, UMTRA sediments, and other DOE contaminated sites; (2) continue development and expansion of a 16S rRNA/rDNA¬-targeted probe suite for microbial community dynamics as new sequences are obtained from DOE-relevant sites; and (3) address the fundamental molecular biology and analytical chemistry associated with the extraction, purification and analysis of functional genes and mRNA in environmental samples. Work on the UW subproject focused on conducting detailed batch and semicontinuous culture reactor experiments with uranium-contaminated FRC Area 2 sediment. The reactor experiments were designed to provide coherent geochemical and microbiological data in support of microarray analyses of microbial communities in Area 2 sediments undergoing biostimulation with ethanol. A total of four major experiments were conducted (one batch and three semicontinuous culture), three of which (the batch and two semicontinuous culture) provided samples for DNA microarray analysis. A variety of other molecular analyses (clone libraries, 16S PhyloChip, RT-PCR, and T-RFLP) were conducted on parallel samples from the various experiments in order to provide independent information on microbial community response to biostimulation.« less

Method for isolating chromosomal DNA in preparation for hybridization in suspension

DOEpatents

Lucas, Joe N.

2000-01-01

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

Detection of Bacillus anthracis DNA in Complex Soil and Air Samples Using Next-Generation Sequencing

PubMed Central

Be, Nicholas A.; Thissen, James B.; Gardner, Shea N.; McLoughlin, Kevin S.; Fofanov, Viacheslav Y.; Koshinsky, Heather; Ellingson, Sally R.; Brettin, Thomas S.; Jackson, Paul J.; Jaing, Crystal J.

2013-01-01

Bacillus anthracis is the potentially lethal etiologic agent of anthrax disease, and is a significant concern in the realm of biodefense. One of the cornerstones of an effective biodefense strategy is the ability to detect infectious agents with a high degree of sensitivity and specificity in the context of a complex sample background. The nature of the B. anthracis genome, however, renders specific detection difficult, due to close homology with B. cereus and B. thuringiensis. We therefore elected to determine the efficacy of next-generation sequencing analysis and microarrays for detection of B. anthracis in an environmental background. We applied next-generation sequencing to titrated genome copy numbers of B. anthracis in the presence of background nucleic acid extracted from aerosol and soil samples. We found next-generation sequencing to be capable of detecting as few as 10 genomic equivalents of B. anthracis DNA per nanogram of background nucleic acid. Detection was accomplished by mapping reads to either a defined subset of reference genomes or to the full GenBank database. Moreover, sequence data obtained from B. anthracis could be reliably distinguished from sequence data mapping to either B. cereus or B. thuringiensis. We also demonstrated the efficacy of a microbial census microarray in detecting B. anthracis in the same samples, representing a cost-effective and high-throughput approach, complementary to next-generation sequencing. Our results, in combination with the capacity of sequencing for providing insights into the genomic characteristics of complex and novel organisms, suggest that these platforms should be considered important components of a biosurveillance strategy. PMID:24039948

Breast Reference Set Application: Karen Anderson-ASU (2014) — EDRN Public Portal

Cancer.gov

In order to increase the predictive value of tumor-specific antibodies for use as immunodiagnostics, our EDRN BDL has developed a novel protein microarray technology, termed Nucleic Acid Protein Programmable Array (NAPPA), which circumvents many of the limitations of traditional protein microarrays. NAPPA arrays are generated by printing full-length cDNAs encoding the target proteins at each feature of the array. The proteins are then transcribed and translated by a cell-free system and immobilized in situ using epitope tags fused to the proteins. Sera are added, and bound IgG is detected by standard secondary reagents. Using a sequential screening strategy to select AAb from 4,988 candidate tumor antigens, we have identified 28 potential AAb biomarkers for the early detection of breast cancer, and here we propose to evaluate these biomarkers using the EDRN Breast Cancer Reference Set.

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.

Method for isolating nucleic acids

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

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

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

Cambridge Healthtech Institute's Third Annual Conference on Lab-on-a-Chip and Microarrays. 22-24 January 2001, Zurich, Switzerland.

PubMed

Jain, K K

2001-02-01

Cambridge Healthtech Institute's Third Annual Conference on Lab-on-a-Chip and Microarray technology covered the latest advances in this technology and applications in life sciences. Highlights of the meetings are reported briefly with emphasis on applications in genomics, drug discovery and molecular diagnostics. There was an emphasis on microfluidics because of the wide applications in laboratory and drug discovery. The lab-on-a-chip provides the facilities of a complete laboratory in a hand-held miniature device. Several microarray systems have been used for hybridisation and detection techniques. Oligonucleotide scanning arrays provide a versatile tool for the analysis of nucleic acid interactions and provide a platform for improving the array-based methods for investigation of antisense therapeutics. A method for analysing combinatorial DNA arrays using oligonucleotide-modified gold nanoparticle probes and a conventional scanner has considerable potential in molecular diagnostics. Various applications of microarray technology for high-throughput screening in drug discovery and single nucleotide polymorphisms (SNP) analysis were discussed. Protein chips have important applications in proteomics. With the considerable amount of data generated by the different technologies using microarrays, it is obvious that the reading of the information and its interpretation and management through the use of bioinformatics is essential. Various techniques for data analysis were presented. Biochip and microarray technology has an essential role to play in the evolving trends in healthcare, which integrate diagnosis with prevention/treatment and emphasise personalised medicines.

Composition for nucleic acid sequencing

DOEpatents

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

2008-08-26

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

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.

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.

A review of current and future molecular diagnostic tests for use in the microbiology laboratory.

PubMed

Jannes, Geert; De Vos, Daniel

2006-01-01

Nucleic acid-based diagnostics gradually are replacing or complementing culture-based, biochemical, and immunological assays in routine microbiology laboratories. Similar to conventional tests, the first-generation deoxyribonucleic acid assays determined only a single analyte. Recent improvements in detection technologies have paved the way for the development of multiparameter assays using macroarrays or micro-arrays, while the introduction of closed-tube real-time polymerase chain reaction systems has resulted in the development of rapid microbial diagnostics with a reduced contamination risk. The use of these new molecular technologies is not restricted to detection and identification of microbial pathogens but also can be used for genotyping, allowing one to determine antibiotic resistance or to perform microbial fingerprinting.

Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

NASA Technical Reports Server (NTRS)

Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Said; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

2002-01-01

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

Parallel Characterization of Anaerobic Toluene- and Ethylbenzene-Degrading Microbial Consortia by PCR-Denaturing Gradient Gel Electrophoresis, RNA-DNA Membrane Hybridization, and DNA Microarray Technology

PubMed Central

Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Saïd; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

2002-01-01

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis. PMID:12088997

Ultrafiltration and Microarray for Detection of Microbial Source Tracking Marker and Pathogen Genes in Riverine and Marine Systems

PubMed Central

Li, Xiang; Harwood, Valerie J.; Nayak, Bina

2016-01-01

Pathogen identification and microbial source tracking (MST) to identify sources of fecal pollution improve evaluation of water quality. They contribute to improved assessment of human health risks and remediation of pollution sources. An MST microarray was used to simultaneously detect genes for multiple pathogens and indicators of fecal pollution in freshwater, marine water, sewage-contaminated freshwater and marine water, and treated wastewater. Dead-end ultrafiltration (DEUF) was used to concentrate organisms from water samples, yielding a recovery efficiency of >95% for Escherichia coli and human polyomavirus. Whole-genome amplification (WGA) increased gene copies from ultrafiltered samples and increased the sensitivity of the microarray. Viruses (adenovirus, bocavirus, hepatitis A virus, and human polyomaviruses) were detected in sewage-contaminated samples. Pathogens such as Legionella pneumophila, Shigella flexneri, and Campylobacter fetus were detected along with genes conferring resistance to aminoglycosides, beta-lactams, and tetracycline. Nonmetric dimensional analysis of MST marker genes grouped sewage-spiked freshwater and marine samples with sewage and apart from other fecal sources. The sensitivity (percent true positives) of the microarray probes for gene targets anticipated in sewage was 51 to 57% and was lower than the specificity (percent true negatives; 79 to 81%). A linear relationship between gene copies determined by quantitative PCR and microarray fluorescence was found, indicating the semiquantitative nature of the MST microarray. These results indicate that ultrafiltration coupled with WGA provides sufficient nucleic acids for detection of viruses, bacteria, protozoa, and antibiotic resistance genes by the microarray in applications ranging from beach monitoring to risk assessment. PMID:26729716

MADGE: scalable distributed data management software for cDNA microarrays.

PubMed

McIndoe, Richard A; Lanzen, Aaron; Hurtz, Kimberly

2003-01-01

The human genome project and the development of new high-throughput technologies have created unparalleled opportunities to study the mechanism of diseases, monitor the disease progression and evaluate effective therapies. Gene expression profiling is a critical tool to accomplish these goals. The use of nucleic acid microarrays to assess the gene expression of thousands of genes simultaneously has seen phenomenal growth over the past five years. Although commercial sources of microarrays exist, investigators wanting more flexibility in the genes represented on the array will turn to in-house production. The creation and use of cDNA microarrays is a complicated process that generates an enormous amount of information. Effective data management of this information is essential to efficiently access, analyze, troubleshoot and evaluate the microarray experiments. We have developed a distributable software package designed to track and store the various pieces of data generated by a cDNA microarray facility. This includes the clone collection storage data, annotation data, workflow queues, microarray data, data repositories, sample submission information, and project/investigator information. This application was designed using a 3-tier client server model. The data access layer (1st tier) contains the relational database system tuned to support a large number of transactions. The data services layer (2nd tier) is a distributed COM server with full database transaction support. The application layer (3rd tier) is an internet based user interface that contains both client and server side code for dynamic interactions with the user. This software is freely available to academic institutions and non-profit organizations at http://www.genomics.mcg.edu/niddkbtc.

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.

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.

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.

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.

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.

Labeled nucleotide phosphate (NP) probes

DOEpatents

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

2009-02-03

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

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

PubMed

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

2016-04-01

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

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.

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.

Thermodynamic framework to assess low abundance DNA mutation detection by hybridization.

PubMed

Willems, Hanny; Jacobs, An; Hadiwikarta, Wahyu Wijaya; Venken, Tom; Valkenborg, Dirk; Van Roy, Nadine; Vandesompele, Jo; Hooyberghs, Jef

2017-01-01

The knowledge of genomic DNA variations in patient samples has a high and increasing value for human diagnostics in its broadest sense. Although many methods and sensors to detect or quantify these variations are available or under development, the number of underlying physico-chemical detection principles is limited. One of these principles is the hybridization of sample target DNA versus nucleic acid probes. We introduce a novel thermodynamics approach and develop a framework to exploit the specific detection capabilities of nucleic acid hybridization, using generic principles applicable to any platform. As a case study, we detect point mutations in the KRAS oncogene on a microarray platform. For the given platform and hybridization conditions, we demonstrate the multiplex detection capability of hybridization and assess the detection limit using thermodynamic considerations; DNA containing point mutations in a background of wild type sequences can be identified down to at least 1% relative concentration. In order to show the clinical relevance, the detection capabilities are confirmed on challenging formalin-fixed paraffin-embedded clinical tumor samples. This enzyme-free detection framework contains the accuracy and efficiency to screen for hundreds of mutations in a single run with many potential applications in molecular diagnostics and the field of personalised medicine.

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.

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.

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

NASA Technical Reports Server (NTRS)

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

2017-01-01

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

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.

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

PubMed

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

2016-10-01

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

A preliminary result of three-dimensional microarray technology to gene analysis with endoscopic ultrasound-guided fine-needle aspiration specimens and pancreatic juices

PubMed Central

2010-01-01

Background Analysis of gene expression and gene mutation may add information to be different from ordinary pathological tissue diagnosis. Since samples obtained endoscopically are very small, it is desired that more sensitive technology is developed for gene analysis. We investigated whether gene expression and gene mutation analysis by newly developed ultra-sensitive three-dimensional (3D) microarray is possible using small amount samples from endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) specimens and pancreatic juices. Methods Small amount samples from 17 EUS-FNA specimens and 16 pancreatic juices were obtained. After nucleic acid extraction, the samples were amplified with labeling and analyzed by the 3D microarray. Results The analyzable rate with the microarray was 46% (6/13) in EUS-FNA specimens of RNAlater® storage, and RNA degradations were observed in all the samples of frozen storage. In pancreatic juices, the analyzable rate was 67% (4/6) in frozen storage samples and 20% (2/10) in RNAlater® storage. EUS-FNA specimens were classified into cancer and non-cancer by gene expression analysis and K-ras codon 12 mutations were also detected using the 3D microarray. Conclusions Gene analysis from small amount samples obtained endoscopically was possible by newly developed 3D microarray technology. High quality RNA from EUS-FNA samples were obtained and remained in good condition only using RNA stabilizer. In contrast, high quality RNA from pancreatic juice samples were obtained only in frozen storage without RNA stabilizer. PMID:20416107

Rapid Microarray Detection of DNA and Proteins in Microliter Volumes with SPR Imaging Measurements

PubMed Central

Seefeld, Ting Hu; Zhou, Wen-Juan; Corn, Robert M.

2011-01-01

A four chamber microfluidic biochip is fabricated for the rapid detection of multiple proteins and nucleic acids from microliter volume samples with the technique of surface plasmon resonance imaging (SPRI). The 18 mm × 18 mm biochip consists of four 3 μL microfluidic chambers attached to an SF10 glass substrate, each of which contains three individually addressable SPRI gold thin film microarray elements. The twelve element (4 × 3) SPRI microarray consists of gold thin film spots (1 mm2 area; 45 nm thickness) each in individually addressable 0.5 μL volume microchannels. Microarrays of single-stranded DNA and RNA (ssDNA and ssRNA respectively) are fabricated by either chemical and/or enzymatic attachment reactions in these microchannels; the SPRI microarrays are then used to detect femtomole amounts (nanomolar concentrations) of DNA and proteins (single stranded DNA binding protein and thrombin via aptamer-protein bioaffinity interactions). Microarrays of ssRNA microarray elements were also used for the ultrasensitive detection of zeptomole amounts (femtomolar concentrations) of DNA via the technique of RNase H-amplified SPRI. Enzymatic removal of ssRNA from the surface due to the hybridization adsorption of target ssDNA is detected as a reflectivity decrease in the SPR imaging measurements. The observed reflectivity loss was proportional to the log of the target ssDNA concentration with a detection limit of 10 fM or 30 zeptomoles (18,000 molecules). This enzymatic amplified ssDNA detection method is not limited by diffusion of ssDNA to the interface, and thus is extremely fast, requiring only 200 seconds in the microliter volume format. PMID:21488682

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.

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.

Invasive cleavage of nucleic acids

DOEpatents

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

1999-01-01

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

Invasive cleavage of nucleic acids

DOEpatents

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

2002-01-01

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

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.

Cleavage of nucleic acids

DOEpatents

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

2000-01-01

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

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.

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.

Rational design of DNA sequences for nanotechnology, microarrays and molecular computers using Eulerian graphs.

PubMed

Pancoska, Petr; Moravek, Zdenek; Moll, Ute M

2004-01-01

Nucleic acids are molecules of choice for both established and emerging nanoscale technologies. These technologies benefit from large functional densities of 'DNA processing elements' that can be readily manufactured. To achieve the desired functionality, polynucleotide sequences are currently designed by a process that involves tedious and laborious filtering of potential candidates against a series of requirements and parameters. Here, we present a complete novel methodology for the rapid rational design of large sets of DNA sequences. This method allows for the direct implementation of very complex and detailed requirements for the generated sequences, thus avoiding 'brute force' filtering. At the same time, these sequences have narrow distributions of melting temperatures. The molecular part of the design process can be done without computer assistance, using an efficient 'human engineering' approach by drawing a single blueprint graph that represents all generated sequences. Moreover, the method eliminates the necessity for extensive thermodynamic calculations. Melting temperature can be calculated only once (or not at all). In addition, the isostability of the sequences is independent of the selection of a particular set of thermodynamic parameters. Applications are presented for DNA sequence designs for microarrays, universal microarray zip sequences and electron transfer experiments.

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

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

Nasarabadi, Shanavaz

2011-01-11

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

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.

Quantitative detection of pathogens in centrifugal microfluidic disks

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

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

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

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.

High processivity polymerases

DOEpatents

Shamoo, Yousif; Sun, Siyang

2014-06-10

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

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.

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.

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.

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.

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.

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.

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

Diff-seq: A high throughput sequencing-based mismatch detection assay for DNA variant enrichment and discovery

PubMed Central

Karas, Vlad O; Sinnott-Armstrong, Nicholas A; Varghese, Vici; Shafer, Robert W; Greenleaf, William J; Sherlock, Gavin

2018-01-01

Abstract Much of the within species genetic variation is in the form of single nucleotide polymorphisms (SNPs), typically detected by whole genome sequencing (WGS) or microarray-based technologies. However, WGS produces mostly uninformative reads that perfectly match the reference, while microarrays require genome-specific reagents. We have developed Diff-seq, a sequencing-based mismatch detection assay for SNP discovery without the requirement for specialized nucleic-acid reagents. Diff-seq leverages the Surveyor endonuclease to cleave mismatched DNA molecules that are generated after cross-annealing of a complex pool of DNA fragments. Sequencing libraries enriched for Surveyor-cleaved molecules result in increased coverage at the variant sites. Diff-seq detected all mismatches present in an initial test substrate, with specific enrichment dependent on the identity and context of the variation. Application to viral sequences resulted in increased observation of variant alleles in a biologically relevant context. Diff-Seq has the potential to increase the sensitivity and efficiency of high-throughput sequencing in the detection of variation. PMID:29361139

Automated Immunomagnetic Separation and Microarray Detection of E. coli O157:H7 from Poultry Carcass Rinse

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

Chandler, Darrell P.; Brown, Jeremy D.; Call, Douglas R.

2001-09-01

We describe the development and application of a novel electromagnetic flow cell and fluidics system for automated immunomagnetic separation of E. coli directly from unprocessed poultry carcass rinse, and the biochemical coupling of automated sample preparation with nucleic acid microarrays without cell growth. Highly porous nickel foam was used as a magnetic flux conductor. Up to 32% recovery efficiency of 'total' E. coli was achieved within the automated system with 6 sec contact times and 15 minute protocol (from sample injection through elution), statistically similar to cell recovery efficiencies in > 1 hour 'batch' captures. The electromagnet flow cell allowedmore » complete recovery of 2.8 mm particles directly from unprocessed poultry carcass rinse whereas the batch system did not. O157:H7 cells were reproducibly isolated directly from unprocessed poultry rinse with 39% recovery efficiency at 103 cells ml-1 inoculum. Direct plating of washed beads showed positive recovery of O 157:H7 directly from carcass rinse at an inoculum of 10 cells ml-1. Recovered beads were used for direct PCR amplification and microarray detection, with a process-level detection limit (automated cell concentration through microarray detection) of < 103 cells ml-1 carcass rinse. The fluidic system and analytical approach described here are generally applicable to most microbial detection problems and applications.« less

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.

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.

Shaping up nucleic acid computation.

PubMed

Chen, Xi; Ellington, Andrew D

2010-08-01

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

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.

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.

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

DOEpatents

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

2004-09-14

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

Nucleic Acid Immunity.

PubMed

Hartmann, G

2017-01-01

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

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.

Gene expression profiling in gill tissues of White spot syndrome virus infected black tiger shrimp Penaeus monodon by DNA microarray.

PubMed

Shekhar, M S; Gomathi, A; Gopikrishna, G; Ponniah, A G

2015-06-01

White spot syndrome virus (WSSV) continues to be the most devastating viral pathogen infecting penaeid shrimp the world over. The genome of WSSV has been deciphered and characterized from three geographical isolates and significant progress has been made in developing various molecular diagnostic methods to detect the virus. However, the information on host immune gene response to WSSV pathogenesis is limited. Microarray analysis was carried out as an approach to analyse the gene expression in black tiger shrimp Penaeus monodon in response to WSSV infection. Gill tissues collected from the WSSV infected shrimp at 6, 24, 48 h and moribund stage were analysed for differential gene expression. Shrimp cDNAs of 40,059 unique sequences were considered for designing the microarray chip. The Cy3-labeled cRNA derived from healthy and WSSV-infected shrimp was subjected to hybridization with all the DNA spots in the microarray which revealed 8,633 and 11,147 as up- and down-regulated genes respectively at different time intervals post infection. The altered expression of these numerous genes represented diverse functions such as immune response, osmoregulation, apoptosis, nucleic acid binding, energy and metabolism, signal transduction, stress response and molting. The changes in gene expression profiles observed by microarray analysis provides molecular insights and framework of genes which are up- and down-regulated at different time intervals during WSSV infection in shrimp. The microarray data was validated by Real Time analysis of four differentially expressed genes involved in apoptosis (translationally controlled tumor protein, inhibitor of apoptosis protein, ubiquitin conjugated enzyme E2 and caspase) for gene expression levels. The role of apoptosis related genes in WSSV infected shrimp is discussed herein.

Protein-protein interactions: an application of Tus-Ter mediated protein microarray system.

PubMed

Sitaraman, Kalavathy; Chatterjee, Deb K

2011-01-01

In this chapter, we present a novel, cost-effective microarray strategy that utilizes expression-ready plasmid DNAs to generate protein arrays on-demand and its use to validate protein-protein interactions. These expression plasmids were constructed in such a way so as to serve a dual purpose of synthesizing the protein of interest as well as capturing the synthesized protein. The microarray system is based on the high affinity binding of Escherichia coli "Tus" protein to "Ter," a 20 bp DNA sequence involved in the regulation of DNA replication. The protein expression is carried out in a cell-free protein synthesis system, with rabbit reticulocyte lysates, and the target proteins are detected either by labeled incorporated tag specific or by gene-specific antibodies. This microarray system has been successfully used for the detection of protein-protein interaction because both the target protein and the query protein can be transcribed and translated simultaneously in the microarray slides. The utility of this system for detecting protein-protein interaction is demonstrated by a few well-known examples: Jun/Fos, FRB/FKBP12, p53/MDM2, and CDK4/p16. In all these cases, the presence of protein complexes resulted in the localization of fluorophores at the specific sites of the immobilized target plasmids. Interestingly, during our interactions studies we also detected a previously unknown interaction between CDK2 and p16. Thus, this Tus-Ter based system of protein microarray can be used for the validation of known protein interactions as well as for identifying new protein-protein interactions. In addition, it can be used to examine and identify targets of nucleic acid-protein, ligand-receptor, enzyme-substrate, and drug-protein interactions.

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.

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

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

Method for nucleic acid isolation using supercritical fluids

DOEpatents

Nivens, David E.; Applegate, Bruce M.

1999-01-01

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

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

Code of Federal Regulations, 2010 CFR

2010-04-01

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

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

Code of Federal Regulations, 2014 CFR

2014-04-01

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

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

Code of Federal Regulations, 2011 CFR

2011-04-01

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

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

Code of Federal Regulations, 2013 CFR

2013-04-01

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

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

Code of Federal Regulations, 2012 CFR

2012-04-01

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

Design of nucleic acid sequences for DNA computing based on a thermodynamic approach

PubMed Central

Tanaka, Fumiaki; Kameda, Atsushi; Yamamoto, Masahito; Ohuchi, Azuma

2005-01-01

We have developed an algorithm for designing multiple sequences of nucleic acids that have a uniform melting temperature between the sequence and its complement and that do not hybridize non-specifically with each other based on the minimum free energy (ΔGmin). Sequences that satisfy these constraints can be utilized in computations, various engineering applications such as microarrays, and nano-fabrications. Our algorithm is a random generate-and-test algorithm: it generates a candidate sequence randomly and tests whether the sequence satisfies the constraints. The novelty of our algorithm is that the filtering method uses a greedy search to calculate ΔGmin. This effectively excludes inappropriate sequences before ΔGmin is calculated, thereby reducing computation time drastically when compared with an algorithm without the filtering. Experimental results in silico showed the superiority of the greedy search over the traditional approach based on the hamming distance. In addition, experimental results in vitro demonstrated that the experimental free energy (ΔGexp) of 126 sequences correlated well with ΔGmin (|R| = 0.90) than with the hamming distance (|R| = 0.80). These results validate the rationality of a thermodynamic approach. We implemented our algorithm in a graphic user interface-based program written in Java. PMID:15701762

Thermodynamic framework to assess low abundance DNA mutation detection by hybridization

PubMed Central

Willems, Hanny; Jacobs, An; Hadiwikarta, Wahyu Wijaya; Venken, Tom; Valkenborg, Dirk; Van Roy, Nadine; Vandesompele, Jo; Hooyberghs, Jef

2017-01-01

The knowledge of genomic DNA variations in patient samples has a high and increasing value for human diagnostics in its broadest sense. Although many methods and sensors to detect or quantify these variations are available or under development, the number of underlying physico-chemical detection principles is limited. One of these principles is the hybridization of sample target DNA versus nucleic acid probes. We introduce a novel thermodynamics approach and develop a framework to exploit the specific detection capabilities of nucleic acid hybridization, using generic principles applicable to any platform. As a case study, we detect point mutations in the KRAS oncogene on a microarray platform. For the given platform and hybridization conditions, we demonstrate the multiplex detection capability of hybridization and assess the detection limit using thermodynamic considerations; DNA containing point mutations in a background of wild type sequences can be identified down to at least 1% relative concentration. In order to show the clinical relevance, the detection capabilities are confirmed on challenging formalin-fixed paraffin-embedded clinical tumor samples. This enzyme-free detection framework contains the accuracy and efficiency to screen for hundreds of mutations in a single run with many potential applications in molecular diagnostics and the field of personalised medicine. PMID:28542229

Recent developments in detection and enumeration of waterborne bacteria: a retrospective minireview.

PubMed

Deshmukh, Rehan A; Joshi, Kopal; Bhand, Sunil; Roy, Utpal

2016-12-01

Waterborne diseases have emerged as global health problems and their rapid and sensitive detection in environmental water samples is of great importance. Bacterial identification and enumeration in water samples is significant as it helps to maintain safe drinking water for public consumption. Culture-based methods are laborious, time-consuming, and yield false-positive results, whereas viable but nonculturable (VBNCs) microorganisms cannot be recovered. Hence, numerous methods have been developed for rapid detection and quantification of waterborne pathogenic bacteria in water. These rapid methods can be classified into nucleic acid-based, immunology-based, and biosensor-based detection methods. This review summarizes the principle and current state of rapid methods for the monitoring and detection of waterborne bacterial pathogens. Rapid methods outlined are polymerase chain reaction (PCR), digital droplet PCR, real-time PCR, multiplex PCR, DNA microarray, Next-generation sequencing (pyrosequencing, Illumina technology and genomics), and fluorescence in situ hybridization that are categorized as nucleic acid-based methods. Enzyme-linked immunosorbent assay (ELISA) and immunofluorescence are classified into immunology-based methods. Optical, electrochemical, and mass-based biosensors are grouped into biosensor-based methods. Overall, these methods are sensitive, specific, time-effective, and important in prevention and diagnosis of waterborne bacterial diseases. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Highly sensitive dual mode electrochemical platform for microRNA detection

NASA Astrophysics Data System (ADS)

Jolly, Pawan; Batistuti, Marina R.; Miodek, Anna; Zhurauski, Pavel; Mulato, Marcelo; Lindsay, Mark A.; Estrela, Pedro

2016-11-01

MicroRNAs (miRNAs) play crucial regulatory roles in various human diseases including cancer, making them promising biomarkers. However, given the low levels of miRNAs present in blood, their use as cancer biomarkers requires the development of simple and effective analytical methods. Herein, we report the development of a highly sensitive dual mode electrochemical platform for the detection of microRNAs. The platform was developed using peptide nucleic acids as probes on gold electrode surfaces to capture target miRNAs. A simple amplification strategy using gold nanoparticles has been employed exploiting the inherent charges of the nucleic acids. Electrochemical impedance spectroscopy was used to monitor the changes in capacitance upon any binding event, without the need for any redox markers. By using thiolated ferrocene, a complementary detection mode on the same sensor was developed where the increasing peaks of ferrocene were recorded using square wave voltammetry with increasing miRNA concentration. This dual-mode approach allows detection of miRNA with a limit of detection of 0.37 fM and a wide dynamic range from 1 fM to 100 nM along with clear distinction from mismatched target miRNA sequences. The electrochemical platform developed can be easily expanded to other miRNA/DNA detection along with the development of microarray platforms.

Nanoplasmonic molecular ruler for nuclease activity and DNA footprinting

DOEpatents

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

2013-10-29

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

Use of CYP52A2A promoter to increase gene expression in yeast

DOEpatents

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

2004-01-06

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

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

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

PubMed Central

Hellman, Lance M.; Fried, Michael G.

2009-01-01

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

Solid phase sequencing of biopolymers

DOEpatents

Cantor, Charles; Koster, Hubert

2010-09-28

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

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.

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

PubMed Central

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

2016-01-01

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

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.

Nucleic Acid-Induced Resistance to Viral Infection

PubMed Central

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

1965-01-01

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

MUSI: an integrated system for identifying multiple specificity from very large peptide or nucleic acid data sets.

PubMed

Kim, Taehyung; Tyndel, Marc S; Huang, Haiming; Sidhu, Sachdev S; Bader, Gary D; Gfeller, David; Kim, Philip M

2012-03-01

Peptide recognition domains and transcription factors play crucial roles in cellular signaling. They bind linear stretches of amino acids or nucleotides, respectively, with high specificity. Experimental techniques that assess the binding specificity of these domains, such as microarrays or phage display, can retrieve thousands of distinct ligands, providing detailed insight into binding specificity. In particular, the advent of next-generation sequencing has recently increased the throughput of such methods by several orders of magnitude. These advances have helped reveal the presence of distinct binding specificity classes that co-exist within a set of ligands interacting with the same target. Here, we introduce a software system called MUSI that can rapidly analyze very large data sets of binding sequences to determine the relevant binding specificity patterns. Our pipeline provides two major advances. First, it can detect previously unrecognized multiple specificity patterns in any data set. Second, it offers integrated processing of very large data sets from next-generation sequencing machines. The results are visualized as multiple sequence logos describing the different binding preferences of the protein under investigation. We demonstrate the performance of MUSI by analyzing recent phage display data for human SH3 domains as well as microarray data for mouse transcription factors.

Artificial mismatch hybridization

DOEpatents

Guo, Zhen; Smith, Lloyd M.

1998-01-01

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

Transgenic cells with increased plastoquinone levels and methods of use

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

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

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

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

PubMed Central

Edberg, S. C.

1985-01-01

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

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

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

Liu, Ling; Xiong, Yi; Gao, Hongyun

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

Detection and isolation of nucleic acid sequences using competitive hybridization probes

DOEpatents

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

1997-01-01

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

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

DOE PAGES

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

2018-04-02

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

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

NASA Technical Reports Server (NTRS)

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

2011-01-01

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

Detection and isolation of nucleic acid sequences using competitive hybridization probes

DOEpatents

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

1997-04-01

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

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

PubMed

Frahm, J L; Muddiman, D C

2005-01-01

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

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

DTIC Science & Technology

1994-12-01

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

European Science Notes, Volume 40, Number 7.

DTIC Science & Technology

1986-07-01

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

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

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

PubMed

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

2017-08-01

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

Optimization of cDNA microarrays procedures using criteria that do not rely on external standards.

