Electrophoretic detection and separation of mutant DNA using replaceable polymer matrices

DOEpatents

Karger, B.L.; Thilly, W.G.; Foret, F.; Khrapko, K.; Koehavong, P.; Cohen, A.S.; Giese, R.W.

1997-05-27

The disclosure relates to a method for resolving double-stranded DNA species differing by at least one base pair. Each of the species is characterized by an iso-melting domain with a unique melting temperature contiguous with a melting domain of higher thermal stability. 18 figs.

Cerium chloride stimulated controlled conversion of B-to-Z DNA in self-assembled nanostructures

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

Bhanjadeo, Madhabi M.; Academy of Scientific & Innovative Research; Nayak, Ashok K.

DNA adopts different conformation not only because of novel base pairs but also while interacting with inorganic or organic compounds. Self-assembled branched DNA (bDNA) structures or DNA origami that change conformation in response to environmental cues hold great promises in sensing and actuation at the nanoscale. Recently, the B-Z transition in DNA is being explored to design various nanomechanical devices. In this communication we have demonstrated that Cerium chloride binds to the phosphate backbone of self-assembled bDNA structure and induce B-to-Z transition at physiological concentration. The mechanism of controlled conversion from right-handed to left-handed has been assayed by various dyemore » binding studies using CD and fluorescence spectroscopy. Three different bDNA structures have been identified to display B-Z transition. This approach provides a rapid and reversible means to change bDNA conformation, which can be used for dynamic and progressive control at the nanoscale. - Highlights: • Cerium-induced B-to-Z DNA transition in self-assembled nanostructures. • Lower melting temperature of Z-DNA than B-DNA confirmed by CD spectroscopy. • Binding mechanism of cerium chloride is explained using fluorescence spectroscopy. • Right-handed to left-handed DNA conformation is also noticed in modified bDNA structure.« less

Modeling DNA bubble formation at the atomic scale

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

Beleva, V; Rasmussen, K. O.; Garcia, A. E.

We describe the fluctuations of double stranded DNA molecules using a minimalist Go model over a wide range of temperatures. Minimalist models allow us to describe, at the atomic level, the opening and formation of bubbles in DNA double helices. This model includes all the geometrical constraints in helix melting imposed by the 3D structure of the molecule. The DNA forms melted bubbles within double helices. These bubbles form and break as a function of time. The equilibrium average number of broken base pairs shows a sharp change as a function of T. We observe a temperature profile of sequencemore » dependent bubble formation similar to those measured by Zeng et al. Long nuclei acid molecules melt partially through the formations of bubbles. It is known that CG rich sequences melt at higher temperatures than AT rich sequences. The melting temperature, however, is not solely determined by the CG content, but by the sequence through base stacking and solvent interactions. Recently, models that incorporate the sequence and nonlinear dynamics of DNA double strands have shown that DNA exhibits a very rich dynamics. Recent extensions of the Bishop-Peyrard model show that fluctuations in the DNA structure lead to opening in localized regions, and that these regions in the DNA are associated with transcription initiation sites. 1D and 2D models of DNA may contain enough information about stacking and base pairing interactions, but lack the coupling between twisting, bending and base pair opening imposed by the double helical structure of DNA that all atom models easily describe. However, the complexity of the energy function used in all atom simulations (including solvent, ions, etc) does not allow for the description of DNA folding/unfolding events that occur in the microsecond time scale.« less

Raman spectroscopy of DNA-metal complexes. II. The thermal denaturation of DNA in the presence of Sr2+, Ba2+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, and Cd2+.

PubMed

Duguid, J G; Bloomfield, V A; Benevides, J M; Thomas, G J

1995-12-01

Differential scanning calorimetry, laser Raman spectroscopy, optical densitometry, and pH potentiometry have been used to investigate DNA melting profiles in the presence of the chloride salts of Ba2+, Sr2+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, and Cd2+. Metal-DNA interactions have been observed for the molar ratio [M2+]/[PO2-] = 0.6 in aqueous solutions containing 5% by weight of 160 bp mononucleosomal calf thymus DNA. All of the alkaline earth metals, plus Mn2+, elevate the melting temperature of DNA (Tm > 75.5 degrees C), whereas the transition metals Co2+, Ni2+, and Cd2+ lower Tm. Calorimetric (delta Hcal) and van't Hoff (delta HVH) enthalpies of melting range from 6.2-8.7 kcal/mol bp and 75.6-188.6 kcal/mol cooperative unit, respectively, and entropies from 17.5 to 24.7 cal/K mol bp. The average number of base pairs in a cooperative melting unit () varied from 11.3 to 28.1. No dichotomy was observed between alkaline earth and transition DNA-metal complexes for any of the thermodynamic parameters other than their effects on Tm. These results complement Raman difference spectra, which reveal decreases in backbone order, base unstacking, distortion of glycosyl torsion angles, and rupture of hydrogen bonds, which occur after thermal denaturation. Raman difference spectroscopy shows that transition metals interact with the N7 atom of guanine in duplex DNA. A broader range of interaction sites with single-stranded DNA includes ionic phosphates, the N1 and N7 atoms of purines, and the N3 atom of pyrimidines. For alkaline earth metals, very little interaction was observed with duplex DNA, whereas spectra of single-stranded complexes are very similar to those of melted DNA without metal. However, difference spectra reveal some metal-specific perturbations at 1092 cm-1 (nPO2-), 1258 cm-1 (dC, dA), and 1668 cm-1 (nC==O, dNH2 dT, dG, dC). Increased spectral intensity could also be observed near 1335 cm-1 (dA, dG) for CaDNA. Optical densitometry, employed to detect DNA aggregation, reveals increased turbidity during the melting transition for all divalent DNA-metal complexes, except SrDNA and BaDNA. Turbidity was not observed for DNA in the absence of metal. A correlation was made between DNA melting, aggregation, and the ratio of Raman intensities I1335/I1374. At room temperature, DNA-metal interactions result in a pH drop of 1.2-2.2 units for alkaline earths and more than 2.5 units for transition metals. Sr2+, Ba2+, and Mg2+ cause protonated sites on the DNA to become thermally labile. These results lead to a model that describes DNA aggregation and denaturation during heating in the presence of divalent metal cations; 1) The cations initially interact with the DNA at phosphate and/or base sites, resulting in proton displacement. 2) A combination of metal-base interactions and heating disrupts the base pairing within the DNA duplex. This allows divalent metals and protons to bind to additional sites on the DNA bases during the aggregation/melting process. 3) Strands whose bases have swung open upon disruption are linked to neighboring strands by metal ion bridges. 4) Near the midpoint of the melting transition, thermal energy breaks up the aggregate. We have no evidence to indicate whether metal ion cross-bridges or direct base-base interactions rupture first. 5) Finally, all cross-links break, resulting in single-stranded DNA complexed with metal ions.

High-Resolution Melting (HRM) of Hypervariable Mitochondrial DNA Regions for Forensic Science.

PubMed

Dos Santos Rocha, Alípio; de Amorim, Isis Salviano Soares; Simão, Tatiana de Almeida; da Fonseca, Adenilson de Souza; Garrido, Rodrigo Grazinoli; Mencalha, Andre Luiz

2018-03-01

Forensic strategies commonly are proceeding by analysis of short tandem repeats (STRs); however, new additional strategies have been proposed for forensic science. Thus, this article standardized the high-resolution melting (HRM) of DNA for forensic analyzes. For HRM, mitochondrial DNA (mtDNA) from eight individuals were extracted from mucosa swabs by DNAzol reagent, samples were amplified by PCR and submitted to HRM analysis to identify differences in hypervariable (HV) regions I and II. To confirm HRM, all PCR products were DNA sequencing. The data suggest that is possible discriminate DNA from different samples by HRM curves. Also, uncommon dual-dissociation was identified in a single PCR product, increasing HRM analyzes by evaluation of melting peaks. Thus, HRM is accurate and useful to screening small differences in HVI and HVII regions from mtDNA and increase the efficiency of laboratory routines based on forensic genetics. © 2017 American Academy of Forensic Sciences.

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

Man, Viet Hoang; Pan, Feng; Sagui, Celeste, E-mail: sagui@ncsu.edu

We explore the use of a fast laser melting simulation approach combined with atomistic molecular dynamics simulations in order to determine the melting and healing responses of B-DNA and Z-DNA dodecamers with the same d(5′-CGCGCGCGCGCG-3′){sub 2} sequence. The frequency of the laser pulse is specifically tuned to disrupt Watson-Crick hydrogen bonds, thus inducing melting of the DNA duplexes. Subsequently, the structures relax and partially refold, depending on the field strength. In addition to the inherent interest of the nonequilibrium melting process, we propose that fast melting by an infrared laser pulse could be used as a technique for a fastmore » comparison of relative stabilities of same-sequence oligonucleotides with different secondary structures with full atomistic detail of the structures and solvent. This could be particularly useful for nonstandard secondary structures involving non-canonical base pairs, mismatches, etc.« less

Mechanism of Promoter Melting by the Xeroderma Pigmentosum Complementation Group B Helicase of Transcription Factor IIH Revealed by Protein-DNA Photo-Cross-Linking

PubMed Central

Douziech, Maxime; Coin, Frédéric; Chipoulet, Jean-Marc; Arai, Yoko; Ohkuma, Yoshiaki; Egly, Jean-Marc; Coulombe, Benoit

2000-01-01

The p89/xeroderma pigmentosum complementation group B (XPB) ATPase-helicase of transcription factor IIH (TFIIH) is essential for promoter melting prior to transcription initiation by RNA polymerase II (RNAPII). By studying the topological organization of the initiation complex using site-specific protein-DNA photo-cross-linking, we have shown that p89/XPB makes promoter contacts both upstream and downstream of the initiation site. The upstream contact, which is in the region where promoter melting occurs (positions −9 to +2), requires tight DNA wrapping around RNAPII. The addition of hydrolyzable ATP tethers the template strand at positions −5 and +1 to RNAPII subunits. A mutation in p89/XPB found in a xeroderma pigmentosum patient impairs the ability of TFIIH to associate correctly with the complex and thereby melt promoter DNA. A model for open complex formation is proposed. PMID:11027286

Structural stability of DNA origami nanostructures in the presence of chaotropic agents

NASA Astrophysics Data System (ADS)

Ramakrishnan, Saminathan; Krainer, Georg; Grundmeier, Guido; Schlierf, Michael; Keller, Adrian

2016-05-01

DNA origami represent powerful platforms for single-molecule investigations of biomolecular processes. The required structural integrity of the DNA origami may, however, pose significant limitations regarding their applicability, for instance in protein folding studies that require strongly denaturing conditions. Here, we therefore report a detailed study on the stability of 2D DNA origami triangles in the presence of the strong chaotropic denaturing agents urea and guanidinium chloride (GdmCl) and its dependence on concentration and temperature. At room temperature, the DNA origami triangles are stable up to at least 24 h in both denaturants at concentrations as high as 6 M. At elevated temperatures, however, structural stability is governed by variations in the melting temperature of the individual staple strands. Therefore, the global melting temperature of the DNA origami does not represent an accurate measure of their structural stability. Although GdmCl has a stronger effect on the global melting temperature, its attack results in less structural damage than observed for urea under equivalent conditions. This enhanced structural stability most likely originates from the ionic nature of GdmCl. By rational design of the arrangement and lengths of the individual staple strands used for the folding of a particular shape, however, the structural stability of DNA origami may be enhanced even further to meet individual experimental requirements. Overall, their high stability renders DNA origami promising platforms for biomolecular studies in the presence of chaotropic agents, including single-molecule protein folding or structural switching.DNA origami represent powerful platforms for single-molecule investigations of biomolecular processes. The required structural integrity of the DNA origami may, however, pose significant limitations regarding their applicability, for instance in protein folding studies that require strongly denaturing conditions. Here, we therefore report a detailed study on the stability of 2D DNA origami triangles in the presence of the strong chaotropic denaturing agents urea and guanidinium chloride (GdmCl) and its dependence on concentration and temperature. At room temperature, the DNA origami triangles are stable up to at least 24 h in both denaturants at concentrations as high as 6 M. At elevated temperatures, however, structural stability is governed by variations in the melting temperature of the individual staple strands. Therefore, the global melting temperature of the DNA origami does not represent an accurate measure of their structural stability. Although GdmCl has a stronger effect on the global melting temperature, its attack results in less structural damage than observed for urea under equivalent conditions. This enhanced structural stability most likely originates from the ionic nature of GdmCl. By rational design of the arrangement and lengths of the individual staple strands used for the folding of a particular shape, however, the structural stability of DNA origami may be enhanced even further to meet individual experimental requirements. Overall, their high stability renders DNA origami promising platforms for biomolecular studies in the presence of chaotropic agents, including single-molecule protein folding or structural switching. Electronic supplementary information (ESI) available: Melting curves without baseline subtraction, AFM images of DNA origami after 24 h incubation, calculated melting temperatures of all staple strands. See DOI: 10.1039/c6nr00835f

Analysis of HIV using a high resolution melting (HRM) diversity assay: automation of HRM data analysis enhances the utility of the assay for analysis of HIV incidence.

PubMed

Cousins, Matthew M; Swan, David; Magaret, Craig A; Hoover, Donald R; Eshleman, Susan H

2012-01-01

HIV diversity may be a useful biomarker for discriminating between recent and non-recent HIV infection. The high resolution melting (HRM) diversity assay was developed to quantify HIV diversity in viral populations without sequencing. In this assay, HIV diversity is expressed as a single numeric HRM score that represents the width of a melting peak. HRM scores are highly associated with diversity measures obtained with next generation sequencing. In this report, a software package, the HRM Diversity Assay Analysis Tool (DivMelt), was developed to automate calculation of HRM scores from melting curve data. DivMelt uses computational algorithms to calculate HRM scores by identifying the start (T1) and end (T2) melting temperatures for a DNA sample and subtracting them (T2 - T1 =  HRM score). DivMelt contains many user-supplied analysis parameters to allow analyses to be tailored to different contexts. DivMelt analysis options were optimized to discriminate between recent and non-recent HIV infection and to maximize HRM score reproducibility. HRM scores calculated using DivMelt were compared to HRM scores obtained using a manual method that is based on visual inspection of DNA melting curves. HRM scores generated with DivMelt agreed with manually generated HRM scores obtained from the same DNA melting data. Optimal parameters for discriminating between recent and non-recent HIV infection were identified. DivMelt provided greater discrimination between recent and non-recent HIV infection than the manual method. DivMelt provides a rapid, accurate method of determining HRM scores from melting curve data, facilitating use of the HRM diversity assay for large-scale studies.

Analysis of HIV Using a High Resolution Melting (HRM) Diversity Assay: Automation of HRM Data Analysis Enhances the Utility of the Assay for Analysis of HIV Incidence

PubMed Central

Cousins, Matthew M.; Swan, David; Magaret, Craig A.; Hoover, Donald R.; Eshleman, Susan H.

2012-01-01

Background HIV diversity may be a useful biomarker for discriminating between recent and non-recent HIV infection. The high resolution melting (HRM) diversity assay was developed to quantify HIV diversity in viral populations without sequencing. In this assay, HIV diversity is expressed as a single numeric HRM score that represents the width of a melting peak. HRM scores are highly associated with diversity measures obtained with next generation sequencing. In this report, a software package, the HRM Diversity Assay Analysis Tool (DivMelt), was developed to automate calculation of HRM scores from melting curve data. Methods DivMelt uses computational algorithms to calculate HRM scores by identifying the start (T1) and end (T2) melting temperatures for a DNA sample and subtracting them (T2–T1 = HRM score). DivMelt contains many user-supplied analysis parameters to allow analyses to be tailored to different contexts. DivMelt analysis options were optimized to discriminate between recent and non-recent HIV infection and to maximize HRM score reproducibility. HRM scores calculated using DivMelt were compared to HRM scores obtained using a manual method that is based on visual inspection of DNA melting curves. Results HRM scores generated with DivMelt agreed with manually generated HRM scores obtained from the same DNA melting data. Optimal parameters for discriminating between recent and non-recent HIV infection were identified. DivMelt provided greater discrimination between recent and non-recent HIV infection than the manual method. Conclusion DivMelt provides a rapid, accurate method of determining HRM scores from melting curve data, facilitating use of the HRM diversity assay for large-scale studies. PMID:23240016

Cockayne syndrome group A and B proteins converge on transcription-linked resolution of non-B DNA.

PubMed

Scheibye-Knudsen, Morten; Tseng, Anne; Borch Jensen, Martin; Scheibye-Alsing, Karsten; Fang, Evandro Fei; Iyama, Teruaki; Bharti, Sanjay Kumar; Marosi, Krisztina; Froetscher, Lynn; Kassahun, Henok; Eckley, David Mark; Maul, Robert W; Bastian, Paul; De, Supriyo; Ghosh, Soumita; Nilsen, Hilde; Goldberg, Ilya G; Mattson, Mark P; Wilson, David M; Brosh, Robert M; Gorospe, Myriam; Bohr, Vilhelm A

2016-11-01

Cockayne syndrome is a neurodegenerative accelerated aging disorder caused by mutations in the CSA or CSB genes. Although the pathogenesis of Cockayne syndrome has remained elusive, recent work implicates mitochondrial dysfunction in the disease progression. Here, we present evidence that loss of CSA or CSB in a neuroblastoma cell line converges on mitochondrial dysfunction caused by defects in ribosomal DNA transcription and activation of the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1). Indeed, inhibition of ribosomal DNA transcription leads to mitochondrial dysfunction in a number of cell lines. Furthermore, machine-learning algorithms predict that diseases with defects in ribosomal DNA (rDNA) transcription have mitochondrial dysfunction, and, accordingly, this is found when factors involved in rDNA transcription are knocked down. Mechanistically, loss of CSA or CSB leads to polymerase stalling at non-B DNA in a neuroblastoma cell line, in particular at G-quadruplex structures, and recombinant CSB can melt G-quadruplex structures. Indeed, stabilization of G-quadruplex structures activates PARP1 and leads to accelerated aging in Caenorhabditis elegans In conclusion, this work supports a role for impaired ribosomal DNA transcription in Cockayne syndrome and suggests that transcription-coupled resolution of secondary structures may be a mechanism to repress spurious activation of a DNA damage response.

Ligand induced stabilization of the melting temperature of the HSV-1 single-strand DNA binding protein using the thermal shift assay.

PubMed

Rupesh, Kanchi Ravi; Smith, Aaron; Boehmer, Paul E

2014-11-28

We have adapted the thermal shift assay to measure the ligand binding properties of the herpes simplex virus-1 single-strand DNA binding protein, ICP8. By measuring SYPRO Orange fluorescence in microtiter plates using a fluorescence-enabled thermal cycler, we have quantified the effects of oligonucleotide ligands on the melting temperature of ICP8. We found that single-stranded oligomers raise the melting temperature of ICP8 in a length- and concentration-dependent manner, ranging from 1°C for (dT)5 to a maximum of 9°C with oligomers ⩾10 nucleotides, with an apparent Kd of <1μM for (dT)20. Specifically, the results indicate that ICP8 is capable of interacting with oligomers as short as 5 nucleotides. Moreover, the observed increases in melting temperature of up to 9°C, indicates that single-strand DNA binding significantly stabilizes the structure of ICP8. This assay may be applied to investigate the ligand binding proteins of other single-strand DNA binding proteins and used as a high-throughput screen to identify compounds with therapeutic potential that inhibit single-strand DNA binding. As proof of concept, the single-strand DNA binding agent ciprofloxacin reduces the ligand induced stabilization of the melting temperature of ICP8 in a dose-dependent manner. Copyright © 2014 Elsevier Inc. All rights reserved.

Simultaneous Profiling of DNA Mutation and Methylation by Melting Analysis Using Magnetoresistive Biosensor Array.

PubMed

Rizzi, Giovanni; Lee, Jung-Rok; Dahl, Christina; Guldberg, Per; Dufva, Martin; Wang, Shan X; Hansen, Mikkel F

2017-09-26

Epigenetic modifications, in particular DNA methylation, are gaining increasing interest as complementary information to DNA mutations for cancer diagnostics and prognostics. We introduce a method to simultaneously profile DNA mutation and methylation events for an array of sites with single site specificity. Genomic (mutation) or bisulphite-treated (methylation) DNA is amplified using nondiscriminatory primers, and the amplicons are then hybridized to a giant magnetoresistive (GMR) biosensor array followed by melting curve measurements. The GMR biosensor platform offers scalable multiplexed detection of DNA hybridization, which is insensitive to temperature variation. The melting curve approach further enhances the assay specificity and tolerance to variations in probe length. We demonstrate the utility of this method by simultaneously profiling five mutation and four methylation sites in human melanoma cell lines. The method correctly identified all mutation and methylation events and further provided quantitative assessment of methylation density validated by bisulphite pyrosequencing.

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

Rapid discrimination between Anopheles gambiae s.s. and Anopheles arabiensis by High-Resolution Melt (HRM) analysis.

PubMed

Zianni, Michael R; Nikbakhtzadeh, Mahmood R; Jackson, Bryan T; Panescu, Jenny; Foster, Woodbridge A

2013-04-01

There is a need for more cost-effective options to more accurately discriminate among members of the Anopheles gambiae complex, particularly An. gambiae and Anopheles arabiensis. These species are morphologically indistinguishable in the adult stage, have overlapping distributions, but are behaviorally and ecologically different, yet both are efficient vectors of malaria in equatorial Africa. The method described here, High-Resolution Melt (HRM) analysis, takes advantage of minute differences in DNA melting characteristics, depending on the number of incongruent single nucleotide polymorphisms in an intragenic spacer region of the X-chromosome-based ribosomal DNA. The two species in question differ by an average of 13 single-nucleotide polymorphisms giving widely divergent melting curves. A real-time PCR system, Bio-Rad CFX96, was used in combination with a dsDNA-specific dye, EvaGreen, to detect and measure the melting properties of the amplicon generated from leg-extracted DNA of selected mosquitoes. Results with seven individuals from pure colonies of known species, as well as 10 field-captured individuals unambiguously identified by DNA sequencing, demonstrated that the method provided a high level of accuracy. The method was used to identify 86 field mosquitoes through the assignment of each to the two common clusters with a high degree of certainty. Each cluster was defined by individuals from pure colonies. HRM analysis is simpler to use than most other methods and provides comparable or more accurate discrimination between the two sibling species but requires a specialized melt-analysis instrument and software.

Rapid Discrimination between Anopheles gambiae s.s. and Anopheles arabiensis by High-Resolution Melt (HRM) Analysis

PubMed Central

Zianni, Michael R.; Nikbakhtzadeh, Mahmood R.; Jackson, Bryan T.; Panescu, Jenny; Foster, Woodbridge A.

2013-01-01

There is a need for more cost-effective options to more accurately discriminate among members of the Anopheles gambiae complex, particularly An. gambiae and Anopheles arabiensis. These species are morphologically indistinguishable in the adult stage, have overlapping distributions, but are behaviorally and ecologically different, yet both are efficient vectors of malaria in equatorial Africa. The method described here, High-Resolution Melt (HRM) analysis, takes advantage of minute differences in DNA melting characteristics, depending on the number of incongruent single nucleotide polymorphisms in an intragenic spacer region of the X-chromosome-based ribosomal DNA. The two species in question differ by an average of 13 single-nucleotide polymorphisms giving widely divergent melting curves. A real-time PCR system, Bio-Rad CFX96, was used in combination with a dsDNA-specific dye, EvaGreen, to detect and measure the melting properties of the amplicon generated from leg-extracted DNA of selected mosquitoes. Results with seven individuals from pure colonies of known species, as well as 10 field-captured individuals unambiguously identified by DNA sequencing, demonstrated that the method provided a high level of accuracy. The method was used to identify 86 field mosquitoes through the assignment of each to the two common clusters with a high degree of certainty. Each cluster was defined by individuals from pure colonies. HRM analysis is simpler to use than most other methods and provides comparable or more accurate discrimination between the two sibling species but requires a specialized melt-analysis instrument and software. PMID:23543777

The Denaturation Transition of DNA in Mixed Solvents

PubMed Central

Hammouda, Boualem; Worcester, David

2006-01-01

The helix-to-coil denaturation transition in DNA has been investigated in mixed solvents at high concentration using ultraviolet light absorption spectroscopy and small-angle neutron scattering. Two solvents have been used: water and ethylene glycol. The “melting” transition temperature was found to be 94°C for 4% mass fraction DNA/d-water and 38°C for 4% mass fraction DNA/d-ethylene glycol. The DNA melting transition temperature was found to vary linearly with the solvent fraction in the mixed solvents case. Deuterated solvents (d-water and d-ethylene glycol) were used to enhance the small-angle neutron scattering signal and 0.1M NaCl (or 0.0058 g/g mass fraction) salt concentration was added to screen charge interactions in all cases. DNA structural information was obtained by small-angle neutron scattering, including a correlation length characteristic of the inter-distance between the hydrogen-containing (desoxyribose sugar-amine base) groups. This correlation length was found to increase from 8.5 to 12.3 Å across the melting transition. Ethylene glycol and water mixed solvents were found to mix randomly in the solvation region in the helix phase, but nonideal solvent mixing was found in the melted coil phase. In the coil phase, solvent mixtures are more effective solvating agents than either of the individual solvents. Once melted, DNA coils behave like swollen water-soluble synthetic polymer chains. PMID:16815902

Force-dependent melting of supercoiled DNA at thermophilic temperatures.

PubMed

Galburt, E A; Tomko, E J; Stump, W T; Ruiz Manzano, A

2014-01-01

Local DNA opening plays an important role in DNA metabolism as the double-helix must be melted before the information contained within may be accessed. Cells finely tune the torsional state of their genomes to strike a balance between stability and accessibility. For example, while mesophilic life forms maintain negatively superhelical genomes, thermophilic life forms use unique mechanisms to maintain relaxed or even positively supercoiled genomes. Here, we use a single-molecule magnetic tweezers approach to quantify the force-dependent equilibrium between DNA melting and supercoiling at high temperatures populated by Thermophiles. We show that negatively supercoiled DNA denatures at 0.5 pN lower tension at thermophilic vs. mesophilic temperatures. This work demonstrates the ability to monitor DNA supercoiling at high temperature and opens the possibility to perform magnetic tweezers assays on thermophilic systems. The data allow for an estimation of the relative energies of base-pairing and DNA bending as a function of temperature and support speculation as to different general mechanisms of DNA opening in different environments. Lastly, our results imply that average in vivo DNA tensions range between 0.3 and 1.1 pN. Copyright © 2014 Elsevier B.V. All rights reserved.

Decoding DNA labels by melting curve analysis using real-time PCR.

PubMed

Balog, József A; Fehér, Liliána Z; Puskás, László G

2017-12-01

Synthetic DNA has been used as an authentication code for a diverse number of applications. However, existing decoding approaches are based on either DNA sequencing or the determination of DNA length variations. Here, we present a simple alternative protocol for labeling different objects using a small number of short DNA sequences that differ in their melting points. Code amplification and decoding can be done in two steps using quantitative PCR (qPCR). To obtain a DNA barcode with high complexity, we defined 8 template groups, each having 4 different DNA templates, yielding 158 (>2.5 billion) combinations of different individual melting temperature (Tm) values and corresponding ID codes. The reproducibility and specificity of the decoding was confirmed by using the most complex template mixture, which had 32 different products in 8 groups with different Tm values. The industrial applicability of our protocol was also demonstrated by labeling a drone with an oil-based paint containing a predefined DNA code, which was then successfully decoded. The method presented here consists of a simple code system based on a small number of synthetic DNA sequences and a cost-effective, rapid decoding protocol using a few qPCR reactions, enabling a wide range of authentication applications.

Localized DNA melting and structural pertubations in the origin of replication, oriC, of Escherichia coli in vitro and in vivo.

PubMed Central

Gille, H; Messer, W

1991-01-01

The leftmost region of the Escherichia coli origin of DNA replication (oriC) contains three tandemly repeated AT-rich 13mers which have been shown to become single-stranded during the early stages of initiation in vitro. Melting is induced by the ATP form of DnaA, the initiator protein of DNA replication. KMnO4 was used to probe for single-stranded regions and altered DNA conformation during the initiation of DNA replication at oriC in vitro and in vivo. Unpairing in the AT-rich 13mer region is thermodynamically stable even in the absence of DnaA protein, but only when divalent cations are omitted from the reaction. In the presence of Mg2+, oriC melting is strictly DnaA dependent. The sensitive region is distinct from that detected in the absence of DnaA as it is located further to the left within the minimal origin. In addition, the DNA is severely distorted between the three 13mers and the IHF binding site in oriC. A change of conformation can also be observed during the initiation of DNA replication in vivo. This is the first in vivo evidence for a structural change at the 13mers during initiation complex formation. Images PMID:2026151

[The study of complex-formation of DNA with the antimicrobial drug decamethoxine].

PubMed

Sorokin, V A; Blagoĭ, Iu P; Valeev, V A; Gladchenko, G O; Sukhodub, L F; Volianskiĭ, Iu L

1990-01-01

The interaction of effective antibacterial drug decametoxyn with natural DNA was studied by UV-spectroscopy. Decametoxyn shows a specificity to nucleotides: it decreases the cooperativity of melting and the thermal stability of DNA parts enriched by AT pairs. The characteristics of the helix-coil transition on the DNA parts enriched by GC-pairs are invariable. Interaction with AT-pairs results in their partial or complete melting at room temperature, followed by intermolecule aggregation. Interacting with phosphates decametoxyn manifests itself not as a dication but as two single-charged ions.

Structural Confirmation of a Bent and Open Model for the Initiation Complex of T7 RNA Polymerase

PubMed Central

Turingan, Rosemary S.; Liu, Cuihua; Hawkins, Mary E.; Martin, Craig T.

2008-01-01

T7 RNA polymerase is known to induce bending of its promoter DNA upon binding, as evidenced by gel-shift assays and by recent end-to-end fluorescence energy transfer distance measurements. Crystal structures of promoter-bound and initially transcribing complexes, however, lack downstream DNA, providing no information on the overall path of the DNA through the protein. Crystal structures of the elongation complex do include downstream DNA and provide valuable guidance in the design of models for the complete melted bubble structure at initiation. In the current study, we test a specific structural model for the initiation complex, obtained by alignment of the C-terminal regions of the protein structures from both initiation and elongation and then simple transferal of the downstream DNA from the elongation complex onto the initiation complex. FRET measurement of distances from a point upstream on the promoter DNA to various points along the downstream helix reproduce the expected helical periodicity in the distances and support the model’s orientation and phasing of the downstream DNA. The model also makes predictions about the extent of melting downstream of the active site. By monitoring fluorescent base analogs incorporated at various positions in the DNA we have mapped the downstream edge of the bubble, confirming the model. The initially melted bubble, in the absence of substrate, encompasses 7–8 bases and is sufficient to allow synthesis of a 3 base transcript before further melting is required. The results demonstrate that despite massive changes in the N-terminal portion of the protein and in the DNA upstream of the active site, the DNA downstream of the active site is virtually identical in both initiation and elongation complexes. PMID:17253774

Dynamics of DNA breathing: weak noise analysis, finite time singularity, and mapping onto the quantum Coulomb problem.