PubMed

Bruland, Torunn; Anderssen, Endre; Doseth, Berit; Bergum, Hallgeir; Beisvag, Vidar; Laegreid, Astrid

2007-10-18

The measurement of gene expression using microarray technology is a complicated process in which a large number of factors can be varied. Due to the lack of standard calibration samples such as are used in traditional chemical analysis it may be a problem to evaluate whether changes done to the microarray procedure actually improve the identification of truly differentially expressed genes. The purpose of the present work is to report the optimization of several steps in the microarray process both in laboratory practices and in data processing using criteria that do not rely on external standards. We performed a cDNA microarry experiment including RNA from samples with high expected differential gene expression termed "high contrasts" (rat cell lines AR42J and NRK52E) compared to self-self hybridization, and optimized a pipeline to maximize the number of genes found to be differentially expressed in the "high contrasts" RNA samples by estimating the false discovery rate (FDR) using a null distribution obtained from the self-self experiment. The proposed high-contrast versus self-self method (HCSSM) requires only four microarrays per evaluation. The effects of blocking reagent dose, filtering, and background corrections methodologies were investigated. In our experiments a dose of 250 ng LNA (locked nucleic acid) dT blocker, no background correction and weight based filtering gave the largest number of differentially expressed genes. The choice of background correction method had a stronger impact on the estimated number of differentially expressed genes than the choice of filtering method. Cross platform microarray (Illumina) analysis was used to validate that the increase in the number of differentially expressed genes found by HCSSM was real. The results show that HCSSM can be a useful and simple approach to optimize microarray procedures without including external standards. Our optimizing method is highly applicable to both long oligo-probe microarrays which have become commonly used for well characterized organisms such as man, mouse and rat, as well as to cDNA microarrays which are still of importance for organisms with incomplete genome sequence information such as many bacteria, plants and fish.

Optimization of cDNA microarrays procedures using criteria that do not rely on external standards

PubMed Central

Bruland, Torunn; Anderssen, Endre; Doseth, Berit; Bergum, Hallgeir; Beisvag, Vidar; Lægreid, Astrid

2007-01-01

Background The measurement of gene expression using microarray technology is a complicated process in which a large number of factors can be varied. Due to the lack of standard calibration samples such as are used in traditional chemical analysis it may be a problem to evaluate whether changes done to the microarray procedure actually improve the identification of truly differentially expressed genes. The purpose of the present work is to report the optimization of several steps in the microarray process both in laboratory practices and in data processing using criteria that do not rely on external standards. Results We performed a cDNA microarry experiment including RNA from samples with high expected differential gene expression termed "high contrasts" (rat cell lines AR42J and NRK52E) compared to self-self hybridization, and optimized a pipeline to maximize the number of genes found to be differentially expressed in the "high contrasts" RNA samples by estimating the false discovery rate (FDR) using a null distribution obtained from the self-self experiment. The proposed high-contrast versus self-self method (HCSSM) requires only four microarrays per evaluation. The effects of blocking reagent dose, filtering, and background corrections methodologies were investigated. In our experiments a dose of 250 ng LNA (locked nucleic acid) dT blocker, no background correction and weight based filtering gave the largest number of differentially expressed genes. The choice of background correction method had a stronger impact on the estimated number of differentially expressed genes than the choice of filtering method. Cross platform microarray (Illumina) analysis was used to validate that the increase in the number of differentially expressed genes found by HCSSM was real. Conclusion The results show that HCSSM can be a useful and simple approach to optimize microarray procedures without including external standards. Our optimizing method is highly applicable to both long oligo-probe microarrays which have become commonly used for well characterized organisms such as man, mouse and rat, as well as to cDNA microarrays which are still of importance for organisms with incomplete genome sequence information such as many bacteria, plants and fish. PMID:17949480

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.

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

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

Cary, Robert E.

2015-12-08

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

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

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

Cary, Robert B.

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

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

PubMed Central

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

2016-01-01

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

RNA-guided transcriptional regulation

DOEpatents

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

2016-02-23

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

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

PubMed

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

2009-08-01

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

Nucleic acid-functionalized transition metal nanosheets for biosensing applications

PubMed Central

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

2017-01-01

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

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.

Introductory Remarks

NASA Astrophysics Data System (ADS)

Lu, Yi; Li, Yingfu

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

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.

Importance of the efficiency of double-stranded DNA formation in cDNA synthesis for the imprecision of microarray expression analysis.

PubMed

Thormar, Hans G; Gudmundsson, Bjarki; Eiriksdottir, Freyja; Kil, Siyoen; Gunnarsson, Gudmundur H; Magnusson, Magnus Karl; Hsu, Jason C; Jonsson, Jon J

2013-04-01

The causes of imprecision in microarray expression analysis are poorly understood, limiting the use of this technology in molecular diagnostics. Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acid molecules on the basis of length and strandness, i.e., double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and RNA·DNA hybrids. We used 2D-SDE to measure the efficiency of cDNA synthesis and its importance for the imprecision of an in vitro transcription-based microarray expression analysis. The relative amount of double-stranded cDNA formed in replicate experiments that used the same RNA sample template was highly variable, ranging between 0% and 72% of the total DNA. Microarray experiments showed an inverse relationship between the difference between sample pairs in probe variance and the relative amount of dsDNA. Approximately 15% of probes showed between-sample variation (P < 0.05) when the dsDNA percentage was between 12% and 35%. In contrast, only 3% of probes showed between-sample variation when the dsDNA percentage was 69% and 72%. Replication experiments of the 35% dsDNA and 72% dsDNA samples were used to separate sample variation from probe replication variation. The estimated SD of the sample-to-sample variation and of the probe replicates was lower in 72% dsDNA samples than in 35% dsDNA samples. Variation in the relative amount of double-stranded cDNA synthesized can be an important component of the imprecision in T7 RNA polymerase-based microarray expression analysis. © 2013 American Association for Clinical Chemistry

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

PubMed

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

2004-01-01

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

EGVII endoglucanase and nucleic acids encoding the same

DOEpatents

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

2014-02-25

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

EGVII endoglucanase and nucleic acids encoding the same

DOEpatents

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

2006-05-16

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

EGVI endoglucanase and nucleic acids encoding the same

DOEpatents

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

2008-04-01

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

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.

EGVIII endoglucanase and nucleic acids encoding the same

DOEpatents

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

2006-05-23

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

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.

EGVI endoglucanase and nucleic acids encoding the same

DOEpatents

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

2006-06-06

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

EGVII endoglucanase and nucleic acids encoding the same

DOEpatents

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

2009-05-05

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

EGVII endoglucanase and nucleic acids encoding the same

DOEpatents

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

2013-07-16

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

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.

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.

Electrically detected displacement assay (EDDA): a practical approach to nucleic acid testing in clinical or medical diagnosis.

PubMed

Liepold, P; Kratzmüller, T; Persike, N; Bandilla, M; Hinz, M; Wieder, H; Hillebrandt, H; Ferrer, E; Hartwich, G

2008-07-01

This paper introduces the electrically detected displacement assay (EDDA), a electrical biosensor detection principle for applications in medical and clinical diagnosis, and compares the method to currently available microarray technologies in this field. The sensor can be integrated into automated systems of routine diagnosis, but may also be used as a sensor that is directly applied to the polymerase chain reaction (PCR) reaction vessel to detect unlabeled target amplicons within a few minutes. Major aspects of sensor assembly like immobilization procedure, accessibility of the capture probes, and prevention from nonspecific target adsorption, that are a prerequisite for a robust and reliable performance of the sensor, are demonstrated. Additionally, exemplary results from a human papillomavirus assay are presented.

eSensor®: A Microarray Technology Based on Electrochemical Detection of Nucleic Acids and Its Application to Cystic Fibrosis Carrier Screening

NASA Astrophysics Data System (ADS)

Reed, Michael R.; Coty, William A.

We have developed a test for identification of carriers for cystic fibrosis using the eSensor® DNA detection technology. Oligonucleotide probes are deposited within self-assembled monolayers on gold electrodes arrayed upon printed circuit boards. These probes allow sequence-specific capture of amplicons containing a panel of mutation sites associated with cystic fibrosis. DNA targets are detected and mutations genotyped using a “sandwich” assay methodology employing electrochemical detection of ferrocene-labeled oligonucleotides for discrimination of carrier and non-carrier alleles. Performance of the cystic fibrosis application demonstrates sufficient accuracy and reliability for clinical diagnostic use, and the procedure can be performed by trained medical technologists available in the hospital laboratory.

Miniaturized multiplex label-free electronic chip for rapid nucleic acid analysis based on carbon nanotube nanoelectrode arrays

NASA Technical Reports Server (NTRS)

Koehne, Jessica E.; Chen, Hua; Cassell, Alan M.; Ye, Qi; Han, Jie; Meyyappan, Meyya; Li, Jun

2004-01-01

BACKGROUND: Reducing cost and time is the major concern in clinical diagnostics, particularly in molecular diagnostics. Miniaturization technologies have been recognized as promising solutions to provide low-cost microchips for diagnostics. With the recent advancement in nanotechnologies, it is possible to further improve detection sensitivity and simplify sample preparation by incorporating nanoscale elements in diagnostics devices. A fusion of micro- and nanotechnologies with biology has great potential for the development of low-cost disposable chips for rapid molecular analysis that can be carried out with simple handheld devices. APPROACH: Vertically aligned multiwalled carbon nanotubes (MWNTs) are fabricated on predeposited microelectrode pads and encapsulated in SiO2 dielectrics with only the very end exposed at the surface to form an inlaid nanoelectrode array (NEA). The NEA is used to collect the electrochemical signal associated with the target molecules binding to the probe molecules, which are covalently attached to the end of the MWNTs. CONTENT: A 3 x 3 microelectrode array is presented to demonstrate the miniaturization and multiplexing capability. A randomly distributed MWNT NEA is fabricated on each microelectrode pad. Selective functionalization of the MWNT end with a specific oligonucleotide probe and passivation of the SiO2 surface with ethylene glycol moieties are discussed. Ru(bpy)2+ -mediator-amplified guanine oxidation is used to directly measure the electrochemical signal associated with target molecules. SUMMARY: The discussed MWNT NEAs have ultrahigh sensitivity in direct electrochemical detection of guanine bases in the nucleic acid target. Fewer than approximately 1000 target nucleic acid molecules can be measured with a single microelectrode pad of approximately 20 x 20 microm2, which approaches the detection limit of laser scanners in fluorescence-based DNA microarray techniques. MWNT NEAs can be easily integrated with microelectronic circuitry and microfluidics for development of a fully automated system for rapid molecular analysis with minimum cost.

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

PubMed

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

2013-12-21

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

Single-stranded nucleic acids promote SAMHD1 complex formation.

PubMed

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

2013-06-01

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

Portable nucleic acid thermocyclers.

PubMed

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

2013-11-21

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

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.

7 CFR 331.1 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-01-01

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

7 CFR 331.1 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-01-01

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

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.

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

PubMed Central

Bujalowski, Wlodzimierz; Jezewska, Maria J.

2011-01-01

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

Nucleic Acid Research Group (NARG) 2009-2010 Study : Optimal Priming Strategies for cDNA Synthesis in Real-Time RT-qPCR

PubMed Central

Hunter, T.C.; Knudtson, K.L.; Nadella, V.; Sol-Church, K.; Taylor, W.L.; Tighe, S.; Yueng, A.T.; Chittur, S.

2010-01-01

r1-1 Real-time reverse transcriptase quantitative PCR (RT-qPCR) is a widely used technique for measuring transcript levels. Priming strategy and reverse transcriptase enzyme are key elements that affect sensitivity and variability of RT-qPCR and microarray results. Previously, the Nucleic Acid Research Group (NARG) had conducted preliminary studies within the group to examine the effects of priming strategy on generating cDNA for use with qPCR. This year's study was an open study in which the qPCR community was invited to participate. Participants received the RT primers and RNA template and were asked to perform the RT reaction using their preferred reaction conditions. Each participating laboratory was provided at least two RNA templates of varying quality. The RT products were returned to the NARG and all RT reactions were used in a qPCR reaction. The qPCR assays looked at three genes of varying abundance, b-actin (high copy), b-glucuronidase (medium copy) and TATA binding protein (low copy) as well as varying distance from the 3? end for each transcript. Results from participating laboratories will be evaluated to determine the impact of priming strategy, assay chemistry and experimental setup on the RT step. Additionally, we will address the impact of RNA integrity on cDNA synthesis.

Solid-Phase Nucleic Acid Sequence-Based Amplification and Length-Scale Effects during RNA Amplification.

PubMed

Ma, Youlong; Teng, Feiyue; Libera, Matthew

2018-06-05

Solid-phase oligonucleotide amplification is of interest because of possible applications to next-generation sequencing, multiplexed microarray-based detection, and cell-free synthetic biology. Its efficiency is, however, less than that of traditional liquid-phase amplification involving unconstrained primers and enzymes, and understanding how to optimize the solid-phase amplification process remains challenging. Here, we demonstrate the concept of solid-phase nucleic acid sequence-based amplification (SP-NASBA) and use it to study the effect of tethering density on amplification efficiency. SP-NASBA involves two enzymes, avian myeloblastosis virus reverse transcriptase (AMV-RT) and RNase H, to convert tethered forward and reverse primers into tethered double-stranded DNA (ds-DNA) bridges from which RNA - amplicons can be generated by a third enzyme, T7 RNA polymerase. We create microgels on silicon surfaces using electron-beam patterning of thin-film blends of hydroxyl-terminated and biotin-terminated poly(ethylene glycol) (PEG-OH, PEG-B). The tethering density is linearly related to the PEG-B concentration, and biotinylated primers and molecular beacon detection probes are tethered to streptavidin-activated microgels. While SP-NASBA is very efficient at low tethering densities, the efficiency decreases dramatically with increasing tethering density due to three effects: (a) a reduced hybridization efficiency of tethered molecular beacon detection probes; (b) a decrease in T7 RNA polymerase efficiency; (c) inhibition of T7 RNA polymerase activity by AMV-RT.

BGL7 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Ward, Michael

2013-01-29

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

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

DOEpatents

Dunn-Coleman, Nigel; Ward, Michael

2012-10-02

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

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

DOEpatents

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

2006-02-28

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

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

DOEpatents

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

2008-03-18

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

BGL6 beta-glucosidase and nucleic acids encoding the same

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

Dunn-Coleman, Nigel; Ward, Michael

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

BGL6 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Ward, Michael

2014-03-04

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

BGL7 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Ward, Michael

2015-04-14

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

BGL7 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Ward, Michael

2014-03-25

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

BGL6 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Ward, Michael

2015-08-11

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

BGL3 beta-glucosidase and nucleic acids encoding the same

DOEpatents

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

2007-09-25

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

BGL3 beta-glucosidase and nucleic acids encoding the same

DOEpatents

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

2008-04-01

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

BGL4 beta-glucosidase and nucleic acids encoding the same

DOEpatents

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

2011-12-06

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

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

DOEpatents

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

2006-05-16

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

BGL3 beta-glucosidase and nucleic acids encoding the same

DOEpatents

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

2011-06-14

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

BGL6 beta-glucosidase and nucleic acids encoding the same

DOEpatents

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

2009-09-01

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

BGL3 beta-glucosidase and nucleic acids encoding the same

DOEpatents

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

2012-10-30

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

BGL4 beta-glucosidase and nucleic acids encoding the same

DOEpatents

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

2008-01-22

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

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

PubMed

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

2015-02-01

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

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

PubMed Central

Kurz, Matthias

2008-01-01

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

Multiplexed microfluidic approach for nucleic acid enrichment

DOEpatents

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

2016-04-26

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

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

PubMed

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

2016-04-07

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

Detection and prevention of mycoplasma hominis infection

DOEpatents

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

1997-01-21

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

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

PubMed Central

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

2017-01-01

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

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

PubMed Central

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

2015-01-01

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

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

PubMed

Kasahara, Yuuya; Kuwahara, Masayasu

2012-01-01

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

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.

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

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

Matthew Mihelic, F.

2010-12-22

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

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

NASA Astrophysics Data System (ADS)

Matthew Mihelic, F.

2010-12-01

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

The illusion of specific capture: surface and solution studies of suboptimal oligonucleotide hybridization

PubMed Central

2013-01-01

Background Hybridization based assays and capture systems depend on the specificity of hybridization between a probe and its intended target. A common guideline in the construction of DNA microarrays, for instance, is that avoiding complementary stretches of more than 15 nucleic acids in a 50 or 60-mer probe will eliminate sequence specific cross-hybridization reactions. Here we present a study of the behavior of partially matched oligonucleotide pairs with complementary stretches starting well below this threshold complementarity length – in silico, in solution, and at the microarray surface. The modeled behavior of pairs of oligonucleotide probes and their targets suggests that even a complementary stretch of sequence 12 nt in length would give rise to specific cross-hybridization. We designed a set of binding partners to a 50-mer oligonucleotide containing complementary stretches from 6 nt to 21 nt in length. Results Solution melting experiments demonstrate that stable partial duplexes can form when only 12 bp of complementary sequence are present; surface hybridization experiments confirm that a signal close in magnitude to full-strength signal can be obtained from hybridization of a 12 bp duplex within a 50mer oligonucleotide. Conclusions Microarray and other molecular capture strategies that rely on a 15 nt lower complementarity bound for eliminating specific cross-hybridization may not be sufficiently conservative. PMID:23445545

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.

Methods for making nucleotide probes for sequencing and synthesis

DOEpatents

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

2014-07-08

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

Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization

PubMed Central

Girard, Laurie D.; Boissinot, Karel; Peytavi, Régis; Boissinot, Maurice; Bergeron, Michel G.

2014-01-01

The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have a high complexity cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotidesequence-dependent segment and a unique “target sequence-independent” 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design of lab-on-chip microfluidic devices, while also reducing consumable costs. At term, it will allow the cost-effective automation of highly multiplexed assays for detection and identification of genetic targets. PMID:25489607

Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization.

PubMed

Girard, Laurie D; Boissinot, Karel; Peytavi, Régis; Boissinot, Maurice; Bergeron, Michel G

2015-02-07

The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have high complexity and cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotide sequence-dependent segment and a unique "target sequence-independent" 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design of lab-on-chip microfluidic devices, while also reducing consumable costs. At term, it will allow the cost-effective automation of highly multiplexed assays for detection and identification of genetic targets.

Open-target sparse sensing of biological agents using DNA microarray

PubMed Central

2011-01-01

Background Current biosensors are designed to target and react to specific nucleic acid sequences or structural epitopes. These 'target-specific' platforms require creation of new physical capture reagents when new organisms are targeted. An 'open-target' approach to DNA microarray biosensing is proposed and substantiated using laboratory generated data. The microarray consisted of 12,900 25 bp oligonucleotide capture probes derived from a statistical model trained on randomly selected genomic segments of pathogenic prokaryotic organisms. Open-target detection of organisms was accomplished using a reference library of hybridization patterns for three test organisms whose DNA sequences were not included in the design of the microarray probes. Results A multivariate mathematical model based on the partial least squares regression (PLSR) was developed to detect the presence of three test organisms in mixed samples. When all 12,900 probes were used, the model correctly detected the signature of three test organisms in all mixed samples (mean(R2)) = 0.76, CI = 0.95), with a 6% false positive rate. A sampling algorithm was then developed to sparsely sample the probe space for a minimal number of probes required to capture the hybridization imprints of the test organisms. The PLSR detection model was capable of correctly identifying the presence of the three test organisms in all mixed samples using only 47 probes (mean(R2)) = 0.77, CI = 0.95) with nearly 100% specificity. Conclusions We conceived an 'open-target' approach to biosensing, and hypothesized that a relatively small, non-specifically designed, DNA microarray is capable of identifying the presence of multiple organisms in mixed samples. Coupled with a mathematical model applied to laboratory generated data, and sparse sampling of capture probes, the prototype microarray platform was able to capture the signature of each organism in all mixed samples with high sensitivity and specificity. It was demonstrated that this new approach to biosensing closely follows the principles of sparse sensing. PMID:21801424

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

NASA Astrophysics Data System (ADS)

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

2017-08-01

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

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.

Promoters and proteins from Clostridium thermocellum and uses thereof

DOEpatents

Wu, J. H. David; Newcomb, Michael

2012-11-13

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

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

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

2007-09-18

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

9 CFR 121.1 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-01-01

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

9 CFR 121.1 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-01-01

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

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

PubMed Central

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

2012-01-01

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

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…

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

DOEpatents

Bearinger, Jane P.; Dugan, Lawrence C.

2016-04-19

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

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

DOEpatents

Bearinger, Jane P.; Dugan, Lawrence C.

2015-12-29

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

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.

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

Code of Federal Regulations, 2010 CFR

2010-04-01

... HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5910 Quality control material for cystic fibrosis nucleic acid assays. (a... cystic fibrosis nucleic acid assays is a device intended to help monitor reliability of a test system by...

Nucleic acids encoding mosaic clade M human immunodeficiency virus type 1 (HIV-1) envelope immunogens

DOEpatents

Korber, Bette T; Fischer, William; Liao, Hua-Xin; Haynes, Barton F; Letvin, Norman; Hahn, Beatrice H

2015-04-21

The present invention relates to nucleic acids encoding mosaic clade M HIV-1 Env polypeptides and to compositions and vectors comprising same. The nucleic acids of the invention are suitable for use in inducing an immune response to HIV-1 in a human.

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.

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.

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.

[Genotoxic modification of nucleic acid bases and biological consequences of it. Review and prospects of experimental and computational investigations

NASA Technical Reports Server (NTRS)

Poltev, V. I.; Bruskov, V. I.; Shuliupina, N. V.; Rein, R.; Shibata, M.; Ornstein, R.; Miller, J.

1993-01-01

The review is presented of experimental and computational data on the influence of genotoxic modification of bases (deamination, alkylation, oxidation) on the structure and biological functioning of nucleic acids. Pathways are discussed for the influence of modification on coding properties of bases, on possible errors of nucleic acid biosynthesis, and on configurations of nucleotide mispairs. The atomic structure of nucleic acid fragments with modified bases and the role of base damages in mutagenesis and carcinogenesis are considered.

Hybridization-based biosensor containing hairpin probes and use thereof

DOEpatents

Miller, Benjamin L.; Strohsahl, Christopher M.

2010-10-12

A sensor chip that includes: a fluorescence quenching surface; a nucleic acid probe that contains first and second ends with the first end bound to the fluorescence quenching surface, and is characterized by being able to self-anneal into a hairpin conformation; and a first fluorophore bound to the second end of the first nucleic acid molecule. When the first nucleic acid molecule is in the hairpin conformation, the fluorescence quenching surface substantially quenches fluorescent emissions by the first fluorophore; and when the first nucleic acid molecule is in a non-hairpin conformation, fluorescent emissions by the fluorophore are substantially free of quenching by the fluorescence quenching surface. Various nucleic acid probes, methods of making the sensor chip, biological sensor devices that contain the sensor chip, and their methods of use are also disclosed.

Nucleic acid duplexes incorporating a dissociable covalent base pair

PubMed Central

Gao, Kui; Orgel, Leslie E.

1999-01-01

We have used molecular modeling techniques to design a dissociable covalently bonded base pair that can replace a Watson-Crick base pair in a nucleic acid with minimal distortion of the structure of the double helix. We introduced this base pair into a potential precursor of a nucleic acid double helix by chemical synthesis and have demonstrated efficient nonenzymatic template-directed ligation of the free hydroxyl groups of the base pair with appropriate short oligonucleotides. The nonenzymatic ligation reactions, which are characteristic of base paired nucleic acid structures, are abolished when the covalent base pair is reduced and becomes noncoplanar. This suggests that the covalent base pair linking the two strands in the duplex is compatible with a minimally distorted nucleic acid double-helical structure. PMID:10611299

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

PubMed

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

2015-02-28

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

Plants having modified response to ethylene by transformation with an ETR nucleic acid

DOEpatents

Meyerowitz, Elliott M.; Chang, Caren; Bleecker, Anthony B.

2001-01-01

The invention includes transformed plants having at least one cell transformed with a modified ETR nucleic acid. Such plants have a phenotype characterized by a decrease in the response of at least one transformed plant cell to ethylene as compared to a plant not containing the transformed plant cell. Tissue and/or temporal specificity for expression of the modified ETR nucleic acid is controlled by selecting appropriate expression regulation sequences to target the location and/or time of expression of the transformed nucleic acid. The plants are made by transforming at least one plant cell with an appropriate modified ETR nucleic acid, regenerating plants from one or more of the transformed plant cells and selecting at least one plant having the desired phenotype.

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.

A Magnetic Nanoparticle Based Nucleic Acid Isolation and Purification Instrument for DNA Extraction of Escherichia Coli O157: H7.

PubMed

Chen, Yahui; Lin, Jianhan; Jiang, Qin; Chen, Qi; Zhang, Shengjun; Li, Li

2016-03-01

The objective of this study was to evaluate the performance of a nucleic acid isolation and purification instrument using Escherichia coli O157:H7 as the model. The instrument was developed with magnetic nanoparticles for efficiently capturing nucleic acids and an intelligent mechanical unit for automatically performing the whole nucleic acid extraction process. A commercial DNA extraction kit from Huier Nano Company was used as reference. Nucleic acids in 1 ml of E. coli O157: H7 at a concentration of 5 x 10(8) CFU/mL were extracted by using this instrument and the kit in parallel and then detected by an ultraviolet spectrophotometer to obtain A260 values and A260/A280 values for the determination of the extracted DNA's quantity and purity, respectively. The A260 values for the instrument and the kit were 0.78 and 0.61, respectively, and the A260/A280 values were 1.98 and 1.93. The coefficient of variations of these parallel tests ranged from 10.5% to 16.7%. The results indicated that this nucleic acid isolation and purification instrument could extract a comparable level of nucleic acid within 50 min compared to the commercial DNA extraction kit.

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.

Identification of Nucleic Acid Binding Sites on Translin-Associated Factor X (TRAX) Protein

PubMed Central

Gupta, Gagan Deep; Kumar, Vinay

2012-01-01

Translin and TRAX proteins play roles in very important cellular processes such as DNA recombination, spatial and temporal expression of mRNA, and in siRNA processing. Translin forms a homomeric nucleic acid binding complex and binds to ssDNA and RNA. However, a mutant translin construct that forms homomeric complex lacking nucleic acid binding activity is able to form fully active heteromeric translin-TRAX complex when co-expressed with TRAX. A substantial progress has been made in identifying translin sites that mediate its binding activity, while TRAX was thought not to bind DNA or RNA on its own. We here for the first time demonstrate nucleic acid binding to TRAX by crosslinking radiolabeled ssDNA to heteromeric translin-TRAX complex using UV-laser. The TRAX and translin, photochemically crosslinked with ssDNA, were individually detected on SDS-PAGE. We mutated two motifs in TRAX and translin, designated B2 and B3, to help define the nucleic acid binding sites in the TRAX sequence. The most pronounced effect was observed in the mutants of B3 motif that impaired nucleic acid binding activity of the heteromeric complexes. We suggest that both translin and TRAX are binding competent and contribute to the nucleic acid binding activity. PMID:22427937

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

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

PubMed Central

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

2017-01-01

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

Methods for molecular surveillance of influenza.

PubMed

Wang, Ruixue; Taubenberger, Jeffery K

2010-05-01

Molecular-based techniques for detecting influenza viruses have become an integral component of human and animal surveillance programs in the last two decades. The recent pandemic of the swine-origin influenza A virus (H1N1) and the continuing circulation of highly pathogenic avian influenza A virus (H5N1) further stress the need for rapid and accurate identification and subtyping of influenza viruses for surveillance, outbreak management, diagnosis and treatment. There has been remarkable progress on the detection and molecular characterization of influenza virus infections in clinical, mammalian, domestic poultry and wild bird samples in recent years. The application of these techniques, including reverse transcriptase-PCR, real-time PCR, microarrays and other nucleic acid sequencing-based amplifications, have greatly enhanced the capability for surveillance and characterization of influenza viruses.

Application of nanomaterials in the bioanalytical detection of disease-related genes.

PubMed

Zhu, Xiaoqian; Li, Jiao; He, Hanping; Huang, Min; Zhang, Xiuhua; Wang, Shengfu

2015-12-15

In the diagnosis of genetic diseases and disorders, nanomaterials-based gene detection systems have significant advantages over conventional diagnostic systems in terms of simplicity, sensitivity, specificity, and portability. In this review, we describe the application of nanomaterials for disease-related genes detection in different methods excluding PCR-related method, such as colorimetry, fluorescence-based methods, electrochemistry, microarray methods, surface-enhanced Raman spectroscopy (SERS), quartz crystal microbalance (QCM) methods, and dynamic light scattering (DLS). The most commonly used nanomaterials are gold, silver, carbon and semiconducting nanoparticles. Various nanomaterials-based gene detection methods are introduced, their respective advantages are discussed, and selected examples are provided to illustrate the properties of these nanomaterials and their emerging applications for the detection of specific nucleic acid sequences. Copyright © 2015. Published by Elsevier B.V.

Thermodynamics of Nucleic Acid ‘Shape Readout’ by an Aminosugar†

PubMed Central

Xi, Hongjuan; Davis, Erik; Ranjan, Nihar; Xue, Liang; Hyde-Volpe, David; Arya, Dev P.

2012-01-01

Recognition of nucleic acids is important for our understanding of nucleic acid structure as well as for our understanding of nucleic acid-protein interactions. In addition to the direct readout mechanisms of nucleic acids such as H-bonding, shape recognition of nucleic acids is being increasingly recognized to play an equally important role in DNA recognition. Competition Dialysis, UV, Flourescent Intercalator displacement (FID), Computational Docking, and calorimetry studies were conducted to study the interaction of neomycin with a variety of nucleic acid conformations (shapes). At pH 5.5, these results suggest: (1) Neomycin binds three RNA structures (16S A site rRNA, poly(rA)•poly(rA), and poly(rA)•poly(rU)) with high affinities, Ka~107M−1. (2) The binding of neomycin to A-form GC-rich oligomer d(A2G15C15T2)2 has comparable affinity to RNA structures. (3) The binding of neomycin to DNA•RNA hybrids shows a three-fold variance attributable to their structural differences (poly(dA) •poly(rU), Ka=9.4×106M−1 and poly(rA)•poly(dT), Ka=3.1×106M−1). (4) The interaction of neomycin with DNA triplex poly(dA)•2poly(dT) yields a binding affinity of Ka=2.4×105M−1. (5) Poly(dA-dT)2 showed the lowest association constant for all nucleic acids studied (Ka=<105). (6) Neomycin binds to G-quadruplexes with Ka~104-105M−1. (7) Computational studies show that the decrease in major groove width in the B to A transition correlates with increasing neomycin affinity. Neomycin’s affinity for various nucleic acid structures can be ranked as follows, RNAs and GC-rich d(A2G15C15T2)2 structures > poly(dA)•poly(rU) > poly(rA)•poly(dT) > T•A-T triplex , G-quadruplexes, B-form AT-rich or GC-rich DNA sequences. The results illustrate the first example of a small molecule based ‘shape readout’ of different nucleic acid conformations. PMID:21863895

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.

Arginine-based poly(ester amide) nanoparticle platform: From structure-property relationship to nucleic acid delivery.

PubMed

You, Xinru; Gu, Zhipeng; Huang, Jun; Kang, Yang; Chu, Chih-Chang; Wu, Jun

2018-05-25

Many different types of polycations have been vigorously studied for nucleic acid delivery, but a systematical investigation of the structure-property relationships of polycations for nucleic acid delivery is still lacking. In this study, a new library of biodegradable and biocompatible arginine-based poly(ester amide) (Arg-PEA) biomaterials was designed and synthesized with a tunable structure for such a comprehensive structure-property research. Nanoparticle (NP) complexes were formed through the electrostatic interactions between the polycationic Arg-PEAs and anionic nucleic acids. The following structure effects of the Arg-PEAs on the transfection efficiency of nucleic acids were investigated: 1) the linker/spacer length (length effect and odd-even effect); 2) salt type of arginine; 3) the side chain; 4) chain stiffness; 5) molecular weight (MW). The data obtained revealed that a slight change in the Arg-PEA structure could finely tune its physicochemical property such as hydrophobicity, and this could subsequently affect the nanoparticle size and zeta potential, which, in turn, regulate the transfection efficiency and silencing outcomes. A further study of the Arg-PEA/CpG oligodeoxynucleotide NP complexes indicated that the polymer structure could precisily regulate the immune response of CpG, thus providing a new potential nano-immunotherapy strategy. The in vitro data have further confirmed that the Arg-PEA NPs showed a satisfactory delivery performance for a variety of nucleic acids. Therefore, the data from the current study provide comprehensive information about the Arg-PEA structure-transfection property relationship; the tunable property of the library of Arg-PEA biomaterials can be one of the promising candidates for nucleic acid delivery and other biomedical applications. Polycations have being intensive utilized for nucleic acid delivery. However, there has not been elucidated about the relationship between polycation's structure and the physicochemical properties/biological function. In this timely report, an arginine based poly(ester amide) (Arg-PEA) library was prepared with finely tunable structure to systematically investigate the structure-property relationships of polycations for nucleic acid delivery. The results revealed that slight change of Arg-PEA structure could finely tune the physicochemical property (such as hydrophobicity), which subsequently affect the size and zeta potential of Arg-PEA/nucleic acid nanoparticles(NPs), and finally regulate the resulting transfection or silencing outcomes. Further study of Arg-PEA/CpG NPs indicated that the polymer structure could precisely regulate immuno response of CpG, providing new potential nano-immunotherapy strategy. In vitro evaluations confirmed that the NPs showed satisfied delivery performance for a variety types of nucleic acids. Therefore, these studies provide comprehensive information of Arg-PEA structure-property relationship, and the tunable properties of Arg-PEAs make them promising candidates for nucleic acid delivery and other biomedical applications. Overall, we have shown enough significance and novelty in terms of nucleic acid delivery, biomaterials, pharmaceutical science and nanomedicine. Copyright © 2018. Published by Elsevier Ltd.

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

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Quality control material for cystic fibrosis... Test Systems § 866.5910 Quality control material for cystic fibrosis nucleic acid assays. (a) Identification. Quality control material for cystic fibrosis nucleic acid assays. A quality control material for...

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...

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

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Quality control material for cystic fibrosis... Test Systems § 866.5910 Quality control material for cystic fibrosis nucleic acid assays. (a) Identification. Quality control material for cystic fibrosis nucleic acid assays. A quality control material for...

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...

Magnetically enhanced nucleic acid delivery. Ten years of magnetofection-progress and prospects.

PubMed

Plank, Christian; Zelphati, Olivier; Mykhaylyk, Olga

2011-11-01

Nucleic acids carry the building plans of living systems. As such, they can be exploited to make cells produce a desired protein, or to shut down the expression of endogenous genes or even to repair defective genes. Hence, nucleic acids are unique substances for research and therapy. To exploit their potential, they need to be delivered into cells which can be a challenging task in many respects. During the last decade, nanomagnetic methods for delivering and targeting nucleic acids have been developed, methods which are often referred to as magnetofection. In this review we summarize the progress and achievements in this field of research. We discuss magnetic formulations of vectors for nucleic acid delivery and their characterization, mechanisms of magnetofection, and the application of magnetofection in viral and nonviral nucleic acid delivery in cell culture and in animal models. We summarize results that have been obtained with using magnetofection in basic research and in preclinical animal models. Finally, we describe some of our recent work and end with some conclusions and perspectives. Copyright © 2011 Elsevier B.V. All rights reserved.

Reduction of Nucleic Acid Content in Candida Yeast Cells by Bovine Pancreatic Ribonuclease A Treatment

PubMed Central

Castro, A. C.; Sinskey, A. J.; Tannenbaum, S. R.

1971-01-01

Yeast as a source of protein for human consumption is limited by its relatively high nucleic acid content. In this study, we developed an enzymatic method of decreasing the nucleic acid content. Candida utilis cells, heat-shocked at 80 C for 30 sec, were treated with bovine pancreatic ribonuclease A. Maximum leakage of nucleic acid was observed when the incubation temperature was between 55 and 65 C, the pH of the system from 6.75 to 8.0, and the enzyme-to-cell ratio 1:10,000 on a weight-by-weight basis. Other factors, such as yeast strain, age of cells, and method of propagation, did not influence the susceptibility of the yeast cells to the action of ribonuclease. Buffers and monovalent cations had no inhibiting effects. Magnesium and calcium ions at concentrations greater than 0.001 m showed marked inhibition on the rate of nucleic acid leakage. This enzymatic method reduced the nucleic acid content of yeast cells from 7.5 to 9.0% to 1.5 to 2.0% with no significant concomitant loss of protein. PMID:5165838

Enabling environmental metagenomics and extremophile discovery through SCODA DNA purification

NASA Astrophysics Data System (ADS)

Lum, T.; Maydan, J.

2016-12-01

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

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).

Nucleic acid polymeric properties and electrostatics: Directly comparing theory and simulation with experiment.

PubMed

Sim, Adelene Y L

2016-06-01

Nucleic acids are biopolymers that carry genetic information and are also involved in various gene regulation functions such as gene silencing and protein translation. Because of their negatively charged backbones, nucleic acids are polyelectrolytes. To adequately understand nucleic acid folding and function, we need to properly describe its i) polymer/polyelectrolyte properties and ii) associating ion atmosphere. While various theories and simulation models have been developed to describe nucleic acids and the ions around them, many of these theories/simulations have not been well evaluated due to complexities in comparison with experiment. In this review, I discuss some recent experiments that have been strategically designed for straightforward comparison with theories and simulation models. Such data serve as excellent benchmarks to identify limitations in prevailing theories and simulation parameters. Copyright © 2015 Elsevier B.V. All rights reserved.

Heat Capacity Changes Associated with Nucleic Acid Folding

PubMed Central

Mikulecky, Peter J.; Feig, Andrew L.

2008-01-01

Whereas heat capacity changes (ΔCPs) associated with folding transitions are commonplace in the literature of protein folding, they have long been considered a minor energetic contributor in nucleic acid folding. Recent advances in the understanding of nucleic acid folding and improved technology for measuring the energetics of folding transitions have allowed a greater experimental window for measuring these effects. We present in this review a survey of current literature that confronts the issue of ΔCPs associated with nucleic acid folding transitions. This work helps to gather the molecular insights that can be gleaned from analysis of ΔCPs and points toward the challenges that will need to be overcome if the energetic contribution of ΔCP terms are to be put to use in improving free energy calculations for nucleic acid structure prediction. PMID:16429398

Fluorogenic, catalytic, photochemical reaction for amplified detection of nucleic acids.

PubMed

Dutta, Subrata; Fülöp, Annabelle; Mokhir, Andriy

2013-09-18

Photochemical, nucleic acid-induced reactions, which are controlled by nontoxic red light, are well-suited for detection of nucleic acids in live cells, since they do not require any additives and can be spatially and temporally regulated. We have recently described the first reaction of this type, in which a phenylselenyl derivative of thymidine (5'-PhSeT-ODNa) is cleaved in the presence of singlet oxygen (Fülöp, A., Peng, X., Greenberg, M. M., Mokhir, A. (2010) A nucleic acid directed, red light-induced chemical reaction. Chem. Commun. 46, 5659-5661). The latter reagent is produced upon exposure of a photosensitizer 3'-PS-ODNb (PS = Indium(III)-pyropheophorbide-a-chloride: InPPa) to >630 nm light. In 2012 we reported on a fluorogenic version of this reaction (Dutta, S., Flottmann, B., Heilemann, M., Mokhir, A. (2012) Hybridization and reaction-based, fluorogenic nucleic acid probes. Chem. Commun. 47, 9664-9666), which is potentially applicable for the detection of nucleic acids in cells. Unfortunately, its yield does not exceed 25% and no catalytic turnover could be observed in the presence of substrate excess. This problem occurs due to the efficient, competing oxidation of the substrate containing an electron rich carbon-carbon double bonds (SCH═CHS) in the presence of singlet oxygen with formation of a noncleavable product (SCH═CHSO). Herein we describe a related, but substantially improved photochemical, catalytic transformation of a fluorogenic, organic substrate, which consists of 9,10-dialkoxyanthracene linked to fluorescein, with formation of a bright fluorescent dye. In highly dilute solution this reaction occurs only in the presence of a nucleic acid template. We developed three types of such a reaction and demonstrated that they are high yielding and generate over 7.7 catalytic turnovers, are sensitive to single mismatches in nucleic acid targets, and can be applied for determination of both the amount of nucleic acids and potentially their localization.

UniPROBE, update 2015: new tools and content for the online database of protein-binding microarray data on protein-DNA interactions.

PubMed

Hume, Maxwell A; Barrera, Luis A; Gisselbrecht, Stephen S; Bulyk, Martha L

2015-01-01

The Universal PBM Resource for Oligonucleotide Binding Evaluation (UniPROBE) serves as a convenient source of information on published data generated using universal protein-binding microarray (PBM) technology, which provides in vitro data about the relative DNA-binding preferences of transcription factors for all possible sequence variants of a length k ('k-mers'). The database displays important information about the proteins and displays their DNA-binding specificity data in terms of k-mers, position weight matrices and graphical sequence logos. This update to the database documents the growth of UniPROBE since the last update 4 years ago, and introduces a variety of new features and tools, including a new streamlined pipeline that facilitates data deposition by universal PBM data generators in the research community, a tool that generates putative nonbinding (i.e. negative control) DNA sequences for one or more proteins and novel motifs obtained by analyzing the PBM data using the BEEML-PBM algorithm for motif inference. The UniPROBE database is available at http://uniprobe.org. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Versatile Gene-Specific Sequence Tags for Arabidopsis Functional Genomics: Transcript Profiling and Reverse Genetics Applications

PubMed Central

Hilson, Pierre; Allemeersch, Joke; Altmann, Thomas; Aubourg, Sébastien; Avon, Alexandra; Beynon, Jim; Bhalerao, Rishikesh P.; Bitton, Frédérique; Caboche, Michel; Cannoot, Bernard; Chardakov, Vasil; Cognet-Holliger, Cécile; Colot, Vincent; Crowe, Mark; Darimont, Caroline; Durinck, Steffen; Eickhoff, Holger; de Longevialle, Andéol Falcon; Farmer, Edward E.; Grant, Murray; Kuiper, Martin T.R.; Lehrach, Hans; Léon, Céline; Leyva, Antonio; Lundeberg, Joakim; Lurin, Claire; Moreau, Yves; Nietfeld, Wilfried; Paz-Ares, Javier; Reymond, Philippe; Rouzé, Pierre; Sandberg, Goran; Segura, Maria Dolores; Serizet, Carine; Tabrett, Alexandra; Taconnat, Ludivine; Thareau, Vincent; Van Hummelen, Paul; Vercruysse, Steven; Vuylsteke, Marnik; Weingartner, Magdalena; Weisbeek, Peter J.; Wirta, Valtteri; Wittink, Floyd R.A.; Zabeau, Marc; Small, Ian

2004-01-01

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics. PMID:15489341

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.

Metal resistant plants and phytoremediation of environmental contamination

DOEpatents

Meagher, Richard B.; Li, Yujing; Dhankher, Om P.

2010-04-20

The present disclosure provides a method of producing transgenic plants which are resistant to at least one metal ion by transforming the plant with a recombinant DNA comprising a nucleic acid encoding a bacterial arsenic reductase under the control of a plant expressible promoter, and a nucleic acid encoding a nucleotide sequence encoding a phytochelatin biosynthetic enzyme under the control of a plant expressible promoter. The invention also relates a method of phytoremediation of a contaminated site by growing in the site a transgenic plant expressing a nucleic acid encoding a bacterial arsenate reductase and a nucleic acid encoding a phytochelatin biosynthetic enzyme.

Lateral flow devices

DOEpatents

Mazumdar, Debapriya; Liu, Juewen; Lu, Yi

2010-09-21

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

Microfluidic device for acoustic cell lysis

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

Branch, Darren W.; Cooley, Erika Jane; Smith, Gennifer Tanabe

2015-08-04

A microfluidic acoustic-based cell lysing device that can be integrated with on-chip nucleic acid extraction. Using a bulk acoustic wave (BAW) transducer array, acoustic waves can be coupled into microfluidic cartridges resulting in the lysis of cells contained therein by localized acoustic pressure. Cellular materials can then be extracted from the lysed cells. For example, nucleic acids can be extracted from the lysate using silica-based sol-gel filled microchannels, nucleic acid binding magnetic beads, or Nafion-coated electrodes. Integration of cell lysis and nucleic acid extraction on-chip enables a small, portable system that allows for rapid analysis in the field.

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.

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

PubMed

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

2016-02-04

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

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.

Logic gates and antisense DNA devices operating on a translator nucleic Acid scaffold.

PubMed

Shlyahovsky, Bella; Li, Yang; Lioubashevski, Oleg; Elbaz, Johann; Willner, Itamar

2009-07-28

A series of logic gates, "AND", "OR", and "XOR", are designed using a DNA scaffold that includes four "footholds" on which the logic operations are activated. Two of the footholds represent input-recognition strands, and these are blocked by complementary nucleic acids, whereas the other two footholds are blocked by nucleic acids that include the horseradish peroxidase (HRP)-mimicking DNAzyme sequence. The logic gates are activated by either nucleic acid inputs that hybridize to the respective "footholds", or by low-molecular-weight inputs (adenosine monophosphate or cocaine) that yield the respective aptamer-substrate complexes. This results in the respective translocation of the blocking nucleic acids to the footholds carrying the HRP-mimicking DNAzyme sequence, and the concomitant release of the respective DNAzyme. The released product-strands then self-assemble into the hemin/G-quadruplex-HRP-mimicking DNAzyme that biocatalyzes the formation of a colored product and provides an output signal for the different logic gates. The principle of the logic operation is, then, implemented as a possible paradigm for future nanomedicine. The nucleic acid inputs that bind to the blocked footholds result in the translocation of the blocking nucleic acids to the respective footholds carrying the antithrombin aptamer. The released aptamer inhibits, then, the hydrolytic activity of thrombin. The system demonstrates the regulation of a biocatalytic reaction by a translator system activated on a DNA scaffold.

Fatty acid-producing hosts

DOEpatents

Pfleger, Brian F; Lennen, Rebecca M

2013-12-31

Described are hosts for overproducing a fatty acid product such as a fatty acid. The hosts include an exogenous nucleic acid encoding a thioesterase and, optionally, an exogenous nucleic acid encoding an acetyl-CoA carboxylase, wherein an acyl-CoA synthetase in the hosts are functionally delected. The hosts prefereably include the nucleic acid encoding the thioesterase at an intermediate copy number. The hosts are preferably recominantly stable and growth-competent at 37.degree. C. Methods of producing a fatty acid product comprising culturing such hosts at 37.degree. C. are also described.

Oxidative Stress and Nucleic Acid Oxidation in Patients with Chronic Kidney Disease

PubMed Central

Sung, Chih-Chien; Hsu, Yu-Chuan; Lin, Yuh-Feng

2013-01-01

Patients with chronic kidney disease (CKD) have high cardiovascular mortality and morbidity and a high risk for developing malignancy. Excessive oxidative stress is thought to play a major role in elevating these risks by increasing oxidative nucleic acid damage. Oxidative stress results from an imbalance between reactive oxygen/nitrogen species (RONS) production and antioxidant defense mechanisms and can cause vascular and tissue injuries as well as nucleic acid damage in CKD patients. The increased production of RONS, impaired nonenzymatic or enzymatic antioxidant defense mechanisms, and other risk factors including gene polymorphisms, uremic toxins (indoxyl sulfate), deficiency of arylesterase/paraoxonase, hyperhomocysteinemia, dialysis-associated membrane bioincompatibility, and endotoxin in patients with CKD can inhibit normal cell function by damaging cell lipids, arachidonic acid derivatives, carbohydrates, proteins, amino acids, and nucleic acids. Several clinical biomarkers and techniques have been used to detect the antioxidant status and oxidative stress/oxidative nucleic acid damage associated with long-term complications such as inflammation, atherosclerosis, amyloidosis, and malignancy in CKD patients. Antioxidant therapies have been studied to reduce the oxidative stress and nucleic acid oxidation in patients with CKD, including alpha-tocopherol, N-acetylcysteine, ascorbic acid, glutathione, folic acid, bardoxolone methyl, angiotensin-converting enzyme inhibitor, and providing better dialysis strategies. This paper provides an overview of radical production, antioxidant defence, pathogenesis and biomarkers of oxidative stress in patients with CKD, and possible antioxidant therapies. PMID:24058721

Red-shifted fluorescent proteins mPlum and mRaspberry and polynucleotides encoding the same

DOEpatents

Tsien, Roger Y [La Jolla, CA; Wang, Lei [San Diego, CA

2008-07-01

Methods using somatic hypermutation (SHM) for producing polypeptide and nucleic acid variants, and nucleic acids encoding such polypeptide variants are disclosed. Such variants may have desired properties. Also disclosed are novel polypeptides, such as improved fluorescent proteins, produced by the novel methods, and nucleic acids, vectors, and host cells comprising such vectors.

Methods for engineering polypeptide variants via somatic hypermutation and polypeptide made thereby

DOEpatents

Tsien, Roger Y; Wang, Lei

2015-01-13

Methods using somatic hypermutation (SHM) for producing polypeptide and nucleic acid variants, and nucleic acids encoding such polypeptide variants are disclosed. Such variants may have desired properties. Also disclosed are novel polypeptides, such as improved fluorescent proteins, produced by the novel methods, and nucleic acids, vectors, and host cells comprising such vectors.

STUDIES OF THE MECHANISM OF ACTION OF URETHANE IN INITIATING PULMONARY ADENOMAS IN MICE

PubMed Central

Rogers, Stanfield

1957-01-01

The process of carcinogenesis following exposure of mice to urethane is demonstrated in the present work to be intimately related to nucleic acid synthesis. Injection of animals with a DNA hydrolysate immediately prior to a single exposure of the animals to urethane markedly reduced the number of pulmonary adenomas initiated. Aminopterin, known to interfere in nucleic acid synthesis (46), potentiated the carcinogenic action of urethane and this potentiation was blocked by injection of a DNA hydrolysate. Of the components and precursors of nucleic acids the pyrimidine series seemed especially concerned. Alterations in the utilization of oxaloacetate, ureidosuccinic acid, dihydro-orotic acid, orotic acid, cytidylic acid, and thymine appeared to be critical steps in the oncogenic process, following upon the primary disorder of cellular metabolism initiated by the carcinogen. All these substances except oxaloacetate profoundly reduced the number of tumors initiated by urethane. Oxaloacetate potentiated the carcinogenic effect. When these results are viewed together and in relation to known facts concerning nucleic acid synthesis they provide evidence suggesting that the point of action of the carcinogen is in the pathway of nucleic acid synthesis below orotic acid and perhaps at the level of ureidosuccinic acid. The potentiating influence of adenine, 4-amino-5-imidazole carboxamide, and aminopterin, the lack of effect of uracil, and the inhibitory influence of thymine together suggest that DNA rather than RNA is the nucleic acid critical to the oncogenic response of mice to urethane. PMID:13416469

Nucleic acid sensing and innate immunity: signaling pathways controlling viral pathogenesis and autoimmunity.

PubMed

Ahlers, Laura R H; Goodman, Alan G

2016-09-01

Innate immunity refers to the body's initial response to curb infection upon exposure to invading organisms. While the detection of pathogen-associated molecules is an ancient form of host defense, if dysfunctional, autoimmune disease may result. The innate immune response during pathogenic infection is initiated through the activation of receptors recognizing conserved molecular patterns, such as nucleic acids from a virus' genome or replicative cycle. Additionally, the host's own nucleic acids are capable of activating an immune response. Therefore, it follows that the nucleic acid-sensing pathways must be tightly controlled to avoid an autoimmune response from recognition of self, yet still be unimpeded to respond to viral infections. In this review, we will describe the nucleic acid sensing pathways and how they respond to virus infection. Moreover, we will discuss autoimmune diseases that develop when these pathways fail to signal properly and identify knowledge gaps that are prime for interrogation.

Gene Therapy for Advanced Melanoma: Selective Targeting and Therapeutic Nucleic Acids

PubMed Central

Viola, Joana R.; Rafael, Diana F.; Wagner, Ernst; Besch, Robert; Ogris, Manfred

2013-01-01

Despite recent advances, the treatment of malignant melanoma still results in the relapse of the disease, and second line treatment mostly fails due to the occurrence of resistance. A wide range of mutations are known to prevent effective treatment with chemotherapeutic drugs. Hence, approaches with biopharmaceuticals including proteins, like antibodies or cytokines, are applied. As an alternative, regimens with therapeutically active nucleic acids offer the possibility for highly selective cancer treatment whilst avoiding unwanted and toxic side effects. This paper gives a brief introduction into the mechanism of this devastating disease, discusses the shortcoming of current therapy approaches, and pinpoints anchor points which could be harnessed for therapeutic intervention with nucleic acids. We bring the delivery of nucleic acid nanopharmaceutics into perspective as a novel antimelanoma therapeutic approach and discuss the possibilities for melanoma specific targeting. The latest reports on preclinical and already clinical application of nucleic acids in melanoma are discussed. PMID:23634303

Nucleic acid and nucleotide-mediated synthesis of inorganic nanoparticles

NASA Astrophysics Data System (ADS)

Berti, Lorenzo; Burley, Glenn A.

2008-02-01

Since the advent of practical methods for achieving DNA metallization, the use of nucleic acids as templates for the synthesis of inorganic nanoparticles (NPs) has become an active area of study. It is now widely recognized that nucleic acids have the ability to control the growth and morphology of inorganic NPs. These biopolymers are particularly appealing as templating agents as their ease of synthesis in conjunction with the possibility of screening nucleotide composition, sequence and length, provides the means to modulate the physico-chemical properties of the resulting NPs. Several synthetic procedures leading to NPs with interesting photophysical properties as well as studies aimed at rationalizing the mechanism of nucleic acid-templated NP synthesis are now being reported. This progress article will outline the current understanding of the nucleic acid-templated process and provides an up to date reference in this nascent field.

Fluorescence enhancement of quercetin complexes by silver nanoparticles and its analytical application

NASA Astrophysics Data System (ADS)

Liu, Ping; Zhao, Liangliang; Wu, Xia; Huang, Fei; Wang, Minqin; Liu, Xiaodan

2014-03-01

It is found that the plasmon effect of silver nanoparticles (AgNPs) helps to enhance the fluorescence intensity of the quercetin (Qu) and nucleic acids system. Qu exhibited strong fluorescence enhancement when it bound to nucleic acids in the presence of AgNPs. Based on this, a sensitive method for the determination of nucleic acids was developed. The detection limits for the nucleic acids (S/N = 3) were reduced to the ng mL-1 level. The interaction mechanism of the AgNPs-fish sperm DNA (fsDNA)-Qu system was also investigated in this paper. This complex system of Qu and AgNPs was also successfully used for the detection of nucleic acids in agarose gel electrophoresis analysis. Preliminary results indicated that AgNPs also helped to improve sensitivity in the fluorescence image analysis of Qu combined with cellular contents in Arabidopsis thaliana protoplasts.

Understanding Nucleic Acid–Ion Interactions

PubMed Central

Lipfert, Jan; Doniach, Sebastian; Das, Rhiju; Herschlag, Daniel

2015-01-01

Ions surround nucleic acids in what is referred to as an ion atmosphere. As a result, the folding and dynamics of RNA and DNA and their complexes with proteins and with each other cannot be understood without a reasonably sophisticated appreciation of these ions’ electrostatic interactions. However, the underlying behavior of the ion atmosphere follows physical rules that are distinct from the rules of site binding that biochemists are most familiar and comfortable with. The main goal of this review is to familiarize nucleic acid experimentalists with the physical concepts that underlie nucleic acid–ion interactions. Throughout, we provide practical strategies for interpreting and analyzing nucleic acid experiments that avoid pitfalls from oversimplified or incorrect models. We briefly review the status of theories that predict or simulate nucleic acid–ion interactions and experiments that test these theories. Finally, we describe opportunities for going beyond phenomenological fits to a next-generation, truly predictive understanding of nucleic acid–ion interactions. PMID:24606136

Exon Microarray Analysis of Human Dorsolateral Prefrontal Cortex in Alcoholism

PubMed Central

Manzardo, Ann M.; Gunewardena, Sumedha; Wang, Kun; Butler, Merlin G.

2014-01-01

Background Alcohol abuse is associated with cellular and biochemical disturbances that impact upon protein and nucleic acid synthesis, brain development, function and behavioral responses. To further characterize the genetic influences in alcoholism and the effects of alcohol consumption on gene expression, we used a highly sensitive exon microarray to examine mRNA expression in human frontal cortex of alcoholics and control males. Methods Messenger RNA was isolated from the dorsolateral prefrontal cortex (dlPFC, Brodmann area 9) of 7 adult Alcoholic (6 males, 1 female, mean age 48 years) and 7 matched controls. Affymetrix Human Exon 1.0 ST Array was performed according to standard procedures and the results analyzed at the gene level. Microarray findings were validated using qRT-PCR, and the ontology of disturbed genes characterized using Ingenuity Pathway Analysis (IPA). Results Decreased mRNA expression was observed for genes involved in cellular adhesion (e.g., CTNNA3, ITGA2), transport (e.g., TF, ABCA8), nervous system development (e.g., LRP2, UGT8, GLDN) and signaling (e.g., RASGRP, LGR5) with influence over lipid and myelin synthesis (e.g., ASPA, ENPP2, KLK6). IPA identified disturbances in network functions associated with neurological disease, and development including cellular assembly and organization impacting on psychological disorders. Conclusions Our data in alcoholism support a reduction in expression of dlPFC mRNA for genes involved with neuronal growth, differentiation and signaling that targets white matter of the brain. PMID:24890784

Exon microarray analysis of human dorsolateral prefrontal cortex in alcoholism.

PubMed

Manzardo, Ann M; Gunewardena, Sumedha; Wang, Kun; Butler, Merlin G

2014-06-01

Alcohol abuse is associated with cellular and biochemical disturbances that impact upon protein and nucleic acid synthesis, brain development, function, and behavioral responses. To further characterize the genetic influences in alcoholism and the effects of alcohol consumption on gene expression, we used a highly sensitive exon microarray to examine mRNA expression in human frontal cortex of alcoholics and control males. Messenger RNA was isolated from the dorsolateral prefrontal cortex (dlPFC; Brodmann area 9) of 7 adult alcoholic (6 males, 1 female, mean age 49 years) and 7 matched controls. Affymetrix Human Exon 1.0 ST array was performed according to standard procedures and the results analyzed at the gene level. Microarray findings were validated using quantitative reverse transcription polymerase chain reaction, and the ontology of disturbed genes characterized using Ingenuity Pathway Analysis (IPA). Decreased mRNA expression was observed for genes involved in cellular adhesion (e.g., CTNNA3, ITGA2), transport (e.g., TF, ABCA8), nervous system development (e.g., LRP2, UGT8, GLDN), and signaling (e.g., RASGRP3, LGR5) with influence over lipid and myelin synthesis (e.g., ASPA, ENPP2, KLK6). IPA identified disturbances in network functions associated with neurological disease and development including cellular assembly and organization impacting on psychological disorders. Our data in alcoholism support a reduction in expression of dlPFC mRNA for genes involved with neuronal growth, differentiation, and signaling that targets white matter of the brain. Copyright © 2014 by the Research Society on Alcoholism.

Nonenzymatic synthesis of RNA and DNA oligomers on hexitol nucleic acid templates: the importance of the A structure

NASA Technical Reports Server (NTRS)

Kozlov, I. A.; Politis, P. K.; Van Aerschot, A.; Busson, R.; Herdewijn, P.; Orgel, L. E.; Bada, J. L. (Principal Investigator); Dolan, M. (Principal Investigator)

1999-01-01

Hexitol nucleic acid (HNA) is an analogue of DNA containing the standard nucleoside bases, but with a phosphorylated 1,5-anhydrohexitol backbone. HNA oligomers form duplexes having the nucleic acid A structure with complementary DNA or RNA oligomers. The HNA decacytidylate oligomer is an efficient template for the oligomerization of the 5'-phosphoroimidazolides of guanosine or deoxyguanosine. Comparison of the oligomerization efficiencies on HNA, RNA, and DNA decacytidylate templates under various conditions suggests strongly that only nucleic acid double helices with the A structure support efficient template-directed synthesis when 5'-phosphoroimidazolides of nucleosides are used as substrates.

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

PubMed

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

2014-05-15

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

[Recombinase Polymerase Amplification and its Applications in Parasite Detection].

PubMed

ZHENG, Wen-bin; WU, Yao-dong; MA, Jian-gang; ZHU, Xing-quan; ZHOU, Dong-hui

2015-10-01

Recombinase polymerase amplification (RPA) is a recently -developed isothermal nucleic-acid-amplification technology that is based on the nucleic acid replication mechanism in T4 bacteriophage. With this technique, nucleic-acid templates can be amplified to measurable levels within 20 min at 37-42 °C. The. RPA process has high sensitivity and specificity, and is simple to operate, thus nucleic acids can be detected rapidly in non-laboratory conditions. Since its development in 2006, the RPA technique has been applied in agriculture, food safety, medicine, transgene detection, etc. In this review, we will give an overview on the research progress of RPA and its application in parasite detection.

Integrated magnetic tweezers and single-molecule FRET for investigating the mechanical properties of nucleic acid.

PubMed

Long, Xi; Parks, Joseph W; Stone, Michael D

2016-08-01

Many enzymes promote structural changes in their nucleic acid substrates via application of piconewton forces over nanometer length scales. Magnetic tweezers (MT) is a single molecule force spectroscopy method widely used for studying the energetics of such mechanical processes. MT permits stable application of a wide range of forces and torques over long time scales with nanometer spatial resolution. However, in any force spectroscopy experiment, the ability to monitor structural changes in nucleic acids with nanometer sensitivity requires the system of interest to be held under high degrees of tension to improve signal to noise. This limitation prohibits measurement of structural changes within nucleic acids under physiologically relevant conditions of low stretching forces. To overcome this challenge, researchers have integrated a spatially sensitive fluorescence spectroscopy method, single molecule-FRET, with MT to allow simultaneous observation and manipulation of nanoscale structural transitions over a wide range of forces. Here, we describe a method for using this hybrid instrument to analyze the mechanical properties of nucleic acids. We expect that this method for analysis of nucleic acid structure will be easily adapted for experiments aiming to interrogate the mechanical responses of other biological macromolecules. Copyright © 2016 Elsevier Inc. All rights reserved.