PubMed

Fogedby, Hans C; Metzler, Ralf

2007-12-01

We study the dynamics of denaturation bubbles in double-stranded DNA on the basis of the Poland-Scheraga model. We show that long time distributions for the survival of DNA bubbles and the size autocorrelation function can be derived from an asymptotic weak noise approach. In particular, below the melting temperature the bubble closure corresponds to a noisy finite time singularity. We demonstrate that the associated Fokker-Planck equation is equivalent to a quantum Coulomb problem. Below the melting temperature, the bubble lifetime is associated with the continuum of scattering states of the repulsive Coulomb potential; at the melting temperature, the Coulomb potential vanishes and the underlying first exit dynamics exhibits a long time power law tail; above the melting temperature, corresponding to an attractive Coulomb potential, the long time dynamics is controlled by the lowest bound state. Correlations and finite size effects are discussed.

Mapping the yeast genome by melting in nanofluidic devices

NASA Astrophysics Data System (ADS)

Welch, Robert L.; Czolkos, Ilja; Sladek, Rob; Reisner, Walter

2012-02-01

Optical mapping of DNA provides large-scale genomic information that can be used to assemble contigs from next-generation sequencing, and to detect re-arrangements between single cells. A recent optical mapping technique called denaturation mapping has the unique advantage of using physical principles rather than the action of enzymes to probe genomic structure. The absence of reagents or reaction steps makes denaturation mapping simpler than other protocols. Denaturation mapping uses fluorescence microscopy to image the pattern of partial melting along a DNA molecule extended in a channel of cross-section ˜100nm at the heart of a nanofluidic device. We successfully aligned melting maps from single DNA molecules to a theoretical map of the yeast genome (11.6Mbp) to identify their location. By aligning hundreds of molecules we assembled a consensus melting map of the yeast genome with 95% coverage.

Kinetic theory for DNA melting with vibrational entropy

NASA Astrophysics Data System (ADS)

Sensale, Sebastian; Peng, Zhangli; Chang, Hsueh-Chia

2017-10-01

By treating DNA as a vibrating nonlinear lattice, an activated kinetic theory for DNA melting is developed to capture the breakage of the hydrogen bonds and subsequent softening of torsional and bending vibration modes. With a coarse-grained lattice model, we identify a key bending mode with GHz frequency that replaces the hydrogen vibration modes as the dominant out-of-phase phonon vibration at the transition state. By associating its bending modulus to a universal in-phase bending vibration modulus at equilibrium, we can hence estimate the entropic change in the out-of-phase vibration from near-equilibrium all-atom simulations. This and estimates of torsional and bending entropy changes lead to the first predictive and sequence-dependent theory with good quantitative agreement with experimental data for the activation energy of melting of short DNA molecules without intermediate hairpin structures.

Detection of somatic mutations by high-resolution DNA melting (HRM) analysis in multiple cancers.

PubMed

Gonzalez-Bosquet, Jesus; Calcei, Jacob; Wei, Jun S; Garcia-Closas, Montserrat; Sherman, Mark E; Hewitt, Stephen; Vockley, Joseph; Lissowska, Jolanta; Yang, Hannah P; Khan, Javed; Chanock, Stephen

2011-01-17

Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.

Detection of Somatic Mutations by High-Resolution DNA Melting (HRM) Analysis in Multiple Cancers

PubMed Central

Gonzalez-Bosquet, Jesus; Calcei, Jacob; Wei, Jun S.; Garcia-Closas, Montserrat; Sherman, Mark E.; Hewitt, Stephen; Vockley, Joseph; Lissowska, Jolanta; Yang, Hannah P.; Khan, Javed; Chanock, Stephen

2011-01-01

Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples. PMID:21264207

Dpb11 may function with RPA and DNA to initiate DNA replication.

PubMed

Bruck, Irina; Dhingra, Nalini; Martinez, Matthew P; Kaplan, Daniel L

2017-01-01

Dpb11 is required for the initiation of DNA replication in budding yeast. We found that Dpb11 binds tightly to single-stranded DNA (ssDNA) or branched DNA structures, while its human homolog, TopBP1, binds tightly to branched-DNA structures. We also found that Dpb11 binds stably to CDK-phosphorylated RPA, the eukaryotic ssDNA binding protein, in the presence of branched DNA. A Dpb11 mutant specifically defective for DNA binding did not exhibit tight binding to RPA in the presence of DNA, suggesting that Dpb11-interaction with DNA may promote the recruitment of RPA to melted DNA. We then characterized a mutant of Dpb11 that is specifically defective in DNA binding in budding yeast cells. Expression of dpb11-m1,2,3,5,ΔC results in a substantial decrease in RPA recruitment to origins, suggesting that Dpb11 interaction with DNA may be required for RPA recruitment to origins. Expression of dpb11-m1,2,3,5,ΔC also results in diminished GINS interaction with Mcm2-7 during S phase, while Cdc45 interaction with Mcm2-7 is like wild-type. The reduced GINS interaction with Mcm2-7 may be an indirect consequence of diminished origin melting. We propose that the tight interaction between Dpb11, CDK-phosphorylated RPA, and branched-DNA may be required for the essential function of stabilizing melted origin DNA in vivo. We also propose an alternative model, wherein Dpb11-DNA interaction is required for some other function in DNA replication initiation, such as helicase activation.

A DNA Melting Exercise for a Large Laboratory Class

ERIC Educational Resources Information Center

Levine, Lauren A.; Junker, Matthew; Stark, Myranda; Greenleaf, Dustin

2015-01-01

A simple and economical experimental setup is described that enables multiple individuals or groups within a laboratory class to measure the thermal melting of double stranded DNA simultaneously. The setup utilizes a basic spectrophotometer capable of measuring absorbance at 260 nm, UV plastic cuvettes, and a stirring hot plate. Students measure…

Rapid discrimination of Isaria javanica and Isaria poprawskii from Isaria spp. using high resolution DNA melting assays

USDA-ARS?s Scientific Manuscript database

The current study evaluates the potential of using high resolution DNA melting assays to discriminate species in the genus, Isaria. The study utilizes a previously identified 103 base pair PCR amplicon, which was reported to be selective for Isaria fumosorosea. Our study finds the amplicon selective...

Trehalose facilitates DNA melting: a single-molecule optical tweezers study.

PubMed

Bezrukavnikov, Sergey; Mashaghi, Alireza; van Wijk, Roeland J; Gu, Chan; Yang, Li Jiang; Gao, Yi Qin; Tans, Sander J

2014-10-07

Using optical tweezers, here we show that the overstretching transition force of double-stranded DNA (dsDNA) is lowered significantly by the addition of the disaccharide trehalose as well as certain polyol osmolytes. This effect is found to depend linearly on the logarithm of the trehalose concentration. We propose an entropic driving mechanism for the experimentally observed destabilization of dsDNA that is rooted in the higher affinity of the DNA bases for trehalose than for water, which promotes base exposure and DNA melting. Molecular dynamics simulation reveals the direct interaction of trehalose with nucleobases. Experiments with other osmolytes confirm that the extent of dsDNA destabilization is governed by the ratio between polar and apolar fractions of an osmolyte.

STR melting curve analysis as a genetic screening tool for crime scene samples.

PubMed

Nguyen, Quang; McKinney, Jason; Johnson, Donald J; Roberts, Katherine A; Hardy, Winters R

2012-07-01

In this proof-of-concept study, high-resolution melt curve (HRMC) analysis was investigated as a postquantification screening tool to discriminate human CSF1PO and THO1 genotypes amplified with mini-STR primers in the presence of SYBR Green or LCGreen Plus dyes. A total of 12 CSF1PO and 11 HUMTHO1 genotypes were analyzed on the LightScanner HR96 and LS-32 systems and were correctly differentiated based upon their respective melt profiles. Short STR amplicon melt curves were affected by repeat number, and single-source and mixed DNA samples were additionally differentiated by the formation of heteroduplexes. Melting curves were shown to be unique and reproducible from DNA quantities ranging from 20 to 0.4 ng and distinguished identical from nonidentical genotypes from DNA derived from different biological fluids and compromised samples. Thus, a method is described which can assess both the quantity and the possible probative value of samples without full genotyping. 2012 American Academy of Forensic Sciences. Published 2012. This article is a U.S. Government work and is in the public domain in the U.S.A.

Identification and discrimination of Toxoplasma gondii, Sarcocystis spp., Neospora spp., and Cryptosporidium spp. by righ-resolution melting analysis

PubMed Central

2017-01-01

The objective of this study was to standardize the high-resolution melting method for identification and discrimination of Toxoplasma gondii, Sarcocystis spp., Neospora spp., and Cryptosporidium spp. by amplification of 18S ribosomal DNA (rDNA) using a single primer pair. The analyses were performed on individual reactions (containing DNA from a single species of a protozoan), on duplex reactions (containing DNA from two species of protozoa in each reaction), and on a multiplex reaction (containing DNA of four parasites in a single reaction). The proposed method allowed us to identify and discriminate the four species by analyzing the derivative, normalized, and difference melting curves, with high reproducibility among and within the experiments, as demonstrated by low coefficients of variation (less than 2.2% and 2.0%, respectively). This is the first study where this method is used for discrimination of these four species of protozoa in a single reaction. PMID:28346485

DNA melting profiles from a matrix method.

PubMed

Poland, Douglas

2004-02-05

In this article we give a new method for the calculation of DNA melting profiles. Based on the matrix formulation of the DNA partition function, the method relies for its efficiency on the fact that the required matrices are very sparse, essentially reducing matrix multiplication to vector multiplication and thus making the computer time required to treat a DNA molecule containing N base pairs proportional to N(2). A key ingredient in the method is the result that multiplication by the inverse matrix can also be reduced to vector multiplication. The task of calculating the melting profile for the entire genome is further reduced by treating regions of the molecule between helix-plateaus, thus breaking the molecule up into independent parts that can each be treated individually. The method is easily modified to incorporate changes in the assignment of statistical weights to the different structural features of DNA. We illustrate the method using the genome of Haemophilus influenzae. Copyright 2003 Wiley Periodicals, Inc.

Detection of plant oil DNA using high resolution melting (HRM) post PCR analysis: a tool for disclosure of olive oil adulteration.

PubMed

Vietina, Michelangelo; Agrimonti, Caterina; Marmiroli, Nelson

2013-12-15

Extra virgin olive oil is frequently subjected to adulterations with addition of oils obtained from plants other than olive. DNA analysis is a fast and economic tool to identify plant components in oils. Extraction and amplification of DNA by PCR was tested in olives, in milled seeds and in oils, to investigate its use in olive oil traceability. DNA was extracted from different oils made of hazelnut, maize, sunflower, peanut, sesame, soybean, rice and pumpkin. Comparing the DNA melting profiles in reference plant materials and in the oils, it was possible to identify any plant components in oils and mixtures of oils. Real-Time PCR (RT-PCR) platform has been added of the new methodology of high resolution melting (HRM), both were used to analyse olive oils mixed with different percentage of other oils. Results showed HRM a cost effective method for efficient detection of adulterations in olive oils. Copyright © 2013 Elsevier Ltd. All rights reserved.

Unlabeled oligonucleotides as internal temperature controls for genotyping by amplicon melting.

PubMed

Seipp, Michael T; Durtschi, Jacob D; Liew, Michael A; Williams, Jamie; Damjanovich, Kristy; Pont-Kingdon, Genevieve; Lyon, Elaine; Voelkerding, Karl V; Wittwer, Carl T

2007-07-01

Amplicon melting is a closed-tube method for genotyping that does not require probes, real-time analysis, or allele-specific polymerase chain reaction. However, correct differentiation of homozygous mutant and wild-type samples by melting temperature (Tm) requires high-resolution melting and closely controlled reaction conditions. When three different DNA extraction methods were used to isolate DNA from whole blood, amplicon Tm differences of 0.03 to 0.39 degrees C attributable to the extractions were observed. To correct for solution chemistry differences between samples, complementary unlabeled oligonucleotides were included as internal temperature controls to shift and scale the temperature axis of derivative melting plots. This adjustment was applied to a duplex amplicon melting assay for the methylenetetrahydrofolate reductase variants 1298A>C and 677C>T. High- and low-temperature controls bracketing the amplicon melting region decreased the Tm SD within homozygous genotypes by 47 to 82%. The amplicon melting assay was 100% concordant to an adjacent hybridization probe (HybProbe) melting assay when temperature controls were included, whereas a 3% error rate was observed without temperature correction. In conclusion, internal temperature controls increase the accuracy of genotyping by high-resolution amplicon melting and should also improve results on lower resolution instruments.

Product differentiation during continuous-flow thermal gradient PCR.

PubMed

Crews, Niel; Wittwer, Carl; Palais, Robert; Gale, Bruce

2008-06-01

A continuous-flow PCR microfluidic device was developed in which the target DNA product can be detected and identified during its amplification. This in situ characterization potentially eliminates the requirement for further post-PCR analysis. Multiple small targets have been amplified from human genomic DNA, having sizes of 108, 122, and 134 bp. With a DNA dye in the PCR mixture, the amplification and unique melting behavior of each sample is observed from a single fluorescent image. The melting behavior of the amplifying DNA, which depends on its molecular composition, occurs spatially in the thermal gradient PCR device, and can be observed with an optical resolution of 0.1 degrees C pixel(-1). Since many PCR cycles are within the field of view of the CCD camera, melting analysis can be performed at any cycle that contains a significant quantity of amplicon, thereby eliminating the cycle-selection challenges typically associated with continuous-flow PCR microfluidics.

Dpb11 may function with RPA and DNA to initiate DNA replication

PubMed Central

Bruck, Irina; Dhingra, Nalini; Martinez, Matthew P.

2017-01-01

Dpb11 is required for the initiation of DNA replication in budding yeast. We found that Dpb11 binds tightly to single-stranded DNA (ssDNA) or branched DNA structures, while its human homolog, TopBP1, binds tightly to branched-DNA structures. We also found that Dpb11 binds stably to CDK-phosphorylated RPA, the eukaryotic ssDNA binding protein, in the presence of branched DNA. A Dpb11 mutant specifically defective for DNA binding did not exhibit tight binding to RPA in the presence of DNA, suggesting that Dpb11-interaction with DNA may promote the recruitment of RPA to melted DNA. We then characterized a mutant of Dpb11 that is specifically defective in DNA binding in budding yeast cells. Expression of dpb11-m1,2,3,5,ΔC results in a substantial decrease in RPA recruitment to origins, suggesting that Dpb11 interaction with DNA may be required for RPA recruitment to origins. Expression of dpb11-m1,2,3,5,ΔC also results in diminished GINS interaction with Mcm2-7 during S phase, while Cdc45 interaction with Mcm2-7 is like wild-type. The reduced GINS interaction with Mcm2-7 may be an indirect consequence of diminished origin melting. We propose that the tight interaction between Dpb11, CDK-phosphorylated RPA, and branched-DNA may be required for the essential function of stabilizing melted origin DNA in vivo. We also propose an alternative model, wherein Dpb11-DNA interaction is required for some other function in DNA replication initiation, such as helicase activation. PMID:28467467

Layered graphene-mica substrates induce melting of DNA origami

NASA Astrophysics Data System (ADS)

Green, Nathaniel S.; Pham, Phi H. Q.; Crow, Daniel T.; Burke, Peter J.; Norton, Michael L.

2018-04-01

Monolayer graphene supported on mica substrates induce melting of cross-shaped DNA origami. This behavior can be contrasted with the case of origami on graphene on graphite, where an expansion or partially re-organized structure is observed. On mica, only well-formed structures are observed. Comparison of the morphological differences observed for these probes after adsorption on these substrates provides insights into the sensitivity of DNA based nanostructures to the properties of the graphene monolayer, as modified by its substrate.

Stability and recovery of DNA origami structure with cation concentration

NASA Astrophysics Data System (ADS)

Chen, Yi; Wang, Ping; Liu, Yang; Liu, Ting; Xu, Yan; Zhu, Shanshan; Zhu, Jun; Ye, Kai; Huang, Guang; Dannong, He

2018-01-01

We synthesized triangular and rectangular DNA origami nanostructures and investigated the stability and recovery of them under low cation concentration. Our results demonstrated that the origami nanostructures would melt when incubated in low cation concentration, and recover whilst kept in the concentration for less than 10 min. However, extending the incubation time would lead to irreversible melting. Our results show the possibility of application of DNA origami nanostructures for things such as a sensor for cation concentration response, etc.

Melting analysis on microbeads in rapid temperature-gradient inside microchannels for single nucleotide polymorphisms detectiona)

PubMed Central

Li, Kan-Chien; Ding, Shih-Torng; Lin, En-Chung; Wang, Lon (Alex); Lu, Yen-Wen

2014-01-01

A continuous-flow microchip with a temperature gradient in microchannels was utilized to demonstrate spatial melting analysis on microbeads for clinical Single Nucleotide Polymorphisms (SNPs) genotyping on animal genomic DNA. The chip had embedded heaters and thermometers, which created a rapid and yet stable temperature gradient between 60 °C and 85 °C in a short distance as the detection region. The microbeads, which served as mobile supports carrying the target DNA and fluorescent dye, were transported across the temperature gradient. As the surrounding temperature increased, the fluorescence signals of the microbeads decayed with this relationship being acquired as the melting curve. Fast DNA denaturation, as a result of the improved heat transfer and thermal stability due to scaling, was also confirmed. Further, each individual microbead could potentially bear different sequences and pass through the detection region, one by one, for a series of melting analysis, with multiplex, high-throughput capability being possible. A prototype was tested with target DNA samples in different genotypes (i.e., wild and mutant types) with a SNP location from Landrace sows. The melting temperatures were obtained and compared to the ones using a traditional tube-based approach. The results showed similar levels of SNP discrimination, validating our proposed technique for scanning homozygotes and heterozygotes to distinguish single base changes for disease research, drug development, medical diagnostics, agriculture, and animal production. PMID:25553186

The nature of the force-induced conformation transition of dsDNA studied by using single molecule force spectroscopy.

PubMed

Liu, Ningning; Bu, Tianjia; Song, Yu; Zhang, Wei; Li, Jinjing; Zhang, Wenke; Shen, Jiacong; Li, Hongbin

2010-06-15

Single-stranded DNA binding proteins (SSB) interact with single-stranded DNA (ssDNA) specifically. Taking advantage of this character, we have employed Bacillus subtilis SSB protein to investigate the nature of force-induced conformation transition of double-stranded DNA (dsDNA) by using AFM-based single molecule force spectroscopy (SMFS) technique. Our results show that, when a dsDNA is stretched beyond its contour length, the dsDNA is partially melted, producing some ssDNA segments which can be captured by SSB proteins. We have also systematically investigated the effects of stretching length, waiting time, and salt concentration on the conformation transition of dsDNA and SSB-ssDNA interactions, respectively. Furthermore, the effect of proflavine, a DNA intercalator, on the SSB-DNA interactions has been investigated, and the results indicate that the proflavine-saturated dsDNA can be stabilized to the extent that the dsDNA will no longer melt into ssDNA under the mechanical force even up to 150 pN, and no SSB-DNA interactions are detectable.

Unlabeled Oligonucleotides as Internal Temperature Controls for Genotyping by Amplicon Melting

PubMed Central

Seipp, Michael T.; Durtschi, Jacob D.; Liew, Michael A.; Williams, Jamie; Damjanovich, Kristy; Pont-Kingdon, Genevieve; Lyon, Elaine; Voelkerding, Karl V.; Wittwer, Carl T.

2007-01-01

Amplicon melting is a closed-tube method for genotyping that does not require probes, real-time analysis, or allele-specific polymerase chain reaction. However, correct differentiation of homozygous mutant and wild-type samples by melting temperature (Tm) requires high-resolution melting and closely controlled reaction conditions. When three different DNA extraction methods were used to isolate DNA from whole blood, amplicon Tm differences of 0.03 to 0.39°C attributable to the extractions were observed. To correct for solution chemistry differences between samples, complementary unlabeled oligonucleotides were included as internal temperature controls to shift and scale the temperature axis of derivative melting plots. This adjustment was applied to a duplex amplicon melting assay for the methylenetetrahydrofolate reductase variants 1298A>C and 677C>T. High- and low-temperature controls bracketing the amplicon melting region decreased the Tm SD within homozygous genotypes by 47 to 82%. The amplicon melting assay was 100% concordant to an adjacent hybridization probe (HybProbe) melting assay when temperature controls were included, whereas a 3% error rate was observed without temperature correction. In conclusion, internal temperature controls increase the accuracy of genotyping by high-resolution amplicon melting and should also improve results on lower resolution instruments. PMID:17591926

Melting of genomic DNA: Predictive modeling by nonlinear lattice dynamics

NASA Astrophysics Data System (ADS)

Theodorakopoulos, Nikos

2010-08-01

The melting behavior of long, heterogeneous DNA chains is examined within the framework of the nonlinear lattice dynamics based Peyrard-Bishop-Dauxois (PBD) model. Data for the pBR322 plasmid and the complete T7 phage have been used to obtain model fits and determine parameter dependence on salt content. Melting curves predicted for the complete fd phage and the Y1 and Y2 fragments of the ϕX174 phage without any adjustable parameters are in good agreement with experiment. The calculated probabilities for single base-pair opening are consistent with values obtained from imino proton exchange experiments.

Dynamical Scaling and Phase Coexistence in Topologically Constrained DNA Melting.

PubMed

Fosado, Y A G; Michieletto, D; Marenduzzo, D

2017-09-15

There is a long-standing experimental observation that the melting of topologically constrained DNA, such as circular closed plasmids, is less abrupt than that of linear molecules. This finding points to an important role of topology in the physics of DNA denaturation, which is, however, poorly understood. Here, we shed light on this issue by combining large-scale Brownian dynamics simulations with an analytically solvable phenomenological Landau mean field theory. We find that the competition between melting and supercoiling leads to phase coexistence of denatured and intact phases at the single-molecule level. This coexistence occurs in a wide temperature range, thereby accounting for the broadening of the transition. Finally, our simulations show an intriguing topology-dependent scaling law governing the growth of denaturation bubbles in supercoiled plasmids, which can be understood within the proposed mean field theory.

Allostery through protein-induced DNA bubbles

DOE PAGES

Traverso, Joseph J.; Manoranjan, Valipuram S.; Bishop, A. R.; ...

2015-03-12

Allostery through DNA is increasingly recognized as an important modulator of DNA functions. Here, we show that the coalescence of protein-induced DNA bubbles can mediate allosteric interactions that drive protein aggregation. We propose that such allostery may regulate DNA's flexibility and the assembly of the transcription machinery. Mitochondrial transcription factor A (TFAM), a dual-function protein involved in mitochondrial DNA (mtDNA) packaging and transcription initiation, is an ideal candidate to test such a hypothesis owing to its ability to locally unwind the double helix. Numerical simulations demonstrate that the coalescence of TFAM-induced bubbles can explain experimentally observed TFAM oligomerization. The resultingmore » melted DNA segment, approximately 10 base pairs long, around the joints of the oligomers act as flexible hinges, which explains the efficiency of TFAM in compacting DNA. Since mitochondrial polymerase (mitoRNAP) is involved in melting the transcription bubble, TFAM may use the same allosteric interaction to both recruit mitoRNAP and initiate transcription.« less

Hybrid analysis (barcode-high resolution melting) for authentication of Thai herbal products, Andrographis paniculata (Burm.f.) Wall.ex Nees.

PubMed

Osathanunkul, Maslin; Suwannapoom, Chatmongkon; Khamyong, Nuttaluck; Pintakum, Danupol; Lamphun, Santisuk Na; Triwitayakorn, Kanokporn; Osathanunkul, Kitisak; Madesis, Panagiotis

2016-01-01

Andrographis paniculata Nees is a medicinal plant with multiple pharmacological properties. It has been used over many centuries as a household remedy. A. paniculata products sold on the markets are in processed forms so it is difficult to authenticate. Therefore buying the herbal products poses a high-risk of acquiring counterfeited, substituted and/or adulterated products. Due to these issues, a reliable method to authenticate products is needed. High resolution melting analysis coupled with DNA barcoding (Bar-HRM) was applied to detect adulteration in commercial herbal products. The rbcL barcode was selected to use in primers design for HRM analysis to produce standard melting profile of A. paniculata species. DNA of the tested commercial products was isolated and their melting profiles were then generated and compared with the standard A. paniculata. The melting profiles of the rbcL amplicons of the three closely related herbal species (A. paniculata, Acanthus ebracteatus and Rhinacanthus nasutus) are clearly separated so that they can be distinguished by the developed method. The method was then used to authenticate commercial herbal products. HRM curves of all 10 samples tested are similar to A. paniculata which indicated that all tested products were contained the correct species as labeled. The method described in this study has been proved to be useful in aiding identification and/or authenticating A. paniculata. This Bar-HRM analysis has allowed us easily to determine the A. paniculata species in herbal products on the markets even they are in processed forms. We propose the use of DNA barcoding combined with High Resolution Melting analysis for authenticating of Andrographis paniculata products.The developed method can be used regardless of the type of the DNA template (fresh or dried tissue, leaf, and stem).rbcL region was chosen for the analysis and work well with our samplesWe can easily determine the A. paniculata species in herbal products tested. Abbreviations used: bp: Base pair, Tm: Melting temperature.

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).

Method for detecting point mutations in DNA utilizing fluorescence energy transfer

DOEpatents

Parkhurst, Lawrence J.; Parkhurst, Kay M.; Middendorf, Lyle

2001-01-01

A method for detecting point mutations in DNA using a fluorescently labeled oligomeric probe and Forster resonance energy transfer (FRET) is disclosed. The selected probe is initially labeled at each end with a fluorescence dye, which act together as a donor/acceptor pair for FRET. The fluorescence emission from the dyes changes dramatically from the duplex stage, wherein the probe is hybridized to the complementary strand of DNA, to the single strand stage, when the probe is melted to become detached from the DNA. The change in fluorescence is caused by the dyes coming into closer proximity after melting occurs and the probe becomes detached from the DNA strand. The change in fluorescence emission as a function of temperature is used to calculate the melting temperature of the complex or T.sub.m. In the case where there is a base mismatch between the probe and the DNA strand, indicating a point mutation, the T.sub.m has been found to be significantly lower than the T.sub.m for a perfectly match probelstand duplex. The present invention allows for the detection of the existence and magnitude of T.sub.m, which allows for the quick and accurate detection of a point mutation in the DNA strand and, in some applications, the determination of the approximate location of the mutation within the sequence.

Spectroscopic studies on the interaction of sodium benzoate, a food preservative, with calf thymus DNA.

PubMed

Zhang, Guowen; Ma, Yadi

2013-11-01

The interaction between sodium benzoate (SB) and calf thymus DNA in simulated physiological buffer (pH 7.4) using acridine orange (AO) dye as a fluorescence probe, was investigated by UV-Vis absorption, fluorescence and circular dichroism (CD) spectroscopy along with DNA melting studies and viscosity measurements. An expanded UV-Vis spectral data matrix was resolved by multivariate curve resolution-alternating least squares (MCR-ALS) approach. The equilibrium concentration profiles and the pure spectra for SB, DNA and DNA-SB complex from the high overlapping composite response were simultaneously obtained. The results indicated that SB could bind to DNA, and hydrophobic interactions and hydrogen bonds played a vital role in the binding process. Moreover, SB was able to quench the fluorescence of DNA-AO complex through a static procedure. The quenching observed was indicative of an intercalative mode of interaction between SB and DNA, which was supported by melting studies, viscosity measurements and CD analysis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Structural stability of DNA origami nanostructures in the presence of chaotropic agents.

PubMed

Ramakrishnan, Saminathan; Krainer, Georg; Grundmeier, Guido; Schlierf, Michael; Keller, Adrian

2016-05-21

DNA origami represent powerful platforms for single-molecule investigations of biomolecular processes. The required structural integrity of the DNA origami may, however, pose significant limitations regarding their applicability, for instance in protein folding studies that require strongly denaturing conditions. Here, we therefore report a detailed study on the stability of 2D DNA origami triangles in the presence of the strong chaotropic denaturing agents urea and guanidinium chloride (GdmCl) and its dependence on concentration and temperature. At room temperature, the DNA origami triangles are stable up to at least 24 h in both denaturants at concentrations as high as 6 M. At elevated temperatures, however, structural stability is governed by variations in the melting temperature of the individual staple strands. Therefore, the global melting temperature of the DNA origami does not represent an accurate measure of their structural stability. Although GdmCl has a stronger effect on the global melting temperature, its attack results in less structural damage than observed for urea under equivalent conditions. This enhanced structural stability most likely originates from the ionic nature of GdmCl. By rational design of the arrangement and lengths of the individual staple strands used for the folding of a particular shape, however, the structural stability of DNA origami may be enhanced even further to meet individual experimental requirements. Overall, their high stability renders DNA origami promising platforms for biomolecular studies in the presence of chaotropic agents, including single-molecule protein folding or structural switching.

Heterozygote PCR product melting curve prediction.

PubMed

Dwight, Zachary L; Palais, Robert; Kent, Jana; Wittwer, Carl T

2014-03-01

Melting curve prediction of PCR products is limited to perfectly complementary strands. Multiple domains are calculated by recursive nearest neighbor thermodynamics. However, the melting curve of an amplicon containing a heterozygous single-nucleotide variant (SNV) after PCR is the composite of four duplexes: two matched homoduplexes and two mismatched heteroduplexes. To better predict the shape of composite heterozygote melting curves, 52 experimental curves were compared with brute force in silico predictions varying two parameters simultaneously: the relative contribution of heteroduplex products and an ionic scaling factor for mismatched tetrads. Heteroduplex products contributed 25.7 ± 6.7% to the composite melting curve, varying from 23%-28% for different SNV classes. The effect of ions on mismatch tetrads scaled to 76%-96% of normal (depending on SNV class) and averaged 88 ± 16.4%. Based on uMelt (www.dna.utah.edu/umelt/umelt.html) with an expanded nearest neighbor thermodynamic set that includes mismatched base pairs, uMelt HETS calculates helicity as a function of temperature for homoduplex and heteroduplex products, as well as the composite curve expected from heterozygotes. It is an interactive Web tool for efficient genotyping design, heterozygote melting curve prediction, and quality control of melting curve experiments. The application was developed in Actionscript and can be found online at http://www.dna.utah.edu/hets/. © 2013 WILEY PERIODICALS, INC.