Recent Advances in Delivery of Drug-Nucleic Acid Combinations for Cancer Treatment

PubMed Central

Li, Jing; Wang, Yan; Zhu, Yu; Oupický, David

2013-01-01

Cancer treatment that uses a combination of approaches with the ability to affect multiple disease pathways has been proven highly effective in the treatment of many cancers. Combination therapy can include multiple chemotherapeutics or combinations of chemotherapeutics with other treatment modalities like surgery or radiation. However, despite the widespread clinical use of combination therapies, relatively little attention has been given to the potential of modern nanocarrier delivery methods, like liposomes, micelles, and nanoparticles, to enhance the efficacy of combination treatments. This lack of knowledge is particularly notable in the limited success of vectors for the delivery of combinations of nucleic acids with traditional small molecule drugs. The delivery of drug-nucleic acid combinations is particularly challenging due to differences in the physicochemical properties of the two types of agents. This review discusses recent advances in the development of delivery methods using combinations of small molecule drugs and nucleic acid therapeutics to treat cancer. This review primarily focuses on the rationale used for selecting appropriate drug-nucleic acid combinations as well as progress in the development of nanocarriers suitable for simultaneous delivery of drug-nucleic acid combinations. PMID:23624358

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.

Nucleic acid aptamer-based methods for diagnosis of infections.

PubMed

Park, Ki Soo

2018-04-15

Infectious diseases are a serious global problem, which not only take an enormous human toll but also incur tremendous economic losses. In combating infectious diseases, rapid and accurate diagnostic tests are required for pathogen identification at the point of care (POC). In this review, investigations of diagnostic strategies for infectious diseases that are based on aptamers, especially nucleic acid aptamers, oligonucleotides that have high affinities and specificities toward their targets, are described. Owing to their unique features including low cost of production, easy chemical modification, high chemical stability, reproducibility, and low levels of immunogenicity and toxicity, aptamers have been widely utilized as bio-recognition elements (bio-receptors) for the development of infection diagnostic systems. We discuss nucleic acid aptamer-based methods that have been developed for diagnosis of infections using a format that organizes discussion according to the target pathogenic analytes including toxins or proteins, whole cells and nucleic acids. Also included is, a summary of recent advances made in the sensitive detection of pathogenic bacteria utilizing the isothermal nucleic acid amplification method. Lastly, a nucleic acid aptamer-based POC system is described and future directions of studies in this area are discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

Recent advances in delivery of drug-nucleic acid combinations for cancer treatment.

PubMed

Li, Jing; Wang, Yan; Zhu, Yu; Oupický, David

2013-12-10

Cancer treatment that uses a combination of approaches with the ability to affect multiple disease pathways has been proven highly effective in the treatment of many cancers. Combination therapy can include multiple chemotherapeutics or combinations of chemotherapeutics with other treatment modalities like surgery or radiation. However, despite the widespread clinical use of combination therapies, relatively little attention has been given to the potential of modern nanocarrier delivery methods, like liposomes, micelles, and nanoparticles, to enhance the efficacy of combination treatments. This lack of knowledge is particularly notable in the limited success of vectors for the delivery of combinations of nucleic acids with traditional small molecule drugs. The delivery of drug-nucleic acid combinations is particularly challenging due to differences in the physicochemical properties of the two types of agents. This review discusses recent advances in the development of delivery methods using combinations of small molecule drugs and nucleic acid therapeutics to treat cancer. This review primarily focuses on the rationale used for selecting appropriate drug-nucleic acid combinations as well as progress in the development of nanocarriers suitable for simultaneous delivery of drug-nucleic acid combinations. Copyright © 2013 Elsevier B.V. All rights reserved.

Integrated magnetic tweezers and single-molecule FRET for investigating the mechanical properties of nucleic acid

PubMed Central

Long, Xi; Parks, Joseph W.; Stone, Michael D.

2017-01-01

Many enzymes promote structural changes in their nucleic acid substrates via application of piconewton forces over nanometer length scales. Magnetic tweezers (MT) is a single molecule force spectroscopy method widely used for studying the energetics of such mechanical processes. MT permits stable application of a wide range of forces and torques over long time scales with nanometer spatial resolution. However, in any force spectroscopy experiment, the ability to monitor structural changes in nucleic acids with nanometer sensitivity requires the system of interest to be held under high degrees of tension to improve signal to noise. This limitation prohibits measurement of structural changes within nucleic acids under physiologically relevant conditions of low stretching forces. To overcome this challenge, researchers have integrated a spatially sensitive fluorescence spectroscopy method, single molecule-FRET, with MT to allow simultaneous observation and manipulation of nanoscale structural transitions over a wide range of forces. Here, we describe a method for using this hybrid instrument to analyze the mechanical properties of nucleic acids. We expect that this method for analysis of nucleic acid structure will be easily adapted for experiments aiming to interrogate the mechanical responses of other biological macromolecules. PMID:27320203

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

THE EFFECT OF $gamma$-RADIATION ON THE EXCHANGE OF NUCLEIC ACIDS IN THE FOOD RESERVES OF PLANTS (in Russian)

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

Metlitskii, L.V.; Korableva, N.P.; Morozova, N.P.

1962-03-01

Two types of onion (Allium cera and Allium sativum), and potato tubers were subjected to doses of 5000 to 60,000 r. The changes in nucleic acid content in the embryonic and pulpy part of the plant were followed. A dose of 10,000 r to potato tubers results in a 50% decrease in the content of guanidine, adenine, cytidine, and uridine in the embryonic parts of the tissue. Observations show that the ordinary onion (Allium cera) is less resistant to radiation than the garlic (Allium sativum). A dose of 5000 r to the ordinary onion results in a 35% decrease inmore » content of nucleic acids in the merismatic parts of the plant, while a dose of 60,000 r caused hardly any change in the garlic. After 6 months storage there is a rise of 30 to 40% in nucleic acid content of the onion and potato bulbs, but the increase in nucleic acid content is much greater for the unirradiated bulbs. The retarding effect of gamma -irradiation on the growth of plants is caused by a disturbance in the nucleic acid exchange. (TTT).« less

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

PubMed

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

2017-07-18

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

Recent Advances in Non-viral Vectors for Gene Delivery

PubMed Central

Guo, Xia; Huang, Leaf

2011-01-01

CONSPECTUS Non-viral vectors, typically based on cationic lipids or polymers, are preferred due to safety concerns with viral vectors. So far, non-viral vectors can proficiently transfect cells in culture, but obtaining efficient nanomedicines is far from evident. To overcome the hurdles associated with non-viral vectors is significant for improving delivery efficiency and therapeutic effect of nucleic acid. The drawbacks include the strong interaction of cationic delivery vehicles with blood components, uptake by the reticuloendothelial system (RES), toxicity, targeting ability of the carriers to the cells of interest, and so on. PEGylation is the predominant method used to reduce the binding of plasma proteins with non-viral vectors and minimize the clearance by RES after intravenous administration. The nanoparticles that are not rapidly cleared from the circulation accumulate in the tumors due to the enhanced permeability and retention effect, and the targeting ligands attached to the distal end of the PEGylated components allow binding to the receptors on the target cell surface. Neutral or anionic liposomes have been also developed for systemic delivery of nucleic acids in experimental animal model. Designing and synthesizing novel cationic lipids and polymers, and binding nucleic acid with peptides, targeting ligands, polymers, or environmentally sensitive moieties also attract many attentions for resolving the problems encountered by non-viral vectors. The application of inorganic nanoparticles in nucleic acid delivery is an emerging field, too. Recently, different classes of non-viral vectors appear to be converging and the features of different classes of non-viral vectors could be combined in one strategy. More hurdles associated with efficient nucleic acid delivery therefore might be expected to be overcome. In this account, we will focus on these novel non-viral vectors, which are classified into multifunctional hybrid nucleic acid vectors, novel membrane/core nanoparticles for nucleic acid delivery and ultrasound-responsive nucleic acid vectors. The systemic delivery studies are highlighted. Finally, we bring forward the prospect for nucleic acid delivery. We think a better understandings of the fate of the nanoparticles inside the cell and of the interactions between the parts of hybrid particles will lead to a delivery system suitable for clinical use. We also underscore the value of sustained release of nucleic acid and presume making vectors targeted to cells with sustained release in vivo should be an interesting research challenge. PMID:21870813

Hepatitis B virus X protein (HBx)-induced abnormalities of nucleic acid metabolism revealed by (1)H-NMR-based metabonomics.

PubMed

Dan Yue; Zhang, Yuwei; Cheng, Liuliu; Ma, Jinhu; Xi, Yufeng; Yang, Liping; Su, Chao; Shao, Bin; Huang, Anliang; Xiang, Rong; Cheng, Ping

2016-04-14

Hepatitis B virus X protein (HBx) plays an important role in HBV-related hepatocarcinogenesis; however, mechanisms underlying HBx-mediated carcinogenesis remain unclear. In this study, an NMR-based metabolomics approach was applied to systematically investigate the effects of HBx on cell metabolism. EdU incorporation assay was conducted to examine the effects of HBx on DNA synthesis, an important feature of nucleic acid metabolism. The results revealed that HBx disrupted metabolism of glucose, lipids, and amino acids, especially nucleic acids. To understand the potential mechanism of HBx-induced abnormalities of nucleic acid metabolism, gene expression profiles of HepG2 cells expressing HBx were investigated. The results showed that 29 genes involved in DNA damage and DNA repair were differentially expressed in HBx-expressing HepG2 cells. HBx-induced DNA damage was further demonstrated by karyotyping, comet assay, Western blotting, immunofluorescence and immunohistochemistry analyses. Many studies have previously reported that DNA damage can induce abnormalities of nucleic acid metabolism. Thus, our results implied that HBx initially induces DNA damage, and then disrupts nucleic acid metabolism, which in turn blocks DNA repair and induces the occurrence of hepatocellular carcinoma (HCC). These findings further contribute to our understanding of the occurrence of HCC.

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

Andersen, G.L.; He, Z.; DeSantis, T.Z.

Microarrays have proven to be a useful and high-throughput method to provide targeted DNA sequence information for up to many thousands of specific genetic regions in a single test. A microarray consists of multiple DNA oligonucleotide probes that, under high stringency conditions, hybridize only to specific complementary nucleic acid sequences (targets). A fluorescent signal indicates the presence and, in many cases, the abundance of genetic regions of interest. In this chapter we will look at how microarrays are used in microbial ecology, especially with the recent increase in microbial community DNA sequence data. Of particular interest to microbial ecologists, phylogeneticmore » microarrays are used for the analysis of phylotypes in a community and functional gene arrays are used for the analysis of functional genes, and, by inference, phylotypes in environmental samples. A phylogenetic microarray that has been developed by the Andersen laboratory, the PhyloChip, will be discussed as an example of a microarray that targets the known diversity within the 16S rRNA gene to determine microbial community composition. Using multiple, confirmatory probes to increase the confidence of detection and a mismatch probe for every perfect match probe to minimize the effect of cross-hybridization by non-target regions, the PhyloChip is able to simultaneously identify any of thousands of taxa present in an environmental sample. The PhyloChip is shown to reveal greater diversity within a community than rRNA gene sequencing due to the placement of the entire gene product on the microarray compared with the analysis of up to thousands of individual molecules by traditional sequencing methods. A functional gene array that has been developed by the Zhou laboratory, the GeoChip, will be discussed as an example of a microarray that dynamically identifies functional activities of multiple members within a community. The recent version of GeoChip contains more than 24,000 50mer oligonucleotide probes and covers more than 10,000 gene sequences in 150 gene categories involved in carbon, nitrogen, sulfur, and phosphorus cycling, metal resistance and reduction, and organic contaminant degradation. GeoChip can be used as a generic tool for microbial community analysis, and also link microbial community structure to ecosystem functioning. Examples of the application of both arrays in different environmental samples will be described in the two subsequent sections.« less

Iron mediates catalysis of nucleic acid processing enzymes: support for Fe(II) as a cofactor before the great oxidation event.

PubMed

Okafor, C Denise; Lanier, Kathryn A; Petrov, Anton S; Athavale, Shreyas S; Bowman, Jessica C; Hud, Nicholas V; Williams, Loren Dean

2017-04-20

Life originated in an anoxic, Fe2+-rich environment. We hypothesize that on early Earth, Fe2+ was a ubiquitous cofactor for nucleic acids, with roles in RNA folding and catalysis as well as in processing of nucleic acids by protein enzymes. In this model, Mg2+ replaced Fe2+ as the primary cofactor for nucleic acids in parallel with known metal substitutions of metalloproteins, driven by the Great Oxidation Event. To test predictions of this model, we assay the ability of nucleic acid processing enzymes, including a DNA polymerase, an RNA polymerase and a DNA ligase, to use Fe2+ in place of Mg2+ as a cofactor during catalysis. Results show that Fe2+ can indeed substitute for Mg2+ in catalytic function of these enzymes. Additionally, we use calculations to unravel differences in energetics, structures and reactivities of relevant Mg2+ and Fe2+ complexes. Computation explains why Fe2+ can be a more potent cofactor than Mg2+ in a variety of folding and catalytic functions. We propose that the rise of O2 on Earth drove a Fe2+ to Mg2+ substitution in proteins and nucleic acids, a hypothesis consistent with a general model in which some modern biochemical systems retain latent abilities to revert to primordial Fe2+-based states when exposed to pre-GOE conditions. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Evolution of functional nucleic acids in the presence of nonheritable backbone heterogeneity.

PubMed

Trevino, Simon G; Zhang, Na; Elenko, Mark P; Lupták, Andrej; Szostak, Jack W

2011-08-16

Multiple lines of evidence support the hypothesis that the early evolution of life was dominated by RNA, which can both transfer information from generation to generation through replication directed by base-pairing, and carry out biochemical activities by folding into functional structures. To understand how life emerged from prebiotic chemistry we must therefore explain the steps that led to the emergence of the RNA world, and in particular, the synthesis of RNA. The generation of pools of highly pure ribonucleotides on the early Earth seems unlikely, but the presence of alternative nucleotides would support the assembly of nucleic acid polymers containing nonheritable backbone heterogeneity. We suggest that homogeneous monomers might not have been necessary if populations of heterogeneous nucleic acid molecules could evolve reproducible function. For such evolution to be possible, function would have to be maintained despite the repeated scrambling of backbone chemistry from generation to generation. We have tested this possibility in a simplified model system, by using a T7 RNA polymerase variant capable of transcribing nucleic acids that contain an approximately 11 mixture of deoxy- and ribonucleotides. We readily isolated nucleotide-binding aptamers by utilizing an in vitro selection process that shuffles the order of deoxy- and ribonucleotides in each round. We describe two such RNA/DNA mosaic nucleic acid aptamers that specifically bind ATP and GTP, respectively. We conclude that nonheritable variations in nucleic acid backbone structure may not have posed an insurmountable barrier to the emergence of functionality in early nucleic acids.

Comparative evaluation of three commercial systems for nucleic acid extraction from urine specimens.

PubMed

Tang, Yi-Wei; Sefers, Susan E; Li, Haijing; Kohn, Debra J; Procop, Gary W

2005-09-01

A nucleic acid extraction system that can handle small numbers of specimens with a short test turnaround time and short hands-on time is desirable for emergent testing. We performed a comparative validation on three systems: the MagNA Pure compact system (Compact), the NucliSens miniMAG extraction instrument (miniMAG), and the BioRobot EZ1 system (EZ1). A total of 75 urine specimens submitted for polyomavirus BK virus detection were used. The human beta-actin gene was detected on 75 (100%), 75 (100%), and 72 (96%) nucleic acid extracts prepared by the miniMAG, EZ1, and Compact, respectively. The miniMAG produced the highest quantity of nucleic acids and the best precision among the three systems. The agreement rate was 100% for BKV detection on nucleic acid extracts prepared by the three extraction systems. When a full panel of specimens was run, the hands-on time and test turnaround time were 105.7 and 121.1 min for miniMAG, 6.1 and 22.6 min for EZ1, and 7.4 and 33.7 min for Compact, respectively. The EZ1 and Compact systems processed automatic nucleic acid extraction properly, providing a good solution to the need for sporadic but emergent specimen detection. The miniMAG yielded the highest quantity of nucleic acids, suggesting that this system would be the best for specimens containing a low number of microorganisms of interest.

Assembly of barcode-like nucleic acid nanostructures.

PubMed

Wang, Pengfei; Tian, Cheng; Li, Xiang; Mao, Chengde

2014-10-15

Barcode-like (BC) nanopatterns from programmed self-assembly of nucleic acids (DNA and RNA) are reported. BC nanostructures are generated by the introduction of open spaces at selected sites to an otherwise closely packed, plain, rectangle nucleic acid nanostructure. This strategy is applied to nanostructures assembled from both origami approach and single stranded tile approach. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of Sorbents for Extraction and Stabilization of Nucleic Acids

DTIC Science & Technology

2016-09-13

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

Adsorption of nucleic acid bases and amino acids on single-walled carbon and boron nitride nanotubes: a first-principles study.

PubMed

Zheng, Jiaxin; Song, Wei; Wang, Lu; Lu, Jing; Luo, Guangfu; Zhou, Jing; Qin, Rui; Li, Hong; Gao, Zhengxiang; Lai, Lin; Li, Guangping; Mei, Wai Ning

2009-11-01

We study the adsorptions of nucleic acid bases adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) and four amino acids phenylalanine, tyrosine, tryptophan, alanine on the single-walled carbon nanotubes (SWCNTs) and boron nitride nanotubes (SWBNNTs) by using density functional theory. We find that the aromatic content plays a critical role in the adsorption. The adsorptions of nucleic acid bases and amino acids on the (7, 7) SWBNNT are stronger than those on the (7, 7) SWCNT. Oxidative treatment of SWCNTs favors the adsorption of biomolecules on nanotubes.

The third annual BRDS on research and development of nucleic acid-based nanomedicines

PubMed Central

Chaudhary, Amit Kumar

2017-01-01

The completion of human genome project, decrease in the sequencing cost, and correlation of genome sequencing data with specific diseases led to the exponential rise in the nucleic acid-based therapeutic approaches. In the third annual Biopharmaceutical Research and Development Symposium (BRDS) held at the Center for Drug Discovery and Lozier Center for Pharmacy Sciences and Education at the University of Nebraska Medical Center (UNMC), we highlighted the remarkable features of the nucleic acid-based nanomedicines, their significance, NIH funding opportunities on nanomedicines and gene therapy research, challenges and opportunities in the clinical translation of nucleic acids into therapeutics, and the role of intellectual property (IP) in drug discovery and development. PMID:27848223

Method and apparatus for purifying nucleic acids and performing polymerase chain reaction assays using an immiscible fluid

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

Koh, Chung-Yan; Light, Yooli Kim; Piccini, Matthew Ernest

Embodiments of the present invention are directed toward devices, systems, and methods for purifying nucleic acids to conduct polymerase chain reaction (PCR) assays. In one example, a method includes generating complexes of silica beads and nucleic acids in a lysis buffer, transporting the complexes through an immiscible fluid to remove interfering compounds from the complexes, further transporting the complexes into a density medium containing components required for PCR where the nucleic acids disassociate from the silica beads, and thermocycling the contents of the density medium to achieve PCR. Signal may be detected from labeling agents in the components required formore » PCR.« less

Recent advances in peptide nucleic acid for cancer bionanotechnology.

PubMed

Wu, Jun-Chen; Meng, Qing-Chun; Ren, Hong-Mei; Wang, Hong-Tao; Wu, Jie; Wang, Qi

2017-06-01

Peptide nucleic acid (PNA) is an oligomer, in which the phosphate backbone has been replaced by a pseudopeptide backbone that is meant to mimic DNA. Peptide nucleic acids are of the utmost importance in the biomedical field because of their ability to hybridize with neutral nucleic acids and their special chemical and biological properties. In recent years, PNAs have emerged in nanobiotechnology for cancer diagnosis and therapy due to their high affinity and sequence selectivity toward corresponding DNA and RNA. In this review, we summarize the recent progresses that have been made in cancer detection and therapy with PNA biotechnology. In addition, we emphasize nanoparticle PNA-based strategies for the efficient delivery of drugs in anticancer therapies.

Generation of Synthetic Copolymer Libraries by Combinatorial Assembly on Nucleic Acid Templates.

PubMed

Kong, Dehui; Yeung, Wayland; Hili, Ryan

2016-07-11

Recent advances in nucleic acid-templated copolymerization have expanded the scope of sequence-controlled synthetic copolymers beyond the molecular architectures witnessed in nature. This has enabled the power of molecular evolution to be applied to synthetic copolymer libraries to evolve molecular function ranging from molecular recognition to catalysis. This Review seeks to summarize different approaches available to generate sequence-defined monodispersed synthetic copolymer libraries using nucleic acid-templated polymerization. Key concepts and principles governing nucleic acid-templated polymerization, as well as the fidelity of various copolymerization technologies, will be described. The Review will focus on methods that enable the combinatorial generation of copolymer libraries and their molecular evolution for desired function.

78 FR 69693 - Draft Guidance for Industry: Recommendations for Premarket Notification (510(k)) Submissions for...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-11-20

...] Draft Guidance for Industry: Recommendations for Premarket Notification (510(k)) Submissions for Nucleic... ``Guidance for Industry: Recommendations for Premarket Notification (510(k)) Submissions for Nucleic Acid... submitters and FDA reviewers in preparing and reviewing 510(k) submissions for nucleic acid-based HLA test...

Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

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

Thompson, David N.; Apel, William A.; Thompson, Vicki S.

A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extractsmore » are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.« less

Tracking fetal development through molecular analysis of maternal biofluids☆

PubMed Central

Edlow, Andrea G.; Bianchi, Diana W.

2015-01-01

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

Enhancing and targeting nucleic acid delivery by magnetic force.

PubMed

Plank, Christian; Anton, Martina; Rudolph, Carsten; Rosenecker, Joseph; Krötz, Florian

2003-08-01

Insufficient contact of inherently highly active nucleic acid delivery systems with target cells is a primary reason for their often observed limited efficacy. Physical methods of targeting can overcome this limitation and reduce the risk of undesired side effects due to non-target site delivery. The authors and others have developed a novel means of physical targeting, exploiting magnetic force acting on nucleic acid vectors associated with magnetic particles in order to mediate the rapid contact of vectors with target cells. Here, the principles of magnetic drug and nucleic acid delivery are reviewed, and the facts and potentials of the technique for research and therapeutic applications are discussed. Magnetically enhanced nucleic acid delivery - magnetofection - is universally applicable to viral and non-viral vectors, is extraordinarily rapid, simple and yields saturation level transfection at low dose in vitro. The method is useful for site-specific vector targeting in vivo. Exploiting the full potential of the technique requires an interdisciplinary research effort in magnetic field physics, magnetic particle chemistry, pharmaceutical formulation and medical application.

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

PubMed Central

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

2010-01-01

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

Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

DOEpatents

Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Ward, Thomas E.

2016-03-22

A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

DOEpatents

Thompson, David N; Apel, William A; Thompson, Vicki S; Ward, Thomas E

2013-07-23

A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

DOEpatents

Thompson, David N; Apel, William A; Thompson, Vicki S; Ward, Thomas E

2014-04-08

A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

Extracellular nucleic acids of the marine bacterium Rhodovulum sulfidophilum and recombinant RNA production technology using bacteria.

PubMed

Kikuchi, Yo; Umekage, So

2018-02-01

Extracellular nucleic acids of high molecular weight are detected ubiquitously in seawater. Recent studies have indicated that these nucleic acids are, at least in part, derived from active production by some bacteria. The marine bacterium Rhodovulum sulfidophilum is one of those bacteria. Rhodovulumsulfidophilum is a non-sulfur phototrophic marine bacterium that is known to form structured communities of cells called flocs, and to produce extracellular nucleic acids in culture media. Recently, it has been revealed that this bacterium produces gene transfer agent-like particles and that this particle production may be related to the extracellular nucleic acid production mechanism. This review provides a summary of recent physiological and genetic studies of these phenomena and also introduces a new method for extracellular production of artificial and biologically functional RNAs using this bacterium. In addition, artificial RNA production using Escherichia coli, which is related to this topic, will also be described. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Primaquine: Modes of Action and Mechanisms of Drug Resistance.

DTIC Science & Technology

1975-06-30

on in vitro protein synthesis, nucleic acid synthesis in vitro and in isolated nuclei, in vitro lipid synthesis, andmembrane transport and permeability...vitro protein synthesis, nucleic acid synthesis in vitro and in isolated nuclei, in vitro lipid synthesis, and membrane transport and permeability. In...protein synthesis. 7 III. The effects of primaquine on nucleic acid synthesis in isolated nuclei. 7 IV. The effects of primaquine on DNA and RNA syntheses

Use of linalool synthase in genetic engineering of scent production

DOEpatents

Pichersky, E.

1998-12-15

A purified S-linalool synthase polypeptide from Clarkia breweri is disclosed as is the recombinant polypeptide and nucleic acid sequences encoding the polypeptide. Also disclosed are antibodies immunoreactive with the purified peptide and with recombinant versions of the polypeptide. Methods of using the nucleic acid sequences, as well as methods of enhancing the smell and the flavor of plants expressing the nucleic acid sequences are also disclosed. 5 figs.

Use of linalool synthase in genetic engineering of scent production

DOEpatents

Pichersky, Eran

1998-01-01

A purified S-linalool synthase polypeptide from Clarkia breweri is disclosed as is the recombinant polypeptide and nucleic acid sequences encoding the polypeptide. Also disclosed are antibodies immunoreactive with the purified peptide and with recombinant versions of the polypeptide. Methods of using the nucleic acid sequences, as well as methods of enhancing the smell and the flavor of plants expressing the nucleic acid sequences are also disclosed.

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

NASA Astrophysics Data System (ADS)

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

2012-03-01

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

Phytoagents for Cancer Management: Regulation of Nucleic Acid Oxidation, ROS, and Related Mechanisms

PubMed Central

Shyur, Lie-Fen

2013-01-01

Accumulation of oxidized nucleic acids causes genomic instability leading to senescence, apoptosis, and tumorigenesis. Phytoagents are known to reduce the risk of cancer development; whether such effects are through regulating the extent of nucleic acid oxidation remains unclear. Here, we outlined the role of reactive oxygen species in nucleic acid oxidation as a driving force in cancer progression. The consequential relationship between genome instability and cancer progression highlights the importance of modulation of cellular redox level in cancer management. Current epidemiological and experimental evidence demonstrate the effects and modes of action of phytoagents in nucleic acid oxidation and provide rationales for the use of phytoagents as chemopreventive or therapeutic agents. Vitamins and various phytoagents antagonize carcinogen-triggered oxidative stress by scavenging free radicals and/or activating endogenous defence systems such as Nrf2-regulated antioxidant genes or pathways. Moreover, metal ion chelation by phytoagents helps to attenuate oxidative DNA damage caused by transition metal ions. Besides, the prooxidant effects of some phytoagents pose selective cytotoxicity on cancer cells and shed light on a new strategy of cancer therapy. The “double-edged sword” role of phytoagents as redox regulators in nucleic acid oxidation and their possible roles in cancer prevention or therapy are discussed in this review. PMID:24454991

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

PubMed

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

2016-01-29

Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3'-5' exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Termite enzymes and uses thereof for in vitro conversion of lignin-containing materials to fermentable products

DOEpatents

Scharf, Michael E; Boucias, Drion G; Tartar, Aurelien; Coy, Monique R; Zhou, Xuguo; Salem, Tamer Ibrahim Zaki; Jadhao, Sanjay B; Wheeler, Marsha M

2013-05-21

The disclosure provides isolated nucleic acid molecules derived from the gut of the termite R flavipes, recombinant nucleic acid molecules comprising a vector and an isolated heterologous nucleic acid molecule operably inserted therein, whereby, when transformed into an appropriate host cell system, the heterologous nucleic acid sequence is expressed as a polypeptide having an activity similar to that when expressed in the gut of the termite R. flavipes. The recombinant nucleic acid molecules can comprise more than one heterologous nucleic acid molecule such that more than one polypeptide may be expressed by the host system. The expressed polypeptides may be substantially purified, or used in a substantially unpurified form, to be admixed with a lignocellulose source to be converted to a fermentable product such as a sugar or a mixture of sugars. One aspect of the present disclosure, therefore, encompasses methods of converting a lignified plant material to a fermentable product, the method comprising obtaining a series of isolated polypeptides of a termite, wherein the series of polypeptides cooperate to convert a plant lignocellulose to a fermentable product; and incubating the series of polypeptides with a source of lignified plant material, under conditions allowing the polypeptides to cooperatively produce a fermentable product from the lignified plant material.

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

PubMed

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

2011-07-01

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

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

PubMed

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

2001-04-01

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

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

PubMed Central

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

2001-01-01

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

Structural aspects of catalytic mechanisms of endonucleases and their binding to nucleic acids

NASA Astrophysics Data System (ADS)

Zhukhlistova, N. E.; Balaev, V. V.; Lyashenko, A. V.; Lashkov, A. A.

2012-05-01

Endonucleases (EC 3.1) are enzymes of the hydrolase class that catalyze the hydrolytic cleavage of deoxyribonucleic and ribonucleic acids at any region of the polynucleotide chain. Endonucleases are widely used both in biotechnological processes and in veterinary medicine as antiviral agents. Medical applications of endonucleases in human cancer therapy hold promise. The results of X-ray diffraction studies of the spatial organization of endonucleases and their complexes and the mechanism of their action are analyzed and generalized. An analysis of the structural studies of this class of enzymes showed that the specific binding of enzymes to nucleic acids is characterized by interactions with nitrogen bases and the nucleotide backbone, whereas the nonspecific binding of enzymes is generally characterized by interactions only with the nucleic-acid backbone. It should be taken into account that the specificity can be modulated by metal ions and certain low-molecular-weight organic compounds. To test the hypotheses about specific and nonspecific nucleic-acid-binding proteins, it is necessary to perform additional studies of atomic-resolution three-dimensional structures of enzyme-nucleic-acid complexes by methods of structural biology.

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

PubMed

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

2017-10-01

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

Novel high-performance purification protocol of recombinant CNBP suitable for biochemical and biophysical characterization.

PubMed

Challier, Emilse; Lisa, María-Natalia; Nerli, Bibiana B; Calcaterra, Nora B; Armas, Pablo

2014-01-01

Cellular nucleic acid binding protein (CNBP) is a highly conserved multi-zinc knuckle protein that enhances c-MYC expression, is related to certain human muscular diseases and is required for proper rostral head development. CNBP binds to single-stranded DNA (ssDNA) and RNA and acts as nucleic acid chaperone. Despite the advances made concerning CNBP biological roles, a full knowledge about the structure-function relationship has not yet been achieved, likely due to difficulty in obtaining pure and tag-free CNBP. Here, we report a fast, simple, reproducible, and high-performance expression and purification protocol that provides recombinant tag-free CNBP from Escherichia coli cultures. We determined that tag-free CNBP binds its molecular targets with higher affinity than tagged-CNBP. Furthermore, fluorescence spectroscopy revealed the presence of a unique and conserved tryptophan, which is exposed to the solvent and involved, directly or indirectly, in nucleic acid binding. Size-exclusion HPLC revealed that CNBP forms homodimers independently of nucleic acid binding and coexist with monomers as non-interconvertible forms or in slow equilibrium. Circular dichroism spectroscopy showed that CNBP has a secondary structure dominated by random-coil and β-sheet coincident with the sequence-predicted repetitive zinc knuckles motifs, which folding is required for CNBP structural stability and biochemical activity. CNBP structural stability increased in the presence of single-stranded nucleic acid targets similar to other unstructured nucleic acid chaperones. Altogether, data suggest that CNBP is a flexible protein with interspersed structured zinc knuckles, and acquires a more rigid structure upon nucleic acid binding. Copyright © 2013 Elsevier Inc. All rights reserved.