Dynamic binding of replication protein a is required for DNA repair

PubMed Central

Chen, Ran; Subramanyam, Shyamal; Elcock, Adrian H.; Spies, Maria; Wold, Marc S.

2016-01-01

Replication protein A (RPA), the major eukaryotic single-stranded DNA (ssDNA) binding protein, is essential for replication, repair and recombination. High-affinity ssDNA-binding by RPA depends on two DNA binding domains in the large subunit of RPA. Mutation of the evolutionarily conserved aromatic residues in these two domains results in a separation-of-function phenotype: aromatic residue mutants support DNA replication but are defective in DNA repair. We used biochemical and single-molecule analyses, and Brownian Dynamics simulations to determine the molecular basis of this phenotype. Our studies demonstrated that RPA binds to ssDNA in at least two modes characterized by different dissociation kinetics. We also showed that the aromatic residues contribute to the formation of the longer-lived state, are required for stable binding to short ssDNA regions and are needed for RPA melting of partially duplex DNA structures. We conclude that stable binding and/or the melting of secondary DNA structures by RPA is required for DNA repair, including RAD51 mediated DNA strand exchange, but is dispensable for DNA replication. It is likely that the binding modes are in equilibrium and reflect dynamics in the RPA–DNA complex. This suggests that dynamic binding of RPA to DNA is necessary for different cellular functions. PMID:27131385

NDI and DAN DNA: nucleic acid-directed assembly of NDI and DAN.

PubMed

Ikkanda, Brian A; Samuel, Stevan A; Iverson, Brent L

2014-03-07

Two novel DNA base surrogate phosphoramidites 1 and 2, based upon relatively electron-rich 1,5-dialkoxynaphthalene (DAN) and relatively electron-deficient 1,4,5,8-naphthalenetetracarboxylic diimide (NDI), respectively, were designed, synthesized, and incorporated into DNA oligonucleotide strands. The DAN and NDI artificial DNA bases were inserted within a three-base-pair region within the interior of a 12-mer oligonucleotide duplex in various sequential arrangements and investigated with CD spectroscopy and UV melting curve analysis. The CD spectra of the modified duplexes indicated B-form DNA topology. Melting curve analyses revealed trends in DNA duplex stability that correlate with the known association of DAN and NDI moieties in aqueous solution as well as the known favorable interactions between NDI and natural DNA base pairs. This demonstrates that DNA duplex stability and specificity can be driven by the electrostatic complementarity between DAN and NDI. In the most favorable case, an NDI-DAN-NDI arrangement in the middle of the DNA duplex was found to be approximately as stabilizing as three A-T base pairs.

Progress in plant protoplast research.

PubMed

Eeckhaut, Tom; Lakshmanan, Prabhu Shankar; Deryckere, Dieter; Van Bockstaele, Erik; Van Huylenbroeck, Johan

2013-12-01

In this review we focus on recent progress in protoplast regeneration, symmetric and asymmetric hybridization and novel technology developments. Regeneration of new species and improved culture techniques opened new horizons for practical breeding in a number of crops. The importance of protoplast sources and embedding systems is discussed. The study of reactive oxygen species effects and DNA (de)condensation, along with thorough phytohormone monitoring, are in our opinion the most promising research topics in the further strive for rationalization of protoplast regeneration. Following, fusion and fragmentation progress is summarized. Genomic, transcriptomic and proteomic studies have led to better insights in fundamental processes such as cell wall formation, cell development and chromosome rearrangements in fusion products, whether or not obtained after irradiation. Advanced molecular screening methods of both genome and cytoplasmome facilitate efficient screening of both symmetric and asymmetric fusion products. We expect that emerging technologies as GISH, high resolution melting and next generation sequencing will pay major contributions to our insights of genome creation and stabilization, mainly after asymmetric hybridization. Finally, we demonstrate agricultural valorization of somatic hybridization through enumerating recent introgression of diverse traits in a number of commercial crops.

Transient kinetics measured with force steps discriminate between double-stranded DNA elongation and melting and define the reaction energetics

PubMed Central

Bongini, Lorenzo; Melli, Luca; Lombardi, Vincenzo; Bianco, Pasquale

2014-01-01

Under a tension of ∼65 pN, double-stranded DNA undergoes an overstretching transition from its basic (B-form) conformation to a 1.7 times longer conformation whose nature is only recently starting to be understood. Here we provide a structural and thermodynamic characterization of the transition by recording the length transient following force steps imposed on the λ-phage DNA with different melting degrees and temperatures (10–25°C). The shortening transient following a 20–35 pN force drop from the overstretching force shows a sequence of fast shortenings of double-stranded extended (S-form) segments and pauses owing to reannealing of melted segments. The lengthening transients following a 2–35 pN stretch to the overstretching force show the kinetics of a two-state reaction and indicate that the whole 70% extension is a B-S transition that precedes and is independent of melting. The temperature dependence of the lengthening transient shows that the entropic contribution to the B-S transition is one-third of the entropy change of thermal melting, reinforcing the evidence for a double-stranded S-form that maintains a significant fraction of the interstrand bonds. The cooperativity of the unitary elongation (22 bp) is independent of temperature, suggesting that structural factors, such as the nucleic acid sequence, control the transition. PMID:24353317

DNA melting analysis: application of the "open tube" format for detection of mutant KRAS.

PubMed

Botezatu, Irina V; Kondratova, Valentina N; Shelepov, Valery P; Lichtenstein, Anatoly V

2011-12-15

High-resolution melting (HRM) analysis is a very effective method for genotyping and mutation scanning that is usually performed just after PCR amplification (the "closed tube" format). Though simple and convenient, the closed tube format makes the HRM dependent on the PCR mix, not generally optimal for DNA melting analysis. Here, the "open tube" format, namely the post-PCR optimization procedure (amplicon shortening and solution chemistry modification), is proposed. As a result, mutation scanning of short amplicons becomes feasible on a standard real-time PCR instrument (not primarily designed for HRM) using SYBR Green I. This approach has allowed us to considerably enhance the sensitivity of detecting mutant KRAS using both low- and high-resolution systems (the Bio-Rad iQ5-SYBR Green I and Bio-Rad CFX96-EvaGreen, respectively). The open tube format, though more laborious than the closed tube one, can be used in situations when maximal sensitivity of the method is needed. It also permits standardization of DNA melting experiments and the introduction of instruments of a "lower level" into the range of those suitable for mutation scanning. Copyright © 2011 Elsevier Inc. All rights reserved.

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

Optical Benson: Following the Impact of Melt Season Progression Using Landsat and Sentinel 2 - Snow Zone Formation Imaged

NASA Astrophysics Data System (ADS)

Fahnestock, M. A.; Shuman, C. A.; Alley, K. E.

2017-12-01

Snow pit observations on a glaciologically-focussed surface traverse in Greenland allowed Benson [1962, SIPRE (now CRREL) Research Report 70] to define a series of snow zones based on the extent of post-depositional diagenesis of the snowpack. At high elevations, Benson found fine-grained "dry snow" where melt (at that time) was absent year-round, followed down-elevation by a "percolation zone" where surface melt penetrated the snowpack, then a "wet snow zone" where firn became saturated during the peak of the melt season, and finally "superimposed ice" and "bare ice" zones where refrozen surface melt and glacier ice were exposed in the melt season. These snow zones can be discriminated in winter synthetic aperture radar (SAR) imagery of the ice sheet (e.g. Fahnestock et al. 2001), but summer melt reduces radar backscatter and makes it difficult to follow the progression of diagenesis beyond the initial indications of surface melting. While some of the impacts of surface melt (especially bands of blue water-saturated firn) are observed from time to time in optical satellite imagery, it has only become possible to map effects of melt over the course of a summer season with the advent of large-data analysis tools such as Google Earth Engine and the inclusion of Landsat and Sentinel-2 data streams in these tools. A map of the maximum extent of this blue saturated zone through the 2016 melt season is shown in the figure. This image is a true color (RGB) composite, but each pixel in the image shows the color of the surface when the "blueness" of the pixel was at a maximum. This means each pixel can be from a different satellite image acquisition than adjacent pixels - but it also means that the maximum extent of the saturated firn (Benson's wet snow zone) is visible. Also visible are percolation, superimposed and bare ice zones. This analysis, using Landsat 8 Operational Land Imager data, was performed using Google Earth Engine to access and analyze the entire melt season's data. Similar spatial analyses for other years in the record, combined with pixel-by-pixel analysis of each time series through the year, can be used to track the progression and overall effect of the melt season in each year. This view of the progression of a melt season provides a new set of tools to help understand changing surface conditions for ice sheets and glaciers globally.

Hybrid analysis (barcode-high resolution melting) for authentication of Thai herbal products, Andrographis paniculata (Burm.f.) Wall.ex Nees

PubMed Central

Osathanunkul, Maslin; Suwannapoom, Chatmongkon; Khamyong, Nuttaluck; Pintakum, Danupol; Lamphun, Santisuk Na; Triwitayakorn, Kanokporn; Osathanunkul, Kitisak; Madesis, Panagiotis

2016-01-01

Background: Andrographis paniculata Nees is a medicinal plant with multiple pharmacological properties. It has been used over many centuries as a household remedy. A. paniculata products sold on the markets are in processed forms so it is difficult to authenticate. Therefore buying the herbal products poses a high-risk of acquiring counterfeited, substituted and/or adulterated products. Due to these issues, a reliable method to authenticate products is needed. Materials and Methods: High resolution melting analysis coupled with DNA barcoding (Bar-HRM) was applied to detect adulteration in commercial herbal products. The rbcL barcode was selected to use in primers design for HRM analysis to produce standard melting profile of A. paniculata species. DNA of the tested commercial products was isolated and their melting profiles were then generated and compared with the standard A. paniculata. Results: The melting profiles of the rbcL amplicons of the three closely related herbal species (A. paniculata, Acanthus ebracteatus and Rhinacanthus nasutus) are clearly separated so that they can be distinguished by the developed method. The method was then used to authenticate commercial herbal products. HRM curves of all 10 samples tested are similar to A. paniculata which indicated that all tested products were contained the correct species as labeled. Conclusion: The method described in this study has been proved to be useful in aiding identification and/or authenticating A. paniculata. This Bar-HRM analysis has allowed us easily to determine the A. paniculata species in herbal products on the markets even they are in processed forms. SUMMARY We propose the use of DNA barcoding combined with High Resolution Melting analysis for authenticating of Andrographis paniculata products.The developed method can be used regardless of the type of the DNA template (fresh or dried tissue, leaf, and stem).rbcL region was chosen for the analysis and work well with our samplesWe can easily determine the A. paniculata species in herbal products tested. Abbreviations used: bp: Base pair, Tm: Melting temperature PMID:27041863

Fluorescence acquisition during hybridization phase in quantitative real-time PCR improves specificity and signal-to-noise ratio.

PubMed

Mehndiratta, Mohit; Palanichamy, Jayanth Kumar; Ramalingam, Pradeep; Pal, Arnab; Das, Prerna; Sinha, Subrata; Chattopadhyay, Parthaprasad

2008-12-01

Quantitative real-time PCR (qPCR) is a standard method used for quantification of specific gene expression. This utilizes either dsDNA binding dyes or probe based chemistry. While dsDNA binding dyes have the advantage of low cost and flexibility, fluorescence due to primer dimers also interferes with the fluorescence of the specific product. Sometimes it is difficult, if not impossible, to standardize conditions and redesign primers in such a way that only specific fluorescence of the products of test and reference genes are acquired. Normally, the fluorescence acquisition in qPCR using dsDNA binding dyes is done during the melting phase of the PCR at a temperature between the melting points of primer dimers and the specific product. We have modified the protocol to acquire fluorescence during the hybridization phase. This significantly increased the signal-to-noise ratio and enabled the use of dsDNA binding dyes for mRNA quantification in situations where it was not possible when measurement was done in the melting phase. We have demonstrated it for three mRNAs, E6, E7, and DNMT1 with beta-actin as the reference gene, and for two miRNAs. This modification broadens the scope of qPCR using dsDNA binding dyes.

Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations.

PubMed

Owczarzy, Richard; Moreira, Bernardo G; You, Yong; Behlke, Mark A; Walder, Joseph A

2008-05-13

Accurate predictions of DNA stability in physiological and enzyme buffers are important for the design of many biological and biochemical assays. We therefore investigated the effects of magnesium, potassium, sodium, Tris ions, and deoxynucleoside triphosphates on melting profiles of duplex DNA oligomers and collected large melting data sets. An empirical correction function was developed that predicts melting temperatures, transition enthalpies, entropies, and free energies in buffers containing magnesium and monovalent cations. The new correction function significantly improves the accuracy of predictions and accounts for ion concentration, G-C base pair content, and length of the oligonucleotides. The competitive effects of potassium and magnesium ions were characterized. If the concentration ratio of [Mg (2+)] (0.5)/[Mon (+)] is less than 0.22 M (-1/2), monovalent ions (K (+), Na (+)) are dominant. Effects of magnesium ions dominate and determine duplex stability at higher ratios. Typical reaction conditions for PCR and DNA sequencing (1.5-5 mM magnesium and 20-100 mM monovalent cations) fall within this range. Conditions were identified where monovalent and divalent cations compete and their stability effects are more complex. When duplexes denature, some of the Mg (2+) ions associated with the DNA are released. The number of released magnesium ions per phosphate charge is sequence dependent and decreases surprisingly with increasing oligonucleotide length.

Optimization of PCR Condition: The First Study of High Resolution Melting Technique for Screening of APOA1 Variance.

PubMed

Wahyuningsih, Hesty; K Cayami, Ferdy; Bahrudin, Udin; A Sobirin, Mochamad; Ep Mundhofir, Farmaditya; Mh Faradz, Sultana; Hisatome, Ichiro

2017-03-01

High resolution melting (HRM) is a post-PCR technique for variant screening and genotyping based on the different melting points of DNA fragments. The advantages of this technique are that it is fast, simple, and efficient and has a high output, particularly for screening of a large number of samples. APOA1 encodes apolipoprotein A1 (apoA1) which is a major component of high density lipoprotein cholesterol (HDL-C). This study aimed to obtain an optimal quantitative polymerase chain reaction (qPCR)-HRM condition for screening of APOA1 variance. Genomic DNA was isolated from a peripheral blood sample using the salting out method. APOA1 was amplified using the RotorGeneQ 5Plex HRM. The PCR product was visualized with the HRM amplification curve and confirmed using gel electrophoresis. The melting profile was confirmed by looking at the melting curve. Five sets of primers covering the translated region of APOA1 exons were designed with expected PCR product size of 100-400 bps. The amplified segments of DNA were amplicons 2, 3, 4A, 4B, and 4C. Amplicons 2, 3 and 4B were optimized at an annealing temperature of 60 °C at 40 PCR cycles. Amplicon 4A was optimized at an annealing temperature of 62 °C at 45 PCR cycles. Amplicon 4C was optimized at an annealing temperature of 63 °C at 50 PCR cycles. In addition to the suitable procedures of DNA isolation and quantification, primer design and an estimated PCR product size, the data of this study showed that appropriate annealing temperature and PCR cycles were important factors in optimization of HRM technique for variant screening in APOA1 .

Optimization of PCR Condition: The First Study of High Resolution Melting Technique for Screening of APOA1 Variance

PubMed Central

Wahyuningsih, Hesty; K Cayami, Ferdy; Bahrudin, Udin; A Sobirin, Mochamad; EP Mundhofir, Farmaditya; MH Faradz, Sultana; Hisatome, Ichiro

2017-01-01

Background High resolution melting (HRM) is a post-PCR technique for variant screening and genotyping based on the different melting points of DNA fragments. The advantages of this technique are that it is fast, simple, and efficient and has a high output, particularly for screening of a large number of samples. APOA1 encodes apolipoprotein A1 (apoA1) which is a major component of high density lipoprotein cholesterol (HDL-C). This study aimed to obtain an optimal quantitative polymerase chain reaction (qPCR)-HRM condition for screening of APOA1 variance. Methods Genomic DNA was isolated from a peripheral blood sample using the salting out method. APOA1 was amplified using the RotorGeneQ 5Plex HRM. The PCR product was visualized with the HRM amplification curve and confirmed using gel electrophoresis. The melting profile was confirmed by looking at the melting curve. Results Five sets of primers covering the translated region of APOA1 exons were designed with expected PCR product size of 100–400 bps. The amplified segments of DNA were amplicons 2, 3, 4A, 4B, and 4C. Amplicons 2, 3 and 4B were optimized at an annealing temperature of 60 °C at 40 PCR cycles. Amplicon 4A was optimized at an annealing temperature of 62 °C at 45 PCR cycles. Amplicon 4C was optimized at an annealing temperature of 63 °C at 50 PCR cycles. Conclusion In addition to the suitable procedures of DNA isolation and quantification, primer design and an estimated PCR product size, the data of this study showed that appropriate annealing temperature and PCR cycles were important factors in optimization of HRM technique for variant screening in APOA1. PMID:28331418

The structure of SV40 large T hexameric helicase in complex with AT-rich origin DNA

PubMed Central

Gai, Dahai; Wang, Damian; Li, Shu-Xing; Chen, Xiaojiang S

2016-01-01

DNA replication is a fundamental biological process. The initial step in eukaryotic DNA replication is the assembly of the pre-initiation complex, including the formation of two head-to-head hexameric helicases around the replication origin. How these hexameric helicases interact with their origin dsDNA remains unknown. Here, we report the co-crystal structure of the SV40 Large-T Antigen (LT) hexameric helicase bound to its origin dsDNA. The structure shows that the six subunits form a near-planar ring that interacts with the origin, so that each subunit makes unique contacts with the DNA. The origin dsDNA inside the narrower AAA+ domain channel shows partial melting due to the compression of the two phosphate backbones, forcing Watson-Crick base-pairs within the duplex to flip outward. This structure provides the first snapshot of a hexameric helicase binding to origin dsDNA, and suggests a possible mechanism of origin melting by LT during SV40 replication in eukaryotic cells. DOI: http://dx.doi.org/10.7554/eLife.18129.001 PMID:27921994

Studies on the interaction of apigenin with calf thymus DNA by spectroscopic methods

NASA Astrophysics Data System (ADS)

Zhang, Shufang; Sun, Xuejun; Kong, Rongmei; Xu, Mingming

2015-02-01

The interaction between apigenin and calf thymus deoxyribonucleic acid (ctDNA) in a pH 7.4 Tris-HCl buffer solution was investigated by UV-Vis spectroscopy, fluorescence spectroscopy, DNA melting techniques, and viscosity measurements. It was found that apigenin molecules could intercalate into the base pairs of DNA, forming a apigenin-DNA complex with a binding constant of K310K = 6.4 × 104 L mol-1. The thermodynamic parameters enthalpy change (ΔH), entropy change (ΔS) and Gibbs free energy (ΔG) were calculated to be 7.36 × 104 J mol-1, 329 J K-1 mol-1 and -2.84 × 104 J mol-1 at 310 K, respectively. Hydrophobic interaction was the predominant intermolecular force in stabilizing the apigenin-DNA complex. Thermal denaturation study suggested that the stabilization of the ctDNA helix was increased when the apigenin binding to ctDNA as indicated by the increase in thermal denaturation temperature of ctDNA at around 5.0 °C in the presence of apigenin. Spectroscopic techniques together with melting techniques and viscosity determination provided evidences of intercalation mode of binding for the interaction between apigenin and ctDNA.

Probing the Characterization of the Interaction of Aflatoxins B1 and G1 with Calf Thymus DNA In Vitro

PubMed Central

Ma, Liang; Wang, Jiaman; Zhang, Yuhao

2017-01-01

The binding characterization of aflatoxins with calf thymus DNA (ctDNA) under physiological conditions was investigated. Multispectroscopic techniques, ctDNA melting, viscosity measurements, and molecular docking techniques were employed to elucidate the binding mechanism of the aflatoxins with DNA. The fluorescence results indicated that both aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1) bound to the ctDNA, forming complexes through hydrogen bonding. The binding constants of AFB1 and AFG1 with ctDNA reached up to 103 L·mol−1 and 104 L·mol−1, respectively, and AFG1 exhibited a higher binding propensity than that of AFB1. Furthermore, both AFB1 and AFG1 bound to the ctDNA through groove binding, as evidenced by the results of the spectroscopic, iodide quenching effect, viscosity, and ctDNA melting measurements. Changes in the circular dichroism signal manifested that both AFB1 and AFG1 induced an increase in the right-handed helicity, but only minimally influenced the base stacking of the DNA. A molecular docking study of the aflatoxin’s binding with the DNA revealed a groove binding mode, which was driven mainly by hydrogen bonding. This study of aflatoxin–ctDNA interaction may provide novel insights into the toxicological effect of the mycotoxins. PMID:28671585

The high stability of the triple helices formed between short purine oligonucleotides and SIV/HIV-2 vpx genes is determined by the targeted DNA structure.

PubMed Central

Svinarchuk, F; Monnot, M; Merle, A; Malvy, C; Fermandjian, S

1995-01-01

In our previous works we have shown that the oligonucleotides 5'-GGGGAGGGGGAGG-3' and 5'-GGAGGGGGAGGGG-3' give very stable and specific triplexes with their target double stranded DNAs [Svinarchuk, F., Bertrand, J.-R. and Malvy, C. (1994) Nucleic Acids Res., 22, 3742-3747; Svinarchuk, F., Paoletti, J. and Malvy, C. (1995) J. Biol. Chem., 270, 14 068-14,071]. The target for the invariable part of these oligonucleotides, 5'-GGAGGGGGAGG-3', is found in a highly conserved 20 bp long purine/pyrimidine tract of the vpx gene of the SIV and HIV-2 viruses and could be a target for oligonucleotide directed antivirus therapy. Here were report on the ability of four purine oligonucleotides with different lengths (11-, 14-, 17- and 20-mer) to form triplexes with the purine/pyrimidine stretch of the vpx gene. Triplex formation was tested by joint dimethyl sulfate (DMS) footprint, gel-retardation assay, circular dichroism (CD) and UV-melting studies. Dimethyl sulfate footprint studies revealed the antiparallel orientation of the third strand to the purine strand of the Watson-Crick duplex. However, the protection of the guanines at the ends of the target sequence decreased as the length of the third strand oligonucleotide increased. Melting temperature studies provided profiles with only one transition for all of the triplexes. The melting temperatures of the triplexes were found to be the same as for the targeted duplex in the case of the 11- and 14-mer third strands while for the 17- and 20-mer third strands the melting temperature of the triplexes were correspondingly 4 and 8 degrees C higher than for the duplex. Heating and cooling melting curves were reversible for all of the tested triplexes except one with the 20-mer third strand oligonucleotide. Circular dichroism spectra showed the ability of the target DNA to adopt an A-like DNA conformation. Upon triplex formation the A-DNA form becomes even more pronounced. This effect depends on the length of the third strand oligonucleotide: the CD spectrum shows a 'classical' A-DNA shape with the 20-mer. This is not observed with the purine/pyrimidine stretch of the HIV-1 DNA which keeps a B-like spectrum even after triplex formation. We suggest, that an A-like duplex DNA is required for the formation of a stable DNA purine(purine-pyrimidine) triplex. Images PMID:7479024

A new method for simultaneous detection and discrimination of Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) using real time PCR with high resolution melting (HRM) analysis.

PubMed

Marin, M S; Quintana, S; Leunda, M R; Recavarren, M; Pagnuco, I; Späth, E; Pérez, S; Odeón, A

2016-01-01

Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are antigenically and genetically similar. The aim of this study was to develop a simple and reliable one-step real time PCR assay with high resolution melting (HRM) analysis for the simultaneous detection and differentiation of BoHV-1 and BoHV-5. Optimization of assay conditions was performed with DNA from reference strains. Then, DNA from field isolates, clinical samples and tissue samples of experimentally infected animals were studied by real time PCR-HRM. An efficient amplification of real time PCR products was obtained, and a clear melting curve and appropriate melting peaks for both viruses were achieved in the HRM curve analysis for BoHV type identification. BoHV was identified in all of the isolates and clinical samples, and BoHV types were properly differentiated. Furthermore, viral DNA was detected in 12/18 and 7/18 samples from BoHV-1- and BoHV-5-infected calves, respectively. Real time PCR-HRM achieved a higher sensitivity compared with virus isolation or conventional PCR. In this study, HRM was used as a novel procedure. This method provides rapid, sensitive, specific and simultaneous detection of bovine alpha-herpesviruses DNA. Thus, this technique is an excellent tool for diagnosis, research and epidemiological studies of these viruses in cattle. Copyright © 2015 Elsevier B.V. All rights reserved.

Structures of RNA Polymerase Closed and Intermediate Complexes Reveal Mechanisms of DNA Opening and Transcription Initiation.

PubMed

Glyde, Robert; Ye, Fuzhou; Darbari, Vidya Chandran; Zhang, Nan; Buck, Martin; Zhang, Xiaodong

2017-07-06

Gene transcription is carried out by RNA polymerases (RNAPs). For transcription to occur, the closed promoter complex (RPc), where DNA is double stranded, must isomerize into an open promoter complex (RPo), where the DNA is melted out into a transcription bubble and the single-stranded template DNA is delivered to the RNAP active site. Using a bacterial RNAP containing the alternative σ 54 factor and cryoelectron microscopy, we determined structures of RPc and the activator-bound intermediate complex en route to RPo at 3.8 and 5.8 Å. Our structures show how RNAP-σ 54 interacts with promoter DNA to initiate the DNA distortions required for transcription bubble formation, and how the activator interacts with RPc, leading to significant conformational changes in RNAP and σ 54 that promote RPo formation. We propose that DNA melting is an active process initiated in RPc and that the RNAP conformations of intermediates are significantly different from that of RPc and RPo. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Analysis of a DNA simulation model through hairpin melting experiments.

PubMed

Linak, Margaret C; Dorfman, Kevin D

2010-09-28

We compare the predictions of a two-bead Brownian dynamics simulation model to melting experiments of DNA hairpins with complementary AT or GC stems and noninteracting loops in buffer A. This system emphasizes the role of stacking and hydrogen bonding energies, which are characteristics of DNA, rather than backbone bending, stiffness, and excluded volume interactions, which are generic characteristics of semiflexible polymers. By comparing high throughput data on the open-close transition of various DNA hairpins to the corresponding simulation data, we (1) establish a suitable metric to compare the simulations to experiments, (2) find a conversion between the simulation and experimental temperatures, and (3) point out several limitations of the model, including the lack of G-quartets and cross stacking effects. Our approach and experimental data can be used to validate similar coarse-grained simulation models.

Highly Sensitive Detection of Isoniazid Heteroresistance in Mycobacterium tuberculosis by DeepMelt Assay.

PubMed

Liang, Bin; Tan, Yaoju; Li, Zi; Tian, Xueshan; Du, Chen; Li, Hui; Li, Guoli; Yao, Xiangyang; Wang, Zhongan; Xu, Ye; Li, Qingge

2018-02-01

Detection of heteroresistance of Mycobacterium tuberculosis remains challenging using current genotypic drug susceptibility testing methods. Here, we described a melting curve analysis-based approach, termed DeepMelt, that can detect less-abundant mutants through selective clamping of the wild type in mixed populations. The singleplex DeepMelt assay detected 0.01% katG S315T in 10 5 M. tuberculosis genomes/μl. The multiplex DeepMelt TB/INH detected 1% of mutant species in the four loci associated with isoniazid resistance in 10 4 M. tuberculosis genomes/μl. The DeepMelt TB/INH assay was tested on a panel of DNA extracted from 602 precharacterized clinical isolates. Using the 1% proportion method as the gold standard, the sensitivity was found to be increased from 93.6% (176/188, 95% confidence interval [CI] = 89.2 to 96.3%) to 95.7% (180/188, 95% CI = 91.8 to 97.8%) compared to the MeltPro TB/INH assay. Further evaluation of 109 smear-positive sputum specimens increased the sensitivity from 83.3% (20/24, 95% CI = 64.2 to 93.3%) to 91.7% (22/24, 95% CI = 74.2 to 97.7%). In both cases, the specificity remained nearly unchanged. All heteroresistant samples newly identified by the DeepMelt TB/INH assay were confirmed by DNA sequencing and even partially by digital PCR. The DeepMelt assay may fill the gap between current genotypic and phenotypic drug susceptibility testing for detecting drug-resistant tuberculosis patients. Copyright © 2018 American Society for Microbiology.

A FLUORESCENCE BASED ASSAY FOR DNA DAMAGE INDUCED BY RADIATION, CHEMICAL MUTAGENS AND ENZYMES

EPA Science Inventory

A simple and rapid assay to detect DNA damage is reported. This novel assay is based on changes in melting/annealing behavior and facilitated using certain dyes that increase their fluorescence upon association with double stranded (ds)DNA. Damage caused by ultraviolet (UV) ra...

The rolling-circle melting-pot model for porcine circovirus DNA replication

USDA-ARS?s Scientific Manuscript database

A stem-loop structure, formed by a pair of inverted repeats during DNA replication, is a conserved feature at the origin of DNA replication (Ori) among plant and animal viruses, bacteriophages and plasmids that replicate their genomes via the rolling-circle replication (RCR) mechanism. Porcine circo...

MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay

PubMed Central

Nagy, Alexander; Černíková, Lenka; Vitásková, Eliška; Křivda, Vlastimil; Dán, Ádám; Dirbáková, Zuzana; Jiřincová, Helena; Procházka, Bohumír; Sedlák, Kamil; Havlíčková, Martina

2016-01-01

In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence. PMID:27031831

Overstretching of B-DNA with various pulling protocols: Appearance of structural polymorphism and S-DNA

NASA Astrophysics Data System (ADS)

Garai, Ashok; Mogurampelly, Santosh; Bag, Saientan; Maiti, Prabal K.