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

NASA Astrophysics Data System (ADS)

Pihlasalo, S.; Mariani, L.; Härmä, H.

2016-03-01

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

A mathematical analysis of multiple-target SELEX.

PubMed

Seo, Yeon-Jung; Chen, Shiliang; Nilsen-Hamilton, Marit; Levine, Howard A

2010-10-01

SELEX (Systematic Evolution of Ligands by Exponential Enrichment) is a procedure by which a mixture of nucleic acids can be fractionated with the goal of identifying those with specific biochemical activities. One combines the mixture with a specific target molecule and then separates the target-NA complex from the resulting reactions. The target-NA complex is separated from the unbound NA by mechanical means (such as by filtration), the NA is eluted from the complex, amplified by PCR (polymerase chain reaction), and the process repeated. After several rounds, one should be left with the nucleic acids that best bind to the target. The problem was first formulated mathematically in Irvine et al. (J. Mol. Biol. 222:739-761, 1991). In Levine and Nilsen-Hamilton (Comput. Biol. Chem. 31:11-25, 2007), a mathematical analysis of the process was given. In Vant-Hull et al. (J. Mol. Biol. 278:579-597, 1998), multiple target SELEX was considered. It was assumed that each target has a single nucleic acid binding site that permits occupation by no more than one nucleic acid. Here, we revisit Vant-Hull et al. (J. Mol. Biol. 278:579-597, 1998) using the same assumptions. The iteration scheme is shown to be convergent and a simplified algorithm is given. Our interest here is in the behavior of the multiple target SELEX process as a discrete "time" dynamical system. Our goal is to characterize the limiting states and their dependence on the initial distribution of nucleic acid and target fraction components. (In multiple target SELEX, we vary the target component fractions, but not their concentrations, as fixed and the initial pool of nucleic acids as a variable starting condition). Given N nucleic acids and a target consisting of M subtarget component species, there is an M × N matrix of affinities, the (i,j) entry corresponding to the affinity of the jth nucleic acid for the ith subtarget. We give a structure condition on this matrix that is equivalent to the following statement: For any initial pool of nucleic acids such that all N species are represented, the dynamical system defined by the multiple target SELEX process will converge to a unique subset of nucleic acids, each of whose concentrations depend only upon the total nucleic acid concentration, the initial fractional target distribution (both of which are assumed to be the same from round to round), and the overall limiting association constant. (The overall association constant is the equilibrium constant for the system of MN reactions when viewed as a composite single reaction). This condition is equivalent to the statement that every member of a certain family of chemical potentials at infinite target dilution can have at most one critical point. (The condition replaces the statement for single target SELEX that the dynamical system generated via the process always converges to a pool that contains only the nucleic acid that binds best to the target). This suggests that the effectiveness of multiple target SELEX as a separation procedure may not be as useful as single target SELEX unless the thermodynamic properties of these chemical potentials are well understood.

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.

Computer-aided visualization and analysis system for sequence evaluation

DOEpatents

Chee, M.S.

1998-08-18

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

Computer-aided visualization and analysis system for sequence evaluation

DOEpatents

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

2004-05-11

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

Computer-aided visualization and analysis system for sequence evaluation

DOEpatents

Chee, Mark S.

1998-08-18

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

Computer-aided visualization and analysis system for sequence evaluation

DOEpatents

Chee, Mark S.

2003-08-19

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

A survey of advancements in nucleic acid-based logic gates and computing for applications in biotechnology and biomedicine.

PubMed

Wu, Cuichen; Wan, Shuo; Hou, Weijia; Zhang, Liqin; Xu, Jiehua; Cui, Cheng; Wang, Yanyue; Hu, Jun; Tan, Weihong

2015-03-04

Nucleic acid-based logic devices were first introduced in 1994. Since then, science has seen the emergence of new logic systems for mimicking mathematical functions, diagnosing disease and even imitating biological systems. The unique features of nucleic acids, such as facile and high-throughput synthesis, Watson-Crick complementary base pairing, and predictable structures, together with the aid of programming design, have led to the widespread applications of nucleic acids (NA) for logic gate and computing in biotechnology and biomedicine. In this feature article, the development of in vitro NA logic systems will be discussed, as well as the expansion of such systems using various input molecules for potential cellular, or even in vivo, applications.

A Survey of Advancements in Nucleic Acid-based Logic Gates and Computing for Applications in Biotechnology and biomedicine

PubMed Central

Wu, Cuichen; Wan, Shuo; Hou, Weijia; Zhang, Liqin; Xu, Jiehua; Cui, Cheng; Wang, Yanyue; Hu, Jun

2015-01-01

Nucleic acid-based logic devices were first introduced in 1994. Since then, science has seen the emergence of new logic systems for mimicking mathematical functions, diagnosing disease and even imitating biological systems. The unique features of nucleic acids, such as facile and high-throughput synthesis, Watson-Crick complementary base pairing, and predictable structures, together with the aid of programming design, have led to the widespread applications of nucleic acids (NA) for logic gating and computing in biotechnology and biomedicine. In this feature article, the development of in vitro NA logic systems will be discussed, as well as the expansion of such systems using various input molecules for potential cellular, or even in vivo, applications. PMID:25597946

Towards XNA nanotechnology: new materials from synthetic genetic polymers

PubMed Central

Pinheiro, Vitor B.; Holliger, Philipp

2014-01-01

Nucleic acids display remarkable properties beyond information storage and propagation. The well-understood base pairing rules have enabled nucleic acids to be assembled into nanostructures of ever increasing complexity. Although nanostructures can be constructed using other building blocks, including peptides and lipids, it is the capacity to evolve that sets nucleic acids apart from all other nanoscale building materials. Nonetheless, the poor chemical and biological stability of DNA and RNA constrain their applications. Recent advances in nucleic acid chemistry and polymerase engineering enable the synthesis, replication, and evolution of a range of synthetic genetic polymers (XNAs) with improved chemical and biological stability. We discuss the impact of this technology on the generation of XNA ligands, enzymes, and nanostructures with tailor-made chemistry. PMID:24745974

The increasing application of multiplex nucleic acid detection tests to the diagnosis of syndromic infections.

PubMed

Gray, J; Coupland, L J

2014-01-01

On 14 January 2013, the US Food and Drug Administration (FDA) announced permission for a multiplex nucleic acid test, the xTAG® Gastrointestinal Pathogen Panel (GPP) (Luminex Corporation, USA), which simultaneously detects 11 common viral, bacterial and parasitic causes of infectious gastroenteritis, to be marketed in the USA. This announcement reflects the current move towards the development and commercialization of detection technologies based on nucleic acid amplification techniques for diagnosis of syndromic infections. We discuss the limitations and advantages of nucleic acid amplification techniques and the recent advances in Conformité Européene - in-vitro diagnostic (CE-IVD)-approved multiplex real-time PCR kits for the simultaneous detection of multiple targets within the clinical diagnostics market.

Compositions and methods for detecting single nucleotide polymorphisms

DOEpatents

Yeh, Hsin-Chih; Werner, James; Martinez, Jennifer S.

2016-11-22

Described herein are nucleic acid based probes and methods for discriminating and detecting single nucleotide variants in nucleic acid molecules (e.g., DNA). The methods include use of a pair of probes can be used to detect and identify polymorphisms, for example single nucleotide polymorphism in DNA. The pair of probes emit a different fluorescent wavelength of light depending on the association and alignment of the probes when hybridized to a target nucleic acid molecule. Each pair of probes is capable of discriminating at least two different nucleic acid molecules that differ by at least a single nucleotide difference. The methods can probes can be used, for example, for detection of DNA polymorphisms that are indicative of a particular disease or condition.

Nucleic acids for the rational design of reaction circuits.

PubMed

Padirac, Adrien; Fujii, Teruo; Rondelez, Yannick

2013-08-01

Nucleic acid-based circuits are rationally designed in vitro assemblies that can perform complex preencoded programs. They can be used to mimic in silico computations. Recent works emphasized the modularity and robustness of these circuits, which allow their scaling-up. Another new development has led to dynamic, time-responsive systems that can display emergent behaviors like oscillations. These are closely related to biological architectures and provide an in vitro model of in vivo information processing. Nucleic acid circuits have already been used to handle various processes for technological or biotechnological purposes. Future applications of these chemical smart systems will benefit from the rapidly growing ability to design, construct, and model nucleic acid circuits of increasing size. Copyright © 2012 Elsevier Ltd. All rights reserved.

Compaction agent clarification of microbial lysates

NASA Technical Reports Server (NTRS)

DeWalt, Brad W.; Murphy, Jason C.; Fox, George E.; Willson, Richard C.

2003-01-01

Recombinant proteins are often purified from microbial lysates containing high concentrations of nucleic acids. Pre-purification steps such as nuclease addition or precipitation with polyethyleneimine or ammonium sulfate are normally required to reduce viscosity and to eliminate competing polyanions before anion exchange chromatography. We report that small polycationic compaction agents such as spermine selectively precipitate nucleic acids during or after Escherichia coli lysis, allowing DNA and RNA to be pelleted with the insoluble cell debris. Analysis by spectrophotometry and protein assay confirmed a significant reduction in the concentration of nucleic acids present, with preservation of protein. Lysate viscosity is greatly reduced, facilitating subsequent processing. We have used 5mM spermine to remove nucleic acids from E. coli lysate in the purification of a hexahistidine-tagged HIV reverse transcriptase.

Plants having modified response to ethylene

DOEpatents

Meyerowitz, Elliott M.; Chang, Caren; Bleecker, Anthony B.

1997-01-01

The invention includes transformed plants having at least one cell transformed with a modified ETR nucleic acid. Such plants have a phenotype characterized by a decrease in the response of at least one transformed plant cell to ethylene as compared to a plant not containing the transformed plant cell. Tissue and/or temporal specificity for expression of the modified ETR nucleic acid is controlled by selecting appropriate expression regulation sequences to target the location and/or time of expression of the transformed nucleic acid. The plants are made by transforming at least one plant cell with an appropriate modified ETR nucleic acid, regenerating plants from one or more of the transformed plant cells and selecting at least one plant having the desired phenotype.

Plants having modified response to ethylene

DOEpatents

Meyerowitz, E.M.; Chang, C.; Bleecker, A.B.

1998-10-20

The invention includes transformed plants having at least one cell transformed with a modified ETR nucleic acid. Such plants have a phenotype characterized by a decrease in the response of at least one transformed plant cell to ethylene as compared to a plant not containing the transformed plant cell. Tissue and/or temporal specificity for expression of the modified ETR nucleic acid is controlled by selecting appropriate expression regulation sequences to target the location and/or time of expression of the transformed nucleic acid. The plants are made by transforming at least one plant cell with an appropriate modified ETR nucleic acid, regenerating plants from one or more of the transformed plant cells and selecting at least one plant having the desired phenotype. 67 figs.

Plants having modified response to ethylene

DOEpatents

Meyerowitz, Elliot M.; Chang, Caren; Bleecker, Anthony B.

1998-01-01

The invention includes transformed plants having at least one cell transformed with a modified ETR nucleic acid. Such plants have a phenotype characterized by a decrease in the response of at least one transformed plant cell to ethylene as compared to a plant not containing the transformed plant cell. Tissue and/or temporal specificity for expression of the modified ETR nucleic acid is controlled by selecting appropriate expression regulation sequences to target the location and/or time of expression of the transformed nucleic acid. The plants are made by transforming at least one plant cell with an appropriate modified ETR nucleic acid, regenerating plants from one or more of the transformed plant cells and selecting at least one plant having the desired phenotype.

Plants having modified response to ethylene

DOEpatents

Meyerowitz, E.M.; Chang, C.; Bleecker, A.B.

1997-11-18

The invention includes transformed plants having at least one cell transformed with a modified ETR nucleic acid. Such plants have a phenotype characterized by a decrease in the response of at least one transformed plant cell to ethylene as compared to a plant not containing the transformed plant cell. Tissue and/or temporal specificity for expression of the modified ETR nucleic acid is controlled by selecting appropriate expression regulation sequences to target the location and/or time of expression of the transformed nucleic acid. The plants are made by transforming at least one plant cell with an appropriate modified ETR nucleic acid, regenerating plants from one or more of the transformed plant cells and selecting at least one plant having the desired phenotype. 31 figs.

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.

NUCLEIC ACID CONCENTRATION AND RADIOSENSITIVITY OF THE SCORPION ANDROCTONUS AMOREUXI AUD. AND SAV (in French)

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

Pascaud, X.; Niaussat, P.

1963-01-01

The concentration of desoxyribonucleic acid and of ribonucleic acid in the soft tissues was determined for the two invertebrates of the arid zone, the scorpion Androctonus amoreuxi Aud. and Sav. and the tenebrionide Pimelia angulata expiata Peyer. The radiosensitivity to gamma rays had been previously determined: LD/sub 50/30// days is 100,000 r for Androctonus and 40,000 for Pimelia. The mean rate of nucleic acids determined in the scorpion was relatively low. A possible relation between the high radioresistance of the scorpion and the low nucleic acid concentration was discussed. (J.S.R.)

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2017-11-01

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

EvOligo: A Novel Software to Design and Group Libraries of Oligonucleotides Applicable for Nucleic Acid-Based Experiments.

PubMed

Milewski, Marek C; Kamel, Karol; Kurzynska-Kokorniak, Anna; Chmielewski, Marcin K; Figlerowicz, Marek

2017-10-01

Experimental methods based on DNA and RNA hybridization, such as multiplex polymerase chain reaction, multiplex ligation-dependent probe amplification, or microarray analysis, require the use of mixtures of multiple oligonucleotides (primers or probes) in a single test tube. To provide an optimal reaction environment, minimal self- and cross-hybridization must be achieved among these oligonucleotides. To address this problem, we developed EvOligo, which is a software package that provides the means to design and group DNA and RNA molecules with defined lengths. EvOligo combines two modules. The first module performs oligonucleotide design, and the second module performs oligonucleotide grouping. The software applies a nearest-neighbor model of nucleic acid interactions coupled with a parallel evolutionary algorithm to construct individual oligonucleotides, and to group the molecules that are characterized by the weakest possible cross-interactions. To provide optimal solutions, the evolutionary algorithm sorts oligonucleotides into sets, preserves preselected parts of the oligonucleotides, and shapes their remaining parts. In addition, the oligonucleotide sets can be designed and grouped based on their melting temperatures. For the user's convenience, EvOligo is provided with a user-friendly graphical interface. EvOligo was used to design individual oligonucleotides, oligonucleotide pairs, and groups of oligonucleotide pairs that are characterized by the following parameters: (1) weaker cross-interactions between the non-complementary oligonucleotides and (2) more uniform ranges of the oligonucleotide pair melting temperatures than other available software products. In addition, in contrast to other grouping algorithms, EvOligo offers time-efficient sorting of paired and unpaired oligonucleotides based on various parameters defined by the user.

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

PubMed

da Cunha Santos, Gilda

2018-03-01

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

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

PubMed

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

2017-03-01

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

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.

Kit for detecting nucleic acid sequences using competitive hybridization probes

DOEpatents

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

2001-01-01

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

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

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

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.

Photochemistry of nucleic acid bases and their thio- and aza-analogues in solution.

PubMed

Pollum, Marvin; Martínez-Fernández, Lara; Crespo-Hernández, Carlos E

2015-01-01

The steady-state and time-resolved photochemistry of the natural nucleic acid bases and their sulfur- and nitrogen-substituted analogues in solution is reviewed. Emphasis is given to the experimental studies performed over the last 3-5 years that showcase topical areas of scientific inquiry and those that require further scrutiny. Significant progress has been made toward mapping the radiative and nonradiative decay pathways of nucleic acid bases. There is a consensus that ultrafast internal conversion to the ground state is the primary relaxation pathway in the nucleic acid bases, whereas the mechanism of this relaxation and the level of participation of the (1)πσ*, (1) nπ*, and (3)ππ* states are still matters of debate. Although impressive research has been performed in recent years, the microscopic mechanism(s) by which the nucleic acid bases dissipate excess vibrational energy to their environment, and the role of the N-glycosidic group in this and in other nonradiative decay pathways, are still poorly understood. The simple replacement of a single atom in a nucleobase with a sulfur or nitrogen atom severely restricts access to the conical intersections responsible for the intrinsic internal conversion pathways to the ground state in the nucleic acid bases. It also enhances access to ultrafast and efficient inter-system crossing pathways that populate the triplet manifold in yields close to unity. Determining the coupled nuclear and electronic pathways responsible for the significantly different photochemistry in these nucleic acid base analogues serves as a convenient platform to examine the current state of knowledge regarding the photodynamic properties of the DNA and RNA bases from both experimental and computational perspectives. Further investigations should also aid in forecasting the prospective use of sulfur- and nitrogen-substituted base analogues in photochemotherapeutic applications.

The in Silico Insight into Carbon Nanotube and Nucleic Acid Bases Interaction.

PubMed

Karimi, Ali Asghar; Ghalandari, Behafarid; Tabatabaie, Seyed Saleh; Farhadi, Mohammad

2016-05-01

To explore practical applications of carbon nanotubes (CNTs) in biomedical fields the properties of their interaction with biomolecules must be revealed. Recent years, the interaction of CNTs with biomolecules is a subject of research interest for practical applications so that previous research explored that CNTs have complementary structure properties with single strand DNA (ssDNA). Hence, the quantum mechanics (QM) method based on ab initio was used for this purpose. Therefore values of binding energy, charge distribution, electronic energy and other physical properties of interaction were studied for interaction of nucleic acid bases and SCNT. In this study, the interaction between nucleic acid bases and a (4, 4) single-walled carbon nanotube (SCNT) were investigated through calculations within quantum mechanics (QM) method at theoretical level of Hartree-Fock (HF) method using 6-31G basis set. Hence, the physical properties such as electronic energy, total dipole moment, charge distributions and binding energy of nucleic acid bases interaction with SCNT were investigated based on HF method. It has been found that the guanine base adsorption is bound stronger to the outer surface of nanotube in comparison to the other bases, consistent with the recent theoretical studies. In the other words, the results explored that guanine interaction with SCNT has optimum level of electronic energy so that their interaction is stable. Also, the calculations illustrated that SCNT interact to nucleic acid bases by noncovalent interaction because of charge distribution an electrostatic area is created in place of interaction. Consequently, small diameter SCNT interaction with nucleic acid bases is noncovalent. Also, the results revealed that small diameter SCNT interaction especially SCNT (4, 4) with nucleic acid bases can be useful in practical application area of biomedical fields such detection and drug delivery.

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

PubMed Central

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

2010-01-01

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

Discrimination of Self and Non-Self Ribonucleic Acids

PubMed Central

Gebhardt, Anna; Laudenbach, Beatrice T.

2017-01-01

Most virus infections are controlled through the innate and adaptive immune system. A surprisingly limited number of so-called pattern recognition receptors (PRRs) have the ability to sense a large variety of virus infections. The reason for the broad activity of PRRs lies in the ability to recognize viral nucleic acids. These nucleic acids lack signatures that are present in cytoplasmic cellular nucleic acids and thereby marking them as pathogen-derived. Accumulating evidence suggests that these signatures, which are predominantly sensed by a class of PRRs called retinoic acid-inducible gene I (RIG-I)-like receptors and other proteins, are not unique to viruses but rather resemble immature forms of cellular ribonucleic acids generated by cellular polymerases. RIG-I-like receptors, and other cellular antiviral proteins, may therefore have mainly evolved to sense nonprocessed nucleic acids typically generated by primitive organisms and pathogens. This capability has not only implications on induction of antiviral immunity but also on the function of cellular proteins to handle self-derived RNA with stimulatory potential. PMID:28475460

Supervised normalization of microarrays

PubMed Central

Mecham, Brigham H.; Nelson, Peter S.; Storey, John D.

2010-01-01

Motivation: A major challenge in utilizing microarray technologies to measure nucleic acid abundances is ‘normalization’, the goal of which is to separate biologically meaningful signal from other confounding sources of signal, often due to unavoidable technical factors. It is intuitively clear that true biological signal and confounding factors need to be simultaneously considered when performing normalization. However, the most popular normalization approaches do not utilize what is known about the study, both in terms of the biological variables of interest and the known technical factors in the study, such as batch or array processing date. Results: We show here that failing to include all study-specific biological and technical variables when performing normalization leads to biased downstream analyses. We propose a general normalization framework that fits a study-specific model employing every known variable that is relevant to the expression study. The proposed method is generally applicable to the full range of existing probe designs, as well as to both single-channel and dual-channel arrays. We show through real and simulated examples that the method has favorable operating characteristics in comparison to some of the most highly used normalization methods. Availability: An R package called snm implementing the methodology will be made available from Bioconductor (http://bioconductor.org). Contact: jstorey@princeton.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:20363728

Computer-aided visualization and analysis system for sequence evaluation

DOEpatents

Chee, Mark S.

1999-10-26

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

Computer-aided visualization and analysis system for sequence evaluation

DOEpatents

Chee, Mark S.

2001-06-05

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

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

DOEpatents

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

2000-01-01

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

Prokaryotic Argonaute proteins: novel genome-editing tools?

PubMed

Hegge, Jorrit W; Swarts, Daan C; van der Oost, John

2018-01-01

Argonaute proteins constitute a highly diverse family of nucleic acid-guided proteins. They were first discovered in eukaryotes as key proteins in RNA interference systems, but homologous prokaryotic Argonaute proteins (pAgos) have also been found in archaea and bacteria. In this Progress article, we focus on long pAgo variants, a class of pAgos that are involved in nucleic acid-guided host defence against invading nucleic acids, and discuss the potential of pAgos in genome editing.

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).

Parallel nucleic acid recognition by the LNA (locked nucleic acid) stereoisomers beta-L-LNA and alpha-D-LNA; studies in the mirror image world.

PubMed

Christensen, Nanna K; Bryld, Torsten; Sørensen, Mads D; Arar, Khalil; Wengel, Jesper; Nielsen, Poul

2004-02-07

Two LNA (locked nucleic acid) stereoisomers (beta-L-LNA and alpha-D-LNA) are evaluated in the mirror-image world, that is by the study of two mixed sequences of LNA and alpha-L-LNA and their L-DNA and L-RNA complements. Both are found to display high-affinity RNA-recognition by the formation of duplexes with parallel strand orientation.

5'to 3' nucleic acid synthesis using 3'-photoremovable protecting group

DOEpatents

Pirrung, Michael C.; Shuey, Steven W.; Bradley, Jean-Claude

1999-01-01

The present invention relates, in general, to a method of synthesizing a nucleic acid, and, in particular, to a method of effecting 5' to 3' nucleic acid synthesis. The method can be used to prepare arrays of oligomers bound to a support via their 5' end. The invention also relates to a method of effecting mutation analysis using such arrays. The invention further relates to compounds and compositions suitable for use in such methods.

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

PubMed Central

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

2004-01-01

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

Signal Amplification Technologies for the Detection of Nucleic Acids: from Cell-Free Analysis to Live-Cell Imaging.

PubMed

Fozooni, Tahereh; Ravan, Hadi; Sasan, Hosseinali

2017-12-01

Due to their unique properties, such as programmability, ligand-binding capability, and flexibility, nucleic acids can serve as analytes and/or recognition elements for biosensing. To improve the sensitivity of nucleic acid-based biosensing and hence the detection of a few copies of target molecule, different modern amplification methodologies, namely target-and-signal-based amplification strategies, have already been developed. These recent signal amplification technologies, which are capable of amplifying the signal intensity without changing the targets' copy number, have resulted in fast, reliable, and sensitive methods for nucleic acid detection. Working in cell-free settings, researchers have been able to optimize a variety of complex and quantitative methods suitable for deploying in live-cell conditions. In this study, a comprehensive review of the signal amplification technologies for the detection of nucleic acids is provided. We classify the signal amplification methodologies into enzymatic and non-enzymatic strategies with a primary focus on the methods that enable us to shift away from in vitro detecting to in vivo imaging. Finally, the future challenges and limitations of detection for cellular conditions are discussed.

Noncanonical structures and their thermodynamics of DNA and RNA under molecular crowding: beyond the Watson-Crick double helix.

PubMed

Sugimoto, Naoki

2014-01-01

How does molecular crowding affect the stability of nucleic acid structures inside cells? Water is the major solvent component in living cells, and the properties of water in the highly crowded media inside cells differ from that in buffered solution. As it is difficult to measure the thermodynamic behavior of nucleic acids in cells directly and quantitatively, we recently developed a cell-mimicking system using cosolutes as crowding reagents. The influences of molecular crowding on the structures and thermodynamics of various nucleic acid sequences have been reported. In this chapter, we discuss how the structures and thermodynamic properties of nucleic acids differ under various conditions such as highly crowded environments, compartment environments, and in the presence of ionic liquids, and the major determinants of the crowding effects on nucleic acids are discussed. The effects of molecular crowding on the activities of ribozymes and riboswitches on noncanonical structures of DNA- and RNA-like quadruplexes that play important roles in transcription and translation are also described. © 2014 Elsevier Inc. All rights reserved.

Nucleic acid-based electrochemical nanobiosensors.

PubMed

Abi, Alireza; Mohammadpour, Zahra; Zuo, Xiaolei; Safavi, Afsaneh

2018-04-15

The detection of biomarkers using sensitive and selective analytical devices is critically important for the early stage diagnosis and treatment of diseases. The synergy between the high specificity of nucleic acid recognition units and the great sensitivity of electrochemical signal transductions has already shown promise for the development of efficient biosensing platforms. Yet nucleic-acid based electrochemical biosensors often rely on target amplification strategies (e.g., polymerase chain reactions) to detect analytes at clinically relevant concentration ranges. The complexity and time-consuming nature of these amplification methods impede moving nucleic acid-based electrochemical biosensors from laboratory-based to point-of-care test settings. Fortunately, advancements in nanotechnology have provided growing evidence that the recruitment of nanoscaled materials and structures can enhance the biosensing performance (particularly in terms of sensitivity and response time) to the level suitable for use in point-of-care diagnostic tools. This Review highlights the significant progress in the field of nucleic acid-based electrochemical nanobiosensing with the focus on the works published during the last five years. Copyright © 2017. Published by Elsevier B.V.

PubDNA Finder: a web database linking full-text articles to sequences of nucleic acids.

PubMed

García-Remesal, Miguel; Cuevas, Alejandro; Pérez-Rey, David; Martín, Luis; Anguita, Alberto; de la Iglesia, Diana; de la Calle, Guillermo; Crespo, José; Maojo, Víctor

2010-11-01

PubDNA Finder is an online repository that we have created to link PubMed Central manuscripts to the sequences of nucleic acids appearing in them. It extends the search capabilities provided by PubMed Central by enabling researchers to perform advanced searches involving sequences of nucleic acids. This includes, among other features (i) searching for papers mentioning one or more specific sequences of nucleic acids and (ii) retrieving the genetic sequences appearing in different articles. These additional query capabilities are provided by a searchable index that we created by using the full text of the 176 672 papers available at PubMed Central at the time of writing and the sequences of nucleic acids appearing in them. To automatically extract the genetic sequences occurring in each paper, we used an original method we have developed. The database is updated monthly by automatically connecting to the PubMed Central FTP site to retrieve and index new manuscripts. Users can query the database via the web interface provided. PubDNA Finder can be freely accessed at http://servet.dia.fi.upm.es:8080/pubdnafinder

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

PubMed

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

2018-05-09

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

Amplified electrochemical detection of nucleic acid hybridization via selective preconcentration of unmodified gold nanoparticles.

PubMed

Li, Yuan; Tian, Rui; Zheng, Xingwang; Huang, Rongfu

2016-08-31

The common drawback of optical methods for rapid detection of nucleic acid by exploiting the differential affinity of single-/double-stranded nucleic acids for unmodified gold nanoparticles (AuNPs) is its relatively low sensitivity. In this article, on the basis of selective preconcentration of AuNPs unprotected by single-stranded DNA (ssDNA) binding, a novel electrochemical strategy for nucleic acid sequence identification assay has been developed. Through detecting the redox signal mediated by AuNPs on 1, 6-hexanedithiol blocked gold electrode, the proposed method is able to ensure substantial signal amplification and a low background current. This strategy is demonstrated for quantitative analysis of the target microRNA (let-7a) in human breast adenocarcinoma cells, and a detection limit of 16 fM is readily achieved with desirable specificity and sensitivity. These results indicate that the selective preconcentration of AuNPs for electrochemical signal readout can offer a promising platform for the detection of specific nucleic acid sequence. Copyright © 2016 Elsevier B.V. All rights reserved.

Visual detection of Ebola virus using reverse transcription loop-mediated isothermal amplification combined with nucleic acid strip detection.

PubMed

Xu, Changping; Wang, Hualei; Jin, Hongli; Feng, Na; Zheng, Xuexing; Cao, Zengguo; Li, Ling; Wang, Jianzhong; Yan, Feihu; Wang, Lina; Chi, Hang; Gai, Weiwei; Wang, Chong; Zhao, Yongkun; Feng, Yan; Wang, Tiecheng; Gao, Yuwei; Lu, Yiyu; Yang, Songtao; Xia, Xianzhu

2016-05-01

Ebola virus (species Zaire ebolavirus) (EBOV) is highly virulent in humans. The largest recorded outbreak of Ebola hemorrhagic fever in West Africa to date was caused by EBOV. Therefore, it is necessary to develop a detection method for this virus that can be easily distributed and implemented. In the current study, we developed a visual assay that can detect EBOV-associated nucleic acids. This assay combines reverse transcription loop-mediated isothermal amplification and nucleic acid strip detection (RT-LAMP-NAD). Nucleic acid amplification can be achieved in a one-step process at a constant temperature (58 °C, 35 min), and the amplified products can be visualized within 2-5 min using a nucleic acid strip detection device. The assay is capable of detecting 30 copies of artificial EBOV glycoprotein (GP) RNA and RNA encoding EBOV GP from 10(2) TCID50 recombinant viral particles per ml with high specificity. Overall, the RT-LAMP-NAD method is simple and has high sensitivity and specificity; therefore, it is especially suitable for the rapid detection of EBOV in African regions.

Non-Viral Nucleic Acid Delivery Strategies to the Central Nervous System

PubMed Central

Tan, James-Kevin Y.; Sellers, Drew L.; Pham, Binhan; Pun, Suzie H.; Horner, Philip J.

2016-01-01

With an increased prevalence and understanding of central nervous system (CNS) injuries and neurological disorders, nucleic acid therapies are gaining promise as a way to regenerate lost neurons or halt disease progression. While more viral vectors have been used clinically as tools for gene delivery, non-viral vectors are gaining interest due to lower safety concerns and the ability to deliver all types of nucleic acids. Nevertheless, there are still a number of barriers to nucleic acid delivery. In this focused review, we explore the in vivo challenges hindering non-viral nucleic acid delivery to the CNS and the strategies and vehicles used to overcome them. Advantages and disadvantages of different routes of administration including: systemic injection, cerebrospinal fluid injection, intraparenchymal injection and peripheral administration are discussed. Non-viral vehicles and treatment strategies that have overcome delivery barriers and demonstrated in vivo gene transfer to the CNS are presented. These approaches can be used as guidelines in developing synthetic gene delivery vectors for CNS applications and will ultimately bring non-viral vectors closer to clinical application. PMID:27847462

Accuracy assessment of the linear Poisson-Boltzmann equation and reparametrization of the OBC generalized Born model for nucleic acids and nucleic acid-protein complexes.

PubMed

Fogolari, Federico; Corazza, Alessandra; Esposito, Gennaro

2015-04-05

The generalized Born model in the Onufriev, Bashford, and Case (Onufriev et al., Proteins: Struct Funct Genet 2004, 55, 383) implementation has emerged as one of the best compromises between accuracy and speed of computation. For simulations of nucleic acids, however, a number of issues should be addressed: (1) the generalized Born model is based on a linear model and the linearization of the reference Poisson-Boltmann equation may be questioned for highly charged systems as nucleic acids; (2) although much attention has been given to potentials, solvation forces could be much less sensitive to linearization than the potentials; and (3) the accuracy of the Onufriev-Bashford-Case (OBC) model for nucleic acids depends on fine tuning of parameters. Here, we show that the linearization of the Poisson Boltzmann equation has mild effects on computed forces, and that with optimal choice of the OBC model parameters, solvation forces, essential for molecular dynamics simulations, agree well with those computed using the reference Poisson-Boltzmann model. © 2015 Wiley Periodicals, Inc.

PrPC has nucleic acid chaperoning properties similar to the nucleocapsid protein of HIV-1.

PubMed

Derrington, Edmund; Gabus, Caroline; Leblanc, Pascal; Chnaidermann, Jonas; Grave, Linda; Dormont, Dominique; Swietnicki, Wieslaw; Morillas, Manuel; Marck, Daniel; Nandi, Pradip; Darlix, Jean-Luc

2002-01-01

The function of the cellular prion protein (PrPC) remains obscure. Studies suggest that PrPC functions in several processes including signal transduction and Cu2+ metabolism. PrPC has also been established to bind nucleic acids. Therefore we investigated the properties of PrPC as a putative nucleic acid chaperone. Surprisingly, PrPC possesses all the nucleic acid chaperoning properties previously specific to retroviral nucleocapsid proteins. PrPC appears to be a molecular mimic of NCP7, the nucleocapsid protein of HIV-1. Thus PrPC, like NCP7, chaperones the annealing of tRNA(Lys) to the HIV-1 primer binding site, the initial step of retrovirus replication. PrPC also chaperones the two DNA strand transfers required for production of a complete proviral DNA with LTRs. Concerning the functions of NCP7 during budding, PrPC also mimices NCP7 by dimerizing the HIV-1 genomic RNA. These data are unprecedented because, although many cellular proteins have been identified as nucleic acid chaperones, none have the properties of retroviral nucleocapsid proteins.

Assessment for Melting Temperature Measurement of Nucleic Acid by HRM.

PubMed

Wang, Jing; Pan, Xiaoming; Liang, Xingguo

2016-01-01

High resolution melting (HRM), with a high sensitivity to distinguish the nucleic acid species with small variations, has been widely applied in the mutation scanning, methylation analysis, and genotyping. For the aim of extending HRM for the evaluation of thermal stability of nucleic acid secondary structures on sequence dependence, we investigated effects of the dye of EvaGreen, metal ions, and impurities (such as dNTPs) on melting temperature ( T m ) measurement by HRM. The accuracy of HRM was assessed as compared with UV melting method, and little difference between the two methods was found when the DNA T m was higher than 40°C. Both insufficiency and excessiveness of EvaGreen were found to give rise to a little bit higher T m , showing that the proportion of dye should be considered for precise T m measurement of nucleic acids. Finally, HRM method was also successfully used to measure T m s of DNA triplex, hairpin, and RNA duplex. In conclusion, HRM can be applied in the evaluation of thermal stability of nucleic acid (DNA or RNA) or secondary structural elements (even when dNTPs are present).

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

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

PubMed

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

2014-01-01

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

A specific scenario for the origin of life and the genetic code based on peptide/oligonucleotide interdependence.

PubMed

Griffith, Robert W

2009-12-01

Among various scenarios that attempt to explain how life arose, the RNA world is currently the most widely accepted scientific hypothesis among biologists. However, the RNA world is logistically implausible and doesn't explain how translation arose and DNA became incorporated into living systems. Here I propose an alternative hypothesis for life's origin based on cooperation between simple nucleic acids, peptides and lipids. Organic matter that accumulated on the prebiotic Earth segregated into phases in the ocean based on density and solubility. Synthesis of complex organic monomers and polymerization reactions occurred within a surface hydrophilic layer and at its aqueous and atmospheric interfaces. Replication of nucleic acids and translation of peptides began at the emulsified interface between hydrophobic and aqueous layers. At the core of the protobiont was a family of short nucleic acids bearing arginine's codon and anticodon that added this amino acid to pre-formed peptides. In turn, the survival and replication of nucleic acid was aided by the peptides. The arginine-enriched peptides served to sequester and transfer phosphate bond energy and acted as cohesive agents, aggregating nucleic acids and keeping them at the interface.

Structural aspects of catalytic mechanisms of endonucleases and their binding to nucleic acids

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

Zhukhlistova, N. E.; Balaev, V. V.; Lyashenko, A. V.

2012-05-15

Endonucleases (EC 3.1) are enzymes of the hydrolase class that catalyze the hydrolytic cleavage of deoxyribonucleic and ribonucleic acids at any region of the polynucleotide chain. Endonucleases are widely used both in biotechnological processes and in veterinary medicine as antiviral agents. Medical applications of endonucleases in human cancer therapy hold promise. The results of X-ray diffraction studies of the spatial organization of endonucleases and their complexes and the mechanism of their action are analyzed and generalized. An analysis of the structural studies of this class of enzymes showed that the specific binding of enzymes to nucleic acids is characterized bymore » interactions with nitrogen bases and the nucleotide backbone, whereas the nonspecific binding of enzymes is generally characterized by interactions only with the nucleic-acid backbone. It should be taken into account that the specificity can be modulated by metal ions and certain low-molecular-weight organic compounds. To test the hypotheses about specific and nonspecific nucleic-acid-binding proteins, it is necessary to perform additional studies of atomic-resolution three-dimensional structures of enzyme-nucleic-acid complexes by methods of structural biology.« less

42 CFR 73.17 - Records.

Code of Federal Regulations, 2013 CFR

2013-10-01

... and/or synthetic nucleic acids, and organisms containing recombinant and/or synthetic nucleic acids... that all records and data bases created under this part are accurate, have controlled access, and that...

42 CFR 73.17 - Records.

Code of Federal Regulations, 2014 CFR

2014-10-01

... and/or synthetic nucleic acids, and organisms containing recombinant and/or synthetic nucleic acids... that all records and data bases created under this part are accurate, have controlled access, and that...

Reactive Derivatives of Nucleic Acids and Their Components as Affinity Reagents

NASA Astrophysics Data System (ADS)

Knorre, Dmitrii G.; Vlasov, Valentin V.

1985-09-01

The review is devoted to derivatives of nucleic acids and their components — nucleotides, nucleoside triphosphates, and oligonucleotides carrying reactive groups. Such derivatives are important tools for the investigation of protein-nucleic acid interactions and the functional topography of complex protein and nucleoprotein structures and can give rise to the prospect of being able to influence in a highly selective manner living organisms, including the nucleic acids and the nucleoproteins of the genetic apparatus. The review considers the principal groups of such reagents, the methods of their synthesis, and their properties which determine the possibility of their use for the selective (affinity) modification of biopolymers. The general principles of the construction of affinity reagents and their applications are analysed in relation to nucleotide affinity reagents. The bibliography includes 121 references.

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

PubMed

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

2010-09-01

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

Computation of statistical secondary structure of nucleic acids.

PubMed Central

Yamamoto, K; Kitamura, Y; Yoshikura, H

1984-01-01

This paper presents a computer analysis of statistical secondary structure of nucleic acids. For a given single stranded nucleic acid, we generated "structure map" which included all the annealing structures in the sequence. The map was transformed into "energy map" by rough approximation; here, the energy level of every pairing structure consisting of more than 2 successive nucleic acid pairs was calculated. By using the "energy map", the probability of occurrence of each annealed structure was computed, i.e., the structure was computed statistically. The basis of computation was the 8-queen problem in the chess game. The validity of our computer programme was checked by computing tRNA structure which has been well established. Successful application of this programme to small nuclear RNAs of various origins is demonstrated. PMID:6198622

In vitro evolution of chemically-modified nucleic acid aptamers: Pros and cons, and comprehensive selection strategies.

PubMed

Lipi, Farhana; Chen, Suxiang; Chakravarthy, Madhuri; Rakesh, Shilpa; Veedu, Rakesh N

2016-12-01

Nucleic acid aptamers are single-stranded DNA or RNA oligonucleotide sequences that bind to a specific target molecule with high affinity and specificity through their ability to adopt 3-dimensional structure in solution. Aptamers have huge potential as targeted therapeutics, diagnostics, delivery agents and as biosensors. However, aptamers composed of natural nucleotide monomers are quickly degraded in vivo and show poor pharmacodynamic properties. To overcome this, chemically-modified nucleic acid aptamers are developed by incorporating modified nucleotides after or during the selection process by Systematic Evolution of Ligands by EXponential enrichment (SELEX). This review will discuss the development of chemically-modified aptamers and provide the pros and cons, and new insights on in vitro aptamer selection strategies by using chemically-modified nucleic acid libraries.

In vitro evolution of chemically-modified nucleic acid aptamers: Pros and cons, and comprehensive selection strategies

PubMed Central

Chen, Suxiang; Chakravarthy, Madhuri; Rakesh, Shilpa; Veedu, Rakesh N.

2016-01-01

ABSTRACT Nucleic acid aptamers are single-stranded DNA or RNA oligonucleotide sequences that bind to a specific target molecule with high affinity and specificity through their ability to adopt 3-dimensional structure in solution. Aptamers have huge potential as targeted therapeutics, diagnostics, delivery agents and as biosensors. However, aptamers composed of natural nucleotide monomers are quickly degraded in vivo and show poor pharmacodynamic properties. To overcome this, chemically-modified nucleic acid aptamers are developed by incorporating modified nucleotides after or during the selection process by Systematic Evolution of Ligands by EXponential enrichment (SELEX). This review will discuss the development of chemically-modified aptamers and provide the pros and cons, and new insights on in vitro aptamer selection strategies by using chemically-modified nucleic acid libraries. PMID:27715478

Sequence-specific label-free nucleic acid biosensor for the detection of the hepatitis C virus genotype 1a using a disposable pencil graphite electrode.

PubMed

Donmez, Soner; Arslan, Fatma; Arslan, Halit

2016-05-01

In this paper, we demonstrate a simple, sensitive, inexpensive, disposable and label-free electrochemical nucleic acid biosensor for the detection of the hepatitis C virus genotype 1a (HCV1a). The nucleic acid biosensor was designed with the amino-linked inosine-substituted 20-mer probes, which were immobilized onto a disposable pencil graphite electrode (PGE) by covalent linking. The proposed nucleic acid biosensor was linear in the range of 0.05 and 0.75 μM, exhibiting a limit of detection of 54.9 nM. The single-stranded synthetic PCR product analogs of HCV1a were also detected with satisfactory results under optimal conditions, showing the potential application of this biosensor.

Hybrids of Nucleic Acids and Carbon Nanotubes for Nanobiotechnology.

PubMed

Umemura, Kazuo

2015-03-12

Recent progress in the combination of nucleic acids and carbon nanotubes (CNTs) has been briefly reviewed here. Since discovering the hybridization phenomenon of DNA molecules and CNTs in 2003, a large amount of fundamental and applied research has been carried out. Among thousands of papers published since 2003, approximately 240 papers focused on biological applications were selected and categorized based on the types of nucleic acids used, but not the types of CNTs. This survey revealed that the hybridization phenomenon is strongly affected by various factors, such as DNA sequences, and for this reason, fundamental studies on the hybridization phenomenon are important. Additionally, many research groups have proposed numerous practical applications, such as nanobiosensors. The goal of this review is to provide perspective on biological applications using hybrids of nucleic acids and CNTs.

Beyond DNA origami: A look on the bright future of nucleic acid nanotechnology

PubMed Central

Michelotti, Nicole; Johnson-Buck, Alexander; Manzo, Anthony J.

2012-01-01

Nucleic acid nanotechnology exploits the programmable molecular recognition properties of natural and synthetic nucleic acids to assemble structures with nanometer-scale precision. In 2006, DNA origami transformed the field by providing a versatile platform for self-assembly of arbitrary shapes from one long DNA strand held in place by hundreds of short, site-specific (spatially addressable) DNA ”staples”. This revolutionary approach has led to the creation of a multitude of 2D and 3D scaffolds that form the basis for functional nanodevices. Not limited to nucleic acids, these nanodevices can incorporate other structural and functional materials, such as proteins and nanoparticles, making them broadly useful for current and future applications in emerging fields such as nanomedicine, nanoelectronics, and alternative energy. PMID:22131292

Codes in the codons: construction of a codon/amino acid periodic table and a study of the nature of specific nucleic acid-protein interactions.

PubMed

Benyo, B; Biro, J C; Benyo, Z

2004-01-01

The theory of "codon-amino acid coevolution" was first proposed by Woese in 1967. It suggests that there is a stereochemical matching - that is, affinity - between amino acids and certain of the base triplet sequences that code for those amino acids. We have constructed a common periodic table of codons and amino acids, where the nucleic acid table showed perfect axial symmetry for codons and the corresponding amino acid table also displayed periodicity regarding the biochemical properties (charge and hydrophobicity) of the 20 amino acids and the position of the stop signals. The table indicates that the middle (2/sup nd/) amino acid in the codon has a prominent role in determining some of the structural features of the amino acids. The possibility that physical contact between codons and amino acids might exist was tested on restriction enzymes. Many recognition site-like sequences were found in the coding sequences of these enzymes and as many as 73 examples of codon-amino acid co-location were observed in the 7 known 3D structures (December 2003) of endonuclease-nucleic acid complexes. These results indicate that the smallest possible units of specific nucleic acid-protein interaction are indeed the stereochemically compatible codons and amino acids.

Dual phase multiplex polymerase chain reaction

DOEpatents

Pemov, Alexander [Charlottesville, VA; Bavykin, Sergei [Darien, IL

2008-10-07

Highly specific and sensitive methods were developed for multiplex amplification of nucleic acids on supports such as microarrays. Based on a specific primer design, methods include five types of amplification that proceed in a reaction chamber simultaneously. These relate to four types of multiplex amplification of a target DNA on a solid support, directed by forward and reverse complex primers immobilized to the support and a fifth type--pseudo-monoplex polymerase chain reaction (PCR) of multiple targets in solution, directed by a single pair of unbound universal primers. The addition of the universal primers in the reaction mixture increases the yield over the traditional "bridge" amplification on a solid support by approximately ten times. Methods that provide multitarget amplification and detection of as little as 0.45-4.5.times.10.sup.-12 g (equivalent to 10.sup.2-10.sup.3 genomes) of a bacterial genomic DNA are disclosed.

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.

Hibiscus cannabinus feruloyl-coa:monolignol transferase

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

Wilkerson, Curtis; Ralph, John; Withers, Saunia

The invention relates to isolated nucleic acids encoding a feruloyl-CoA:monolignol transferase and feruloyl-CoA:monolignol transferase enzymes. The isolated nucleic acids and/or the enzymes enable incorporation of monolignol ferulates into the lignin of plants, where such monolignol ferulates include, for example, p-coumaryl ferulate, coniferyl ferulate, and/or sinapyl ferulate. The invention also includes methods and plants that include nucleic acids encoding a feruloyl-CoA:monolignol transferase enzyme and/or feruloyl-CoA:monolignol transferase enzymes.

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[prime] to 3[prime] nucleic acid synthesis using 3[prime]-photoremovable protecting group

DOEpatents

Pirrung, M.C.; Shuey, S.W.; Bradley, J.C.

1999-06-01

The present invention relates, in general, to a method of synthesizing a nucleic acid, and, in particular, to a method of effecting 5[prime] to 3[prime] nucleic acid synthesis. The method can be used to prepare arrays of oligomers bound to a support via their 5[prime] end. The invention also relates to a method of effecting mutation analysis using such arrays. The invention further relates to compounds and compositions suitable for use in such methods.

Photocrosslinking and Photodamage in Protein-Nucleic Acid Systems Resulting from UV and IR Radiation.

NASA Astrophysics Data System (ADS)

Kozub, John Andrew

1995-01-01

Photocrosslinking of protein-nucleic acid complexes with low intensity UV has frequently been used to study biological systems. We have investigated the photochemistry of protein-nucleic acid systems using nanosecond UV pulses from a Nd:YAG-pumped dye laser system, low-intensity continuous UV from a typical germicidal lamp, and high-intensity mid -IR pulses from the Vanderbilt Free Electron Laser. Quantum yields for UV-induced nucleic acid damage from laser pulses and the germicidal lamp were found to be nearly equivalent. We have demonstrated the general applicability of the laser to this technique by successfully crosslinking hnRNP protein to RNA, yeast TATA-binding protein to dsDNA, and gene 32 protein to ssDNA with UV laser pulses. Our results indicate that UV-crosslinking has an intrinsic specificity for nucleic acid sites containing thymidine (or uridine), forcing a distinction between preferred binding sites and favorable crosslinking sites. We have found in each system that protein and nucleic acid photodamage competes with crosslinking, limits the yield, and may interfere with subsequent analysis. The distribution of photoproducts in the gene 32 protein-ssDNA system was investigated as a function of the total dose of UV radiation and the intensity of UV laser pulses. It was found that laser pulses providing up to 50 photons per nucleic acid base induce a linear response from the system; the absolute and relative yields of photoproducts depend only on the total dose of UV and not on the rate of delivery. At higher intensities, the yield of crosslinks per incident photon was reduced. A single pulse at the optimum intensity (about 100-200 photons per nucleic acid base) induced roughly 80% of the maximum attainable yield of crosslinks in this system. The early results of our search for photochemistry induced by Free Electron Laser pulses indicate the potential to induce a unique photoreaction in the gene 32 protein -ssDNA system. The yield is apparently enhanced by simultaneous exposure to UV pulses. Future experiments will test the potential of IR and UV irradiations to increase the specificity for photocrosslinks.

Similarities and differences in the nucleic acid chaperone activity of HIV-2 and HIV-1 nucleocapsid proteins in vitro.

PubMed

Pachulska-Wieczorek, Katarzyna; Stefaniak, Agnieszka K; Purzycka, Katarzyna J

2014-07-03

The nucleocapsid domain of Gag and mature nucleocapsid protein (NC) act as nucleic acid chaperones and facilitate folding of nucleic acids at critical steps of retroviral replication cycle. The basic N-terminus of HIV-1 NC protein was shown most important for the chaperone activity. The HIV-2 NC (NCp8) and HIV-1 NC (NCp7) proteins possess two highly conserved zinc fingers, flanked by basic residues. However, the NCp8 N-terminal domain is significantly shorter and contains less positively charged residues. This study characterizes previously unknown, nucleic acid chaperone activity of the HIV-2 NC protein. We have comparatively investigated the in vitro nucleic acid chaperone properties of the HIV-2 and HIV-1 NC proteins. Using substrates derived from the HIV-1 and HIV-2 genomes, we determined the ability of both proteins to chaperone nucleic acid aggregation, annealing and strand exchange in duplex structures. Both NC proteins displayed comparable, high annealing activity of HIV-1 TAR DNA and its complementary nucleic acid. Interesting differences between the two NC proteins were discovered when longer HIV substrates, particularly those derived from the HIV-2 genome, were used in chaperone assays. In contrast to NCp7, NCp8 weakly facilitates annealing of HIV-2 TAR RNA to its complementary TAR (-) DNA. NCp8 is also unable to efficiently stimulate tRNALys3 annealing to its respective HIV-2 PBS motif. Using truncated NCp8 peptide, we demonstrated that despite the fact that the N-terminus of NCp8 differs from that of NCp7, this domain is essential for NCp8 activity. Our data demonstrate that the HIV-2 NC protein displays reduced nucleic acid chaperone activity compared to that of HIV-1 NC. We found that NCp8 activity is limited by substrate length and stability to a greater degree than that of NCp7. This is especially interesting in light of the fact that the HIV-2 5'UTR is more structured than that of HIV-1. The reduced chaperone activity observed with NCp8 may influence the efficiency of reverse transcription and other key steps of the HIV-2 replication cycle.

Trichoderma .beta.-glucosidase

DOEpatents

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

2006-01-03

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

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

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

PubMed

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

2013-04-01

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

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.

RBscore&NBench: a high-level web server for nucleic acid binding residues prediction with a large-scale benchmarking database.

PubMed

Miao, Zhichao; Westhof, Eric

2016-07-08

RBscore&NBench combines a web server, RBscore and a database, NBench. RBscore predicts RNA-/DNA-binding residues in proteins and visualizes the prediction scores and features on protein structures. The scoring scheme of RBscore directly links feature values to nucleic acid binding probabilities and illustrates the nucleic acid binding energy funnel on the protein surface. To avoid dataset, binding site definition and assessment metric biases, we compared RBscore with 18 web servers and 3 stand-alone programs on 41 datasets, which demonstrated the high and stable accuracy of RBscore. A comprehensive comparison led us to develop a benchmark database named NBench. The web server is available on: http://ahsoka.u-strasbg.fr/rbscorenbench/. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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.

A review of room temperature storage of biospecimen tissue and nucleic acids for anatomic pathology laboratories and biorepositories

PubMed Central

Lou, Jerry J; Mirsadraei, Leili; Sanchez, Desiree E; Wilson, Ryan W; Shabihkhani, Maryam; Lucey, Gregory M; Wei, Bowen; Singer, Elyse J; Mareninov, Sergey; Yong, William H

2014-01-01

Frozen biospecimens are crucial for translational research and contain well preserved nucleic acids and protein. However, the risk for catastrophic freezer failure as well as space, cost, and environmental concerns argue for evaluating long-term room temperature storage alternatives. Formalin-fixed paraffin embedded (FFPE) tissues have great value but their use is limited by cross-linking and fragmentation of nucleic acids, as well as loss of enzymatic activity. Stabilization solutions can now robustly preserve fresh tissue for up to 7 days at room temperature. For longer term storage, commercial vendors of chemical matrices claim real time stability of nucleic acids of over 2 years and their accelerated aging studies to date suggest stability for 12 years for RNA and 60 years for DNA. However, anatomic pathology biorepositories store mostly frozen tissue rather than nucleic acids. Small quantities of tissue can be directly placed on some chemical matrices to stabilize DNA, however RNA and proteins are not preserved. Current lyophilization approaches can preserve histomorphology, DNA, RNA, and proteins though RNA shows moderate degradation after 1–2 years. Formalin free fixatives show improved but varying abilities to preserve nucleic acids and face validation as well as cost barriers in replacing FFPE specimens. The paraffin embedding process can degrade RNA. Development of robust long-term room temperature biospecimen tissue storage technology can potentially reduce costs for the biomedical community in the face of growing targeted therapy needs and decreasing budgets. PMID:24362270

Sodium sulphite inhibition of potato and cherry polyphenolics in nucleic acid extraction for virus detection by RT-PCR.

PubMed

Singh, R P; Nie, X; Singh, M; Coffin, R; Duplessis, P

2002-01-01

Phenolic compounds from plant tissues inhibit reverse transcription-polymerase chain reaction (RT-PCR). Multiple-step protocols using several additives to inhibit polyphenolic compounds during nucleic acid extraction are common, but time consuming and laborious. The current research highlights that the inclusion of 0.65 to 0.70% of sodium sulphite in the extraction buffer minimizes the pigmentation of nucleic acid extracts and improves the RT-PCR detection of Potato virus Y (PVY) and Potato leafroll virus (PLRV) in potato (Solanum tuberosum) tubers and Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV) in leaves and bark in the sweet cherry (Prunus avium) tree. Substituting sodium sulphite in the nucleic acid extraction buffer eliminated the use of proteinase K during extraction. Reagents phosphate buffered saline (PBS)-Tween 20 and polyvinylpyrrolidone (PVP) were also no longer required during RT or PCR phase. The resultant nucleic acid extracts were suitable for both duplex and multiplex RT-PCR. This simple and less expensive nucleic acid extraction protocol has proved very effective for potato cv. Russet Norkotah, which contains a high amount of polyphenolics. Comparing commercially available RNA extraction kits (Catrimox and RNeasy), the sodium sulphite based extraction protocol yielded two to three times higher amounts of RNA, while maintaining comparable virus detection by RT-PCR. The sodium sulphite based extraction protocol was equally effective in potato tubers, and in leaves and bark from the cherry tree.

Method for altering antibody light chain interactions

DOEpatents

Stevens, Fred J.; Stevens, Priscilla Wilkins; Raffen, Rosemarie; Schiffer, Marianne

2002-01-01

A method for recombinant antibody subunit dimerization including modifying at least one codon of a nucleic acid sequence to replace an amino acid occurring naturally in the antibody with a charged amino acid at a position in the interface segment of the light polypeptide variable region, the charged amino acid having a first polarity; and modifying at least one codon of the nucleic acid sequence to replace an amino acid occurring naturally in the antibody with a charged amino acid at a position in an interface segment of the heavy polypeptide variable region corresponding to a position in the light polypeptide variable region, the charged amino acid having a second polarity opposite the first polarity. Nucleic acid sequences which code for novel light chain proteins, the latter of which are used in conjunction with the inventive method, are also provided.

.beta.-glucosidase 5 (BGL5) compositions

DOEpatents

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

2010-06-01

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

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

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

PubMed

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

2016-09-01

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

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.

A pliable electroporation patch (ep-Patch) for efficient delivery of nucleic acid molecules into animal tissues with irregular surface shapes.

PubMed

Wei, Zewen; Huang, Yuanyu; Zhao, Deyao; Hu, Zhiyuan; Li, Zhihong; Liang, Zicai

2015-01-05

Delivery of nucleic acids into animal tissues by electroporation is an appealing approach for various types of gene therapy, but efficiency of existing methodsis not satisfactory. Here we present the validation of novel electroporation patch (ep-Patch) for efficient delivery of DNA and siRNA into mouse tissues. Using micromachining technology, closely spaced gold electrodes were made on the pliable parylene substrate to form a patch-like electroporation metrics. It enabled large coverage of the target tissues and close surface contact between the tissues and electrodes, thus providing a uniform electric field to deliver nucleic acids into tissues, even beneath intact skin. Using this ep-Patch for efficiently delivery of both DNA and siRNA, non-invasive electroporation of healthy mouse muscle tissue was successfully achieved. Delivery of these nucleic acids was performed to intact tumors with satisfactory results. Silencing of tumor genes using the ep-Patch was also demonstrated on mice. This pliable electroporation patch method constitutes a novel way of in vivo delivery of siRNA and DNA to certain tissues or organs to circumvent the disadvantages of existing methodologies for in vivo delivery of nucleic acid molecules.

Thermophoretic melting curves quantify the conformation and stability of RNA and DNA

PubMed Central

Wienken, Christoph J.; Baaske, Philipp; Duhr, Stefan; Braun, Dieter

2011-01-01

Measuring parameters such as stability and conformation of biomolecules, especially of nucleic acids, is important in the field of biology, medical diagnostics and biotechnology. We present a thermophoretic method to analyse the conformation and thermal stability of nucleic acids. It relies on the directed movement of molecules in a temperature gradient that depends on surface characteristics of the molecule, such as size, charge and hydrophobicity. By measuring thermophoresis of nucleic acids over temperature, we find clear melting transitions and resolve intermediate conformational states. These intermediate states are indicated by an additional peak in the thermophoretic signal preceding most melting transitions. We analysed single nucleotide polymorphisms, DNA modifications, conformational states of DNA hairpins and microRNA duplexes. The method is validated successfully against calculated melting temperatures and UV absorbance measurements. Interestingly, the methylation of DNA is detected by the thermophoretic amplitude even if it does not affect the melting temperature. In the described setup, thermophoresis is measured all-optical in a simple setup using a reproducible capillary format with only 250 nl probe consumption. The thermophoretic analysis of nucleic acids shows the technique’s versatility for the investigation of nucleic acids relevant in cellular processes like RNA interference or gene silencing. PMID:21297115

Nanopore analysis of polymers in solution.

NASA Astrophysics Data System (ADS)

Deamer, David

2002-03-01

Nanopores represent a novel approach for investigating macromolecules in solution. Polymers that have been analyzed by this technique include polyethylene glycol (PEG), certain proteins and nucleic acids. The a-hemolysin pore inserted into lipid bilayers provides continuous non-gated ion current through a pore diameter of approximately 1.5 - 2 nm. Nucleic acid molecules can be driven through the pore by imposing a voltage across the supporting membrane. Single stranded, but not double stranded nucleic acids pass through in strict linear sequence from one end of the molecule to the other. While in the pore, the molecule reduces ionic current, and properties of the ionic current blockade such as duration, mean amplitude and modulations of amplitude provide information about structure and composition of the nucleic acid. For a given molecular species, the duration of the blockade is a function of chain length, and the rate of blockades is linearly related to concentration. More recent studies have shown that the a-hemolysin nanopore can discriminate between synthetic DNA molecules differing by a single base pair or even a single nucleotide. These results indicate that a nanopore may have the resolution required for nucleic acid sequencing applications.

The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro.

PubMed

Kuciak, Monika; Gabus, Caroline; Ivanyi-Nagy, Roland; Semrad, Katharina; Storchak, Roman; Chaloin, Olivier; Muller, Sylviane; Mély, Yves; Darlix, Jean-Luc

2008-06-01

The human immunodeficiency virus type 1 (HIV-1) is a primate lentivirus that causes the acquired immunodeficiency syndrome (AIDS). In addition to the virion structural proteins and enzyme precursors, that are Gag, Env and Pol, HIV-1 encodes several regulatory proteins, notably a small nuclear transcriptional activator named Tat. The Tat protein is absolutely required for virus replication since it controls proviral DNA transcription to generate the full-length viral mRNA. Tat can also regulate mRNA capping and splicing and was recently found to interfere with the cellular mi- and siRNA machinery. Because of its extensive interplay with nucleic acids, and its basic and disordered nature we speculated that Tat had nucleic acid-chaperoning properties. This prompted us to examine in vitro the nucleic acid-chaperoning activities of Tat and Tat peptides made by chemical synthesis. Here we report that Tat has potent nucleic acid-chaperoning activities according to the standard DNA annealing, DNA and RNA strand exchange, RNA ribozyme cleavage and trans-splicing assays. The active Tat(44-61) peptide identified here corresponds to the smallest known sequence with DNA/RNA chaperoning properties.