2017-12-01

We report a structural polymorphism of the S-DNA when a canonical B-DNA is stretched under different pulling protocols and provide a fundamental molecular understanding of the DNA stretching mechanism. Extensive all atom molecular dynamics simulations reveal a clear formation of S-DNA when the B-DNA is stretched along the 3' directions of the opposite strands (OS3) and is characterized by the changes in the number of H-bonds, entropy, and free energy. Stretching along the 5' directions of the opposite strands (OS5) leads to force induced melting form of the DNA. Interestingly, stretching along the opposite ends of the same strand leads to a coexistence of both the S- and melted M-DNA structures. We also do the structural characterization of the S-DNA by calculating various helical parameters. We find that the S-DNA has a twist of ˜10° which corresponds to a helical repeat length of ˜36 base pairs in close agreement with the previous experimental results. Moreover, we find that the free energy barrier between the canonical and overstretched states of DNA is higher for the same termini pulling protocol in comparison to all other protocols considered in this work. Overall, our observations not only reconcile with the available experimental results qualitatively but also enhance the understanding of different overstretched DNA structures.

High resolution melting (HRM) analysis of DNA--its role and potential in food analysis.

PubMed

Druml, Barbara; Cichna-Markl, Margit

2014-09-01

DNA based methods play an increasing role in food safety control and food adulteration detection. Recent papers show that high resolution melting (HRM) analysis is an interesting approach. It involves amplification of the target of interest in the presence of a saturation dye by the polymerase chain reaction (PCR) and subsequent melting of the amplicons by gradually increasing the temperature. Since the melting profile depends on the GC content, length, sequence and strand complementarity of the product, HRM analysis is highly suitable for the detection of single-base variants and small insertions or deletions. The review gives an introduction into HRM analysis, covers important aspects in the development of an HRM analysis method and describes how HRM data are analysed and interpreted. Then we discuss the potential of HRM analysis based methods in food analysis, i.e. for the identification of closely related species and cultivars and the identification of pathogenic microorganisms. Copyright © 2014 Elsevier Ltd. All rights reserved.

Impact melt breccias at the Apollo 17 landing site

NASA Technical Reports Server (NTRS)

Ryder, Graham

1992-01-01

Impact melt breccias are by far the most common highland rock type collected on the Apollo 17 mission. They tend to be fine grained, with virtually no clast-free impact melt rocks having been identified. All the highland boulders sampled are impact melt breccia, with the possible exception of one South Massif boulder that might have a friable matrix (but nonetheless consists dominantly of impact melt) and a shocked igneous norite boulder from the North Massif. The impact melt breccias were originally described as metaclastic, but their melt origin became apparent as work progressed. Chemical compositions appear to allow natural groupings of the impact melt breccias. Various groupings of the impact melt breccias are discussed.

Screening the sequence selectivity of DNA-binding molecules using a gold nanoparticle-based colorimetric approach.

PubMed

Hurst, Sarah J; Han, Min Su; Lytton-Jean, Abigail K R; Mirkin, Chad A

2007-09-15

We have developed a novel competition assay that uses a gold nanoparticle (Au NP)-based, high-throughput colorimetric approach to screen the sequence selectivity of DNA-binding molecules. This assay hinges on the observation that the melting behavior of DNA-functionalized Au NP aggregates is sensitive to the concentration of the DNA-binding molecule in solution. When short, oligomeric hairpin DNA sequences were added to a reaction solution consisting of DNA-functionalized Au NP aggregates and DNA-binding molecules, these molecules may either bind to the Au NP aggregate interconnects or the hairpin stems based on their relative affinity for each. This relative affinity can be measured as a change in the melting temperature (Tm) of the DNA-modified Au NP aggregates in solution. As a proof of concept, we evaluated the selectivity of 4',6-diamidino-2-phenylindone (an AT-specific binder), ethidium bromide (a nonspecific binder), and chromomycin A (a GC-specific binder) for six sequences of hairpin DNA having different numbers of AT pairs in a five-base pair variable stem region. Our assay accurately and easily confirmed the known trends in selectivity for the DNA binders in question without the use of complicated instrumentation. This novel assay will be useful in assessing large libraries of potential drug candidates that work by binding DNA to form a drug/DNA complex.

FIBER OPTIC BIOSENSOR FOR DNA DAMAGE

EPA Science Inventory

This paper describes a fiber optic biosensor for the rapid and sensitive detection of radiation-induced or chemically-induced oxidative DNA damage. The assay is based on the hybridization and temperature-induced dissociation (melting curves) of synthetic oligonucleotides. The...

ESI-MS Investigation of an Equilibrium between a Bimolecular Quadruplex DNA and a Duplex DNA/RNA Hybrid

NASA Astrophysics Data System (ADS)

Birrento, Monica L.; Bryan, Tracy M.; Samosorn, Siritron; Beck, Jennifer L.

2015-07-01

Electrospray ionization mass spectrometry (ESI-MS) conditions were optimized for simultaneous observation of a bimolecular qDNA and a Watson-Crick base-paired duplex DNA/RNA hybrid. The DNA sequence used was telomeric DNA, and the RNA contained the template for telomerase-mediated telomeric DNA synthesis. Addition of RNA to the quadruplex DNA (qDNA) resulted in formation of the duplex DNA/RNA hybrid. Melting profiles obtained using circular dichroism spectroscopy confirmed that the DNA/RNA hybrid exhibited greater thermal stability than the bimolecular qDNA in solution. Binding of a 13-substituted berberine ( 1) derivative to the bimolecular qDNA stabilized its structure as evidenced by an increase in its stability in the mass spectrometer, and an increase in its circular dichroism (CD) melting temperature of 10°C. The DNA/RNA hybrid did not bind the ligand extensively and its thermal stability was unchanged in the presence of ( 1). The qDNA-ligand complex resisted unfolding in the presence of excess RNA, limiting the formation of the DNA/RNA hybrid. Previously, it has been proposed that DNA secondary structures, such as qDNA, may be involved in the telomerase mechanism. DNA/RNA hybrid structures occur at the active site of telomerase. The results presented in the current work show that if telomeric DNA was folded into a qDNA structure, it is possible for a DNA/RNA hybrid to form as is required during template alignment. The discrimination of ligand ( 1) for binding to the bimolecular qDNA over the DNA/RNA hybrid positions it as a useful compound for probing the role(s), if any, of antiparallel qDNA in the telomerase mechanism.

Variability of Surface Temperature and Melt on the Greenland Ice Sheet, 2000-2011

NASA Technical Reports Server (NTRS)

Hall, Dorothy K.; Comiso, Josefino, C.; Shuman, Christopher A.; Koenig, Lora S.; DiGirolamo, Nicolo E.

2012-01-01

Enhanced melting along with surface-temperature increases measured using infrared satellite data, have been documented for the Greenland Ice Sheet. Recently we developed a climate-quality data record of ice-surface temperature (IST) of the Greenland Ice Sheet using the Moderate-Resolution Imaging Spectroradiometer (MODIS) 1ST product -- http://modis-snow-ice.gsfc.nasa.gov. Using daily and mean monthly MODIS 1ST maps from the data record we show maximum extent of melt for the ice sheet and its six major drainage basins for a 12-year period extending from March of 2000 through December of 2011. The duration of the melt season on the ice sheet varies in different drainage basins with some basins melting progressively earlier over the study period. Some (but not all) of the basins also show a progressively-longer duration of melt. The short time of the study period (approximately 12 years) precludes an evaluation of statistically-significant trends. However the dataset provides valuable information on natural variability of IST, and on the ability of the MODIS instrument to capture changes in IST and melt conditions indifferent drainage basins of the ice sheet.

When Is Melting Not Really Melting?

ERIC Educational Resources Information Center

Mangiaracina, Mike

2017-01-01

This 5E cycle of lessons takes students through a fun and thorough study of Silly Putty's properties, progressing from an initial observation of a "melting snowman" toy in the Engage phase to making and "marketing" their own homemade putty in the Evaluate phase. Along the way, students use evidence to construct their own…

Use of the melting curve assay as a means for high-throughput quantification of Illumina sequencing libraries.

PubMed

Shinozuka, Hiroshi; Forster, John W

2016-01-01

Background. Multiplexed sequencing is commonly performed on massively parallel short-read sequencing platforms such as Illumina, and the efficiency of library normalisation can affect the quality of the output dataset. Although several library normalisation approaches have been established, none are ideal for highly multiplexed sequencing due to issues of cost and/or processing time. Methods. An inexpensive and high-throughput library quantification method has been developed, based on an adaptation of the melting curve assay. Sequencing libraries were subjected to the assay using the Bio-Rad Laboratories CFX Connect(TM) Real-Time PCR Detection System. The library quantity was calculated through summation of reduction of relative fluorescence units between 86 and 95 °C. Results.PCR-enriched sequencing libraries are suitable for this quantification without pre-purification of DNA. Short DNA molecules, which ideally should be eliminated from the library for subsequent processing, were differentiated from the target DNA in a mixture on the basis of differences in melting temperature. Quantification results for long sequences targeted using the melting curve assay were correlated with those from existing methods (R (2) > 0.77), and that observed from MiSeq sequencing (R (2) = 0.82). Discussion.The results of multiplexed sequencing suggested that the normalisation performance of the described method is equivalent to that of another recently reported high-throughput bead-based method, BeNUS. However, costs for the melting curve assay are considerably lower and processing times shorter than those of other existing methods, suggesting greater suitability for highly multiplexed sequencing applications.

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

PubMed Central

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

2011-01-01

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

Rapid detection of G6PD mutations by multicolor melting curve analysis.

PubMed

Xia, Zhongmin; Chen, Ping; Tang, Ning; Yan, Tizhen; Zhou, Yuqiu; Xiao, Qizhi; Huang, Qiuying; Li, Qingge

2016-09-01

The MeltPro G6PD assay is the first commercial genetic test for glucose-6-phosphate dehydrogenase (G6PD) deficiency. This multicolor melting curve analysis-based real-time PCR assay is designed to genotype 16 G6PD mutations prevalent in the Chinese population. We comprehensively evaluated both the analytical and clinical performances of this assay. All 16 mutations were accurately genotyped, and the standard deviation of the measured Tm was <0.3°C. The limit of detection was 1.0ng/μL human genomic DNA. The assay could be run on four mainstream models of real-time PCR machines. The shortest running time (150min) was obtained with LightCycler 480 II. A clinical study using 763 samples collected from three hospitals indicated that, of 433 samples with reduced G6PD activity, the MeltPro assay identified 423 samples as mutant, yielding a clinical sensitivity of 97.7% (423/433). Of the 117 male samples with normal G6PD activity, the MeltPro assay confirmed that 116 samples were wild type, yielding a clinical specificity of 99.1% (116/117). Moreover, the MeltPro assay demonstrated 100% concordance with DNA sequencing for all targeted mutations. We concluded that the MeltPro G6PD assay is useful as a diagnostic or screening tool for G6PD deficiency in clinical settings. Copyright © 2016 Elsevier Inc. All rights reserved.

Quality assessment of DNA derived from up to 30 years old formalin fixed paraffin embedded (FFPE) tissue for PCR-based methylation analysis using SMART-MSP and MS-HRM.

PubMed

Kristensen, Lasse S; Wojdacz, Tomasz K; Thestrup, Britta B; Wiuf, Carsten; Hager, Henrik; Hansen, Lise Lotte

2009-12-21

The High Resolution Melting (HRM) technology has recently been introduced as a rapid and robust analysis tool for the detection of DNA methylation. The methylation status of multiple tumor suppressor genes may serve as biomarkers for early cancer diagnostics, for prediction of prognosis and for prediction of response to treatment. Therefore, it is important that methodologies for detection of DNA methylation continue to evolve. Sensitive Melting Analysis after Real Time - Methylation Specific PCR (SMART-MSP) and Methylation Sensitive - High Resolution Melting (MS-HRM) are two methods for single locus DNA methylation detection based on HRM. Here, we have assessed the quality of DNA extracted from up to 30 years old Formalin Fixed Paraffin Embedded (FFPE) tissue for DNA methylation analysis using SMART-MSP and MS-HRM. The quality assessment was performed on DNA extracted from 54 Non-Small Cell Lung Cancer (NSCLC) samples derived from FFPE tissue, collected over 30 years and grouped into five years intervals. For each sample, the methylation levels of the CDKN2A (p16) and RARB promoters were estimated using SMART-MSP and MS-HRM assays designed to assess the methylation status of the same CpG positions. This allowed for a direct comparison of the methylation levels estimated by the two methods for each sample. CDKN2A promoter methylation levels were successfully determined by SMART-MSP and MS-HRM in all 54 samples. Identical methylation estimates were obtained by the two methods in 46 of the samples. The methylation levels of the RARB promoter were successfully determined by SMART-MSP in all samples. When using MS-HRM to assess RARB methylation five samples failed to amplify and 15 samples showed a melting profile characteristic for heterogeneous methylation. Twenty-seven of the remaining 34 samples, for which the methylation level could be estimated, gave the same result as observed when using SMART-MSP. MS-HRM and SMART-MSP can be successfully used for single locus methylation studies using DNA derived from up to 30 years old FFPE tissue. Furthermore, it can be expected that MS-HRM and SMART-MSP will provide similar methylation estimates when assays are designed to analyze the same CpG positions.

Heat-transfer resistance at solid-liquid interfaces: a tool for the detection of single-nucleotide polymorphisms in DNA.

PubMed

van Grinsven, Bart; Vanden Bon, Natalie; Strauven, Hannelore; Grieten, Lars; Murib, Mohammed; Monroy, Kathia L Jiménez; Janssens, Stoffel D; Haenen, Ken; Schöning, Michael J; Vermeeren, Veronique; Ameloot, Marcel; Michiels, Luc; Thoelen, Ronald; De Ceuninck, Ward; Wagner, Patrick

2012-03-27

In this article, we report on the heat-transfer resistance at interfaces as a novel, denaturation-based method to detect single-nucleotide polymorphisms in DNA. We observed that a molecular brush of double-stranded DNA grafted onto synthetic diamond surfaces does not notably affect the heat-transfer resistance at the solid-to-liquid interface. In contrast to this, molecular brushes of single-stranded DNA cause, surprisingly, a substantially higher heat-transfer resistance and behave like a thermally insulating layer. This effect can be utilized to identify ds-DNA melting temperatures via the switching from low- to high heat-transfer resistance. The melting temperatures identified with this method for different DNA duplexes (29 base pairs without and with built-in mutations) correlate nicely with data calculated by modeling. The method is fast, label-free (without the need for fluorescent or radioactive markers), allows for repetitive measurements, and can also be extended toward array formats. Reference measurements by confocal fluorescence microscopy and impedance spectroscopy confirm that the switching of heat-transfer resistance upon denaturation is indeed related to the thermal on-chip denaturation of DNA. © 2012 American Chemical Society

High-resolution Melting Analysis for Gene Scanning of Adenomatous Polyposis Coli (APC) Gene With Oral Squamous Cell Carcinoma Samples.

PubMed

Chang, Ya-Sian; Lin, Chien-Yu; Yang, Shu-Fen; Ho, Cheng Mao; Chang, Jan-Gowth

2016-02-01

There have been many different mutations reported for the large adenomatous polyposis coli (APC) tumor suppressor gene. APC mutations result in inactivation of APC tumor suppressor action, allowing the progression of tumorigenesis. The present study utilized a highly efficient method to identify APC mutations and investigated the association between the APC genetic variants Y486Y, A545A, T1493T, and D1822V and susceptibility to oral squamous cell carcinoma (OSCC). High-resolution melting (HRM) analysis was used to characterize APC mutations. Genomic DNA was extracted from 83 patient specimens of OSCC and 50 blood samples from healthy control subjects. The 14 exons and mutation cluster region of exon 15 were screened by HRM analysis. All mutations were confirmed by direct DNA sequencing. Three mutations and 4 single nucleotide polymorphisms (SNPs) were found in this study. The mutations were c.573T>C (Y191Y) in exon 5, c.1005A>G (L335L) in exon 9, and c.1488A>T (T496T) in exon 11. Two SNPs, c.4479G>A (T1493T) and c.5465A>T (D1822V), were located in exon 15, whereas c.1458T>C (Y486Y) and c.1635G>A (A545A) were located in exon 11 and 13, respectively. There was no observed association between OSCC risk and genotype for any of the 4 APC SNPs. The mutation of APC is rare in Taiwanese patients with OSCC. HRM analysis is a reliable, accurate, and fast screening method for APC mutations.

Evaluating Snowmelt Runoff Processes Using Stable Isotopes in a Permafrost Hillslope

NASA Astrophysics Data System (ADS)

Carey, S. K.

2004-05-01

Conceptual understanding of runoff generation in permafrost regions have been derived primarily from hydrometric information, with isotope and hydrochemical data having only limited application in delineating sources and pathways of water. Furthermore, when stable isotope data are used to infer runoff processes, it often provides conflicting results from hydrometric measurements. In a small subarctic alpine catchment within the Wolf Creek Research Basin, Yukon, Canada, experiments were conducted during the melt period of 2002 and 2003 to trace the stable isotopic signature (d18O) of meltwater from a melting snowpack into permafrost soils and laterally to the stream to identify runoff processes and evaluate sources of error for traditional hydrograph separation studies in snowmelt-dominated permafrost basins. Isotopic variability in the snowpack was recorded at 0.1 m depth intervals during the melt period and compared with the meltwater isotopic signature at the snowpack base collected in lysimeters. Throughout the melt period in both years, there was an isotopic enrichment of meltwater as the season progressed. A downslope transect of wells and piezometers were used to evaluate the influence of infiltrating meltwater and thawing ground on the subsurface d18O signature. As melt began, meltwater infiltrated the frozen porous organic layer, leading to liquid water saturation in the unsaturated pore spaces. Water sampled during this initial melt stage show soil water d18O mirroring that of the meltwater signal. As the melt season progressed, frozen soil began to melt, mixing enriched pre-melt soil water with meltwater. This mixing increased the overall value of d18O obtained from the soil, which gradually increased as thaw progressed. At the end of snowmelt, soil water had a d18O value similar to values from the previous fall, suggesting that much of the initial snowmelt water had been flushed from the hillslope. Results from the hillslope scale are compared with two-component hydrograph separations and sources of error are discussed.

Microfludic Device for Creating Ionic Strength Gradients over DNA Microarrays for Efficient DNA Melting Studies and Assay Development

PubMed Central

Petersen, Jesper; Poulsen, Lena; Birgens, Henrik; Dufva, Martin

2009-01-01

The development of DNA microarray assays is hampered by two important aspects: processing of the microarrays is done under a single stringency condition, and characteristics such as melting temperature are difficult to predict for immobilized probes. A technical solution to these limitations is to use a thermal gradient and information from melting curves, for instance to score genotypes. However, application of temperature gradients normally requires complicated equipment, and the size of the arrays that can be investigated is restricted due to heat dissipation. Here we present a simple microfluidic device that creates a gradient comprising zones of defined ionic strength over a glass slide, in which each zone corresponds to a subarray. Using this device, we demonstrated that ionic strength gradients function in a similar fashion as corresponding thermal gradients in assay development. More specifically, we noted that (i) the two stringency modulators generated melting curves that could be compared, (ii) both led to increased assay robustness, and (iii) both were associated with difficulties in genotyping the same mutation. These findings demonstrate that ionic strength stringency buffers can be used instead of thermal gradients. Given the flexibility of design of ionic gradients, these can be created over all types of arrays, and encompass an attractive alternative to temperature gradients, avoiding curtailment of the size or spacing of subarrays on slides associated with temperature gradients. PMID:19277213

Microfludic device for creating ionic strength gradients over DNA microarrays for efficient DNA melting studies and assay development.

PubMed

Petersen, Jesper; Poulsen, Lena; Birgens, Henrik; Dufva, Martin

2009-01-01

The development of DNA microarray assays is hampered by two important aspects: processing of the microarrays is done under a single stringency condition, and characteristics such as melting temperature are difficult to predict for immobilized probes. A technical solution to these limitations is to use a thermal gradient and information from melting curves, for instance to score genotypes. However, application of temperature gradients normally requires complicated equipment, and the size of the arrays that can be investigated is restricted due to heat dissipation. Here we present a simple microfluidic device that creates a gradient comprising zones of defined ionic strength over a glass slide, in which each zone corresponds to a subarray. Using this device, we demonstrated that ionic strength gradients function in a similar fashion as corresponding thermal gradients in assay development. More specifically, we noted that (i) the two stringency modulators generated melting curves that could be compared, (ii) both led to increased assay robustness, and (iii) both were associated with difficulties in genotyping the same mutation. These findings demonstrate that ionic strength stringency buffers can be used instead of thermal gradients. Given the flexibility of design of ionic gradients, these can be created over all types of arrays, and encompass an attractive alternative to temperature gradients, avoiding curtailment of the size or spacing of subarrays on slides associated with temperature gradients.

Lactase persistence genotyping on whole blood by loop-mediated isothermal amplification and melting curve analysis.

PubMed

Abildgaard, Anders; Tovbjerg, Sara K; Giltay, Axel; Detemmerman, Liselot; Nissen, Peter H

2018-03-26

The lactase persistence phenotype is controlled by a regulatory enhancer region upstream of the Lactase (LCT) gene. In northern Europe, specifically the -13910C > T variant has been associated with lactase persistence whereas other persistence variants, e.g. -13907C > G and -13915 T > G, have been identified in Africa and the Middle East. The aim of the present study was to compare a previously developed high resolution melting assay (HRM) with a novel method based on loop-mediated isothermal amplification and melting curve analysis (LAMP-MC) with both whole blood and DNA as input material. To evaluate the LAMP-MC method, we used 100 whole blood samples and 93 DNA samples in a two tiered study. First, we studied the ability of the LAMP-MC method to produce specific melting curves for several variants of the LCT enhancer region. Next, we performed a blinded comparison between the LAMP-MC method and our existing HRM method with clinical samples of unknown genotype. The LAMP-MC method produced specific melting curves for the variants at position -13909, -13910, -13913 whereas the -13907C > G and -13915 T > G variants produced indistinguishable melting profiles. The LAMP-MC assay is a simple method for lactase persistence genotyping and compares well with our existing HRM method. Copyright © 2018. Published by Elsevier B.V.

Triplex-mediated analysis of cytosine methylation at CpA sites in DNA.

PubMed

Johannsen, Marie W; Gerrard, Simon R; Melvin, Tracy; Brown, Tom

2014-01-18

Modified triplex-forming oligonucleotides distinguish 5-methyl cytosine from unmethylated cytosine in DNA duplexes by differences in triplex melting temperatures. The discrimination is sequence-specific; dramatic differences in stabilisation are seen for CpA methylation, whereas CpG methylation is not detected. This direct detection of DNA methylation constitutes a new approach for epigenetic analysis.

Cumulative mtDNA damage and mutations contribute to the progressive loss of RGCs in a rat model of glaucoma

PubMed Central

Nickerson, John M.; Gao, Feng-juan; Sun, Zhongmou; Chen, Xin-ya; Zhang, Shu-jie; Gao, Feng; Chen, Jun-yi; Luo, Yi; Wang, Yan; Sun, Xing-huai

2015-01-01

Glaucoma is a chronic neurodegenerative disease characterized by the progressive loss of retinal ganglion cells (RGCs). Mitochondrial DNA (mtDNA) alterations have been documented as a key component of many neurodegenerative disorders. However, whether mtDNA alterations contribute to the progressive loss of RGCs and the mechanism whereby this phenomenon could occur are poorly understood. We investigated mtDNA alterations in RGCs using a rat model of chronic intraocular hypertension and explored the mechanisms underlying progressive RGC loss. We demonstrate that the mtDNA damage and mutations triggered by intraocular pressure (IOP) elevation are initiating, crucial events in a cascade leading to progressive RGC loss. Damage to and mutation of mtDNA, mitochondrial dysfunction, reduced levels of mtDNA repair/replication enzymes, and elevated reactive oxygen species form a positive feedback loop that produces irreversible mtDNA damage and mutation and contributes to progressive RGC loss, which occurs even after a return to normal IOP. Furthermore, we demonstrate that mtDNA damage and mutations increase the vulnerability of RGCs to elevated IOP and glutamate levels, which are among the most common glaucoma insults. This study suggests that therapeutic approaches that target mtDNA maintenance and repair and that promote energy production may prevent the progressive death of RGCs. PMID:25478814

Continuous method for manufacturing grain-oriented magnetostrictive bodies

DOEpatents

Gibson, Edwin D.; Verhoeven, John D.; Schmidt, Frederick A.; McMasters, O. Dale

1988-01-01

The invention comprises a continuous casting and crystallization method for manufacturing grain-oriented magnetostrictive bodies. A magnetostrictive alloy is melted in a crucible having a bottom outlet. The melt is discharged through the bottom of the crucible and deposited in an elongated mold. Heat is removed from the deposited melt through the lower end portion of the mold to progressively solidify the melt. The solid-liquid interface of the melt moves directionally upwardly from the bottom to the top of the mold, to produce the axial grain orientation.

Rapid detection of Opisthorchis viverrini and Strongyloides stercoralis in human fecal samples using a duplex real-time PCR and melting curve analysis.

PubMed

Janwan, Penchom; Intapan, Pewpan M; Thanchomnang, Tongjit; Lulitanond, Viraphong; Anamnart, Witthaya; Maleewong, Wanchai

2011-12-01

Human opisthorchiasis caused by the liver fluke Opisthorchis viverrini is an endemic disease in Southeast Asian countries including the Lao People's Democratic Republic, Cambodia, Vietnam, and Thailand. Infection with the soil-transmitted roundworm Strongyloides stercoralis is an important problem worldwide. In some areas, both parasitic infections are reported as co-infections. A duplex real-time fluorescence resonance energy transfer (FRET) PCR merged with melting curve analysis was developed for the rapid detection of O. viverrini and S. stercoralis in human fecal samples. Duplex real-time FRET PCR is based on fluorescence melting curve analysis of a hybrid of amplicons generated from two genera of DNA elements: the 162 bp pOV-A6 DNA sequence specific to O. viverrini and the 244 bp 18S rRNA sequence specific to S. stercoralis, and two pairs of specific fluorophore-labeled probes. Both O. viverrini and S. stercoralis can be differentially detected in infected human fecal samples by this process through their different fluorescence channels and melting temperatures. Detection limit of the method was as little as two O. viverrini eggs and four S. stercoralis larvae in 100 mg of fecal sample. The assay could distinguish the DNA of both parasites from the DNA of negative fecal samples and fecal samples with other parasite materials, as well as from the DNA of human leukocytes and other control parasites. The technique showed 100% sensitivity and specificity. The introduced duplex real-time FRET PCR can reduce labor time and reagent costs and is not prone to carry over contamination. The method is important for simultaneous detection especially in areas where both parasites overlap incidence and is useful as the screening tool in the returning travelers and immigrants to industrialized countries where number of samples in the diagnostic units will become increasing.

[Features of binding of proflavine to DNA at different DNA-ligand concentration ratios].

PubMed

Berezniak, E G; gladkovskaia, N A; Khrebtova, A S; Dukhopel'nikov, E V; Zinchenko, A V

2009-01-01

The binding of proflavine to calf thymus DNA has been studied using the methods of differential scanning calorimetry and spectrophotometry. It was shown that proflavine can interact with DNA by at least 3 binding modes. At high DNA-ligand concentration ratios (P/D), proflavine intercalates into both GC- and AT-sites, with a preference to GC-rich sequences. At low P/D ratios proflavine interacts with DNA by the external binding mode. From spectrophotometric concentration dependences, the parameters of complexing of proflavine with DNA were calculated. Thermodynamic parameters of DNA melting were calculated from differential scanning calorimetry data.

Core-melt source reduction system

DOEpatents

Forsberg, C.W.; Beahm, E.C.; Parker, G.W.

1995-04-25

A core-melt source reduction system for ending the progression of a molten core during a core-melt accident and resulting in a stable solid cool matrix. The system includes alternating layers of a core debris absorbing material and a barrier material. The core debris absorbing material serves to react with and absorb the molten core such that containment overpressurization and/or failure does not occur. The barrier material slows the progression of the molten core debris through the system such that the molten core has sufficient time to react with the core absorbing material. The system includes a provision for cooling the glass/molten core mass after the reaction such that a stable solid cool matrix results. 4 figs.

Core-melt source reduction system

DOEpatents

Forsberg, Charles W.; Beahm, Edward C.; Parker, George W.

1995-01-01

A core-melt source reduction system for ending the progression of a molten core during a core-melt accident and resulting in a stable solid cool matrix. The system includes alternating layers of a core debris absorbing material and a barrier material. The core debris absorbing material serves to react with and absorb the molten core such that containment overpressurization and/or failure does not occur. The barrier material slows the progression of the molten core debris through the system such that the molten core has sufficient time to react with the core absorbing material. The system includes a provision for cooling the glass/molten core mass after the reaction such that a stable solid cool matrix results.

Authenticity analyses of Rhizoma Paridis using barcoding coupled with high resolution melting (Bar-HRM) analysis to control its quality for medicinal plant product.

PubMed

Duan, Bao-Zhong; Wang, Ya-Ping; Fang, Hai-Lan; Xiong, Chao; Li, Xi-Wen; Wang, Ping; Chen, Shi-Lin

2018-01-01

Rhizoma Paridis (Chonglou) is a commonly used and precious traditional Chinese medicine. Paris polyphylla Smith var. yunnanensis (Franch.) Hand. -Mazz. and Paris polyphylla Smith var . chinensis (Franch.) Hara are the two main sources of Chonglou under the monograph of Rhizoma Paridis in Chinese Pharmacopoeia. In the local marketplace, however, this medicine is prone to be accidentally contaminated, deliberately substituted or admixed with other species that are similar to Rhizoma Paridis in shape and color. Consequently, these adulterations might compromise quality control and result in considerable health concerns for consumers. This study aims to develop a rapid and sensitive method for accurate identification of Rhizoma Paridis and its common adulterants. DNA barcoding coupled with high resolution melting analysis was applied in this research to distinguish Rhizoma Paridis from its adulteration. The internal transcribed spacer 2 (ITS2) barcode was selected for HRM analysis to produce standard melting profile of the selected species. DNA of the tested herbal medicines was isolated and their melting profiles were generated and compared with the standard melting profile of P. polyphylla var. chinensis . The results indicate that the ITS2 molecular regions coupled with HRM analysis can effectively differentiate nine herbal species, including two authentic origins of Chonglou and their seven common adulterants. Ten herbal medicines labeled "Chonglou" obtained from a local market were collected and identified with our methods, and their sequence information was analyzed to validate the accuracy of HRM analysis. DNA barcoding coupled with HRM analysis is a accurate, reliable, rapid, cost-effective and robust tool, which could contribute to the quality control of Rhizoma Paridis in the supply chain of the natural health product industry (NHP).

Using surface-enhanced Raman spectroscopy and electrochemically driven melting to discriminate Yersinia pestis from Y. pseudotuberculosis based on single nucleotide polymorphisms within unpurified polymerase chain reaction amplicons.

PubMed

Papadopoulou, Evanthia; Goodchild, Sarah A; Cleary, David W; Weller, Simon A; Gale, Nittaya; Stubberfield, Michael R; Brown, Tom; Bartlett, Philip N

2015-02-03

The development of sensors for the detection of pathogen-specific DNA, including relevant species/strain level discrimination, is critical in molecular diagnostics with major impacts in areas such as bioterrorism and food safety. Herein, we use electrochemically driven denaturation assays monitored by surface-enhanced Raman spectroscopy (SERS) to target single nucleotide polymorphisms (SNPs) that distinguish DNA amplicons generated from Yersinia pestis, the causative agent of plague, from the closely related species Y. pseudotuberculosis. Two assays targeting SNPs within the groEL and metH genes of these two species have been successfully designed. Polymerase chain reaction (PCR) was used to produce Texas Red labeled single-stranded DNA (ssDNA) amplicons of 262 and 251 bases for the groEL and metH targets, respectively. These amplicons were used in an unpurified form to hybridize to immobilized probes then subjected to electrochemically driven melting. In all cases electrochemically driven melting was able to discriminate between fully homologous DNA and that containing SNPs. The metH assay was particularly challenging due to the presence of only a single base mismatch in the middle of the 251 base long PCR amplicon. However, manipulation of assay conditions (conducting the electrochemical experiments at 10 °C) resulted in greater discrimination between the complementary and mismatched DNA. Replicate data were collected and analyzed for each duplex on different days, using different batches of PCR product and different sphere segment void (SSV) substrates. Despite the variability introduced by these differences, the assays are shown to be reliable and robust providing a new platform for strain discrimination using unpurified PCR samples.

Molecular spectroscopic studies on the interaction of ferulic acid with calf thymus DNA

NASA Astrophysics Data System (ADS)

Zhang, Shufang; Sun, Xuejun; Qu, Fengli; Kong, Rongmei

2013-08-01

The interaction between ferulic acid and calf thymus deoxyribonucleic acid (ctDNA) under physiological conditions (Tris-HCl buffer solutions, pH 7.4) was investigated by UV-Vis spectroscopy, fluorescence spectroscopy, DNA melting techniques, and viscosity measurements. Results indicated that a complex of ferulic acid with ctDNA was formed with a binding constant of K290K = 7.60 × 104 L mol-1 and K310K = 4.90 × 104 L mol-1. The thermodynamic parameters enthalpy change (ΔH°), entropy change (ΔS°) and Gibbs free energy (ΔG°) were calculated to be -1.69 × 104 J mol-1, 35.36 J K-1 mol-1 and -2.79 × 104 J mol-1 at 310 K, respectively. The acting forces between ferulic acid and DNA mainly included hydrophobic interaction and hydrogen bonds. Acridine orange displacement studies revealed that ferulic acid can substitute for AO probe in the AO-DNA complex which was indicative of intercalation binding. Thermal denaturation study suggested that the interaction of ferulic acid with DNA could result in the increase of the denaturation temperature, which indicated that the stabilization of the DNA helix was increased in the presence of ferulic acid. Spectroscopic techniques together with melting techniques and viscosity determination provided evidences of intercalation mode of binding for the interaction between ferulic acid and ctDNA.

Probing the interaction of the phytochemical 6-gingerol from the spice ginger with DNA.

PubMed

Haris, Poovvathingal; Mary, Varughese; Sudarsanakumar, Chellappanpillai

2018-07-01

6-Gingerol [5-hydroxy-1-(4-hydroxy-3-methoxyphenyl) decan-3-one], the bio-active ingredient of the popular Indian spice ginger (Zingiber officinale Roscoe), is well-known for its pharmacological and physiological actions. The potent antioxidant, antiemetic, antiulcer, antimicrobial, analgesic, hypoglycemic, antihypertensive, antihyperlipidemic, immunostimulant, anti-inflammatory, cardiotonic, and cancer prevention activities of 6-Gingerol has been investigated and explored. 6-Gingerol is a good candidate for the treatment of various cancers including prostrate, pancreatic, breast, skin, gastrointestinal, pulmonary, and renal cancer. In this study we report for the first time the molecular recognition of 6-Gingerol with calf thymus DNA (ctDNA) through experimental and molecular modeling techniques confirming a minor groove binding mode of 6-Gingerol with ctDNA. Fluorescence and UV-vis spectroscopic studies confirm the complex formation of 6-gingerol with ctDNA. The energetics and thermodynamics of the interaction of 6-Gingerol with ctDNA was explored by Isothermal Titration Calorimetry (ITC) and Differential Scanning Calorimetry (DSC). The ctDNA helix melting upon 6-Gingerol binding was examined by melting temperature T m analysis. Further the electrophoretic mobility shift assay confirms a possible groove binding of 6-Gingerol with ctDNA. Molecular docking and Molecular dynamics (MD) studies provide a detailed understanding on the interaction of 6-Gingerol binding in the minor groove of DNA which supports experimental results. Copyright © 2018. Published by Elsevier B.V.

Effect of Backbone Design on Hybridization Thermodynamics of Oligo-nucleic Acids: A Coarse-Grained Molecular Dynamics Simulation Study

NASA Astrophysics Data System (ADS)

Ghobadi, Ahmadreza F.; Jayaraman, Arthi

DNA hybridization is the basis of various bio-nano technologies, such as DNA origami and assembly of DNA-functionalized nanoparticles. A hybridized double stranded (ds) DNA is formed when complementary nucleobases on hybridizing strands exhibit specific and directional hydrogen bonds through canonical Watson-Crick base-pairing interactions. In recent years, the need for cheaper alternatives and significant synthetic advances have driven design of DNA mimics with new backbone chemistries. However, a fundamental understanding of how these backbone modifications in the oligo-nucleic acids impact the hybridization and melting behavior of the duplex is still lacking. In this talk, we present our recent findings on impact of varying backbone chemistry on hybridization of oligo-nucleic acid duplexes. We use coarse-grained molecular dynamics simulations to isolate the effect of strand flexibility, electrostatic interactions and nucleobase spacing on the melting curves for duplexes with various strand sequences and concentrations. Since conjugation of oligo-nucleic acids with polymers serve as building blocks for thermo-responsive polymer networks and gels, we also present the effect of such conjugation on hybridization thermodynamics and polymer conformation.

Cytotoxicity and DNA interaction of brucine and strychnine-Two alkaloids of semen strychni.

PubMed

Liu, Fei; Wang, Xiaolin; Han, Xu; Tan, Xiaoxin; Kang, Weijun

2015-01-01

The cytotoxicities of the two alkaloids strychnine and brucine from the seed of Strychnos nux-vomica and their interaction with DNA were investigated. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrasolium bromide (MTT) assay was used to examine the growth inhibitory effects of these alkaloids on Vero cells after 24, 48 and 72h of incubation. The cytotoxicities of strychnine and brucine were found to be time- and concentration-dependent. Strychnine was determined to be more toxic to Vero cells than brucine. At the same time, the interactions of strychnine and brucine with DNA were investigated using neutral red (NR) dye as a probe by UV-vis spectroscopy, fluorescence spectroscopy, and an examination of the ionic strength effect, and the effects of alkaloids on DNA melting were also examined. The results indicated that a DNA-brucine mixture but not a DNA-strychnine mixture could be extracted from Vero cells after treatment with brucine and strychnine, respectively. Brucine competitively intercalated into the DNA double-helix causing fluorescence quenching of the DNA-NR system. UV absorption spectroscopy and the melting temperature (Tm) curve also provided evidence that brucine interacted with DNA through intercalation. Furthermore, the results of the ionic strength effect experiment suggested that electrostatic interactions between brucine and phosphate groups in the DNA backbone might also play an important role in the binding of brucine to DNA. Copyright © 2015 Elsevier B.V. All rights reserved.

Isolation and Characterization of Chloroplast DNA from the Duckweed Spirodela oligorrhiza

PubMed Central

van Ee, Jan H.; Veld, Willem A. Man In'T; Planta, Rudi J.

1980-01-01

Chloroplast DNA of the duckweed Spirodela oligorrhiza, isolated by CsCl gradient centrifugation, was characterized by its buoyant density, guanine + cytosine content, melting behavior, circularity, and contour length. In all these characteristics, chloroplast DNA of S. oligorrhiza is similar to the chloroplast genomes of other higher plants, except that it has a significantly larger size. Images PMID:16661479

Cumulative mtDNA damage and mutations contribute to the progressive loss of RGCs in a rat model of glaucoma.

PubMed

Wu, Ji-Hong; Zhang, Sheng-Hai; Nickerson, John M; Gao, Feng-Juan; Sun, Zhongmou; Chen, Xin-Ya; Zhang, Shu-Jie; Gao, Feng; Chen, Jun-Yi; Luo, Yi; Wang, Yan; Sun, Xing-Huai

2015-02-01

Glaucoma is a chronic neurodegenerative disease characterized by the progressive loss of retinal ganglion cells (RGCs). Mitochondrial DNA (mtDNA) alterations have been documented as a key component of many neurodegenerative disorders. However, whether mtDNA alterations contribute to the progressive loss of RGCs and the mechanism whereby this phenomenon could occur are poorly understood. We investigated mtDNA alterations in RGCs using a rat model of chronic intraocular hypertension and explored the mechanisms underlying progressive RGC loss. We demonstrate that the mtDNA damage and mutations triggered by intraocular pressure (IOP) elevation are initiating, crucial events in a cascade leading to progressive RGC loss. Damage to and mutation of mtDNA, mitochondrial dysfunction, reduced levels of mtDNA repair/replication enzymes, and elevated reactive oxygen species form a positive feedback loop that produces irreversible mtDNA damage and mutation and contributes to progressive RGC loss, which occurs even after a return to normal IOP. Furthermore, we demonstrate that mtDNA damage and mutations increase the vulnerability of RGCs to elevated IOP and glutamate levels, which are among the most common glaucoma insults. This study suggests that therapeutic approaches that target mtDNA maintenance and repair and that promote energy production may prevent the progressive death of RGCs. Copyright © 2014 Elsevier Inc. All rights reserved.

Kinetics of Melting and Dissolution in Lunar Materials

NASA Technical Reports Server (NTRS)

Hess, Paul C.

2002-01-01

An understanding of the petrogenesis of lunar magmas, particularly mare basalts and the parent magmas to the Mg-rich suite, remains an unfulfilled goal. The fact is not surprising given the complexity of the problem. On the Moon, the source region for lunar magmas is not primitive mantle but rather a series of cumulate rocks that vary widely in both minerology and major and minor element contents. The stratigraphy of the cumulate mantle is not likely to be very regular given that the culumate pile is formed initially in an unstable configuration and subsequent thermal and compositional heterogeneities on a number of length scales. These lithologic heterogeneities, the large range of pressures and temperatures over which melts are generated on the Moon, and the close juxtaposition of cumulate rock with widely varying solidii introduce significant complications to the nature of the melting relations that control melt generation. These factors, coupled with the likelihood that polybaric fractional melting of varying efficiencies ultimately control the composition of planetary progress, are ample reasons why the lunar magmas remain the enigma they are. To make progress, phase equilibria studies must be coupled with a detailed understanding of the time scales and the dynamics of crystal and melt reequilibration processes.

Application of a coarse-grained model for DNA to homo- and heterogeneous melting equilibria

NASA Astrophysics Data System (ADS)

Tito, Nicholas B.; Stubbs, John M.

2010-01-01

Configurational-bias Monte Carlo simulations were carried out on deoxyribonucleic acid (DNA) decamers using a coarse-grained molecular model. The effects of single mutations on the melting transition were investigated as were heterogeneous systems with immobilization of one strand on a surface, both with and without a spacer. The destabilizing effect of an internal mutation is attributed to a lack of cooperativity, which acts through a hydrogen bonding nucleotide's restriction of the conformational freedom of neighboring bases. A surface-oligomer spacer is necessary for duplex stability with the destabilizing effect of the surface coinciding with the volume it excludes.

Automated Microfluidic Platform for Serial Polymerase Chain Reaction and High-Resolution Melting Analysis.

PubMed

Cao, Weidong; Bean, Brian; Corey, Scott; Coursey, Johnathan S; Hasson, Kenton C; Inoue, Hiroshi; Isano, Taisuke; Kanderian, Sami; Lane, Ben; Liang, Hongye; Murphy, Brian; Owen, Greg; Shinoda, Nobuhiko; Zeng, Shulin; Knight, Ivor T

2016-06-01

We report the development of an automated genetic analyzer for human sample testing based on microfluidic rapid polymerase chain reaction (PCR) with high-resolution melting analysis (HRMA). The integrated DNA microfluidic cartridge was used on a platform designed with a robotic pipettor system that works by sequentially picking up different test solutions from a 384-well plate, mixing them in the tips, and delivering mixed fluids to the DNA cartridge. A novel image feedback flow control system based on a Canon 5D Mark II digital camera was developed for controlling fluid movement through a complex microfluidic branching network without the use of valves. The same camera was used for measuring the high-resolution melt curve of DNA amplicons that were generated in the microfluidic chip. Owing to fast heating and cooling as well as sensitive temperature measurement in the microfluidic channels, the time frame for PCR and HRMA was dramatically reduced from hours to minutes. Preliminary testing results demonstrated that rapid serial PCR and HRMA are possible while still achieving high data quality that is suitable for human sample testing. © 2015 Society for Laboratory Automation and Screening.

Methylation-Sensitive High Resolution Melting (MS-HRM).

PubMed

Hussmann, Dianna; Hansen, Lise Lotte

2018-01-01

Methylation-Sensitive High Resolution Melting (MS-HRM) is an in-tube, PCR-based method to detect methylation levels at specific loci of interest. A unique primer design facilitates a high sensitivity of the assays enabling detection of down to 0.1-1% methylated alleles in an unmethylated background.Primers for MS-HRM assays are designed to be complementary to the methylated allele, and a specific annealing temperature enables these primers to anneal both to the methylated and the unmethylated alleles thereby increasing the sensitivity of the assays. Bisulfite treatment of the DNA prior to performing MS-HRM ensures a different base composition between methylated and unmethylated DNA, which is used to separate the resulting amplicons by high resolution melting.The high sensitivity of MS-HRM has proven useful for detecting cancer biomarkers in a noninvasive manner in urine from bladder cancer patients, in stool from colorectal cancer patients, and in buccal mucosa from breast cancer patients. MS-HRM is a fast method to diagnose imprinted diseases and to clinically validate results from whole-epigenome studies. The ability to detect few copies of methylated DNA makes MS-HRM a key player in the quest for establishing links between environmental exposure, epigenetic changes, and disease.

Capturing a DNA duplex under near-physiological conditions

NASA Astrophysics Data System (ADS)

Zhang, Huijuan; Xu, Wei; Liu, Xiaogang; Stellacci, Francesco; Thong, John T. L.

2010-10-01

We report in situ trapping of a thiolated DNA duplex with eight base pairs into a polymer-protected gold nanogap device under near-physiological conditions. The double-stranded DNA was captured by electrophoresis and covalently attached to the nanogap electrodes through sulfur-gold bonding interaction. The immobilization of the DNA duplex was confirmed by direct electrical measurements under near-physiological conditions. The conductance of the DNA duplex was estimated to be 0.09 μS. We also demonstrate the control of DNA dehybridization by heating the device to temperatures above the melting point of the DNA.

Petrogenesis of melt rocks, Manicouagan impact structure, Quebec

NASA Technical Reports Server (NTRS)

Simonds, C. H.; Floran, R. J.; Mcgee, P. E.; Phinney, W. C.; Warner, J. L.

1978-01-01

It is suggested, on the basis of previous theoretical studies of shock waves, that the Manicouagan melt formed in 1 or 2 s in a 5-km-radius hemisphere near the point of impact. The melt and the less shocked debris surrounding it flowed downward and outward for a few minutes until the melt formed a lining of a 5- to 8-km deep, 15- to 22-km-radius cavity. Extremely turbulent flow thoroughly homogenized the melt and promoted the incorporation and progressive digestion of debris that had been finely fragmented (but not melted) to grain sizes of less than one mm by the passage of the shock waves. The equilibration of clasts and melt, plagioclase nucleation, and readjustment of the crater floor are discussed.

Divergence, differential methylation and interspersion of melon satellite DNA sequences.

PubMed Central

Shmookler Reis, R; Timmis, J N; Ingle, J

1981-01-01

Melon (Cucumis melo) satellite DNA consists of two components, Q and S, each with a buoyant density in CsCl of 1.707 g/ml, but differing by 9 degrees C in "melting" temperature. These physical properties appear to be in contradiction, since both depend on G + C content. In order to resolve this anomaly, base compositions were directly determined for isolated fractions. the low-"melting" component S contains 41.8% G + C, with 6% of C present as 5-methylcytosine, whereas Q DNA contains 54% G + C, with 41% of C methylated. Analyses of restriction site loss agreed well with the direct determinations of methylation and divergence, and indicated some clustering of methylated sites in Q DNA. Analysis of restricted main-band DNA by hydridization with RNA complementary to Q satellite DNA ("Southern transfer") showed satellite Q tandem arrays interspersed in DNA of main-band density. Sequence divergence and extent of methylation did not appear to depend on whether a repeat array was present as satellite or interspersed in main-band DNA. Hydridization in situ indicated considerable heterogeneity in the genomic proportion of the Q-DNA sequences in melon fruit nuclei, implying over- and under-representation consistent with extensive unequal recombination in satellite Q tandem arrays. The cucumber, Cucumis sativus, contains less than 8% as much Q-homologous DNA per genome as the melon, suggesting rapid evolutionary gain or loss of these tandem repeat sequences. Images Fig. 2. PLATE 1 Fig. 4. Fig. 10. PMID:6172117

Deciphering the groove binding modes of tau-fluvalinate and flumethrin with calf thymus DNA

NASA Astrophysics Data System (ADS)

Tao, Mo; Zhang, Guowen; Pan, Junhui; Xiong, Chunhong

2016-02-01

Tau-fluvalinate (TFL) and flumethrin (FL), widely used in agriculture and a class of synthetic pyrethroid pesticides with a similar structure, may cause a potential security risk. Herein, the modes of binding in vitro of TFL and FL with calf thymus DNA (ctDNA) were characterized by fluorescence, UV-vis absorption, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy with the aid of viscosity measurements, melting analyses and molecular docking studies. The fluorescence titration indicated that both TFL and FL bound to ctDNA forming complexes through hydrogen bonding and van der Waals forces. The binding constants of TFL and FL with ctDNA were in the range of 104 L mol- 1, and FL exhibited a higher binding propensity than TFL. The iodide quenching effect, single/double-stranded DNA effects, and ctDNA melting and viscosity measurements demonstrated that the binding of both TFL and FL to ctDNA was groove mode. The FT-IR analyses suggested the A-T region of the minor groove of ctDNA as the preferential binding for TFL and FL, which was confirmed by the displacement assays with Hoechst 33258 probe, and the molecular docking visualized the specific binding. The changes in CD spectra indicated that both FL and TFL induced the perturbation on the base stacking and helicity of B-DNA, but the disturbance caused by FL was more obvious. Gel electrophoresis analyses indicated that both TFL and FL did not cause significant DNA cleavage. This study provides novel insights into the binding properties of TFL/FL with ctDNA and its toxic mechanisms.

Partitioning of Mg, Ca, and Na between carbonatite melt and hydrous fluid at 0.1-0.2 GPa

NASA Astrophysics Data System (ADS)

Veksler, Ilya V.; Keppler, Hans

Experimental studies of the element distribution between carbonatite melts and hydrous fluids are hampered by the fact that neither the fluid nor the melt can be isochemically quenched in conventional high-pressure vessels. In order to overcome this problem, we used a double-capsule technique to separate immiscible fluid and melt phases during and after the runs. The inner platinum capsules were charged with carbonate mixtures (CaCO3, MgCO3 and Na2CO3) and placed inside the outer capsules charged with distilled water and diamond powder. The latter was used as an inert trap for solids precipitating from the fluid on quenching. Carbonate melt and hydrous fluid equilibrated through a small hole left in the upper end of the inner capsule. The runs were performed in rapid-quench cold-seal pressure vessels at 0.1-0.2 GPa and 700-900°C in the two-phase (fluid+melt) stability region. Both quenched melt and quenched fluid were dissolved in dilute HCl and analysed by inductively coupled plasma atomic emission spectroscopy. The results show that under all conditions investigated, fluid/melt partition coefficients for Ca and Mg are similar and several times smaller than those for Na. At 0.1 GPa and a water/carbonatite ratio of 1 (by weight), the partition coefficients are DNa= 0.35+/- 0.02, DCa=0.09+/-0.02, and DMg=0.13+/- 0.01. Between 700 and 900°C, the effect of temperature on partitioning is negligible. However, DNa increases significantly with decreasing water/carbonatite ratio in the system. Our data show that the release of a hydrous fluid enriched in sodium and simultaneous crystallisation of calcite can transform an alkaline, vapour-saturated carbonatite melt into a body of pure calcite surrounded by zones of sodium metasomatism. Thus, it is quite possible that carbonate magmas with substantial amounts of alkalies were common parental liquids of plutonic carbonatites.

50 years of DNA ‘Breathing’: Reflections on Old and New Approaches

PubMed Central

von Hippel, Peter H.; Johnson, Neil P.; Marcus, Andrew H.

2015-01-01

Summary The coding sequences for genes, and much other regulatory information involved in genome expression, are located ‘inside’ the DNA duplex. Thus the ‘macromolecular machines’ that read-out this information from the base sequence of the DNA must somehow access the DNA ‘interior’. Double-stranded (ds) DNA is a highly structured and cooperatively stabilized system at physiological temperatures, but is also only marginally stable and undergoes a cooperative ‘melting phase transition’ at temperatures not far above physiological. Furthermore, due to its length and heterogeneous sequence, with AT-rich segments being less stable than GC-rich segments, the DNA genome ‘melts’ in a multistate fashion. Therefore the DNA genome must also manifest thermally driven structural (‘breathing’) fluctuations at physiological temperatures that should reflect the heterogeneity of the dsDNA stability near the melting temperature. Thus many of the breathing fluctuations of dsDNA are likely also to be sequence dependent, and could well contain information that should be ‘readable’ and useable by regulatory proteins and protein complexes in site-specific binding reactions involving dsDNA ‘opening’. Our laboratory has been involved in studying the breathing fluctuations of duplex DNA for about 50 years. In this ‘Reflections’ article we present a relatively chronological overview of these studies, starting with the use of simple chemical probes (such as hydrogen exchange, formaldehyde and simple DNA ‘melting’ proteins) to examine the local stability of the dsDNA structure, and culminating in sophisticated spectroscopic approaches that can be used to monitor the breathing-dependent interactions of regulatory complexes with their duplex DNA targets in ‘real time’. PMID:23840028

Enabling fiber optic serotyping of pathogenic bacteria through improved anti-fouling functional surfaces

NASA Astrophysics Data System (ADS)

Janssen, K. P. F.; Knez, K.; Vanysacker, L.; Schrooten, J.; Spasic, D.; Lammertyn, J.

2012-06-01

Significant research efforts are continually being directed towards the development of sensitive and accurate surface plasmon resonance biosensors for sequence specific DNA detection. These sensors hold great potential for applications in healthcare and diagnostics. However, the performance of these sensors in practical usage scenarios is often limited due to interference from the sample matrix. This work shows how the co-immobilization of glycol (PEG) diluents or ‘back filling’ of the DNA sensing layer can successfully address these problems. A novel SPR based melting assay is used for the analysis of a synthetic oligomer target as well as PCR amplified genomic DNA extracted from Legionella pneumophila. The benefits of sensing layer back filling on the assay performance are first demonstrated through melting analysis of the oligomer target and it is shown how back filling enables accurate discrimination of Legionella pneumophila serogroups directly from the PCR reaction product with complete suppression of sensor fouling.

A rule of seven in Watson-Crick base-pairing of mismatched sequences.

PubMed

Cisse, Ibrahim I; Kim, Hajin; Ha, Taekjip

2012-05-13

Sequence recognition through base-pairing is essential for DNA repair and gene regulation, but the basic rules governing this process remain elusive. In particular, the kinetics of annealing between two imperfectly matched strands is not well characterized, despite its potential importance in nucleic acid-based biotechnologies and gene silencing. Here we use single-molecule fluorescence to visualize the multiple annealing and melting reactions of two untethered strands inside a porous vesicle, allowing us to precisely quantify the annealing and melting rates. The data as a function of mismatch position suggest that seven contiguous base pairs are needed for rapid annealing of DNA and RNA. This phenomenological rule of seven may underlie the requirement for seven nucleotides of complementarity to seed gene silencing by small noncoding RNA and may help guide performance improvement in DNA- and RNA-based bio- and nanotechnologies, in which off-target effects can be detrimental.

Molecular spectroscopic studies on the interaction of ferulic acid with calf thymus DNA.

PubMed

Zhang, Shufang; Sun, Xuejun; Qu, Fengli; Kong, Rongmei

2013-08-01

The interaction between ferulic acid and calf thymus deoxyribonucleic acid (ctDNA) under physiological conditions (Tris-HCl buffer solutions, pH 7.4) was investigated by UV-Vis spectroscopy, fluorescence spectroscopy, DNA melting techniques, and viscosity measurements. Results indicated that a complex of ferulic acid with ctDNA was formed with a binding constant of K(290K)=7.60×10(4) L mol(-1) and K(310K)=4.90×10(4) L mol(-1). The thermodynamic parameters enthalpy change (ΔH°), entropy change (ΔS°) and Gibbs free energy (ΔG°) were calculated to be -1.69×10(4) J mol(-1), 35.36 J K(-1) mol(-1) and -2.79×10(4) J mol(-1) at 310 K, respectively. The acting forces between ferulic acid and DNA mainly included hydrophobic interaction and hydrogen bonds. Acridine orange displacement studies revealed that ferulic acid can substitute for AO probe in the AO-DNA complex which was indicative of intercalation binding. Thermal denaturation study suggested that the interaction of ferulic acid with DNA could result in the increase of the denaturation temperature, which indicated that the stabilization of the DNA helix was increased in the presence of ferulic acid. Spectroscopic techniques together with melting techniques and viscosity determination provided evidences of intercalation mode of binding for the interaction between ferulic acid and ctDNA. Copyright © 2013 Elsevier B.V. All rights reserved.

Sperm DNA damage or progressive motility: which one is the better predictor of fertilization in vitro?

PubMed

Simon, Luke; Lewis, Sheena E M

2011-06-01

Sperm progressive motility has been reported to be one of the key factors influencing in vitro fertilization rates. However, recent studies have shown that sperm DNA fragmentation is a more robust predictor of assisted reproductive outcomes including reduced fertilization rates, embryo quality, and pregnancy rates. This study aimed to compare the usefulness of sperm progressive motility and DNA damage as predictive tools of in vitro fertilization rates. Here, 136 couples provided 1,767 eggs with an overall fertilization rate of 64.2%. The fertilization rate in vitro correlated with both sperm progressive motility (r² = 0.236; P = 0.002) and DNA fragmentation (r² = -0.318; P < 0.001). The relative risk of a poor fertilization rate was 9.5 times higher in sperm of men with high DNA fragmentation (>40%) compared with 2.6 times in sperm with poor motility (<40%). Further, sperm DNA fragmentation gave a higher specificity (93.3%) in predicting the fertilization rate than progressive motility (77.8%). Finally, the odds ratio to determine fertilization rate (>70%) was 4.81 (1.89-12.65) using progressive motility compared with 24.18 (5.21-154.51) using DNA fragmentation. This study shows that fertilization rates are directly dependent upon both sperm progressive motility and DNA fragmentation, but sperm DNA fragmentation is a much stronger test.

Physicochemical and histological changes in the arterial wall of nonhuman primates during progression and regression of atherosclerosis.