The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro

PubMed Central

Kuciak, Monika; Gabus, Caroline; Ivanyi-Nagy, Roland; Semrad, Katharina; Storchak, Roman; Chaloin, Olivier; Muller, Sylviane; Mély, Yves; Darlix, Jean-Luc

2008-01-01

The human immunodeficiency virus type 1 (HIV-1) is a primate lentivirus that causes the acquired immunodeficiency syndrome (AIDS). In addition to the virion structural proteins and enzyme precursors, that are Gag, Env and Pol, HIV-1 encodes several regulatory proteins, notably a small nuclear transcriptional activator named Tat. The Tat protein is absolutely required for virus replication since it controls proviral DNA transcription to generate the full-length viral mRNA. Tat can also regulate mRNA capping and splicing and was recently found to interfere with the cellular mi- and siRNA machinery. Because of its extensive interplay with nucleic acids, and its basic and disordered nature we speculated that Tat had nucleic acid-chaperoning properties. This prompted us to examine in vitro the nucleic acid-chaperoning activities of Tat and Tat peptides made by chemical synthesis. Here we report that Tat has potent nucleic acid-chaperoning activities according to the standard DNA annealing, DNA and RNA strand exchange, RNA ribozyme cleavage and trans-splicing assays. The active Tat(44–61) peptide identified here corresponds to the smallest known sequence with DNA/RNA chaperoning properties. PMID:18442994

25 years and still going strong: 2'-O-(pyren-1-yl)methylribonucleotides - versatile building blocks for applications in molecular biology, diagnostics and materials science.

PubMed

Hrdlicka, Patrick J; Karmakar, Saswata

2017-11-29

Oligonucleotides (ONs) modified with 2'-O-(pyren-1-yl)methylribonucleotides have been explored for a range of applications in molecular biology, nucleic acid diagnostics, and materials science for more than 25 years. The first part of this review provides an overview of synthetic strategies toward 2'-O-(pyren-1-yl)methylribonucleotides and is followed by a summary of biophysical properties of nucleic acid duplexes modified with these building blocks. Insights from structural studies are then presented to rationalize the reported properties. In the second part, applications of ONs modified with 2'-O-(pyren-1-yl)methyl-RNA monomers are reviewed, which include detection of RNA targets, discrimination of single nucleotide polymorphisms, formation of self-assembled pyrene arrays on nucleic acid scaffolds, the study of charge transfer phenomena in nucleic acid duplexes, and sequence-unrestricted recognition of double-stranded DNA. The predictable binding mode of the pyrene moiety, coupled with the microenvironment-dependent properties and synthetic feasibility, render 2'-O-(pyren-1-yl)methyl-RNA monomers as a promising class of pyrene-functionalized nucleotide building blocks for new applications in molecular biology, nucleic acid diagnostics, and materials science.

Assessment for Melting Temperature Measurement of Nucleic Acid by HRM

PubMed Central

2016-01-01

High resolution melting (HRM), with a high sensitivity to distinguish the nucleic acid species with small variations, has been widely applied in the mutation scanning, methylation analysis, and genotyping. For the aim of extending HRM for the evaluation of thermal stability of nucleic acid secondary structures on sequence dependence, we investigated effects of the dye of EvaGreen, metal ions, and impurities (such as dNTPs) on melting temperature (T m) measurement by HRM. The accuracy of HRM was assessed as compared with UV melting method, and little difference between the two methods was found when the DNA T m was higher than 40°C. Both insufficiency and excessiveness of EvaGreen were found to give rise to a little bit higher T m, showing that the proportion of dye should be considered for precise T m measurement of nucleic acids. Finally, HRM method was also successfully used to measure T ms of DNA triplex, hairpin, and RNA duplex. In conclusion, HRM can be applied in the evaluation of thermal stability of nucleic acid (DNA or RNA) or secondary structural elements (even when dNTPs are present). PMID:27833775

Ordered Self-Assembled Monolayers of Peptide Nucleic Acids with DNA Recognition Capability

NASA Astrophysics Data System (ADS)

Briones, C.; Mateo-Marti, E.; Gómez-Navarro, C.; Parro, V.; Román, E.; Martín-Gago, J. A.

2004-11-01

We report on the formation of ordered self-assembled monolayers (SAMs) of single-stranded peptide nucleic acids (ssPNA). In spite of their remarkable length (7nm) thiolated PNAs assemble standing up on gold surfaces similarly to the SAMs of short alkanethiols. SAMs of ssPNA recognize complementary nucleic acids, acting as specific biosensors that discriminate even a point mutation in target ssDNA. These results are obtained by surface characterization techniques that avoid labeling of the target molecule: x-ray photoemission, x-ray absorption and atomic force microscopy.

Cloning and sequencing of pyruvate decarboxylase (PDC) genes from bacteria and uses therefor

DOEpatents

Maupin-Furlow, Julie A [Gainesville, FL; Talarico, Lee Ann [Gainesville, FL; Raj, Krishnan Chandra [Tamil Nadu, IN; Ingram, Lonnie O [Gainesville, FL

2008-02-05

The invention provides isolated nucleic acids molecules which encode pyruvate decarboxylase enzymes having improved decarboxylase activity, substrate affinity, thermostability, and activity at different pH. The nucleic acids of the invention also have a codon usage which allows for high expression in a variety of host cells. Accordingly, the invention provides recombinant expression vectors containing such nucleic acid molecules, recombinant host cells comprising the expression vectors, host cells further comprising other ethanologenic enzymes, and methods for producing useful substances, e.g., acetaldehyde and ethanol, using such host cells.

Methods and kits for nucleic acid analysis using fluorescence resonance energy transfer

DOEpatents

Kwok, Pui-Yan; Chen, Xiangning

1999-01-01

A method for detecting the presence of a target nucleotide or sequence of nucleotides in a nucleic acid is disclosed. The method is comprised of forming an oligonucleotide labeled with two fluorophores on the nucleic acid target site. The doubly labeled oligonucleotide is formed by addition of a singly labeled dideoxynucleoside triphosphate to a singly labeled polynucleotide or by ligation of two singly labeled polynucleotides. Detection of fluorescence resonance energy transfer upon denaturation indicates the presence of the target. Kits are also provided. The method is particularly applicable to genotyping.

Content and synthesis of nucleic acids in the cartilage in chondromalacia patellae.

PubMed

Lund, F; Telhag, H

1978-12-01

The content and the synthesis of nucleic acids in chondromalacian, osteoarthritis and normal cartilage was compared. The chondromalacian cartilage differed from osteoarthritis in that the content of nucleic acids was less. Also, the cell density was less in chondromalacian than in normal cartilage as opposed to previous findings in osteoarthritis. The synthesis of DNA was greater in chondromalacian than in normal cartilage but less than in osteoarthritis. With regard to the RNA synthesis, however, the chondromalacian cartilage showed a higher rate than both normal and osteoarthritic cartilage.

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.

Extending Immunological Profiling in the Gilthead Sea Bream, Sparus aurata, by Enriched cDNA Library Analysis, Microarray Design and Initial Studies upon the Inflammatory Response to PAMPs.

PubMed

Boltaña, Sebastian; Castellana, Barbara; Goetz, Giles; Tort, Lluis; Teles, Mariana; Mulero, Victor; Novoa, Beatriz; Figueras, Antonio; Goetz, Frederick W; Gallardo-Escarate, Cristian; Planas, Josep V; Mackenzie, Simon

2017-02-03

This study describes the development and validation of an enriched oligonucleotide-microarray platform for Sparus aurata (SAQ) to provide a platform for transcriptomic studies in this species. A transcriptome database was constructed by assembly of gilthead sea bream sequences derived from public repositories of mRNA together with reads from a large collection of expressed sequence tags (EST) from two extensive targeted cDNA libraries characterizing mRNA transcripts regulated by both bacterial and viral challenge. The developed microarray was further validated by analysing monocyte/macrophage activation profiles after challenge with two Gram-negative bacterial pathogen-associated molecular patterns (PAMPs; lipopolysaccharide (LPS) and peptidoglycan (PGN)). Of the approximately 10,000 EST sequenced, we obtained a total of 6837 EST longer than 100 nt, with 3778 and 3059 EST obtained from the bacterial-primed and from the viral-primed cDNA libraries, respectively. Functional classification of contigs from the bacterial- and viral-primed cDNA libraries by Gene Ontology (GO) showed that the top five represented categories were equally represented in the two libraries: metabolism (approximately 24% of the total number of contigs), carrier proteins/membrane transport (approximately 15%), effectors/modulators and cell communication (approximately 11%), nucleoside, nucleotide and nucleic acid metabolism (approximately 7.5%) and intracellular transducers/signal transduction (approximately 5%). Transcriptome analyses using this enriched oligonucleotide platform identified differential shifts in the response to PGN and LPS in macrophage-like cells, highlighting responsive gene-cassettes tightly related to PAMP host recognition. As observed in other fish species, PGN is a powerful activator of the inflammatory response in S. aurata macrophage-like cells. We have developed and validated an oligonucleotide microarray (SAQ) that provides a platform enriched for the study of gene expression in S. aurata with an emphasis upon immunity and the immune response.

Experience With Rapid Microarray-Based Diagnostic Technology and Antimicrobial Stewardship for Patients With Gram-Positive Bacteremia.

PubMed

Neuner, Elizabeth A; Pallotta, Andrea M; Lam, Simon W; Stowe, David; Gordon, Steven M; Procop, Gary W; Richter, Sandra S

2016-11-01

OBJECTIVE To describe the impact of rapid diagnostic microarray technology and antimicrobial stewardship for patients with Gram-positive blood cultures. DESIGN Retrospective pre-intervention/post-intervention study. SETTING A 1,200-bed academic medical center. PATIENTS Inpatients with blood cultures positive for Staphylococcus aureus, Enterococcus faecalis, E. faecium, Streptococcus pneumoniae, S. pyogenes, S. agalactiae, S. anginosus, Streptococcus spp., and Listeria monocytogenes during the 6 months before and after implementation of Verigene Gram-positive blood culture microarray (BC-GP) with an antimicrobial stewardship intervention. METHODS Before the intervention, no rapid diagnostic technology was used or antimicrobial stewardship intervention was undertaken, except for the use of peptide nucleic acid fluorescent in situ hybridization and MRSA agar to identify staphylococcal isolates. After the intervention, all Gram-positive blood cultures underwent BC-GP microarray and the antimicrobial stewardship intervention consisting of real-time notification and pharmacist review. RESULTS In total, 513 patients with bacteremia were included in this study: 280 patients with S. aureus, 150 patients with enterococci, 82 patients with stretococci, and 1 patient with L. monocytogenes. The number of antimicrobial switches was similar in the pre-BC-GP (52%; 155 of 300) and post-BC-GP (50%; 107 of 213) periods. The time to antimicrobial switch was significantly shorter in the post-BC-GP group than in the pre-BC-GP group: 48±41 hours versus 75±46 hours, respectively (P<.001). The most common antimicrobial switch was de-escalation and time to de-escalation, was significantly shorter in the post-BC-GP group than in the pre-BC-GP group: 53±41 hours versus 82±48 hours, respectively (P<.001). There was no difference in mortality or hospital length of stay as a result of the intervention. CONCLUSIONS The combination of a rapid microarray diagnostic test with an antimicrobial stewardship intervention improved time to antimicrobial switch, especially time to de-escalation to optimal therapy, in patients with Gram-positive blood cultures. Infect Control Hosp Epidemiol 2016;1-6.

Artificial Informational Polymers and Nanomaterials from Ring-Opening Metathesis Polymerization

NASA Astrophysics Data System (ADS)

James, Carrie Rae

Inspired by naturally occurring polymers (DNA, polypeptides, polysaccharides, etc.) that can self-assemble on the nanoscale into complex, information-rich architectures, we have synthesized nucleic acid based polymers using ROMP. These polymers were synthesized using a graft-through strategy, whereby nucleic acids bearing a strained cyclic olefin were directly polymerized. This is the first example of the graft-through polymerization of nucleic acids. Our approach takes advantage of non-charged peptide nucleic acids (PNAs) as elements to incorporate into ROMP polymer backbones. PNA is a synthetic nucleic acid analogue known for its increased affinity and specificity for complementary DNA or RNA. To accomplish the graft-through polymerization of PNA, we conjugated PNA to strained cyclic olefins using solid phase peptide conjugation chemistry. These PNA monomers were then directly polymerized into homo and block copolymers forming brushes, or comb-like arrangements, of information. Block copolymer amphiphiles of these materials, where the PNA brush served as the hydrophilic portion, were capable of self-assembly into spherical nanoparticles (PNA NPs). These PNA NPs were then studied with respect to their ability to hybridize complementary DNA sequences, as well as their ability to undergo cellular internalization. PNA NPs consisting of densely packed brushes of nucleic acids possessed increased thermal stability when mixed with their complementary DNA sequence, indicating a greater DNA binding affinity over their unpolymerized PNA counterparts. In addition, by arranging the PNA into dense brushes at the surface of the nanoparticle, Cy5.5 labeled PNA NPs were able to undergo cellular internalization into HeLa cells without the need for an additional cellular delivery device. Importantly, cellular internalization of PNA has remained a significant challenge in the literature due to the neutrally charged amino-ethyl glycine backbone of PNA. Therefore, this represents a novel way of facilitating cellular uptake of PNA. This materials strategy represents the first direct polymerization of nucleic acids, and presents a novel method for arranging biological information on the nanoscale at high density in order to confer novel attributes.

A MATHEMATICAL ANALYSIS OF SELEX

PubMed Central

Levine, Howard A.; Nilsen-Hamilton, Marit

2007-01-01

SELEX (Systematic Evolution of Ligands by Exponential Enrichment) is a procedure by which a mixture of nucleic acids can be separated into pure components with the goal of isolating those with specific biochemical activities. The basic idea is to combine the mixture with a specific target molecule and then separate the target-NA complex from the resulting reaction. The target-NA complex is then separated by mechanical means (for example by nitrocellulose filtration), the NA is then eluted from the complex, amplified by PCR (polymerase chain reaction) and the process repeated. After several rounds, one should be left with a pool of [NA]that consists mostly of the species in the original pool that best binds to the target. In Irvine et al. (1991) a mathematical analysis of this process was given. In this paper we revisit Irvine et al. (1991). By rewriting the equations for the SELEX process, we considerably reduce the labor of computing the round to round distribution of nucleic acid fractions. We also establish necessary and sufficient conditions for the SELEX process to converge to a pool consisting solely of the best binding nucleic acid to a fixed target in a manner that maximizes the percentage of bound target. The assumption is that there is a single nucleic acid binding site on the target that permits occupation by no more than one nucleic acid. We analyze the case for which there is no background loss, (no support losses and no free [NA] left on the support.) We then examine the case in which such there are such losses. The significance of the analysis is that it suggests an experimental approach for the SELEX process as defined in Irvine et al. (1991) to converge to a pool consisting of a single best binding nucleic acid without recourse to any a-priori information about the nature of the binding constants or the distribution of the individual nucleic acid fragments. PMID:17218151

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. © The Author(s) 2016. Published by Oxford University Press.

Engineering nucleic acid structures for programmable molecular circuitry and intracellular biocomputation

NASA Astrophysics Data System (ADS)

Li, Jiang; Green, Alexander A.; Yan, Hao; Fan, Chunhai

2017-11-01

Nucleic acids have attracted widespread attention due to the simplicity with which they can be designed to form discrete structures and programmed to perform specific functions at the nanoscale. The advantages of DNA/RNA nanotechnology offer numerous opportunities for in-cell and in-vivo applications, and the technology holds great promise to advance the growing field of synthetic biology. Many elegant examples have revealed the potential in integrating nucleic acid nanostructures in cells and in vivo where they can perform important physiological functions. In this Review, we summarize the current abilities of DNA/RNA nanotechnology to realize applications in live cells and then discuss the key problems that must be solved to fully exploit the useful properties of nanostructures. Finally, we provide viewpoints on how to integrate the tools provided by DNA/RNA nanotechnology and related new technologies to construct nucleic acid nanostructure-based molecular circuitry for synthetic biology.

Assessment of Damage to Nucleic Acids and Repair Machinery in Salmonella typhimurium Exposed to Chlorine

PubMed Central

Phe, M. H.; Hajj Chehade, M.; Guilloteau, H.; Merlin, C.; Block, J. C.

2009-01-01

Water disinfection is usually evaluated using mandatory methods based on cell culturability. However, such methods do not consider the potential of cells to recover, which should also be kept as low as possible. In this paper, we hypothesized that a successful disinfection is achieved only when the applied chlorine leads to both intracellular nucleic acid damage and strong alterations of the DNA repair machinery. Monitoring the SOS system responsiveness with a umuC’-‘lacZ reporter fusion, we found that the expression of this important cellular machinery was altered after the beginning of membrane permeabilization but prior to the total decline of both the cell culturability and the nucleic acid integrity as revealed by Sybr-II staining. Rapid measurement of such nucleic acid alterations by fluorochrome-based staining could be used as an alternative method for assessing the effectiveness of disinfection with chlorine. PMID:19936107

Methods of staining target chromosomal DNA employing high complexity nucleic acid probes

DOEpatents

Gray, Joe W.; Pinkel, Daniel; Kallioniemi, Ol'li-Pekka; Kallioniemi, Anne; Sakamoto, Masaru

2006-10-03

Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods and reagents are provided for the detection of genetic rearrangements. Probes and test kits are provided for use in detecting genetic rearrangements, particularly for use in tumor cytogenetics, in the detection of disease related loci, specifically cancer, such as chronic myelogenous leukemia (CML), retinoblastoma, ovarian and uterine cancers, and for biological dosimetry. Methods and reagents are described for cytogenetic research, for the differentiation of cytogenetically similar but genetically different diseases, and for many prognostic and diagnostic applications.

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.

Label-free functional nucleic acid sensors for detecting target agents

DOEpatents

Lu, Yi; Xiang, Yu

2015-01-13

A general methodology to design label-free fluorescent functional nucleic acid sensors using a vacant site approach and an abasic site approach is described. In one example, a method for designing label-free fluorescent functional nucleic acid sensors (e.g., those that include a DNAzyme, aptamer or aptazyme) that have a tunable dynamic range through the introduction of an abasic site (e.g., dSpacer) or a vacant site into the functional nucleic acids. Also provided is a general method for designing label-free fluorescent aptamer sensors based on the regulation of malachite green (MG) fluorescence. A general method for designing label-free fluorescent catalytic and molecular beacons (CAMBs) is also provided. The methods demonstrated here can be used to design many other label-free fluorescent sensors to detect a wide range of analytes. Sensors and methods of using the disclosed sensors are also provided.

Key Aspects of Nucleic Acid Library Design for in Vitro Selection

PubMed Central

Vorobyeva, Maria A.; Davydova, Anna S.; Vorobjev, Pavel E.; Pyshnyi, Dmitrii V.; Venyaminova, Alya G.

2018-01-01

Nucleic acid aptamers capable of selectively recognizing their target molecules have nowadays been established as powerful and tunable tools for biospecific applications, be it therapeutics, drug delivery systems or biosensors. It is now generally acknowledged that in vitro selection enables one to generate aptamers to almost any target of interest. However, the success of selection and the affinity of the resulting aptamers depend to a large extent on the nature and design of an initial random nucleic acid library. In this review, we summarize and discuss the most important features of the design of nucleic acid libraries for in vitro selection such as the nature of the library (DNA, RNA or modified nucleotides), the length of a randomized region and the presence of fixed sequences. We also compare and contrast different randomization strategies and consider computer methods of library design and some other aspects. PMID:29401748

Method for high-volume sequencing of nucleic acids: random and directed priming with libraries of oligonucleotides

DOEpatents

Studier, F. William

1995-04-18

Random and directed priming methods for determining nucleotide sequences by enzymatic sequencing techniques, using libraries of primers of lengths 8, 9 or 10 bases, are disclosed. These methods permit direct sequencing of nucleic acids as large as 45,000 base pairs or larger without the necessity for subcloning. Individual primers are used repeatedly to prime sequence reactions in many different nucleic acid molecules. Libraries containing as few as 10,000 octamers, 14,200 nonamers, or 44,000 decamers would have the capacity to determine the sequence of almost any cosmid DNA. Random priming with a fixed set of primers from a smaller library can also be used to initiate the sequencing of individual nucleic acid molecules, with the sequence being completed by directed priming with primers from the library. In contrast to random cloning techniques, a combined random and directed priming strategy is far more efficient.

Method for high-volume sequencing of nucleic acids: random and directed priming with libraries of oligonucleotides

DOEpatents

Studier, F.W.

1995-04-18

Random and directed priming methods for determining nucleotide sequences by enzymatic sequencing techniques, using libraries of primers of lengths 8, 9 or 10 bases, are disclosed. These methods permit direct sequencing of nucleic acids as large as 45,000 base pairs or larger without the necessity for subcloning. Individual primers are used repeatedly to prime sequence reactions in many different nucleic acid molecules. Libraries containing as few as 10,000 octamers, 14,200 nonamers, or 44,000 decamers would have the capacity to determine the sequence of almost any cosmid DNA. Random priming with a fixed set of primers from a smaller library can also be used to initiate the sequencing of individual nucleic acid molecules, with the sequence being completed by directed priming with primers from the library. In contrast to random cloning techniques, a combined random and directed priming strategy is far more efficient. 2 figs.

Exciton-controlled fluorescence: application to hybridization-sensitive fluorescent DNA probe.

PubMed

Okamoto, Akimitsu; Ikeda, Shuji; Kubota, Takeshi; Yuki, Mizue; Yanagisawa, Hiroyuki

2009-01-01

A hybridization-sensitive fluorescent probe has been designed for nucleic acid detection, using the concept of fluorescence quenching caused by the intramolecular excitonic interaction of fluorescence dyes. We synthesized a doubly thiazole orange-labeled nucleotide showing high fluorescence intensity for a hybrid with the target nucleic acid and effective quenching for the single-stranded state. This exciton-controlled fluorescent probe was applied to living HeLa cells using microinjection to visualize intracellular mRNA localization. Immediately after injection of the probe into the cell, fluorescence was observed from the probe hybridizing with the target RNA. This fluorescence rapidly decreased upon addition of a competitor DNA. Multicoloring of this probe resulted in the simple simultaneous detection of plural target nucleic acid sequences. This probe realized a large, rapid, reversible change in fluorescence intensity in sensitive response to the amount of target nucleic acid, and facilitated spatiotemporal monitoring of the behavior of intracellular RNA.

Laboratory procedures to generate viral metagenomes.

PubMed

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

2009-01-01

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

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

Simple Approaches to Minimally-Instrumented, Microfluidic-Based Point-of-Care Nucleic Acid Amplification Tests

PubMed Central

Mauk, Michael G.; Song, Jinzhao; Liu, Changchun; Bau, Haim H.

2018-01-01

Designs and applications of microfluidics-based devices for molecular diagnostics (Nucleic Acid Amplification Tests, NAATs) in infectious disease testing are reviewed, with emphasis on minimally instrumented, point-of-care (POC) tests for resource-limited settings. Microfluidic cartridges (‘chips’) that combine solid-phase nucleic acid extraction; isothermal enzymatic nucleic acid amplification; pre-stored, paraffin-encapsulated lyophilized reagents; and real-time or endpoint optical detection are described. These chips can be used with a companion module for separating plasma from blood through a combined sedimentation-filtration effect. Three reporter types: Fluorescence, colorimetric dyes, and bioluminescence; and a new paradigm for end-point detection based on a diffusion-reaction column are compared. Multiplexing (parallel amplification and detection of multiple targets) is demonstrated. Low-cost detection and added functionality (data analysis, control, communication) can be realized using a cellphone platform with the chip. Some related and similar-purposed approaches by others are surveyed. PMID:29495424

[Evaluation of viral etiology in central nervous system infections from a university hospital point of view in Izmir based on seven years data].

PubMed

Zeytinoğlu, Ayşın; Erensoy, Selda; Sertöz, Rüçhan; Altuğlu, İmre; Çiçek, Candan; Kayın, Münevver; Şirin, Hadiye; Taner, Şafak

2017-04-01

The serious diseases of the central nervous system (CNS); encephalitis and meningitis, have high mortality and morbidity rate especially not diagnosed and treated in time. Nucleic acid testing (NAT) is the tool of choice for viral diagnosis in CNS infections. In this study, viral etiological agents found in cerebrospinal fluid (CSF) samples sent to our university hospital virology laboratory for laboratory diagnosis of CNS infections were retrospectively evaluated and results were compared with other reports from our country. Viral etiological agents found in cerebrospinal fluid (CSF) samples sent to Ege University Faculty of Medicine Department of Medical Microbiology Virology Laboratories for laboratory diagnosis of CNS infection between 01.01.2009-31.12.2015 were evaluated retrospectively. A total of 3778 CSF tests were performed for cell culture of enterovirus (EV) in 487 samples and 3291 tests for nucleic acid testing (NAT) by real time polymerase chain reaction (PCR) in herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpes virus 6 (HHV6) and EV. VZV and EV NAT's were performed during the last one and five years period, respectively. NAT positive results for HSV1, HSV2, CMV, EBV, VZV, HHV6 and EV were 1.80% (24/1333), 0.08% (1/1333), 3.28% (19/580), 4.35% (22/506), 0.46% (1/216), 1.05% (5/478) and 3.37% (6/178), respectively. EV was isolated in 30 (6.20%) of 487 CSF samples by viral culture. Positive samples were mainly from pediatric, neurology and infectious diseases clinics as expected. The number of higher positive results were found in samples sentin december (35.3%), july (12.9%) and november (10.6%). Overall 80% of positive samples belonged to patients over 18 years old. When the results of other studies reported from Turkey are examined, although the positivity rates are generally similar, it is seen that the rates specific to certain factors are higher in selected smaller patient groups like HSV1 and EV. Rapid nucleic acid tests like multiplex PCR and microarray will provide more practical and effective laboratory diagnosis approach in CNS infections, since many more microorganisms may be causative agents.

Differentiation of respiratory syncytial virus subgroups with cDNA probes in a nucleic acid hybridization assay.

PubMed Central

Sullender, W M; Anderson, L J; Anderson, K; Wertz, G W

1990-01-01

A new approach to respiratory syncytial (RS) virus subgroup determination was developed by using a simple nucleic acid filter hybridization technique. By this method, virus-infected cells are bound and fixed in a single step, and the viral RNA in the fixed-cell preparation is characterized directly by its ability to hybridize to cDNA probes specific for either the A or B subgroups of RS virus. The subgroup-specific probes were constructed from cDNA clones that corresponded to a portion of the extracellular domain of the RS virus G protein of either a subgroup B RS virus (8/60) or a subgroup A RS virus (A2). The cDNA probes were labeled with 32P and used to analyze RS virus isolates collected over a period of three decades. Replicate templates of infected cell preparations were hybridized with either the subgroup A or B probe. The subgroup assignments of 40 viruses tested by nucleic acid hybridization were in agreement with the results of subgroup determinations based on their reactivities with monoclonal antibodies, which previously has been the only method available for determining the subgroup classification of RS virus isolates. The nucleic acid hybridization assay has the advantage of providing broad-based discrimination of the two subgroups on the basis of nucleic acid homology, irrespective of minor antigenic differences that are detected in assays in which monoclonal antibodies are used. The nucleic acid hybridization technique provides a reliable method for RS virus subgroup characterization. Images PMID:2118548

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.

Programming the Assembly of Unnatural Materials with Nucleic Acids

NASA Astrophysics Data System (ADS)

Mirkin, Chad

Nature directs the assembly of enormously complex and highly functional materials through an encoded class of biomolecules, nucleic acids. The establishment of a similarly programmable code for the construction of synthetic, unnatural materials would allow researchers to impart functionality by precisely positioning all material components. Although it is exceedingly difficult to control the complex interactions between atomic and molecular species in such a manner, interactions between nanoscale components can be directed through the ligands attached to their surface. Our group has shown that nucleic acids can be used as highly programmable surface ligands to control the spacing and symmetry of nanoparticle building blocks in structurally sophisticated and functional materials. These nucleic acids function as programmable ``bonds'' between nanoparticle ``atoms,'' analogous to a nanoscale genetic code for assembling materials. The sequence and length tunability of nucleic acid bonds has allowed us to define a powerful set of design rules for the construction of nanoparticle superlattices with more than 30 unique lattice symmetries, tunable defect structures and interparticle spacings, and several well-defined crystal habits. Further, the nature of the nucleic acid bond enables an additional level of structural control: temporal regulation of dynamic material response to external biomolecular and chemical stimuli. This control allows for the reversible transformation between thermodynamic states with different crystal symmetries, particle stoichiometries, thermal stabilities, and interparticle spacings on demand. Notably, our unique genetic approach affords functional nanoparticle architectures that, among many other applications, can be used to systematically explore and manipulate optoelectronic material properties, such as tunable interparticle plasmonic interactions, microstructure-directed energy emission, and coupled plasmonic and photonic modes.

RNA:DNA Ratio and Other Nucleic Acid Derived Indices in Marine Ecology

PubMed Central

Chícharo, Maria Alexandra; Chícharo, Luis

2008-01-01

Some of most used indicators in marine ecology are nucleic acid-derived indices. They can be divided by target levels in three groups: 1) at the organism level as ecophysiologic indicators, indicators such as RNA:DNA ratios, DNA:dry weight and RNA:protein, 2) at the population level, indicators such as growth rate, starvation incidence or fisheries impact indicators, and 3) at the community level, indicators such as trophic interactions, exergy indices and prey identification. The nucleic acids derived indices, especially RNA:DNA ratio, have been applied with success as indicators of nutritional condition, well been and growth in marine organisms. They are also useful as indicators of natural or anthropogenic impacts in marine population and communities, such as upwelling or dredge fisheries, respectively. They can help in understanding important issues of marine ecology such as trophic interactions in marine environment, fish and invertebrate recruitment failure and biodiversity changes, without laborious work of counting, measuring and identification of small marine organisms. Besides the objective of integrate nucleic acid derived indices across levels of organization, the paper will also include a general characterization of most used nucleic acid derived indices in marine ecology and also advantages and limitations of them. We can conclude that using indicators, such RNA:DNA ratios and other nucleic acids derived indices concomitantly with organism and ecosystems measures of responses to climate change (distribution, abundance, activity, metabolic rate, survival) will allow for the development of more rigorous and realistic predictions of the effects of anthropogenic climate change on marine systems. PMID:19325815

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

PubMed

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

2016-02-07

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

Thermal and acid tolerant beta-xylosidases, genes encoding, related organisms, and methods

DOEpatents

Thompson, David N [Idaho Falls, ID; Thompson, Vicki S [Idaho Falls, ID; Schaller, Kastli D [Ammon, ID; Apel, William A [Jackson, WY; Lacey, Jeffrey A [Idaho Falls, ID; Reed, David W [Idaho Falls, ID

2011-04-12

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius and variations thereof are provided. Further provided are methods of at least partially degrading xylotriose and/or xylobiose using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius and variations thereof.

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

PubMed Central

Pawlowski, David R.; Karalus, Richard J.

2012-01-01

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

Electricity-free, sequential nucleic acid and protein isolation.

PubMed

Pawlowski, David R; Karalus, Richard J

2012-05-15

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

Chip-Based Sensors for Disease Diagnosis

NASA Astrophysics Data System (ADS)

Fang, Zhichao

Nucleic acid analysis is one of the most important disease diagnostic approaches in medical practice, and has been commonly used in cancer biomarker detection, bacterial speciation and many other fields in laboratory. Currently, the application of powerful research methods for genetic analysis, including the polymerase chain reaction (PCR), DNA sequencing, and gene expression profiling using fluorescence microarrays, are not widely used in hospitals and extended-care units due to high-cost, long detection times, and extensive sample preparation. Bioassays, especially chip-based electrochemical sensors, may be suitable for the next generation of rapid, sensitive, and multiplexed detection tools. Herein, we report three different microelectrode platforms with capabilities enabled by nano- and microtechnology: nanoelectrode ensembles (NEEs), nanostructured microelectrodes (NMEs), and hierarchical nanostructured microelectrodes (HNMEs), all of which are able to directly detect unpurified RNA in clinical samples without enzymatic amplification. Biomarkers that are cancer and infectious disease relevant to clinical medicine were chosen to be the targets. Markers were successfully detected with clinically-relevant sensitivity. Using peptide nucleic acids (PNAs) as probes and an electrocatalytic reporter system, NEEs were able to detect prostate cancer-related gene fusions in tumor tissue samples with 100 ng of RNA. The development of NMEs improved the sensitivity of the assay further to 10 aM of DNA target, and multiplexed detection of RNA sequences of different prostate cancer-related gene fusion types was achieved on the chip-based NMEs platform. An HNMEs chip integrated with a bacterial lysis device was able to detect as few as 25 cfu bacteria in 30 minutes and monitor the detection in real time. Bacterial detection could also be performed in neat urine samples. The development of these versatile clinical diagnostic tools could be extended to the detection of various cancers, genetic, and infectious diseases.

Studies related to primitive chemistry. A proton and nitrogen-14 nuclear magnetic resonance amino acid and nucleic acid constituents and a and their possible relation to prebiotic

NASA Technical Reports Server (NTRS)

Manatt, S. L.; Cohen, E. A.; Shiller, A. M.; Chan, S. I.

1973-01-01

Preliminary proton nuclear magnetic resonance (NMR) studies were made to determine the applicability of this technique for the study of interactions between monomeric and polymeric amino acids with monomeric nucleic acid bases and nucleotides. Proton NMR results for aqueous solutions (D2O) demonstrated interactions between the bases cytosine and adenine and acidic and aromatic amino acids. Solutions of 5'-AMP admixed with amino acids exhibited more complex behavior but stacking between aromatic rings and destacking at high amino acids concentration was evident. The multisite nature of 5'-AMP was pointed out. Chemical shift changes for adenine and 5'-AMP with three water soluble polypeptides demonstrated that significant interactions exist. It was found that the linewidth-pH profile of each amino acid is unique. It is concluded that NMR techniques can give significant and quantitative data on the association of amino acid and nucleic acid constituents.

Triptycene: A Nucleic Acid Three-Way Junction Binder Scaffold

NASA Astrophysics Data System (ADS)

Yoon, Ina

Nucleic acids play a critical role in many biological processes such as gene regulation and replication. The development of small molecules that modulate nucleic acids with sequence or structure specificity would provide new strategies for regulating disease states at the nucleic acid level. However, this remains challenging mainly because of the nonspecific interactions between nucleic acids and small molecules. Three-way junctions are critical structural elements of nucleic acids. They are present in many important targets such as trinucleotide repeat junctions related to Huntington's disease, a temperature sensor sigma32 in E. coli, Dengue virus, and HIV. Triptycene-derived small molecules have been shown to bind to nucleic acid three-way junctions, resulting from their shape complementary. To develop a better understanding of designing molecules for targeting different junctions, a rapid screening of triptycene-based small molecules is needed. We envisioned that the installation of a linker at C9 position of the bicyclic core would allow for a rapid solid phase diversification. To achieve this aim, we synthesized 9-substituted triptycene scaffolds by using two different synthetic routes. The first synthetic route installed the linker from the amidation reaction between carboxylic acid at C9 position of the triptycene and an amine linker, beta-alanine ethyl ester. This new 9-substituted triptycene scaffold was then attached to a 2-chlorotrityl chloride resin for solid-phase diversification. This enabled a rapid diversification and an easy purification of mono-, di-, and tri-peptide triptycene derivatives. The binding affinities of these compounds were investigated towards a (CAG)˙(CTG) trinucleotide repeat junction. In the modified second synthetic route, we utilized a combined Heck coupling/benzyne Diels-Alder strategy. This improved synthetic strategy reduced the number of steps and total reaction times, increased the overall yield, improved solubilities of intermediates, and provided a new regioisomer that was not observed in the previous synthesis. Through this investigation, we discovered new high-affinity lead compounds towards a d(CAG)·(CTG) trinucleotide repeat junction. In addition, we turned our attention to sigma 32 mRNA, which contains a RNA three-way junction in E. coli. We demonstrated that triptycene-based small molecules can modulate the heat shock response in E. coli..

Automated detection and quantitation of bacterial RNA by using electrical microarrays.

PubMed

Elsholz, B; Wörl, R; Blohm, L; Albers, J; Feucht, H; Grunwald, T; Jürgen, B; Schweder, T; Hintsche, Rainer

2006-07-15

Low-density electrical 16S rRNA specific oligonucleotide microarrays and an automated analysis system have been developed for the identification and quantitation of pathogens. The pathogens are Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus epidermidis, which are typically involved in urinary tract infections. Interdigitated gold array electrodes (IDA-electrodes), which have structures in the nanometer range, have been used for very sensitive analysis. Thiol-modified oligonucleotides are immobilized on the gold IDA as capture probes. They mediate the specific recognition of the target 16S rRNA by hybridization. Additionally three unlabeled oligonucleotides are hybridized in close proximity to the capturing site. They are supporting molecules, because they improve the RNA hybridization at the capturing site. A biotin labeled detector oligonucleotide is also allowed to hybridize to the captured RNA sequence. The biotin labels enable the binding of avidin alkaline phophatase conjugates. The phosphatase liberates the electrochemical mediator p-aminophenol from its electrically inactive phosphate derivative. The electrical signals were generated by amperometric redox cycling and detected by a unique multipotentiostat. The read out signals of the microarray are position specific current and change over time in proportion to the analyte concentration. If two additional biotins are introduced into the affinity binding complex via the supporting oligonucleotides, the sensitivity of the assays increase more than 60%. The limit of detection of Escherichia coli total RNA has been determined to be 0.5 ng/microL. The control of fluidics for variable assay formats as well as the multichannel electrical read out and data handling have all been fully automated. The fast and easy procedure does not require any amplification of the targeted nucleic acids by PCR.

Lipid Microarray Biosensor for Biotoxin Detection.

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

Singh, Anup K.; Throckmorton, Daniel J.; Moran-Mirabal, Jose C.

2006-05-01

We present the use of micron-sized lipid domains, patterned onto planar substrates and within microfluidic channels, to assay the binding of bacterial toxins via total internal reflection fluorescence microscopy (TIRFM). The lipid domains were patterned using a polymer lift-off technique and consisted of ganglioside-populated DSPC:cholesterol supported lipid bilayers (SLBs). Lipid patterns were formed on the substrates by vesicle fusion followed by polymer lift-off, which revealed micron-sized SLBs containing either ganglioside GT1b or GM1. The ganglioside-populated SLB arrays were then exposed to either Cholera toxin subunit B (CTB) or Tetanus toxin fragment C (TTC). Binding was assayed on planar substrates bymore » TIRFM down to 1 nM concentration for CTB and 100 nM for TTC. Apparent binding constants extracted from three different models applied to the binding curves suggest that binding of a protein to a lipid-based receptor is strongly affected by the lipid composition of the SLB and by the substrate on which the bilayer is formed. Patterning of SLBs inside microfluidic channels also allowed the preparation of lipid domains with different compositions on a single device. Arrays within microfluidic channels were used to achieve segregation and selective binding from a binary mixture of the toxin fragments in one device. The binding and segregation within the microfluidic channels was assayed with epifluorescence as proof of concept. We propose that the method used for patterning the lipid microarrays on planar substrates and within microfluidic channels can be easily adapted to proteins or nucleic acids and can be used for biosensor applications and cell stimulation assays under different flow conditions. KEYWORDS. Microarray, ganglioside, polymer lift-off, cholera toxin, tetanus toxin, TIRFM, binding constant.4« less

Modulating lignin in plants

DOEpatents

Apuya, Nestor; Bobzin, Steven Craig; Okamuro, Jack; Zhang, Ke

2013-01-29

Materials and methods for modulating (e.g., increasing or decreasing) lignin content in plants are disclosed. For example, nucleic acids encoding lignin-modulating polypeptides are disclosed as well as methods for using such nucleic acids to generate transgenic plants having a modulated lignin content.

[To Protect Corneal Transparency against Diseases].

PubMed

Usui, Tomohiko

2016-03-01

To protect corneal transparency, we tried to develop a new therapeutic strategy for corneal neovascularization, corneal scar, and TGFBI-related corneal dystrophy using nucleic acid drug. 1. The expression of angiopietin-like protein 2 (Angptl2) markedly increased in the neovascularized corneas compared to the normal cornea, and Angtpl2 was(a potent inducer of inflammatory corneal neovascularization. We have produced a single-stranded proline-modified short hairpin anti-Angptl2 ribonucleric acid interference (RNAi) molecule that is carried in a lipid nanoparticle for topical application. We have found this agent can penetrate all layers of the cornea. Angptl2 mRNA expression and corneal neovascularization were inhibited in a mouse alkari injury model by topical application of this agent. Thus, this modified RNAi agent is a new topical formulation for use against corneal neovascularization and scar. 2. Human umbilical vein endothelial cells (HUVECs) were cultured with human corneal keratocytes under serum-free conditions. We performed microarray gene-expression analysis in the coculture system and selected angiopoietin-like protein 7 (Angptl7). In vivo, intrastromal injections of an anti-Angptl7 RNAi agent into the avascular corneal stroma of mice resulted in the growth of blood vessels. Further, we examined the effects of Angptl7 on corneal nerves using culture rat trigeminal cells and this molecule had neurotrophic property on the cornea. Thus, Angpt17 is a unique molecule, which contain its bilateral character (anti-angiogenic and neurotrophic) in the cornea; an agonistic nucleic acid drug for Angptl7 may be a new therapeutic tool for protecting corneal transparency. 3. We examined local gene editing for TGFBI-related corneal dystrophy using CRISPR-Cas9 mediated homology directed repair (HDR). Cultured corneal keratocytes were obtained from a patient of R124H granular dystrophy. The R124H gene arrangement was corrected by a tranfection of guide RNA and HDR repair template single strand DNA in vitro. Thus, CRISPR-Cas9 medi-ated HDR could be a future radical treatment for TGFBI-related corneal dystrophy.

Procedure for normalization of cDNA libraries

DOEpatents

Bonaldo, Maria DeFatima; Soares, Marcelo Bento

1997-01-01

This invention provides a method to normalize a cDNA library constructed in a vector capable of being converted to single-stranded circles and capable of producing complementary nucleic acid molecules to the single-stranded circles comprising: (a) converting the cDNA library in single-stranded circles; (b) generating complementary nucleic acid molecules to the single-stranded circles; (c) hybridizing the single-stranded circles converted in step (a) with complementary nucleic acid molecules of step (b) to produce partial duplexes to an appropriate Cot; (e) separating the unhybridized single-stranded circles from the hybridized single-stranded circles, thereby generating a normalized cDNA library.

Efficacy of peptide nucleic acid and selected conjugates against specific cellular pathologies of amyotrophic lateral sclerosis.

PubMed

Browne, Elisse C; Parakh, Sonam; Duncan, Luke F; Langford, Steven J; Atkin, Julie D; Abbott, Belinda M

2016-04-01

Cellular studies have been undertaken on a nonamer peptide nucleic acid (PNA) sequence, which binds to mRNA encoding superoxide dismutase 1, and a series of peptide nucleic acids conjugated to synthetic lipophilic vitamin analogs including a recently prepared menadione (vitamin K) analog. Reduction of both mutant superoxide dismutase 1 inclusion formation and endoplasmic reticulum stress, two of the key cellular pathological hallmarks in amyotrophic lateral sclerosis, by two of the prepared PNA oligomers is reported for the first time. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Modification of nucleic acids by azobenzene derivatives and their applications in biotechnology and nanotechnology.

PubMed

Li, Jing; Wang, Xingyu; Liang, Xingguo

2014-12-01

Azobenzene has been widely used as a photoregulator due to its reversible photoisomerization, large structural change between E and Z isomers, high photoisomerization yield, and high chemical stability. On the other hand, some azobenzene derivatives can be used as universal quenchers for many fluorophores. Nucleic acid is a good candidate to be modified because it is not only the template of gene expression but also widely used for building well-organized nanostructures and nanodevices. Because the size and polarity distribution of the azobenzene molecule is similar to a nucleobase pair, the introduction of azobenzene into nucleic acids has been shown to be an ingenious molecular design for constructing light-switching biosystems or light-driven nanomachines. Here we review recent advances in azobenzene-modified nucleic acids and their applications for artificial regulation of gene expression and enzymatic reactions, construction of photoresponsive nanostructures and nanodevices, molecular beacons, as well as obtaining structural information using the introduced azobenzene as an internal probe. In particular, nucleic acids bearing multiple azobenzenes can be used as a novel artificial nanomaterial with merits of high sequence specificity, regular duplex structure, and high photoregulation efficiency. The combination of functional groups with biomolecules may further advance the development of chemical biotechnology and biomolecular engineering. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

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

2017-06-01

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

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

PubMed

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

2007-03-01

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

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. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

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

2015-11-01

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

Optical nano-biosensing interface via nucleic acid amplification strategy: construction and application.

PubMed

Zhou, Hong; Liu, Jing; Xu, Jing-Juan; Zhang, Shu-Sheng; Chen, Hong-Yuan

2018-03-21

Modern optical detection technology plays a critical role in current clinical detection due to its high sensitivity and accuracy. However, higher requirements such as extremely high detection sensitivity have been put forward due to the clinical needs for the early finding and diagnosing of malignant tumors which are significant for tumor therapy. The technology of isothermal amplification with nucleic acids opens up avenues for meeting this requirement. Recent reports have shown that a nucleic acid amplification-assisted modern optical sensing interface has achieved satisfactory sensitivity and accuracy, high speed and specificity. Compared with isothermal amplification technology designed to work completely in a solution system, solid biosensing interfaces demonstrated better performances in stability and sensitivity due to their ease of separation from the reaction mixture and the better signal transduction on these optical nano-biosensing interfaces. Also the flexibility and designability during the construction of these nano-biosensing interfaces provided a promising research topic for the ultrasensitive detection of cancer diseases. In this review, we describe the construction of the burgeoning number of optical nano-biosensing interfaces assisted by a nucleic acid amplification strategy, and provide insightful views on: (1) approaches to the smart fabrication of an optical nano-biosensing interface, (2) biosensing mechanisms via the nucleic acid amplification method, (3) the newest strategies and future perspectives.

Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Triggered Isothermal Amplification for Site-Specific Nucleic Acid Detection.

PubMed

Huang, Mengqi; Zhou, Xiaoming; Wang, Huiying; Xing, Da

2018-02-06

A novel CRISPR/Cas9 triggered isothermal exponential amplification reaction (CAS-EXPAR) strategy based on CRISPR/Cas9 cleavage and nicking endonuclease (NEase) mediated nucleic acids amplification was developed for rapid and site-specific nucleic acid detection. CAS-EXPAR was primed by the target DNA fragment produced by cleavage of CRISPR/Cas9, and the amplification reaction performed cyclically to generate a large number of DNA replicates which were detected using a real-time fluorescence monitoring method. This strategy that combines the advantages of CRISPR/Cas9 and exponential amplification showed high specificity as well as rapid amplification kinetics. Unlike conventional nucleic acids amplification reactions, CAS-EXPAR does not require exogenous primers, which often cause target-independent amplification. Instead, primers are first generated by Cas9/sgRNA directed site-specific cleavage of target and accumulated during the reaction. It was demonstrated this strategy gave a detection limit of 0.82 amol and showed excellent specificity in discriminating single-base mismatch. Moreover, the applicability of this method to detect DNA methylation and L. monocytogenes total RNA was also verified. Therefore, CAS-EXPAR may provide a new paradigm for efficient nucleic acid amplification and hold the potential for molecular diagnostic applications.

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

Direct Detection of Nucleic Acid with Minimizing Background and Improving Sensitivity Based on a Conformation-Discriminating Indicator.

PubMed

Zhu, Lixuan; Qing, Zhihe; Hou, Lina; Yang, Sheng; Zou, Zhen; Cao, Zhong; Yang, Ronghua

2017-08-25

As is well-known, the nucleic acid indicator-based strategy is one of the major approaches to monitor the nucleic acid hybridization-mediated recognition events in biochemical analysis, displaying obvious advantages including simplicity, low cost, convenience, and generality. However, conventional indicators either hold strong self-fluorescence or can be lighted by both ssDNA and dsDNA, lacking absolute selectivity for a certain conformation, always with high background interference and low sensitivity in sensing; and additional processing (e.g., nanomaterial-mediated background suppression, and enzyme-catalyzed signal amplification) is generally required to improve the detection performance. In this work, a carbazole derivative, EBCB, has been synthesized and screened as a dsDNA-specific fluorescent indicator. Compared with conventional indicators under the same conditions, EBCB displayed a much higher selective coefficient for dsDNA, with little self-fluorescence and negligible effect from ssDNA. Based on its superior capability in DNA conformation-discrimination, high sensitivity with minimizing background interference was demonstrated for direct detection of nucleic acid, and monitoring nucleic acid-based circuitry with good reversibity, resulting in low detection limit and high capability for discriminating base-mismatching. Thus, we expect that this highly specific DNA conformation-discriminating indicator will hold good potential for application in biochemical sensing and molecular logic switching.

NMR studies of protein-nucleic acid interactions.

PubMed

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

2004-01-01

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

Analysis of nucleic acid chaperoning by the prion protein and its inhibition by oligonucleotides.

PubMed

Guichard, Cécile; Ivanyi-Nagy, Roland; Sharma, Kamal Kant; Gabus, Caroline; Marc, Daniel; Mély, Yves; Darlix, Jean-Luc

2011-10-01

Prion diseases are unique neurodegenerative illnesses associated with the conversion of the cellular prion protein (PrP(C)) into the aggregated misfolded scrapie isoform, named PrP(Sc). Recent studies on the physiological role of PrP(C) revealed that this protein has probably multiple functions, notably in cell-cell adhesion and signal transduction, and in assisting nucleic acid folding. In fact, in vitro findings indicated that the human PrP (huPrP) possesses nucleic acid binding and annealing activities, similarly to nucleic acid chaperone proteins that play essential roles in cellular DNA and RNA metabolism. Here, we show that a peptide, representing the N-terminal domain of huPrP, facilitates nucleic acid annealing by two parallel pathways nucleated through the stem termini. We also show that PrP of human or ovine origin facilitates DNA strand exchange, ribozyme-directed cleavage of an RNA template and RNA trans-splicing in a manner similar to the nucleocapsid protein of HIV-1. In an attempt to characterize inhibitors of PrP-chaperoning in vitro we discovered that the thioaptamer 5'-GACACAAGCCGA-3' was extensively inhibiting the PrP chaperoning activities. At the same time a recently characterized methylated oligoribonucleotide inhibiting the chaperoning activity of the HIV-1 nucleocapsid protein was poorly impairing the PrP chaperoning activities.

Fluorogenic PNA probes

PubMed Central

2018-01-01

Fluorogenic oligonucleotide probes that can produce a change in fluorescence signal upon binding to specific biomolecular targets, including nucleic acids as well as non-nucleic acid targets, such as proteins and small molecules, have applications in various important areas. These include diagnostics, drug development and as tools for studying biomolecular interactions in situ and in real time. The probes usually consist of a labeled oligonucleotide strand as a recognition element together with a mechanism for signal transduction that can translate the binding event into a measurable signal. While a number of strategies have been developed for the signal transduction, relatively little attention has been paid to the recognition element. Peptide nucleic acids (PNA) are DNA mimics with several favorable properties making them a potential alternative to natural nucleic acids for the development of fluorogenic probes, including their very strong and specific recognition and excellent chemical and biological stabilities in addition to their ability to bind to structured nucleic acid targets. In addition, the uncharged backbone of PNA allows for other unique designs that cannot be performed with oligonucleotides or analogues with negatively-charged backbones. This review aims to introduce the principle, showcase state-of-the-art technologies and update recent developments in the areas of fluorogenic PNA probes during the past 20 years. PMID:29507634

Real-time electrochemical monitoring of isothermal helicase-dependent amplification of nucleic acids.

PubMed

Kivlehan, Francine; Mavré, François; Talini, Luc; Limoges, Benoît; Marchal, Damien

2011-09-21

We described an electrochemical method to monitor in real-time the isothermal helicase-dependent amplification of nucleic acids. The principle of detection is simple and well-adapted to the development of portable, easy-to-use and inexpensive nucleic acids detection technologies. It consists of monitoring a decrease in the electrochemical current response of a reporter DNA intercalating redox probe during the isothermal DNA amplification. The method offers the possibility to quantitatively analyze target nucleic acids in less than one hour at a single constant temperature, and to perform at the end of the isothermal amplification a DNA melt curve analysis for differentiating between specific and non-specific amplifications. To illustrate the potentialities of this approach for the development of a simple, robust and low-cost instrument with high throughput capability, the method was validated with an electrochemical system capable of monitoring up to 48 real-time isothermal HDA reactions simultaneously in a disposable microplate consisting of 48-electrochemical microwells. Results obtained with this approach are comparable to that obtained with a well-established but more sophisticated and expensive fluorescence-based method. This makes for a promising alternative detection method not only for real-time isothermal helicase-dependent amplification of nucleic acid, but also for other isothermal DNA amplification strategies.

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

Synergistic Combination of Unquenching and Plasmonic Fluorescence Enhancement in Fluorogenic Nucleic Acid Hybridization Probes.

PubMed

Vietz, Carolin; Lalkens, Birka; Acuna, Guillermo P; Tinnefeld, Philip

2017-10-11

Fluorogenic nucleic acid hybridization probes are widely used for detecting and quantifying nucleic acids. The achieved sensitivity strongly depends on the contrast between a quenched closed form and an unquenched opened form with liberated fluorescence. So far, this contrast was improved by improving the quenching efficiency of the closed form. In this study, we modularly combine these probes with optical antennas used for plasmonic fluorescence enhancement and study the effect of the nanophotonic structure on the fluorescence of the quenched and the opened form. As quenched fluorescent dyes are usually enhanced more by fluorescence enhancement, a detrimental reduction of the contrast between closed and opened form was anticipated. In contrast, we could achieve a surprising increase of the contrast with full additivity of quenching of the dark form and fluorescence enhancement of the bright form. Using single-molecule experiments, we demonstrate that the additivity of the two mechanisms depends on the perfect quenching in the quenched form, and we delineate the rules for new nucleic acid probes for enhanced contrast and absolute brightness. Fluorogenic hybridization probes optimized not only for quenching but also for the brightness of the open form might find application in nucleic acid assays with PCR avoiding detection schemes.

Nuclemeter: A Reaction-Diffusion Column for Quantifying Nucleic Acids Undergoing Enzymatic Amplification

NASA Astrophysics Data System (ADS)

Bau, Haim; Liu, Changchun; Killawala, Chitvan; Sadik, Mohamed; Mauk, Michael

2014-11-01

Real-time amplification and quantification of specific nucleic acid sequences plays a major role in many medical and biotechnological applications. In the case of infectious diseases, quantification of the pathogen-load in patient specimens is critical to assessing disease progression, effectiveness of drug therapy, and emergence of drug-resistance. Typically, nucleic acid quantification requires sophisticated and expensive instruments, such as real-time PCR machines, which are not appropriate for on-site use and for low resource settings. We describe a simple, low-cost, reactiondiffusion based method for end-point quantification of target nucleic acids undergoing enzymatic amplification. The number of target molecules is inferred from the position of the reaction-diffusion front, analogous to reading temperature in a mercury thermometer. We model the process with the Fisher Kolmogoroff Petrovskii Piscounoff (FKPP) Equation and compare theoretical predictions with experimental observations. The proposed method is suitable for nucleic acid quantification at the point of care, compatible with multiplexing and high-throughput processing, and can function instrument-free. C.L. was supported by NIH/NIAID K25AI099160; M.S. was supported by the Pennsylvania Ben Franklin Technology Development Authority; C.K. and H.B. were funded, in part, by NIH/NIAID 1R41AI104418-01A1.

Thermal and acid tolerant beta xylosidases, arabinofuranosidases, genes encoding, related organisms, and methods

DOEpatents

Thompson, David N; Thompson, Vicki S; Schaller, Kastli D; Apel, William A; Reed, David W; Lacey, Jeffrey A

2013-04-30

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius and variations thereof are provided. Further provided are methods of at least partially degrading xylotriose, xylobiose, and/or arabinofuranose-substituted xylan using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius and variations thereof.

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

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-28

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

Fitting new technologies into the safety paradigm: use of microarrays in transfusion.

PubMed

Fournier-Wirth, C; Coste, J

2007-01-01

Until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. The recent emergence of Nucleic acid Amplification Technologies (NAT) has revolutionised viral diagnosis, not only by increasing the level of sensitivity but also by facilitating the detection of several viruses in parallel by multiplexing specific primers. In more complex biological situations, when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. High throughput systems, such as DNA Arrays, permit a conceptually new approach. These miniaturised micro systems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, a reduction in the number of confirmation tests and a simplification of data interpretation. However, the systems currently available require additional instrumentation and reagents for sample preparation and target amplification prior to detection on the DNA array. A major challenge in the area of DNA detection is the development of methods that do not rely on target amplification systems. Likewise, the advances of protein microarrays have lagged because of poor stability of proteins, complex coupling chemistry and weak detection signals. Emerging technologies like Biosensors and nano-particle based DNA or Protein Bio-Barcode Amplification Assays are promising diagnostic tools for a wide range of clinical applications, including blood donation screening.

Human Immunodeficiency Virus type 1 group M consensus and mosaic envelope glycoproteins

DOEpatents

Korber, Bette T.; Fischer, William; Liao, Hua-Xin; Haynes, Barton F.; Letvin, Norman; Hahn, Beatrice H.

2017-11-21

The disclosure relates to nucleic acids mosaic clade M HIV-1 Env polypeptides and to compositions and vectors comprising same. The nucleic acids are suitable for use in inducing an immune response to HIV-1 in a human.

9 CFR 121.4 - Overlap select agents and toxins.

Code of Federal Regulations, 2010 CFR

2010-01-01

... in paragraph (b) of this section that have been genetically modified. (d) Overlap select agents or... OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS POSSESSION, USE... elements, recombinant nucleic acids, and recombinant organisms: (1) Nucleic acids that can produce...

Optimized catalytic DNA-cleaving ribozymes

NASA Technical Reports Server (NTRS)

Joyce, Gerald F. (Inventor)

1996-01-01

The present invention discloses nucleic acid enzymes capable of cleaving nucleic acid molecules, including single-stranded DNA, in a site-specific manner under physiologic conditions, as well as compositions including same. The present invention also discloses methods of making and using the disclosed enzymes and compositions.

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

Reiser, Steven E.; Somerville, Chris R.

The present invention relates to bacterial enzymes, in particular to an acyl-CoA reductase and a gene encoding an acyl-CoA reductase, the amino acid and nucleic acid sequences corresponding to the reductase polypeptide and gene, respectively, and to methods of obtaining such enzymes, amino acid sequences and nucleic acid sequences. The invention also relates to the use of such sequences to provide transgenic host cells capable of producing fatty alcohols and fatty aldehydes.

Depletion of Unwanted Nucleic Acid Templates by Selective Cleavage: LNAzymes, Catalytically Active Oligonucleotides Containing Locked Nucleic Acids, Open a New Window for Detecting Rare Microbial Community Members

PubMed Central

Dolinšek, Jan; Dorninger, Christiane; Lagkouvardos, Ilias; Wagner, Michael

2013-01-01

Many studies of molecular microbial ecology rely on the characterization of microbial communities by PCR amplification, cloning, sequencing, and phylogenetic analysis of genes encoding rRNAs or functional marker enzymes. However, if the established clone libraries are dominated by one or a few sequence types, the cloned diversity is difficult to analyze by random clone sequencing. Here we present a novel approach to deplete unwanted sequence types from complex nucleic acid mixtures prior to cloning and downstream analyses. It employs catalytically active oligonucleotides containing locked nucleic acids (LNAzymes) for the specific cleavage of selected RNA targets. When combined with in vitro transcription and reverse transcriptase PCR, this LNAzyme-based technique can be used with DNA or RNA extracts from microbial communities. The simultaneous application of more than one specific LNAzyme allows the concurrent depletion of different sequence types from the same nucleic acid preparation. This new method was evaluated with defined mixtures of cloned 16S rRNA genes and then used to identify accompanying bacteria in an enrichment culture dominated by the nitrite oxidizer “Candidatus Nitrospira defluvii.” In silico analysis revealed that the majority of publicly deposited rRNA-targeted oligonucleotide probes may be used as specific LNAzymes with no or only minor sequence modifications. This efficient and cost-effective approach will greatly facilitate tasks such as the identification of microbial symbionts in nucleic acid preparations dominated by plastid or mitochondrial rRNA genes from eukaryotic hosts, the detection of contaminants in microbial cultures, and the analysis of rare organisms in microbial communities of highly uneven composition. PMID:23263968

An update on the Enzyme Portal: an integrative approach for exploring enzyme knowledge

PubMed Central

Onwubiko, J.; Zaru, R.; Rosanoff, S.; Antunes, R.; Bingley, M.; Watkins, X.; O'Donovan, C.; Martin, M. J.

2017-01-01

Abstract Enzymes are a key part of life processes and are increasingly important for various areas of research such as medicine, biotechnology, bioprocessing and drug research. The goal of the Enzyme Portal is to provide an interface to all European Bioinformatics Institute (EMBL-EBI) data about enzymes (de Matos, P., et al., (2013), BMC Bioinformatics, 14 (1), 103). These data include enzyme function, sequence features and family classification, protein structure, reactions, pathways, small molecules, diseases and the associated literature. The sources of enzyme data are: the UniProt Knowledgebase (UniProtKB) (UniProt Consortium, 2015), the Protein Data Bank in Europe (PDBe), (Valenkar, S., et al., Nucleic Acids Res.2016; 44, D385–D395) Rhea—a database of enzyme-catalysed reactions (Morgat, A., et al., Nucleic Acids Res. 2015; 43, D459-D464), Reactome—a database of biochemical pathways (Fabregat, A., et al., Nucleic Acids Res. 2016; 44, D481–D487), IntEnz—a resource with enzyme nomenclature information (Fleischmann, A., et al., Nucleic Acids Res. 2004 32, D434–D437) and ChEBI (Hastings, J., et al., Nucleic Acids Res. 2013) and ChEMBL (Bento, A. P., et al., Nucleic Acids Res. 201442, 1083–1090)—resources which contain information about small-molecule chemistry and bioactivity. This article describes the redesign of Enzyme Portal and the increased functionality added to maximise integration and interpretation of these data. Use case examples of the Enzyme Portal and the versatile workflows its supports are illustrated. We welcome the suggestion of new resources for integration. PMID:28158609

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

PubMed

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

2010-08-01

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