PubMed Central

Small, D M; Bond, M G; Waugh, D; Prack, M; Sawyer, J K

1984-01-01

To identify the temporal changes occurring during progression and regression of atherosclerosis in nonhuman primates, we have studied the physicochemical and histological characteristics of arterial wall lesions during a 30-mo progression period of diet-induced hypercholesterolemia and during a 12-mo period of regression. Three groups of cynomolgous monkeys (Macaca fascicularis) were studied. Control groups were fed a basal chow diet for 18, 24, and 30 mo and were compared with progression groups that were fed a high-cholesterol-containing diet for up to 30 mo. Regression groups were fed a high-cholesterol diet for 18 mo to induce atherosclerosis and then fed monkey chow for up to 12 mo. The progression group monkeys were killed at 6, 12, 18, 24, and 30 mo, and the regression animals were killed at 24 and 30 mo (i.e., after 6 and 12 mo of being fed a noncholesterol-containing chow diet). Histology and morphometry, physical microscopy for cholesterol monohydrate crystals, foam cell and droplet melting points and chemical composition studies were completed on a large number of individual arterial lesions. Control animals had very little cholesterol ester, rare foam cells, and no extracellular cholesterol ester droplets or cholesterol crystals. During progression, the arteries first increased cholesterol ester content to produce high melting (approximately 45 degrees C) foam cell-rich lesions essentially devoid of cholesterol crystals. With time, the number of cholesterol crystals increased so that by 30 mo large numbers were present. Foam cells decreased with time but their melting temperature remained high while that of extracellular droplets fell to approximately 38 degrees C. Between 18 and 30 mo necrosis appeared and worsened. After 6-mo regression, unexpected changes occurred in the lesions. Compared with 24-mo progression, the chemical composition showed a relative increase in free cholesterol, a decrease in cholesterol ester and microscopy revealed large numbers of cholesterol crystals. Concomitantly, foam cells decreased and the melting temperature of both intra- and extracellular cholesterol ester markedly decreased. After 12-mo regression cholesterol decreased, cholesterol crystals and necrosis diminished and collagen appeared increased. Thus, during progression there is initially an increase in the number of foam cells containing very high-melting intracellular cholesterol ester droplets. By 30 mo, cholesterol crystals and necrosis dominate and high-melting foam cells appear only at lesion margins, suggesting that the initial process continues at the lesion edge. The lower melting point of extracellular esters indicates a lipid composition different from intracellular droplets. Thus, the changes observed in these animals generally reflect those predicted for progression of human atherosclerosis. During the initial 6 mo of regression, necrosis remains, the number of foam cell decreases, and cholesterol ester content decreases; however the relative proportion of free cholesterol content increases, and large numbers of cholesterol content are formed. Thus, large and rapid decreases in serum cholesterol concentration to produce regression in fact may result in the precipitation of cholesterol monohydrate and an apparent worsening of the lesions. More prolonged regression (12-mo) tends to return the lipid composition of the artery wall towards normal, partially reduces cholesterol crystals, and results in an improved but scarred intima. Images PMID:6725553

Next-Generation Sequencing-Based Detection of Circulating Tumour DNA After Allogeneic Stem Cell Transplantation for Lymphoma

PubMed Central

Herrera, Alex F.; Kim, Haesook T.; Kong, Katherine A.; Faham, Malek; Sun, Heather; Sohani, Aliyah R.; Alyea, Edwin P.; Carlton, Victoria E.; Chen, Yi-Bin; Cutler, Corey S.; Ho, Vincent T.; Koreth, John; Kotwaliwale, Chitra; Nikiforow, Sarah; Ritz, Jerome; Rodig, Scott J.; Soiffer, Robert J.; Antin, Joseph H.; Armand, Philippe

2016-01-01

Summary Next-generation sequencing (NGS)-based circulating tumour DNA (ctDNA) detection is a promising monitoring tool for lymphoid malignancies. We evaluated whether the presence of ctDNA was associated with outcome after allogeneic haematopoietic stem cell transplantation (HSCT) in lymphoma patients. We studied 88 patients drawn from a phase 3 clinical trial of reduced-intensity conditioning HSCT in lymphoma. Conventional restaging and collection of peripheral blood samples occurred at pre-specified time points before and after HSCT and were assayed for ctDNA by sequencing of the immunoglobulin or T-cell receptor genes. Tumour clonotypes were identified in 87% of patients with adequate tumour samples. Sixteen of 19 (84%) patients with disease progression after HSCT had detectable ctDNA prior to progression at a median of 3.7 months prior to relapse/progression. Patients with detectable ctDNA 3 months after HSCT had inferior progression-free survival (PFS) (2-year PFS 58% versus 84% in ctDNA-negative patients, p=0.033). In multivariate models, detectable ctDNA was associated with increased risk of progression/death (Hazard ratio 3.9, p=0.003) and increased risk of relapse/progression (Hazard ratio 10.8, p=0.0006). Detectable ctDNA is associated with an increased risk of relapse/progression, but further validation studies are necessary to confirm these findings and determine the clinical utility of NGS-based minimal residual disease monitoring in lymphoma patients after HSCT. PMID:27711974

Normalized cDNA libraries

DOEpatents

Soares, Marcelo B.; Efstratiadis, Argiris

1997-01-01

This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library.

Normalized cDNA libraries

DOEpatents

Soares, M.B.; Efstratiadis, A.

1997-06-10

This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3{prime} noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. 4 figs.

Detection of Orthopoxvirus DNA by Real-Time PCR and Identification of Variola Virus DNA by Melting Analysis

PubMed Central

Nitsche, Andreas; Ellerbrok, Heinz; Pauli, Georg

2004-01-01

Although variola virus was eradicated by the World Health Organization vaccination program in the 1970s, the diagnosis of smallpox infection has attracted great interest in the context of a possible deliberate release of variola virus in bioterrorist attacks. Obviously, fast and reliable diagnostic tools are required to detect variola virus and to distinguish it from orthopoxviruses that have identical morphological characteristics, including vaccinia virus. The advent of real-time PCR for the clinical diagnosis of viral infections has facilitated the detection of minute amounts of viral nucleic acids in a fast, safe, and precise manner, including the option to quantify and to genotype the target reliably. In this study a complete set of four hybridization probe-based real-time PCR assays for the specific detection of orthopoxvirus DNA is presented. Melting analysis following PCR enables the identification of variola virus by the PCR product's characteristic melting temperature, permitting the discrimination of variola virus from other orthopoxviruses. In addition, an assay for the specific amplification of variola virus DNA is presented. All assays can be performed simultaneously in the same cycler, and results of a PCR run are obtained in less than 1 h. The application of more than one assay for the same organism significantly contributes to the diagnostic reliability, reducing the risk of false-negative results due to unknown sequence variations. In conclusion, the assays presented will improve the speed and reliability of orthopoxvirus diagnostics and variola virus identification. PMID:15004077

Self-Assembly of DNA-Coated Particles: Experiment, Simulation and Theory

NASA Astrophysics Data System (ADS)

Song, Minseok

The bottom-up assembly of material architectures with tunable complexity, function, composition, and structure is a long sought goal in rational materials design. One promising approach aims to harnesses the programmability and specificity of DNA hybridization in order to direct the assembly of oligonucleotide-functionalized nano- and micro-particles by tailoring, in part, interparticle interactions. DNA-programmable assembly into three-dimensionally ordered structures has attracted extensive research interest owing to emergent applications in photonics, plasmonics and catalysis and potentially many other areas. Progress on the rational design of DNA-mediated interactions to create useful two-dimensional structures (e.g., structured films), on the other hand, has been rather slow. In this thesis, we establish strategies to engineer a diversity of 2D crystalline arrangements by designing and exploiting DNA-programmable interparticle interactions. We employ a combination of simulation, theory and experiments to predict and confirm accessibility of 2D structural diversity in an effort to establish a rational approach to 2D DNA-mediated particle assembly. We start with the experimental realization of 2D DNA-mediated assembly by decorating micron-sized silica particles with covalently attached single-stranded DNA through a two-step reaction. Subsequently, we elucidate sensitivity and ultimate controllability of DNA-mediated assembly---specifically the melting transition from dispersed singlet particles to aggregated or assembled structures---through control of the concentration of commonly employed nonionic surfactants. We relate the observed tunability to an apparent coupling with the critical micelle temperature in these systems. Also, both square and hexagonal 2D ordered particle arrangements are shown to evolve from disordered aggregates under appropriate annealing conditions defined based upon pre-established melting profiles. Subsequently, the controlled mixing of complementary ssDNA functionality on individual particles ('multi-flavoring') as opposed to functionalization of particles with the same type of ssDNA ('uni-flavoring') is explored as a possible design handle for tuning interparticle interactions and, thereby, accessing diverse structures. We employ a combination of simulations, theory, and experimental validation toward establishing 'multi-flavoring' as a rational design strategy. Firstly, MD simulations are carried out using effective pair potentials to describe interparticle interactions that are representative of different degrees of ssDNA 'multi-flavoring'. These simulations reveal the template-free assembly of a diversity of 2D crystal polymorphs that is apparently tunable by controlling the relative attractive strengths between like and unlike functionalized particles. The resulting phase diagrams predict conditions (i.e., strengths of relative interparticle interactions) for obtaining crystalline phases with lattice symmetries ranging among square, alternating string hexagonal, random hexagonal, rhombic, honeycomb, and even kagome. Finally, these model findings are translated to experiments, in which binary microparticles are decorated with a tailored mixture of two different complementary ssDNA strands as a straight-forward means to realize tunable particle interactions. Guided by simple statistical mechanics and the detailed MD simulations, 'multi-flavoring' and control of solution phase particle stoichiometry resulted in experimental realization of structurally diverse 2D microparticle assemblies consistent with predictions, such as square, pentagonal and hexagonal lattices (honeycomb, kagome). The combined simulation, theory, and experimental findings reveal how control of interparticle interactions via DNA-functionalized particle "multi-flavoring" can lead to an even wider range of accessible colloidal crystal structures. The 2D experiments coupled with the model predictions may be used to provide new fundamental insight into nano- or microparticle assembly in three dimensions.

A correlated Walks' theory for DNA denaturation

NASA Astrophysics Data System (ADS)

Mejdani, R.

1994-08-01

We have shown that by using a correlated Walks' theory for the lattice gas model on a one-dimensional lattice, we can study, beside the saturation curves obtained before for the enzyme kinetics, also the DNA denaturation process. In the limit of no interactions between sites the equation for melting curves of DNA reduces to the random model equation. Thus our leads naturally to this classical equation in the limiting case.

Isoquinoline alkaloids and their binding with DNA: calorimetry and thermal analysis applications.

PubMed

Bhadra, Kakali; Kumar, Gopinatha Suresh

2010-11-01

Alkaloids are a group of natural products with unmatched chemical diversity and biological relevance forming potential quality pools in drug screening. The molecular aspects of their interaction with many cellular macromolecules like DNA, RNA and proteins are being currently investigated in order to evolve the structure activity relationship. Isoquinolines constitute an important group of alkaloids. They have extensive utility in cancer therapy and a large volume of data is now emerging in the literature on their mode, mechanism and specificity of binding to DNA. Thermodynamic characterization of the binding of these alkaloids to DNA may offer key insights into the molecular aspects that drive complex formation and these data can provide valuable information about the balance of driving forces. Various thermal techniques have been conveniently used for this purpose and modern calorimetric instrumentation provides direct and quick estimation of thermodynamic parameters. Thermal melting studies and calorimetric techniques like isothermal titration calorimetry and differential scanning calorimetry have further advanced the field by providing authentic, reliable and sensitive data on various aspects of temperature dependent structural analysis of the interaction. In this review we present the application of various thermal techniques, viz. isothermal titration calorimetry, differential scanning calorimetry and optical melting studies in the characterization of drug-DNA interactions with particular emphasis on isoquinoline alkaloid-DNA interaction.

High resolution melting analysis for epidermal growth factor receptor mutations in formalin-fixed paraffin-embedded tissue and plasma free DNA from non-small cell lung cancer patients.

PubMed

Jing, Chang-Wen; Wang, Zhuo; Cao, Hai-Xia; Ma, Rong; Wu, Jian-Zhong

2014-01-01

The aim of the research was to explore a cost effective, fast, easy to perform, and sensitive method for epidermal growth factor receptor (EGFR) mutation testing. High resolution melting analysis (HRM) was introduced to evaluate the efficacy of the analysis for dectecting EGFR mutations in exons 18 to 21 using formalin-fixed paraffin-embedded (FFPE) tissues and plasma free DNA from 120 patients. The total EGFR mutation rate was 37.5% (45/120) detected by direct sequencing. There were 48 mutations in 120 FFPE tissues assessed by HRM. For plasma free DNA, the EGFR mutation rate was 25.8% (31/120). The sensitivity of HRM assays in FFPE samples was 100% by HRM. There was a low false-positive mutation rate but a high false-negative rate in plasma free DNA detected by HRM. Our results show that HRM analysis has the advantage of small tumor sample need. HRM applied with plasma free DNA showed a high false-negative rate but a low false-positive rate. Further research into appropriate methods and analysis needs to be performed before HRM for plasma free DNA could be accepted as an option in diagnostic or screening settings.

DNA Barcoding Coupled with High Resolution Melting Analysis Enables Rapid and Accurate Distinction of Aspergillus species.

PubMed

Fidler, Gabor; Kocsube, Sandor; Leiter, Eva; Biro, Sandor; Paholcsek, Melinda

2017-08-01

We describe a high-resolution melting (HRM) analysis method that is rapid, reproducible, and able to identify reference strains and further 40 clinical isolates of Aspergillus fumigatus (14), A. lentulus (3), A. terreus (7), A. flavus (8), A. niger (2), A. welwitschiae (4), and A. tubingensis (2). Asp1 and Asp2 primer sets were designed to amplify partial sequences of the Aspergillus benA (beta-tubulin) genes in a closed-, single-tube system. Human placenta DNA, further Aspergillus (3), Candida (9), Fusarium (6), and Scedosporium (2) nucleic acids from type strains and clinical isolates were also included in this study to evaluate cross reactivity with other relevant pathogens causing invasive fungal infections. The barcoding capacity of this method proved to be 100% providing distinctive binomial scores; 14, 34, 36, 35, 25, 15, 26 when tested among species, while the within-species distinction capacity of the assay proved to be 0% based on the aligned thermodynamic profiles of the Asp1, Asp2 melting clusters allowing accurate species delimitation of all tested clinical isolates. The identification limit of this HRM assay was also estimated on Aspergillus reference gDNA panels where it proved to be 10-102 genomic equivalents (GE) except the A. fumigatus panel where it was 103 only. Furthermore, misidentification was not detected with human genomic DNA or with Candida, Fusarium, and Scedosporium strains. Our DNA barcoding assay introduced here provides results within a few hours, and it may possess further diagnostic utility when analyzing standard cultures supporting adequate therapeutic decisions. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The "COLD-PCR approach" for early and cost-effective detection of tyrosine kinase inhibitor resistance mutations in EGFR-positive non-small cell lung cancer.

PubMed

Mairinger, Fabian D; Vollbrecht, Claudia; Streubel, Anna; Roth, Andreas; Landt, Olfert; Walter, Henry F R; Kollmeier, Jens; Mairinger, Thomas

2014-01-01

Activating epidermal growth factor receptor (EGFR) gene mutations can be successfully treated by EGFR tyrosine kinase inhibitors (EGFR-TKIs), but nearly 50% of all patients' exhibit progression of the disease until treatment because of T790M mutations. It is proposed that this is mostly caused by therapy-resistant tumor clones harboring a T790M mutation. Until now no cost-effective routine-diagnostic method for EGFR-resistance mutation status analysis is available leaving long-time response to TKI treatment to chance. Unambiguous identification of T790M EGFR mutations is mandatory to optimize initial treatment strategies. Artificial EGFR T790M mutations and human wild-type gDNA were prepared in several dilution series. Preferential amplification using coamplification at lower denaturation temperature-PCR (COLD-PCR) of the mutant sequence and subsequent HybProbe melting curve detection or pyrosequencing were performed in comparison to normal processing. COLD-PCR-based amplification allowed the detection of 0.125% T790M mutant DNA in a background of wild-type DNA in comparison to 5% while normal processing. These results were reproducible. COLD-PCR is a powerful and cost-effective tool for routine diagnostic to detect underrepresented tumor clones in clinical samples. A diagnostic tool for unambiguous identification of T790M-mutated minor tumor clones is now available enabling optimized therapy.

Multiplexed Elimination of Wild-Type DNA and High-Resolution Melting Prior to Targeted Resequencing of Liquid Biopsies.

PubMed

Ladas, Ioannis; Fitarelli-Kiehl, Mariana; Song, Chen; Adalsteinsson, Viktor A; Parsons, Heather A; Lin, Nancy U; Wagle, Nikhil; Makrigiorgos, G Mike

2017-10-01

The use of clinical samples and circulating cell-free DNA (cfDNA) collected from liquid biopsies for diagnostic and prognostic applications in cancer is burgeoning, and improved methods that reduce the influence of excess wild-type (WT) portion of the sample are desirable. Here we present enrichment of mutation-containing sequences using enzymatic degradation of WT DNA. Mutation enrichment is combined with high-resolution melting (HRM) performed in multiplexed closed-tube reactions as a rapid, cost-effective screening tool before targeted resequencing. We developed a homogeneous, closed-tube approach to use a double-stranded DNA-specific nuclease for degradation of WT DNA at multiple targets simultaneously. The No Denaturation Nuclease-assisted Minor Allele Enrichment with Probe Overlap (ND-NaME-PrO) uses WT oligonucleotides overlapping both strands on putative DNA targets. Under conditions of partial denaturation (DNA breathing), the oligonucleotide probes enhance double-stranded DNA-specific nuclease digestion at the selected targets, with high preference toward WT over mutant DNA. To validate ND-NaME-PrO, we used multiplexed HRM, digital PCR, and MiSeq targeted resequencing of mutated genomic DNA and cfDNA. Serial dilution of KRAS mutation-containing DNA shows mutation enrichment by 10- to 120-fold and detection of allelic fractions down to 0.01%. Multiplexed ND-NaME-PrO combined with multiplexed PCR-HRM showed mutation scanning of 10-20 DNA amplicons simultaneously. ND-NaME-PrO applied on cfDNA from clinical samples enables mutation enrichment and HRM scanning over 10 DNA targets. cfDNA mutations were enriched up to approximately 100-fold (average approximately 25-fold) and identified via targeted resequencing. Closed-tube homogeneous ND-NaME-PrO combined with multiplexed HRM is a convenient approach to efficiently enrich for mutations on multiple DNA targets and to enable prescreening before targeted resequencing. © 2017 American Association for Clinical Chemistry.

High resolution melt curve analysis based on methylation status for human semen identification.

PubMed

Fachet, Caitlyn; Quarino, Lawrence; Karnas, K Joy

2017-03-01

A high resolution melt curve assay to differentiate semen from blood, saliva, urine, and vaginal fluid based on methylation status at the Dapper Isoform 1 (DACT1) gene was developed. Stains made from blood, saliva, urine, semen, and vaginal fluid were obtained from volunteers and DNA was isolated using either organic extraction (saliva, urine, and vaginal fluid) or Chelex ® 100 extraction (blood and semen). Extracts were then subjected to bisulfite modification in order to convert unmethylated cytosines to uracil, consequently creating sequences whose amplicons have melt curves that vary depending on their initial methylation status. When primers designed to amplify the promoter region of the DACT1 gene were used, DNA from semen samples was distinguishable from other fluids by a having a statistically significant lower melting temperature. The assay was found to be sperm-significant since semen from a vasectomized man produced a melting temperature similar to the non-semen body fluids. Blood and semen stains stored up to 5 months and tested at various intervals showed little variation in melt temperature indicating the methylation status was stable during the course of the study. The assay is a more viable method for forensic science practice than most molecular-based methods for body fluid stain identification since it is time efficient and utilizes instrumentation common to forensic biology laboratories. In addition, the assay is advantageous over traditional presumptive chemical methods for body fluid identification since results are confirmatory and the assay offers the possibility of multiplexing which may test for multiple body fluids simultaneously.

High Resolution Melting Analysis is Very Useful to Identify BRCA1 c.4964_4982del19 (rs80359876) Founder Calabrian Pathogenic Variant on Peripheral Blood and Buccal Swab DNA.

PubMed

Minucci, Angelo; De Bonis, Maria; De Paolis, Elisa; Gentile, Leonarda; Santonocito, Concetta; Concolino, Paola; Mignone, Flavio; Capoluongo, Ettore

2017-04-01

Detection of pathogenic variants in hereditary breast and ovarian cancer-related breast cancer type 1 and type 2 susceptibility proteins (BRCA1/2) genes is an effective strategy in cancer prevention and treatment. Some ethnic and geographical regions show different BRCA1/2 mutation spectrum and prevalence. In Italy, elucidation of founder effect in BRCA1/2 genes can have an impact on the management of hereditary cancer families on a healthcare system level, making genetic testing more affordable and cost effective in certain regions. The purpose of this paper is to develop a rapid, low-cost, high-throughput single-tube technology for genotyping the Italian founder mutation c.4964_4982del19 (rs80359876) in the BRCA1 gene, starting from peripheral blood and/or buccal swab DNA. Heterozygote samples for c.4964_4982del19 variant were easily and unambiguously identified by the altered shape of the melting curves and were clearly distinguished by a change in melting temperature that differed by approximately 5 °C. The same results were obtained both with DNA from peripheral blood than buccal swab. We provide evidence about application of high-resolution melting analysis (HRMA) in unambiguously genotyping of the founder BRCA1 c.4964_4982del19 variant (rs80359876) in individuals from the Calabria region of Italy. In fact, HRMA was confirmed to be particularly suitable for the identification of BRCA1 c.4964_4982del19 variant, making this approach useful in clinical molecular diagnostics.

Process modelling for space station experiments

NASA Technical Reports Server (NTRS)

Rosenberger, Franz; Alexander, J. Iwan D.

1988-01-01

The work performed during the first year 1 Oct. 1987 to 30 Sept. 1988 involved analyses of crystal growth from the melt and from solution. The particular melt growth technique under investigation is directional solidification by the Bridgman-Stockbarger method. Two types of solution growth systems are also being studied. One involves growth from solution in a closed container, the other concerns growth of protein crystals by the hanging drop method. Following discussions with Dr. R. J. Naumann of the Low Gravity Science Division at MSFC it was decided to tackle the analysis of crystal growth from the melt earlier than originally proposed. Rapid progress was made in this area. Work is on schedule and full calculations were underway for some time. Progress was also made in the formulation of the two solution growth models.

Non-Homologous End Joining and Homology Directed DNA Repair Frequency of Double-Stranded Breaks Introduced by Genome Editing Reagents.

PubMed

Zaboikin, Michail; Zaboikina, Tatiana; Freter, Carl; Srinivasakumar, Narasimhachar

2017-01-01

Genome editing using transcription-activator like effector nucleases or RNA guided nucleases allows one to precisely engineer desired changes within a given target sequence. The genome editing reagents introduce double stranded breaks (DSBs) at the target site which can then undergo DNA repair by non-homologous end joining (NHEJ) or homology directed recombination (HDR) when a template DNA molecule is available. NHEJ repair results in indel mutations at the target site. As PCR amplified products from mutant target regions are likely to exhibit different melting profiles than PCR products amplified from wild type target region, we designed a high resolution melting analysis (HRMA) for rapid identification of efficient genome editing reagents. We also designed TaqMan assays using probes situated across the cut site to discriminate wild type from mutant sequences present after genome editing. The experiments revealed that the sensitivity of the assays to detect NHEJ-mediated DNA repair could be enhanced by selection of transfected cells to reduce the contribution of unmodified genomic DNA from untransfected cells to the DNA melting profile. The presence of donor template DNA lacking the target sequence at the time of genome editing further enhanced the sensitivity of the assays for detection of mutant DNA molecules by excluding the wild-type sequences modified by HDR. A second TaqMan probe that bound to an adjacent site, outside of the primary target cut site, was used to directly determine the contribution of HDR to DNA repair in the presence of the donor template sequence. The TaqMan qPCR assay, designed to measure the contribution of NHEJ and HDR in DNA repair, corroborated the results from HRMA. The data indicated that genome editing reagents can produce DSBs at high efficiency in HEK293T cells but a significant proportion of these are likely masked by reversion to wild type as a result of HDR. Supplying a donor plasmid to provide a template for HDR (that eliminates a PCR amplifiable target) revealed these cryptic DSBs and facilitated the determination of the true efficacy of genome editing reagents. The results indicated that in HEK293T cells, approximately 40% of the DSBs introduced by genome editing, were available for participation in HDR.

DNA Replication Profiling Using Deep Sequencing.

PubMed

Saayman, Xanita; Ramos-Pérez, Cristina; Brown, Grant W

2018-01-01

Profiling of DNA replication during progression through S phase allows a quantitative snap-shot of replication origin usage and DNA replication fork progression. We present a method for using deep sequencing data to profile DNA replication in S. cerevisiae.

Next-generation sequencing-based detection of circulating tumour DNA After allogeneic stem cell transplantation for lymphoma.

PubMed

Herrera, Alex F; Kim, Haesook T; Kong, Katherine A; Faham, Malek; Sun, Heather; Sohani, Aliyah R; Alyea, Edwin P; Carlton, Victoria E; Chen, Yi-Bin; Cutler, Corey S; Ho, Vincent T; Koreth, John; Kotwaliwale, Chitra; Nikiforow, Sarah; Ritz, Jerome; Rodig, Scott J; Soiffer, Robert J; Antin, Joseph H; Armand, Philippe

2016-12-01

Next-generation sequencing (NGS)-based circulating tumour DNA (ctDNA) detection is a promising monitoring tool for lymphoid malignancies. We evaluated whether the presence of ctDNA was associated with outcome after allogeneic haematopoietic stem cell transplantation (HSCT) in lymphoma patients. We studied 88 patients drawn from a phase 3 clinical trial of reduced-intensity conditioning HSCT in lymphoma. Conventional restaging and collection of peripheral blood samples occurred at pre-specified time points before and after HSCT and were assayed for ctDNA by sequencing of the immunoglobulin or T-cell receptor genes. Tumour clonotypes were identified in 87% of patients with adequate tumour samples. Sixteen of 19 (84%) patients with disease progression after HSCT had detectable ctDNA prior to progression at a median of 3·7 months prior to relapse/progression. Patients with detectable ctDNA 3 months after HSCT had inferior progression-free survival (PFS) (2-year PFS 58% vs. 84% in ctDNA-negative patients, P = 0·033). In multivariate models, detectable ctDNA was associated with increased risk of progression/death (Hazard ratio 3·9, P = 0·003) and increased risk of relapse/progression (Hazard ratio 10·8, P = 0·0006). Detectable ctDNA is associated with an increased risk of relapse/progression, but further validation studies are necessary to confirm these findings and determine the clinical utility of NGS-based minimal residual disease monitoring in lymphoma patients after HSCT. © 2016 John Wiley & Sons Ltd.

Detection and Identification of Psilocybe cubensis DNA Using a Real-Time Polymerase Chain Reaction High Resolution Melt Assay.

PubMed

Cowan, Ashley F; Elkins, Kelly M

2017-12-01

Psilocybe cubensis, or "magic mushroom," is the most common species of fungus with psychedelic characteristics. Two primer sets were designed to target Psilocybe DNA using web-based software and NBCI gene sequences. DNA was extracted from eighteen samples, including twelve mushroom species, using the Qiagen DNeasy ® Plant Mini Kit. The DNA was amplified by the polymerase chain reaction (PCR) using the primers and a master mix containing either a SYBR ® Green I, Radiant™ Green, or LCGreen Plus ® intercalating dye; amplicon size was determined using agarose gel electrophoresis. The PCR assays were tested for amplifiability, specificity, reproducibility, robustness, sensitivity, and multiplexing with primers that target marijuana. The observed high resolution melt (HRM) temperatures for primer sets 1 and 7 were 78.85 ± 0.31°C and 73.22 ± 0.61°C, respectively, using SYBR ® Green I dye and 81.67 ± 0.06°C and 76.04 ± 0.11°C, respectively, using Radiant™ Green dye. © 2017 American Academy of Forensic Sciences.

The Potential Power of Bar-HRM Technology in Herbal Medicine Identification

PubMed Central

Sun, Wei; Li, Jing-jian; Xiong, Chao; Zhao, Bo; Chen, Shi-lin

2016-01-01

The substitution of low-cost or adulterated herbal products for high-priced herbs makes it important to be able to identify and trace herbal plant species and their processed products in the drug supply chain. PCR-based methods play an increasing role in monitoring the safety of herbal medicines by detecting adulteration. Recent studies have shown the potential of DNA barcoding combined with high resolution melting (Bar-HRM) analysis in herbal medicine identification. This method involves precisely monitoring the change in fluorescence caused by the release of an intercalating DNA dye from a DNA duplex as it is denatured by a gradual increase in temperature. Since the melting profile depends on the GC content, length, and strand complementarity of the amplification product, Bar-HRM analysis opens up the possibility of detecting single-base variants or species-specific differences in a short region of DNA. This review summarizes key factors affecting Bar-HRM analysis and describes how Bar-HRM is performed. We then discuss advances in Bar-HRM analysis of medicinal plant ingredients (herbal materia medica) as a contribution toward safe and effective herbal medicines. PMID:27066026

The Potential Power of Bar-HRM Technology in Herbal Medicine Identification.

PubMed

Sun, Wei; Li, Jing-Jian; Xiong, Chao; Zhao, Bo; Chen, Shi-Lin

2016-01-01

The substitution of low-cost or adulterated herbal products for high-priced herbs makes it important to be able to identify and trace herbal plant species and their processed products in the drug supply chain. PCR-based methods play an increasing role in monitoring the safety of herbal medicines by detecting adulteration. Recent studies have shown the potential of DNA barcoding combined with high resolution melting (Bar-HRM) analysis in herbal medicine identification. This method involves precisely monitoring the change in fluorescence caused by the release of an intercalating DNA dye from a DNA duplex as it is denatured by a gradual increase in temperature. Since the melting profile depends on the GC content, length, and strand complementarity of the amplification product, Bar-HRM analysis opens up the possibility of detecting single-base variants or species-specific differences in a short region of DNA. This review summarizes key factors affecting Bar-HRM analysis and describes how Bar-HRM is performed. We then discuss advances in Bar-HRM analysis of medicinal plant ingredients (herbal materia medica) as a contribution toward safe and effective herbal medicines.

Porosity and environment

NASA Technical Reports Server (NTRS)

Piwonka, T. S.

1984-01-01

Significant progress was achieved when it was realized that porosity could be analyzed successfully by considering not only heat flow, but also fluid flow within the solidifying casting. Sound castings may be produced by lowering pressure during melting (to allow dissolved gas to escape the melt) and increasing pressure during solidification (to force liquid metal into the mushy zone to feed shrinkage). Such techniques are especially effective if they are combined with chilling of parts of the casting to produce progressive solidification, which shortens the mushy zone and, hence, the distance that metal must travel to feed porosity.

High-resolution melting analysis of the single nucleotide polymorphism hot-spot region in the rpoB gene as an indicator of reduced susceptibility to rifaximin in Clostridium difficile.

PubMed

Pecavar, Verena; Blaschitz, Marion; Hufnagl, Peter; Zeinzinger, Josef; Fiedler, Anita; Allerberger, Franz; Maass, Matthias; Indra, Alexander

2012-06-01

Clostridium difficile, a Gram-positive, spore-forming, anaerobic bacterium, is the main causative agent of hospital-acquired diarrhoea worldwide. In addition to metronidazole and vancomycin, rifaximin, a rifamycin derivative, is a promising antibiotic for the treatment of recurring C. difficile infections (CDI). However, exposure of C. difficile to this antibiotic has led to the development of rifaximin-resistance due to point mutations in the β-subunit of the RNA polymerase (rpoB) gene. In the present study, 348 C. difficile strains with known PCR-ribotypes were investigated for respective single nucleotide polymorphisms (SNPs) within the proposed rpoB hot-spot region by using high-resolution melting (HRM) analysis. This method allows the detection of SNPs by comparing the altered melting behaviour of dsDNA with that of wild-type DNA. Discrimination between wild-type and mutant strains was enhanced by creating heteroduplexes by mixing sample DNA with wild-type DNA, leading to characteristic melting curve shapes from samples containing SNPs in the respective rpoB section. In the present study, we were able to identify 16 different rpoB sequence-types (ST) by sequencing analysis of a 325 bp fragment. The 16 PCR STs displayed a total of 24 different SNPs. Fifteen of these 24 SNPs were located within the proposed 151 bp SNP hot-spot region, resulting in 11 different HRM curve profiles (CP). Eleven SNPs (seven of which were within the proposed hot-spot region) led to amino acid substitutions associated with reduced susceptibility to rifaximin and 13 SNPs (eight of which were within the hot-spot region) were synonymous. This investigation clearly demonstrates that HRM analysis of the proposed SNP hot-spot region in the rpoB gene of C. difficile is a fast and cost-effective method for the identification of C. difficile samples with reduced susceptibility to rifaximin and even allows simultaneous SNP subtyping of the respective C. difficile isolates.

[Novel Approaches in DNA Methylation Studies - MS-HRM Analysis and Electrochemistry].

PubMed

Bartošík, M; Ondroušková, E

Cytosine methylation in DNA is an epigenetic mechanism regulating gene expression and plays a vital role in cell differentiation or proliferation. Tumor cells often exhibit aberrant DNA methylation, e.g. hypermethylation of tumor suppressor gene promoters. New methods, capable of determining methylation status of specific DNA sequences, are thus being developed. Among them, MS-HRM (methylation-specific high resolution melting) and electrochemistry offer relatively inexpensive instrumentation, fast assay times and possibility of screening multiple samples/DNA regions simultaneously. MS-HRM is due to its sensitivity and simplicity an interesting alternative to already established techniques, including methylation-specific PCR or bisulfite sequencing. Electrochemistry, when combined with suitable electroactive labels and electrode surfaces, has been applied in several unique strategies for discrimination of cytosines and methylcytosines. Both techniques were successfully tested in analysis of DNA methylation within promoters of important tumor suppressor genes and could thus help in achieving more precise diagnostics and prognostics of cancer. Aberrant methylation of promoters has already been described in hundreds of genes associated with tumorigenesis and could serve as important biomarker if new methods applicable into clinical practice are sufficiently advanced.Key words: DNA methylation - 5-methylcytosine - HRM analysis - melting temperature - DNA duplex - electrochemistry - nucleic acid hybridizationThis work was supported by MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 6. 5. 2016Accepted: 16. 5. 2016.

The initiation of segmented buoyancy-driven melting during continental breakup

PubMed Central

Gallacher, Ryan J.; Keir, Derek; Harmon, Nicholas; Stuart, Graham; Leroy, Sylvie; Hammond, James O. S.; Kendall, J-Michael; Ayele, Atalay; Goitom, Berhe; Ogubazghi, Ghebrebrhan; Ahmed, Abdulhakim

2016-01-01

Melting of the mantle during continental breakup leads to magmatic intrusion and volcanism, yet our understanding of the location and dominant mechanisms of melt generation in rifting environments is impeded by a paucity of direct observations of mantle melting. It is unclear when during the rifting process the segmented nature of magma supply typical of seafloor spreading initiates. Here, we use Rayleigh-wave tomography to construct a high-resolution absolute three-dimensional shear-wave velocity model of the upper 250 km beneath the Afar triple junction, imaging the mantle response during progressive continental breakup. Our model suggests melt production is highest and melting depths deepest early during continental breakup. Elevated melt production during continental rifting is likely due to localized thinning and melt focusing when the rift is narrow. In addition, we interpret segmented zones of melt supply beneath the rift, suggesting that buoyancy-driven active upwelling of the mantle initiates early during continental rifting. PMID:27752044

Helix-coil transition of a four-way DNA junction observed by multiple fluorescence parameters.

PubMed

Vámosi, György; Clegg, Robert M

2008-10-16

The thermal denaturation of immobile four-way DNA ("Holliday-") junctions with 17 base pair arms was studied via fluorescence spectroscopic measurements. Two arms of the molecule were labeled at the 5'-end with fluorescein and tetramethylrhodamine, respectively. Melting was monitored by the fluorescence intensity of the dyes, the fluorescence anisotropy of tetramethylrhodamine, and Forster resonance energy transfer (FRET) between fluorescein and rhodamine. To fit the thermal denaturation curves of the four-way junctions, two basic thermodynamic models were tested: (1) all-or-none transitions assuming a molecularity of one, two, or four and (2) a statistical "zipper" model. The all-or-none models correspond to reaction mechanisms assuming that the cooperative melting unit (that is, the structure changing from complete helix to complete coil) consists of (1) one arm, (2) two neighboring arms (which have one continuous strand common to the two arms), or (3) all four arms. In each case, the melting of the cooperative unit takes place in a single step. The tetramolecular reaction model (four-arm melting) yielded unrealistically low van't Hoff enthalpy and entropy values, whereas the monomolecular model (one-arm melting) resulted in a poor fit to the experimental data. The all-or-none bimolecular (two neighboring arm model) fit gave intermediate standard enthalpy change (Delta H) values between those expected for the melting of a duplex with a total length between the helix lengths of one and two arms (17 and 34 base pairs). Simulations according to the zipper model fit the experimental curves best when the length of the simulated duplex was assumed to be 34 base pairs, the length of a single strand. This suggests that the most important parameter determining the melting behavior of the molecule is the end-to-end distance of the strands (34 bases) rather than the length of the individual arms (17 base pairs) and that the equilibrium concentration of partially denatured intermediate states has to be taken into account. These findings are in good agreement with results obtained for three-way DNA junctions ( Stuhmeier, F. ; Lilley, D. M. ; Clegg, R. M. Biochemistry 1997, 36, 13539 ). An interesting result is that the extent-of-melting curves derived from the fluorescence intensity and anisotropy nearly agree, whereas the curve derived from the FRET data shows a change prior to the melting. This may be an indication of a conformational change leaving the double-stranded structure intact but changing the end-to-end distance of the different arms in a way consistent with the transition to the extended square configuration ( Clegg, R. M. ; Murchie, A. I. ; Lilley, D. M. Biophys. J. 1994, 66, 99 ) of this branched molecule.

Interaction study of some macrocyclic inorganic schiff base complexes with calf thymus DNA using spectroscopic and voltammetric methods

NASA Astrophysics Data System (ADS)

Bordbar, Maryam; Tavoosi, Fariba; Yeganeh-Faal, Ali; Zebarjadian, Mohammad Hasan

2018-01-01

The interaction of Cd(II), Zn(II) and Mn(II)-L (4,8-bis(2-pyridylmethyl)-4,8-diazaundecane-1,11-diamine) transition metal complexes with calf thymus DNA (CT-DNA) has been investigated using electronic, fluorescence and circular dichroism (CD) spectroscopy, thermal denaturation and cyclic voltammetry (CV). Based on the UV-Vis study, binding constants of the complexes with CT-DNA were calculated. Changes in the band of the CD spectrum, DNA melting temperature and in the ipa and ipc of the complexes in the presenceCT-DNA, overall, showed that the studied complex exhibited good DNA interaction ability with partial intercalation mode.

Solving traveling salesman problems with DNA molecules encoding numerical values.

PubMed

Lee, Ji Youn; Shin, Soo-Yong; Park, Tai Hyun; Zhang, Byoung-Tak

2004-12-01

We introduce a DNA encoding method to represent numerical values and a biased molecular algorithm based on the thermodynamic properties of DNA. DNA strands are designed to encode real values by variation of their melting temperatures. The thermodynamic properties of DNA are used for effective local search of optimal solutions using biochemical techniques, such as denaturation temperature gradient polymerase chain reaction and temperature gradient gel electrophoresis. The proposed method was successfully applied to the traveling salesman problem, an instance of optimization problems on weighted graphs. This work extends the capability of DNA computing to solving numerical optimization problems, which is contrasted with other DNA computing methods focusing on logical problem solving.

Sequence-selective binding of C8-conjugated pyrrolobenzodiazepines (PBDs) to DNA.

PubMed

Basher, Mohammad A; Rahman, Khondaker Miraz; Jackson, Paul J M; Thurston, David E; Fox, Keith R

2017-11-01

DNA footprinting and melting experiments have been used to examine the sequence-specific binding of C8-conjugates of pyrrolobenzodiazepines (PBDs) and benzofused rings including benzothiophene and benzofuran, which are attached using pyrrole- or imidazole-containing linkers. The conjugates modulate the covalent attachment points of the PBDs, so that they bind best to guanines flanked by A/T-rich sequences on either the 5'- or 3'-side. The linker affects the binding, and pyrrole produces larger changes than imidazole. Melting studies with 14-mer oligonucleotide duplexes confirm covalent attachment of the conjugates, which show a different selectivity to anthramycin and reveal that more than one ligand molecule can bind to each duplex. Copyright © 2017 Elsevier B.V. All rights reserved.

[Research progress of probe design software of oligonucleotide microarrays].

PubMed

Chen, Xi; Wu, Zaoquan; Liu, Zhengchun

2014-02-01

DNA microarray has become an essential medical genetic diagnostic tool for its high-throughput, miniaturization and automation. The design and selection of oligonucleotide probes are critical for preparing gene chips with high quality. Several sets of probe design software have been developed and are available to perform this work now. Every set of the software aims to different target sequences and shows different advantages and limitations. In this article, the research and development of these sets of software are reviewed in line with three main criteria, including specificity, sensitivity and melting temperature (Tm). In addition, based on the experimental results from literatures, these sets of software are classified according to their applications. This review will be helpful for users to choose an appropriate probe-design software. It will also reduce the costs of microarrays, improve the application efficiency of microarrays, and promote both the research and development (R&D) and commercialization of high-performance probe design software.

Method for construction of normalized cDNA libraries

DOEpatents

Soares, Marcelo B.; Efstratiadis, Argiris

1996-01-01

This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library.

Method for construction of normalized cDNA libraries

DOEpatents

Soares, M.B.; Efstratiadis, A.

1996-01-09

This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form. The method comprises: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3` noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. 4 figs.

Monitoring of KRAS-mutated ctDNA to discriminate pseudo-progression from true progression during anti-PD-1 treatment of lung adenocarcinoma

PubMed Central

Guibert, Nicolas; Mazieres, Julien; Delaunay, Myriam; Casanova, Anne; Farella, Magali; Keller, Laura; Favre, Gilles; Pradines, Anne

2017-01-01

Objectives Pseudo-progression is a rare but worrying situation for both clinicians and patients during immunotherapy. Dedicated ir-RECIST criteria have been established to improve this situation. However, this can be sometimes considered inadequate and patients experiencing true progression may then receive inefficient treatments. Additional reliable tools to discriminate pseudo from true progression are thus needed. So far, no biomarker has been identified to distinguish pseudo from true progression. We hypothesize that biomarkers associated with the molecular characteristics of the tumor may be of interest. To avoid a tumor re-biopsy, circulating markers appear to be a less invasive and reproducible procedure. As ctDNA kinetics correlate with the response to treatment in KRAS-mutated adenocarcinoma, we anticipated that this analysis could be of interest. Materials and methods We monitored the level of KRAS-mutated ctDNA by digital droplet PCR in serial plasma samples from two patients who had experienced pseudo-progression and compared the variations with those from of a patient that had true progression. Results ctDNA showed rapid and dramatic decreases in pseudo-progressive patients, whereas it was strongly increased in the progressive patient. Conclusions ddPCR of ctDNA may thus be an additional tool to discriminate pseudo-progression from true progression for tumors that harbor an oncogenic addiction. PMID:28445137

Melt-inclusion-hosted excess 40Ar in quartz crystals of the Bishop and Bandelier magma systems

USGS Publications Warehouse

Winick, J.A.; McIntosh, W.C.; Dunbar, N.W.

2001-01-01

40Ar/39Ar experiments on melt-inclusion-bearing quartz (MIBQ) from the Bishop and Bandelier Tuff Plinian deposits indicate high concentrations of excess 40Ar in melt inclusions. Two rhyolite glass melt inclusion populations are present in quartz; exposed melt inclusions and trapped melt inclusions. Air-abrasion mill grinding and hydrofluoric acid treatments progressively remove exposed melt inclusions while leaving trapped melt inclusions unaffected. Laser step-heating of MIBQ yields increasing apparent ages as a function of exposed melt inclusion removal, reflecting the higher nonatmospheric 40Ar concentrations hosted in trapped melt inclusions. Exposed melt inclusion-free MIBQ from the Bishop, Upper Bandelier, and Lower Bandelier Tufts yield total-gas ages of 3.70 ?? 1.00 Ma, 11.54 ?? 0.87 Ma, and 14.60 ?? 1.50 Ma, respectively. We interpret these old apparent ages as compelling evidence for the presence of excess 40Ar in MIBQ. Trapped melt inclusions in sanidine phenocrysts may contain excess 40Ar concentrations similar to those in MIBQ. This excess 40Ar has the potential to increase single-crystal laser-fusion ages of sanidine by tens of thousands of years, relative to the actual eruption age.

Experimental test of the viscous anisotropy hypothesis for partially molten rocks

PubMed Central

Qi, Chao; Kohlstedt, David L.; Katz, Richard F.; Takei, Yasuko

2015-01-01

Chemical differentiation of rocky planets occurs by melt segregation away from the region of melting. The mechanics of this process, however, are complex and incompletely understood. In partially molten rocks undergoing shear deformation, melt pockets between grains align coherently in the stress field; it has been hypothesized that this anisotropy in microstructure creates an anisotropy in the viscosity of the aggregate. With the inclusion of anisotropic viscosity, continuum, two-phase-flow models reproduce the emergence and angle of melt-enriched bands that form in laboratory experiments. In the same theoretical context, these models also predict sample-scale melt migration due to a gradient in shear stress. Under torsional deformation, melt is expected to segregate radially inward. Here we present torsional deformation experiments on partially molten rocks that test this prediction. Microstructural analyses of the distribution of melt and solid reveal a radial gradient in melt fraction, with more melt toward the center of the cylinder. The extent of this radial melt segregation grows with progressive strain, consistent with theory. The agreement between theoretical prediction and experimental observation provides a validation of this theory. PMID:26417107

Experimental test of the viscous anisotropy hypothesis for partially molten rocks.

PubMed

Qi, Chao; Kohlstedt, David L; Katz, Richard F; Takei, Yasuko

2015-10-13

Chemical differentiation of rocky planets occurs by melt segregation away from the region of melting. The mechanics of this process, however, are complex and incompletely understood. In partially molten rocks undergoing shear deformation, melt pockets between grains align coherently in the stress field; it has been hypothesized that this anisotropy in microstructure creates an anisotropy in the viscosity of the aggregate. With the inclusion of anisotropic viscosity, continuum, two-phase-flow models reproduce the emergence and angle of melt-enriched bands that form in laboratory experiments. In the same theoretical context, these models also predict sample-scale melt migration due to a gradient in shear stress. Under torsional deformation, melt is expected to segregate radially inward. Here we present torsional deformation experiments on partially molten rocks that test this prediction. Microstructural analyses of the distribution of melt and solid reveal a radial gradient in melt fraction, with more melt toward the center of the cylinder. The extent of this radial melt segregation grows with progressive strain, consistent with theory. The agreement between theoretical prediction and experimental observation provides a validation of this theory.

Observation of melt onset on multiyear Arctic sea ice using the ERS 1 synthetic aperture radar

NASA Technical Reports Server (NTRS)

Winebrenner, D. P.; Nelson, E. D.; Colony, R.; West, R. D.

1994-01-01

We present nearly coincident observations of backscattering from the Earth Remote-Sensing Satellite (ERS) 1 synthetic aperture radar (SAR) and of near-surface temperature from six drifting buoys in the Beaufort Sea, showing that the onset of melting in snow on multiyear sea ice is clearly detectable in the SAR data. Melt onset is marked by a clean, steep decrease in the backscattering cross section of multiyear ice at 5.3 GHz and VV polarization. We investigate the scattering physics responsible for the signature change and find that the cross section decrease is due solely to the appearance of liquid water in the snow cover overlying the ice. A thin layer of moist snow is sufficient to cause the observed decrease. We present a prototype algorithm to estimate the date of melt onset using the ERS 1 SAR and apply the algorithm first to the SAR data for which we have corresponding buoy temperatures. The melt onset dates estimated by the SAR algorithm agree with those obtained independently from the temperature data to within 4 days or less, with the exception of one case in which temperatures oscillated about 0 C for several weeks. Lastly, we apply the algorithm to the entire ERS 1 SAR data record acquired by the Alaska SAR Facility for the Beaufort Sea north of 73 deg N during the spring of 1992, to produce a map of the dates of melt onset over an area roughly 1000 km on a side. The progression of melt onset is primarily poleward but shows a weak meridional dependence at latitudes of approximately 76 deg-77 deg N. Melting begins in the southern part of the study region on June 13 and by June 20 has progressed to the northermost part of the region.

Archaeal Genome Organization and Stress Responses: Implications for the Origin and Evolution of Cellular Life

NASA Astrophysics Data System (ADS)

Musgrave, David; Zhang, Xiaoying; Dinger, Marcel

2002-08-01

For DNA to be used as an informational molecule it must exist in the cell on the edge of stability because all genomic processes require local controlled melting. This presents mechanistic opportunities and problems for genomic DNA from hyperthermophilic organisms, whose unpackaged DNA could melt at optimal temperatures for growth. Hyperthermophiles are suggested to employ the novel positively supercoiling topoisomerase enzyme reverse gyrase (RG) to form positively supercoiled DNA that is intrinsically resistant to thermal denaturation. RG is presently the only archaeal gene that is uniquely found in hyperthermophiles and therefore is central to hypotheses suggesting a hypothermophilic origin of life. However, the suggestion that RG has evolved by the fusion of two pre-existing enzymes has led to hypotheses for a lower temperature for the origin of life. In addition to the action of topoisomerases, DNA packaging and the intracellular ionic environment can also manipulate DNA topology significantly. In the Euryarchaeota, nucleosomes containing minimal histones can adopt two alternate DNA topologies in a salt-dependent manner. From this we hypothesize that since internal salt concentrations are increased following an increase in temperature, the genomic effects of temperature fluctuations could also be accommodated by changes in nucleosome organization. In addition, stress-induced changes in the nucleoid proteins could also play a role in maintaining the genome in the optimal topological state in changing environments. The function of these systems could therefore be central to temperature adaptation and thus be implicated in origin of life scenarios involving hyperthermophiles.

Genotyping of beta thalassemia trait by high-resolution DNA melting analysis.

PubMed

Saetung, Rattika; Ongchai, Siriwan; Charoenkwan, Pimlak; Sanguansermsri, Torpong

2013-11-01

Beta thalassemia is a common hereditary hemalogogical disease in Thailand, with a prevalence of 5-8%. In this study, we evaluated the high resolution DNA melting (HRM) assay to identify beta thalassemia mutation in samples from 143 carriers of the beta thalassemia traits in at risk couples. The DNA was isolated from venous blood samples and tested for mutation under a series of 5 PCR-HRM (A, B, C, D and E primers) protocols. The A primers were for detection of beta thalassemia mutations in the HBB promoter region, the B primers for mutations in exon I, the C primers for exon II, the D primers for exon III and the E primers for the 3.4 kb deletion mutation. The mutations were diagnosed by comparing the complete melting curve profiles of a wild type control with those for each mutant sample. With the PCR-HRM technique, fourteen types of beta thalassemia mutations were detected. Each mutation had a unique and specific melting profile. The mutations included 36.4% (52 cases) codon 41/42-CTTT, 26.6% (38 cases) codon 17 A-T, 11.2% (16 cases) IVS1-1 G-T, 8.4% (12 cases) codon 71/72 +A, 8.4% (12 cases) of the 3.4 kb deletion and 3.5% (5 cases) -28 A-G. The remainder included one instance each of -87 C-A, -31 A-C, codon 27/28 +C, codon 30 G-A, IVS1-5 G-C, codon 35 C-A, codon 41-C and IVSII -654 C-T. Of the total cases, 85.8% of the mutations could be detected by primers B and C. The PCR-HRM method provides a rapid, simple and highly feasible strategy for mutation screening of beta thalassemia traits.

Mechanisms of Surface-Mediated DNA Hybridization

PubMed Central

2015-01-01

Single-molecule total internal reflection fluorescence microscopy was employed in conjunction with resonance energy transfer (RET) to observe the dynamic behavior of donor-labeled ssDNA at the interface between aqueous solution and a solid surface decorated with complementary acceptor-labeled ssDNA. At least 100 000 molecular trajectories were determined for both complementary strands and negative control ssDNA. RET was used to identify trajectory segments corresponding to the hybridized state. The vast majority of molecules from solution adsorbed nonspecifically to the surface, where a brief two-dimensional search was performed with a 7% chance of hybridization. Successful hybridization events occurred with a characteristic search time of ∼0.1 s, and unsuccessful searches resulted in desorption from the surface, ultimately repeating the adsorption and search process. Hybridization was reversible, and two distinct modes of melting (i.e., dehybridization) were observed, corresponding to long-lived (∼15 s) and short-lived (∼1.4 s) hybridized time intervals. A strand that melted back onto the surface could rehybridize after a brief search or desorb from the interface. These mechanistic observations provide guidance for technologies that involve DNA interactions in the near-surface region, suggesting a need to design surfaces that both enhance the complex multidimensional search process and stabilize the hybridized state. PMID:24708278

Long-Range Vibrational Dynamics Are Directed by Watson-Crick Base Pairing in Duplex DNA.

PubMed

Hithell, Gordon; Shaw, Daniel J; Donaldson, Paul M; Greetham, Gregory M; Towrie, Michael; Burley, Glenn A; Parker, Anthony W; Hunt, Neil T

2016-05-05

Ultrafast two-dimensional infrared (2D-IR) spectroscopy of a 15-mer A-T DNA duplex in solution has revealed structure-dependent vibrational coupling and energy transfer processes linking bases with the sugar-phosphate backbone. Duplex melting induces significant changes in the positions of off-diagonal peaks linking carbonyl and ring-stretching vibrational modes of the adenine and thymine bases with vibrations of the phosphate group and phosphodiester linkage. These indicate that Watson-Crick hydrogen bonding and helix formation lead to a unique vibrational coupling arrangement of base vibrational modes with those of the phosphate unit. On the basis of observations from time-resolved 2D-IR data, we conclude that rapid energy transfer processes occur between base and backbone, mediated by additional modes located on the deoxyribose moiety within the same nucleotide. These relaxation dynamics are insensitive to duplex melting, showing that efficient intramolecular energy relaxation to the solvent via the phosphate groups is the key to excess energy dissipation in both single- and double-stranded DNA.

Rapid identification and classification of bacteria by 16S rDNA restriction fragment melting curve analyses (RFMCA).

PubMed

Rudi, Knut; Kleiberg, Gro H; Heiberg, Ragnhild; Rosnes, Jan T

2007-08-01

The aim of this work was to evaluate restriction fragment melting curve analyses (RFMCA) as a novel approach for rapid classification of bacteria during food production. RFMCA was evaluated for bacteria isolated from sous vide food products, and raw materials used for sous vide production. We identified four major bacterial groups in the material analysed (cluster I-Streptococcus, cluster II-Carnobacterium/Bacillus, cluster III-Staphylococcus and cluster IV-Actinomycetales). The accuracy of RFMCA was evaluated by comparison with 16S rDNA sequencing. The strains satisfying the RFMCA quality filtering criteria (73%, n=57), with both 16S rDNA sequence information and RFMCA data (n=45) gave identical group assignments with the two methods. RFMCA enabled rapid and accurate classification of bacteria that is database compatible. Potential application of RFMCA in the food or pharmaceutical industry will include development of classification models for the bacteria expected in a given product, and then to build an RFMCA database as a part of the product quality control.

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.

Development and characterization of a microheater array device for real-time DNA mutation detection

NASA Astrophysics Data System (ADS)

Williams, Layne; Okandan, Murat; Chagovetz, Alex; Blair, Steve

2008-04-01

DNA analysis, specifically single nucleotide polymorphism (SNP) detection, is becoming increasingly important in rapid diagnostics and disease detection. Temperature is often controlled to help speed reaction rates and perform melting of hybridized oligonucleotides. The difference in melting temperatures, Tm, between wild-type and SNP sequences, respectively, to a given probe oligonucleotide, is indicative of the specificity of the reaction. We have characterized Tm's in solution and on a solid substrate of three sequences from known mutations associated with Cystic Fibrosis. Taking advantage of Tm differences, a microheater array device was designed to enable individual temperature control of up to 18 specific hybridization events. The device was fabricated at Sandia National Laboratories using surface micromachining techniques. The microheaters have been characterized using an IR camera at Sandia and show individual temperature control with minimal thermal cross talk. Development of the device as a real-time DNA detection platform, including surface chemistry and associated microfluidics, is described.

Development and characterization of a microheater array device for real-time DNA mutation detection

NASA Astrophysics Data System (ADS)

Williams, Layne; Okandan, Murat; Chagovetz, Alex; Blair, Steve

2008-02-01

DNA analysis, specifically single nucleotide polymorphism (SNP) detection, is becoming increasingly important in rapid diagnostics and disease detection. Temperature is often controlled to help speed reaction rates and perform melting of hybridized oligonucleotides. The difference in melting temperatures, Tm, between wild-type and SNP sequences, respectively, to a given probe oligonucleotide, is indicative of the specificity of the reaction. We have characterized Tm's in solution and on a solid substrate of three sequences from known mutations associated with Cystic Fibrosis. Taking advantage of Tm differences, a microheater array device was designed to enable individual temperature control of up to 18 specific hybridization events. The device was fabricated at Sandia National Laboratories using surface micromachining techniques. The microheaters have been characterized using an IR camera at Sandia and show individual temperature control with minimal thermal cross talk. Development of the device as a real-time DNA detection platform, including surface chemistry and associated microfluidics, is described.

Adult cases of mitochondrial DNA depletion due to TK2 defect: an expanding spectrum.

PubMed

Béhin, A; Jardel, C; Claeys, K G; Fagart, J; Louha, M; Romero, N B; Laforêt, P; Eymard, B; Lombès, A

2012-02-28

In this study we aim to demonstrate the occurrence of adult forms of TK2 mutations causing progressive mitochondrial myopathy with significant muscle mitochondrial DNA (mtDNA) depletion. Patients' investigations included serum creatine kinase, blood lactate, electromyographic, echocardiographic, and functional respiratory analyses as well as TK2 gene sequencing and TK2 activity measurement. Mitochondrial activities and mtDNA were analyzed in the patients' muscle biopsy. The 3 adult patients with TK2 mutations presented with slowly progressive myopathy compatible with a fairly normal life during decades. Apart from its much slower progression, these patients' phenotype closely resembled that of pediatric cases including early onset, absence of CNS symptoms, generalized muscle weakness predominating on axial and proximal muscles but affecting facial, ocular, and respiratory muscles, typical mitochondrial myopathy with a mosaic pattern of COX-negative and ragged-red fibers, combined mtDNA-dependent respiratory complexes deficiency and mtDNA depletion. In accordance with the disease's relatively slow progression, the residual mtDNA content was higher than that observed in pediatric cases. That difference was not explained by the type of the TK2 mutations or by the residual TK2 activity. TK2 mutations can cause mitochondrial myopathy with a slow progression. Comparison of patients with similar mutations but different disease progression might address potential mechanisms of mtDNA maintenance modulation.

Influence of PCR reagents on DNA polymerase extension rates measured on real-time PCR instruments.

PubMed

Montgomery, Jesse L; Wittwer, Carl T

2014-02-01

Radioactive DNA polymerase activity methods are cumbersome and do not provide initial extension rates. A simple extension rate assay would enable study of basic assumptions about PCR and define the limits of rapid PCR. A continuous assay that monitors DNA polymerase extension using noncovalent DNA dyes on common real-time PCR instruments was developed. Extension rates were measured in nucleotides per second per molecule of polymerase. To initiate the reaction, a nucleotide analog was heat activated at 95 °C for 5 min, the temperature decreased to 75 °C, and fluorescence monitored until substrate exhaustion in 30-90 min. The assay was linear with time for over 40% of the reaction and for polymerase concentrations over a 100-fold range (1-100 pmol/L). Extension rates decreased continuously with increasing monovalent cation concentrations (lithium, sodium, potassium, cesium, and ammonium). Melting-temperature depressors had variable effects. DMSO increased rates up to 33%, whereas glycerol had little effect. Betaine, formamide, and 1,2-propanediol decreased rates with increasing concentrations. Four common noncovalent DNA dyes inhibited polymerase extension. Heat-activated nucleotide analogs were 92% activated after 5 min, and hot start DNA polymerases were 73%-90% activated after 20 min. Simple DNA extension rate assays can be performed on real-time PCR instruments. Activity is decreased by monovalent cations, DNA dyes, and most melting temperature depressors. Rational inclusion of PCR components on the basis of their effects on polymerase extension is likely to be useful in PCR, particularly rapid-cycle or fast PCR.

Unlabeled probes for the detection and typing of herpes simplex virus.

PubMed

Dames, Shale; Pattison, David C; Bromley, L Kathryn; Wittwer, Carl T; Voelkerding, Karl V

2007-10-01

Unlabeled probe detection with a double-stranded DNA (dsDNA) binding dye is one method to detect and confirm target amplification after PCR. Unlabeled probes and amplicon melting have been used to detect small deletions and single-nucleotide polymorphisms in assays where template is in abundance. Unlabeled probes have not been applied to low-level target detection, however. Herpes simplex virus (HSV) was chosen as a model to compare the unlabeled probe method to an in-house reference assay using dual-labeled, minor groove binding probes. A saturating dsDNA dye (LCGreen Plus) was used for real-time PCR. HSV-1, HSV-2, and an internal control were differentiated by PCR amplicon and unlabeled probe melting analysis after PCR. The unlabeled probe technique displayed 98% concordance with the reference assay for the detection of HSV from a variety of archived clinical samples (n = 182). HSV typing using unlabeled probes was 99% concordant (n = 104) to sequenced clinical samples and allowed for the detection of sequence polymorphisms in the amplicon and under the probe. Unlabeled probes and amplicon melting can be used to detect and genotype as few as 10 copies of target per reaction, restricted only by stochastic limitations. The use of unlabeled probes provides an attractive alternative to conventional fluorescence-labeled, probe-based assays for genotyping and detection of HSV and might be useful for other low-copy targets where typing is informative.

Physics and capabilities of terahertz spectroscopy to study the water-biomolecule interaction

NASA Astrophysics Data System (ADS)

Vezzoli, G. C.

2007-09-01

We have conducted the first study of the use of terahertz radiation to precisely identify pre-melting, melting, polymerization, depolymerization and the influence of polar water in sulfur by scanning frequency as a parametric function of temperature, and including identifying precursor and intermediate states. This spectroscopic study has also identified the orthorhombic-monoclinic phase transformation, and the melting of the superheated orthorhombic phase. This work also reports detection of a water absorption indicating a perturbation of the water molecules, associated with solvation spheres of the inter-chain dynamics, as a precursor to a transition, and supporting our earlier results showing the transducing capabilities of conglomerates of water molecules. Through a study of the fine structure of the water absorption, we are able to determine information about local polarization effects which contribute to the transducing properties of water relative to a ligand. The above inorganic polymer study is applied to the understanding of the response of biomolecules to thermal and chemical influences, and data are included giving optical, electrical, and pH properties of the DNA-water system, showing a major conformational transition at ~43°C, and various forms of reconformation of DNA macromolecule due to chemical perturbation. Our results include findings aimed at complementing existing inhibitors that are intended to prevent retrovirus/phage invasion of the host cell DNA.

Moving beyond Watson-Crick models of coarse grained DNA dynamics.

PubMed

Linak, Margaret C; Tourdot, Richard; Dorfman, Kevin D

2011-11-28

DNA produces a wide range of structures in addition to the canonical B-form of double-stranded DNA. Some of these structures are stabilized by Hoogsteen bonds. We developed an experimentally parameterized, coarse-grained model that incorporates such bonds. The model reproduces many of the microscopic features of double-stranded DNA and captures the experimental melting curves for a number of short DNA hairpins, even when the open state forms complicated secondary structures. We demonstrate the utility of the model by simulating the folding of a thrombin aptamer, which contains G-quartets, and strand invasion during triplex formation. Our results highlight the importance of including Hoogsteen bonding in coarse-grained models of DNA.

Enantiospecific recognition of DNA sequences by a proflavine Tröger base.

PubMed

Bailly, C; Laine, W; Demeunynck, M; Lhomme, J

2000-07-05

The DNA interaction of a chiral Tröger base derived from proflavine was investigated by DNA melting temperature measurements and complementary biochemical assays. DNase I footprinting experiments demonstrate that the binding of the proflavine-based Tröger base is both enantio- and sequence-specific. The (+)-isomer poorly interacts with DNA in a non-sequence-selective fashion. In sharp contrast, the corresponding (-)-isomer recognizes preferentially certain DNA sequences containing both A. T and G. C base pairs, such as the motifs 5'-GTT. AAC and 5'-ATGA. TCAT. This is the first experimental demonstration that acridine-type Tröger bases can be used for enantiospecific recognition of DNA sequences. Copyright 2000 Academic Press.

Application of differential scanning calorimetry to measure the differential binding of ions, water and protons in the unfolding of DNA molecules.

PubMed

Olsen, Chris M; Shikiya, Ronald; Ganugula, Rajkumar; Reiling-Steffensmeier, Calliste; Khutsishvili, Irine; Johnson, Sarah E; Marky, Luis A

2016-05-01

The overall stability of DNA molecules globally depends on base-pair stacking, base-pairing, polyelectrolyte effect and hydration contributions. In order to understand how they carry out their biological roles, it is essential to have a complete physical description of how the folding of nucleic acids takes place, including their ion and water binding. To investigate the role of ions, water and protons in the stability and melting behavior of DNA structures, we report here an experimental approach i.e., mainly differential scanning calorimetry (DSC), to determine linking numbers: the differential binding of ions (Δnion), water (ΔnW) and protons (ΔnH(+)) in the helix-coil transition of DNA molecules. We use DSC and temperature-dependent UV spectroscopic techniques to measure the differential binding of ions, water, and protons for the unfolding of a variety of DNA molecules: salmon testes DNA (ST-DNA), one dodecamer, one undecamer and one decamer duplexes, nine hairpin loops, and two triplexes. These methods can be applied to any conformational transition of a biomolecule. We determined complete thermodynamic profiles, including all three linking numbers, for the unfolding of each molecule. The favorable folding of a DNA helix results from a favorable enthalpy-unfavorable entropy compensation. DSC thermograms and UV melts as a function of salt, osmolyte and proton concentrations yielded releases of ions and water. Therefore, the favorable folding of each DNA molecule results from the formation of base-pair stacks and uptake of both counterions and water molecules. In addition, the triplex with C(+)GC base triplets yielded an uptake of protons. Furthermore, the folding of a DNA duplex is accompanied by a lower uptake of ions and a similar uptake of four water molecules as the DNA helix gets shorter. In addition, the oligomer duplexes and hairpin thermodynamic data suggest ion and water binding depends on the DNA sequence rather than DNA composition. Copyright © 2015. Published by Elsevier B.V.

Method for construction of normalized cDNA libraries

DOEpatents

Soares, Marcelo B.; Efstratiadis, Argiris

1998-01-01

This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to appropriate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. This invention also provides normalized cDNA libraries generated by the above-described method and uses of the generated libraries.

Method for construction of normalized cDNA libraries

DOEpatents

Soares, M.B.; Efstratiadis, A.

1998-11-03

This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3` noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to appropriate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. This invention also provides normalized cDNA libraries generated by the above-described method and uses of the generated libraries. 19 figs.

simulation of the DNA force-extension curve

NASA Astrophysics Data System (ADS)

Shinaberry, Gregory; Mikhaylov, Ivan; Balaeff, Alexander

A molecular dynamics simulation study of the force-extension curve of double-stranded DNA is presented. Extended simulations of the DNA at multiple points along the force-extension curve are conducted with DNA end-to-end length constrained at each point. The calculated force-extension curve qualitatively reproduces the experimental one. The DNA conformational ensemble at each extension shows that the famous plateau of the force-extension curve results from B-DNA melting, whereas the formation of the earlier-predicted novel DNA conformation called 'zip-DNA' takes place at extensions past the plateau. An extensive analysis of the DNA conformational ensemble in terms of base configuration, backbone configuration, solvent interaction energy, etc., is conducted in order to elucidate the physical origin of DNA elasticity and the main interactions responsible for the shape of the force-extension curve.

Comparative thermal and thermodynamic study of DNA chemically modified with antitumor drug cisplatin and its inactive analog transplatin.

PubMed

Lando, Dmitri Y; Chang, Chun-Ling; Fridman, Alexander S; Grigoryan, Inessa E; Galyuk, Elena N; Hsueh, Ya-Wei; Hu, Chin-Kun

2014-08-01

Antitumor activity of cisplatin is exerted by covalent binding to DNA. For comparison, studies of cisplatin-DNA complexes often employ the very similar but inactive transplatin. In this work, thermal and thermodynamic properties of DNA complexes with these compounds were studied using differential scanning calorimetry (DSC) and computer modeling. DSC demonstrates that cisplatin decreases thermal stability (melting temperature, Tm) of long DNA, and transplatin increases it. At the same time, both compounds decrease the enthalpy and entropy of the helix-coil transition, and the impact of transplatin is much higher. From Pt/nucleotide molar ratio rb=0.001, both compounds destroy the fine structure of DSC profile and increase the temperature melting range (ΔT). For cisplatin and transplatin, the dependences δTm vs rb differ in sign, while δΔT vs rb are positive for both compounds. The change in the parameter δΔT vs rb demonstrates the GC specificity in the location of DNA distortions. Our experimental results and calculations show that 1) in contrast to [Pt(dien)Cl]Cl, monofunctional adducts formed by transplatin decrease the thermal stability of long DNA at [Na(+)]>30mM; 2) interstrand crosslinks of cisplatin and transplatin only slightly increase Tm; 3) the difference in thermal stability of DNA complexes with cisplatin vs DNA complexes with transplatin mainly arises from the different thermodynamic properties of their intrastrand crosslinks. This type of crosslink appears to be responsible for the antitumor activity of cisplatin. At any [Na(+)] from interval 10-210mM, cisplatin and transplatin intrastrand crosslinks give rise to destabilization and stabilization, respectively. Copyright © 2014 Elsevier Inc. All rights reserved.

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.

Preliminary morphological and X-ray diffraction studies of the crystals of the DNA cetyltrimethylammonium salt.

PubMed

Osica, V D; Pyatigorskaya, T L; Polyvtsev, O F; Dembo, A T; Kliya, M O; Vasilchenko, V N; Verkin, B I; Sukharevskya, B Y

1977-04-01

Double-stranded DNA molecules (molecular weight 2.5 X 10(5) - 5 X 10(5) daltons) have been crystallized from water-salt solutions as cetyltrimethylammonium salts (CTA-DNA). Variation of crystallization conditions results in a production of different types of CTA-DNA crystals: spherulits, dendrites, needle-shaped and faceted rhombic crystals, the latter beeing up to 0.3 mm on a side. X-ray diffraction data indicate that DNA molecules in the crystals form a hexagonal lattice which parameters vary slightly with the morphological type of the crystal. Comparison of the melting curves of the DNA preparation before and after crystallization suggests that DNA molecules are partially fractionated in the course of crystallization. Crystals of the CTA-DNA-proflavine complex have also been obtained.

Preliminary morphological and X-ray diffraction studies of the crystals of the DNA cetyltrimethylammonium salt.

PubMed Central

Osica, V D; Pyatigorskaya, T L; Polyvtsev, O F; Dembo, A T; Kliya, M O; Vasilchenko, V N; Verkin, B I; Sukharevskya, B Y

1977-01-01

Double-stranded DNA molecules (molecular weight 2.5 X 10(5) - 5 X 10(5) daltons) have been crystallized from water-salt solutions as cetyltrimethylammonium salts (CTA-DNA). Variation of crystallization conditions results in a production of different types of CTA-DNA crystals: spherulits, dendrites, needle-shaped and faceted rhombic crystals, the latter beeing up to 0.3 mm on a side. X-ray diffraction data indicate that DNA molecules in the crystals form a hexagonal lattice which parameters vary slightly with the morphological type of the crystal. Comparison of the melting curves of the DNA preparation before and after crystallization suggests that DNA molecules are partially fractionated in the course of crystallization. Crystals of the CTA-DNA-proflavine complex have also been obtained. Images PMID:866188

Measuring the Surface Temperature of the Cryosphere using Remote Sensing

NASA Technical Reports Server (NTRS)

Hall, Dorothy K.

2012-01-01

A general description of the remote sensing of cryosphere surface temperatures from satellites will be provided. This will give historical information on surface-temperature measurements from space. There will also be a detailed description of measuring the surface temperature of the Greenland Ice Sheet using Moderate-Resolution Imaging Spectroradiometer (MODIS) data which will be the focus of the presentation. Enhanced melting of the Greenland Ice Sheet has been documented in recent literature along with surface-temperature increases measured using infrared satellite data since 1981. Using a recently-developed climate data record, trends in the clear-sky ice-surface temperature (IST) of the Greenland Ice Sheet have been studied using the MODIS IST product. Daily and monthly MODIS ISTs of the Greenland Ice Sheet beginning on 1 March 2000 and continuing through 31 December 2010 are now freely available to download at 6.25-km spatial resolution on a polar stereographic grid. Maps showing the maximum extent of melt for the entire ice sheet and for the six major drainage basins have been developed from the MODIS IST dataset. Twelve-year trends of the duration of the melt season on the ice sheet vary in different drainage basins with some basins melting progressively earlier over the course of the study period. Some (but not all) of the basins also show a progressively-longer duration of melt. The consistency of this IST record, with temperature and melt records from other sources will be discussed.

An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents

PubMed Central

Chang, Shy-Shin; Hsu, Hsung-Ling; Cheng, Ju-Chien; Tseng, Ching-Ping

2011-01-01

Background Bacterial DNA contamination in PCR reagents has been a long standing problem that hampers the adoption of broad-range PCR in clinical and applied microbiology, particularly in detection of low abundance bacteria. Although several DNA decontamination protocols have been reported, they all suffer from compromised PCR efficiency or detection limits. To date, no satisfactory solution has been found. Methodology/Principal Findings We herein describe a method that solves this long standing problem by employing a broad-range primer extension-PCR (PE-PCR) strategy that obviates the need for DNA decontamination. In this method, we first devise a fusion probe having a 3′-end complementary to the template bacterial sequence and a 5′-end non-bacterial tag sequence. We then hybridize the probes to template DNA, carry out primer extension and remove the excess probes using an optimized enzyme mix of Klenow DNA polymerase and exonuclease I. This strategy allows the templates to be distinguished from the PCR reagent contaminants and selectively amplified by PCR. To prove the concept, we spiked the PCR reagents with Staphylococcus aureus genomic DNA and applied PE-PCR to amplify template bacterial DNA. The spiking DNA neither interfered with template DNA amplification nor caused false positive of the reaction. Broad-range PE-PCR amplification of the 16S rRNA gene was also validated and minute quantities of template DNA (10–100 fg) were detectable without false positives. When adapting to real-time and high-resolution melting (HRM) analytical platforms, the unique melting profiles for the PE-PCR product can be used as the molecular fingerprints to further identify individual bacterial species. Conclusions/Significance Broad-range PE-PCR is simple, efficient, and completely obviates the need to decontaminate PCR reagents. When coupling with real-time and HRM analyses, it offers a new avenue for bacterial species identification with a limited source of bacterial DNA, making it suitable for use in clinical and applied microbiology laboratories. PMID:21637859

Use of molecular hybridization to explore genetic relationships. Progress report, July 1, 1975--March 31, 1976. [Primates, mice, Drosophila

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

Atwood, K.C.

Progress is reported on the following research projects: distribution of rDNA in lymphocyte chromosomes of the gibbon; site of 55 DNA in chromosomes of the baboon; satellite associations and rDNA; polymorphisms in rDNA of mouse chromosomes; effect of prephotographing on hybridization; histone and immunoglobulin gene mapping; and rDNA magnification in Drosophila. (HLW)

A Prospective Evaluation of Circulating Tumor Cells and Cell-Free DNA in EGFR-Mutant Non-Small Cell Lung Cancer Patients Treated with Erlotinib on a Phase II Trial.

PubMed

Yanagita, Masahiko; Redig, Amanda J; Paweletz, Cloud P; Dahlberg, Suzanne E; O'Connell, Allison; Feeney, Nora; Taibi, Myriam; Boucher, David; Oxnard, Geoffrey R; Johnson, Bruce E; Costa, Daniel B; Jackman, David M; Jänne, Pasi A

2016-12-15

Genotype-directed therapy is the standard of care for advanced non-small cell lung cancer (NSCLC), but obtaining tumor tissue for genotyping remains a challenge. Circulating tumor cell (CTC) or cell-free DNA (cfDNA) analysis may allow for noninvasive evaluation. This prospective trial evaluated CTCs and cfDNA in EGFR-mutant NSCLC patients treated with erlotinib until progression. EGFR-mutant NSCLC patients were enrolled in a phase II trial of erlotinib. Blood was collected at baseline, every 2 months on study, and at disease progression. Plasma genotyping was performed by droplet digital PCR for EGFR19del, L858R, and T790M. CTCs were isolated by CellSave, enumerated, and analyzed by immunofluorescence for CD45 and pan-cytokeratin and EGFR and MET FISH were also performed. Rebiopsy was performed at disease progression. Sixty patients were enrolled; 44 patients discontinued therapy for disease progression. Rebiopsy occurred in 35 of 44 patients (80%), with paired CTC/cfDNA analysis in 41 of 44 samples at baseline and 36 of 44 samples at progression. T790M was identified in 23 of 35 (66%) tissue biopsies and 9 of 39 (23%) cfDNA samples. CTC analysis at progression identified MET amplification in 3 samples in which tissue analysis could not be performed. cfDNA analysis identified T790M in 2 samples in which rebiopsy was not possible. At diagnosis, high levels of cfDNA but not high levels of CTCs correlated with progression-free survival. cfDNA and CTCs are complementary, noninvasive assays for evaluation of acquired resistance to first-line EGFR TKIs and may expand the number of patients in whom actionable genetic information can be obtained at acquired resistance. Serial cfDNA monitoring may offer greater clinical utility than serial monitoring of CTCs. Clin Cancer Res; 22(24); 6010-20. ©2016 AACR. ©2016 American Association for Cancer Research.

Ultrasensitive Quantification of Hepatitis B Virus A1762T/G1764A Mutant by a SimpleProbe PCR Using a Wild-Type-Selective PCR Blocker and a Primer-Blocker-Probe Partial-Overlap Approach ▿

PubMed Central

Nie, Hui; Evans, Alison A.; London, W. Thomas; Block, Timothy M.; Ren, Xiangdong David

2011-01-01

Hepatitis B virus (HBV) carrying the A1762T/G1764A double mutation in the basal core promoter (BCP) region is associated with HBe antigen seroconversion and increased risk of liver cirrhosis and hepatocellular carcinoma (HCC). Quantification of the mutant viruses may help in predicting the risk of HCC. However, the viral genome tends to have nucleotide polymorphism, which makes it difficult to design hybridization-based assays including real-time PCR. Ultrasensitive quantification of the mutant viruses at the early developmental stage is even more challenging, as the mutant is masked by excessive amounts of the wild-type (WT) viruses. In this study, we developed a selective inhibitory PCR (siPCR) using a locked nucleic acid-based PCR blocker to selectively inhibit the amplification of the WT viral DNA but not the mutant DNA. At the end of siPCR, the proportion of the mutant could be increased by about 10,000-fold, making the mutant more readily detectable by downstream applications such as real-time PCR and DNA sequencing. We also describe a primer-probe partial overlap approach which significantly simplified the melting curve patterns and minimized the influence of viral genome polymorphism on assay accuracy. Analysis of 62 patient samples showed a complete match of the melting curve patterns with the sequencing results. More than 97% of HBV BCP sequences in the GenBank database can be correctly identified by the melting curve analysis. The combination of siPCR and the SimpleProbe real-time PCR enabled mutant quantification in the presence of a 100,000-fold excess of the WT DNA. PMID:21562108

Thermal Diffusivity for III-VI Semiconductor Melts at Different Temperatures

NASA Technical Reports Server (NTRS)

Ban, H.; Li, C.; Lin, B.; Emoto, K.; Scripa, R. N.; Su, C.-H.; Lehoczky, S. L.

2004-01-01

The change of the thermal properties of semiconductor melts reflects the structural changes inside the melts, and a fundamental understanding of this structural transformation is essential for high quality semiconductor crystal growth process. This paper focused on the technical development and the measurement of thermal properties of III-VI semiconductor melts at high temperatures. Our previous work has improved the laser flash method for the specialized quartz sample cell. In this paper, we reported the results of our recent progress in further improvements of the measurement system by minimizing the free convection of the melt, adding a front IR detector, and placing the sample cell in a vacuum environment. The results for tellurium and selenium based compounds, some of which have never been reported in the literature, were obtained at different temperatures as a function of time. The data were compared with other measured thermophysical properties to shed light on the structural transformations of the melt.

Melting of superheated molecular crystals

NASA Astrophysics Data System (ADS)

Cubeta, Ulyana; Bhattacharya, Deepanjan; Sadtchenko, Vlad

2017-07-01

Melting dynamics of micrometer scale, polycrystalline samples of isobutane, dimethyl ether, methyl benzene, and 2-propanol were investigated by fast scanning calorimetry. When films are superheated with rates in excess of 105 K s-1, the melting process follows zero-order, Arrhenius-like kinetics until approximately half of the sample has transformed. Such kinetics strongly imply that melting progresses into the bulk via a rapidly moving solid-liquid interface that is likely to originate at the sample's surface. Remarkably, the apparent activation energies for the phase transformation are large; all exceed the enthalpy of vaporization of each compound and some exceed it by an order of magnitude. In fact, we find that the crystalline melting kinetics are comparable to the kinetics of dielectric α-relaxation in deeply supercooled liquids. Based on these observations, we conclude that the rate of non-isothermal melting for superheated, low-molecular-weight crystals is limited by constituent diffusion into an abnormally dense, glass-like, non-crystalline phase.

Differentiation of BHV-1 isolates from vaccine virus by high-resolution melting analysis.

PubMed

Ostertag-Hill, Claire; Fang, Liang; Izume, Satoko; Lee, Megan; Reed, Aimee; Jin, Ling

2015-02-16

An efficacious bovine herpesvirus type-1 (BHV-1) vaccine has been used for many years. However, in the past few years, abortion and respiratory diseases have occurred after administration of the modified live vaccine. To investigate whether BHV-1 isolates from disease outbreaks are identical to those of the vaccines used, selected regions of the BHV-1 genome were investigated by high-resolution melting (HRM) analysis and PCR-DNA sequencing. When a target region within the thymidine kinase (TK) gene was examined by HRM analysis, 6 out of the 11 isolates from abortion cases and 22 out of the 25 isolates from bovine respiratory disease (BRD) cases had different melting curves compared to the vaccine virus. Surprisingly, when a conserved region within the US6 gene that encodes glycoprotein D (gD) was examined by HRM analysis, 5 out of the 11 abortion isolates and 18 out of the 23 BRD isolates had different melting curves from the vaccine virus. To determine whether SNPs within the coding regions of glycoprotein E (gE) and TK genes can be used to differentiate the isolates from the vaccine virus, PCR-DNA sequencing was used to examine these SNPs in all the isolates. This revealed that only 1 out of 11 of the abortion isolates and 4 out of 24 of the BRD isolates are different in the target region of gE from the vaccine virus, while 5 out of 11 abortion isolates and 4 out of 22 BRD isolates are different in the target region of TK from the vaccine virus. No DNA sequence differences were observed in glycoprotein G (gG) region between disease and vaccine isolates. Our study demonstrated that many disease isolates had genetic differences from the vaccine virus in regions examined by HRM and PCR-DNA sequencing analysis. In addition, many isolates contained more than one type of mutation and were composed of mixed variants. Our study suggests that a mixture of variants were present in isolates collected post-vaccination. HRM is a rapid diagnostic method that can be used for rapid differentiation of clinical isolates from vaccine strains. Copyright © 2014 Elsevier B.V. All rights reserved.

TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism.

PubMed

Harley, Margaret E; Murina, Olga; Leitch, Andrea; Higgs, Martin R; Bicknell, Louise S; Yigit, Gökhan; Blackford, Andrew N; Zlatanou, Anastasia; Mackenzie, Karen J; Reddy, Kaalak; Halachev, Mihail; McGlasson, Sarah; Reijns, Martin A M; Fluteau, Adeline; Martin, Carol-Anne; Sabbioneda, Simone; Elcioglu, Nursel H; Altmüller, Janine; Thiele, Holger; Greenhalgh, Lynn; Chessa, Luciana; Maghnie, Mohamad; Salim, Mahmoud; Bober, Michael B; Nürnberg, Peter; Jackson, Stephen P; Hurles, Matthew E; Wollnik, Bernd; Stewart, Grant S; Jackson, Andrew P

2016-01-01

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

Development of a novel device to trap heavy metal cations: application of the specific interaction between heavy metal cation and mismatch DNA base pair.

PubMed

Torigoe, Hidetaka; Miyakawa, Yukako; Fukushi, Miyako; Ono, Akira; Kozasa, Tetsuo

2009-01-01

We have already found that Hg(II) cation specifically binds to T:T mismatch base pair in heteroduplex DNA, which increases the melting temperature of heteroduplex DNA involving T:T mismatch base pair by about 4 degrees C. We have also found that Ag(I) cation specifically binds to C:C mismatch base pair in heteroduplex DNA, which increases the melting temperature of heteroduplex DNA involving C:C mismatch base pair by about 4 degrees C. Using the specific interaction, we developed a novel device to trap each of Hg(II) and Ag(I) cation. The device is composed of 5'-biotinylated T-rich or C-rich DNA oligonucleotides, BIO-T20: 5'-Bio-T(20)-3' or BIO-C20: 5'-Bio-C(20)-3' (Bio is a biotin), immobilized on streptavidin-coated polystylene beads. When the BIO-T20-immobilized beads were added to a solution containing Hg(II) cation, and the beads trapping Hg(II) cation were collected by centrifugation, almost all of Hg(II) cation were removed from the solution. Also, when the BIO-C20-immobilized beads were added to a solution containing Ag(I) cation, and the beads trapping Ag(I) cation were collected by centrifugation, almost all of Ag(I) cation were removed from the solution. We conclude that, using the novel device developed in this study, Hg(II) and Ag(I) cation can be effectively removed from the solution.

Fructosylation induced structural changes in mammalian DNA examined by biophysical techniques

NASA Astrophysics Data System (ADS)

Zaman, Asif; Arif, Zarina; Alam, Khursheed

2017-03-01

Glycosylation of DNA, proteins, lipids, etc. by reducing sugars, can lead to the formation of advanced glycation end products (AGEs). These products may accumulate and involve in the pathogenesis of a number of diseases, contributing to tissue injury via several mechanisms. In this study, fructosylation of calf thymus dsDNA was carried out with varying concentrations of fructose. The neo-structure of fructosylated-DNA was studied by various biophysical techniques and morphological characterization. Fructosylated-DNA showed hyperchromicity, increase in fluorescence intensity and decrease in melting temperature. The CD signal of modified-DNA shifted in the direction of higher wavelength indicative of structural changes in DNA. FTIR results indicated shift in specific band positions in fructosylated-DNA. Morphological characterization of fructosylated-DNA exhibited strand breakage and aggregation. The results suggest that the structure and conformation of DNA may be altered under high concentrations of fructose.

A new general model for predicting melting thermodynamics of complementary and mismatched B-form duplexes containing locked nucleic acids: application to probe design for digital PCR detection of somatic mutations.

PubMed

Hughesman, Curtis; Fakhfakh, Kareem; Bidshahri, Roza; Lund, H Louise; Haynes, Charles

2015-02-17

Advances in real-time polymerase chain reaction (PCR), as well as the emergence of digital PCR (dPCR) and useful modified nucleotide chemistries, including locked nucleic acids (LNAs), have created the potential to improve and expand clinical applications of PCR through their ability to better quantify and differentiate amplification products, but fully realizing this potential will require robust methods for designing dual-labeled hydrolysis probes and predicting their hybridization thermodynamics as a function of their sequence, chemistry, and template complementarity. We present here a nearest-neighbor thermodynamic model that accurately predicts the melting thermodynamics of a short oligonucleotide duplexed either to its perfect complement or to a template containing mismatched base pairs. The model may be applied to pure-DNA duplexes or to duplexes for which one strand contains any number and pattern of LNA substitutions. Perturbations to duplex stability arising from mismatched DNA:DNA or LNA:DNA base pairs are treated at the Gibbs energy level to maintain statistical significance in the regressed model parameters. This approach, when combined with the model's accounting of the temperature dependencies of the melting enthalpy and entropy, permits accurate prediction of T(m) values for pure-DNA homoduplexes or LNA-substituted heteroduplexes containing one or two independent mismatched base pairs. Terms accounting for changes in solution conditions and terminal addition of fluorescent dyes and quenchers are then introduced so that the model may be used to accurately predict and thereby tailor the T(m) of a pure-DNA or LNA-substituted hydrolysis probe when duplexed either to its perfect-match template or to a template harboring a noncomplementary base. The model, which builds on classic nearest-neighbor thermodynamics, should therefore be of use to clinicians and biologists who require probes that distinguish and quantify two closely related alleles in either a quantitative PCR or dPCR assay. This potential is demonstrated by using the model to design allele-specific probes that completely discriminate and quantify clinically relevant mutant alleles (BRAF V600E and KIT D816V) in a dPCR assay.

High-resolution melting (HRM) re-analysis of a polyposis patients cohort reveals previously undetected heterozygous and mosaic APC gene mutations.

PubMed

Out, Astrid A; van Minderhout, Ivonne J H M; van der Stoep, Nienke; van Bommel, Lysette S R; Kluijt, Irma; Aalfs, Cora; Voorendt, Marsha; Vossen, Rolf H A M; Nielsen, Maartje; Vasen, Hans F A; Morreau, Hans; Devilee, Peter; Tops, Carli M J; Hes, Frederik J

2015-06-01

Familial adenomatous polyposis is most frequently caused by pathogenic variants in either the APC gene or the MUTYH gene. The detection rate of pathogenic variants depends on the severity of the phenotype and sensitivity of the screening method, including sensitivity for mosaic variants. For 171 patients with multiple colorectal polyps without previously detectable pathogenic variant, APC was reanalyzed in leukocyte DNA by one uniform technique: high-resolution melting (HRM) analysis. Serial dilution of heterozygous DNA resulted in a lowest detectable allelic fraction of 6% for the majority of variants. HRM analysis and subsequent sequencing detected pathogenic fully heterozygous APC variants in 10 (6%) of the patients and pathogenic mosaic variants in 2 (1%). All these variants were previously missed by various conventional scanning methods. In parallel, HRM APC scanning was applied to DNA isolated from polyp tissue of two additional patients with apparently sporadic polyposis and without detectable pathogenic APC variant in leukocyte DNA. In both patients a pathogenic mosaic APC variant was present in multiple polyps. The detection of pathogenic APC variants in 7% of the patients, including mosaics, illustrates the usefulness of a complete APC gene reanalysis of previously tested patients, by a supplementary scanning method. HRM is a sensitive and fast pre-screening method for reliable detection of heterozygous and mosaic variants, which can be applied to leukocyte and polyp derived DNA.

Human ribonuclease H1 resolves R-loops and thereby enables progression of the DNA replication fork.

PubMed

Parajuli, Shankar; Teasley, Daniel C; Murali, Bhavna; Jackson, Jessica; Vindigni, Alessandro; Stewart, Sheila A

2017-09-15

Faithful DNA replication is essential for genome stability. To ensure accurate replication, numerous complex and redundant replication and repair mechanisms function in tandem with the core replication proteins to ensure DNA replication continues even when replication challenges are present that could impede progression of the replication fork. A unique topological challenge to the replication machinery is posed by RNA-DNA hybrids, commonly referred to as R-loops. Although R-loops play important roles in gene expression and recombination at immunoglobulin sites, their persistence is thought to interfere with DNA replication by slowing or impeding replication fork progression. Therefore, it is of interest to identify DNA-associated enzymes that help resolve replication-impeding R-loops. Here, using DNA fiber analysis, we demonstrate that human ribonuclease H1 (RNH1) plays an important role in replication fork movement in the mammalian nucleus by resolving R-loops. We found that RNH1 depletion results in accumulation of RNA-DNA hybrids, slowing of replication forks, and increased DNA damage. Our data uncovered a role for RNH1 in global DNA replication in the mammalian nucleus. Because accumulation of RNA-DNA hybrids is linked to various human cancers and neurodegenerative disorders, our study raises the possibility that replication fork progression might be impeded, adding to increased genomic instability and contributing to disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Detection of Babesia canis vogeli and Hepatozoon canis in canine blood by a single-tube real-time fluorescence resonance energy transfer polymerase chain reaction assay and melting curve analysis.

PubMed

Kongklieng, Amornmas; Intapan, Pewpan M; Boonmars, Thidarut; Thanchomnang, Tongjit; Janwan, Penchom; Sanpool, Oranuch; Lulitanond, Viraphong; Taweethavonsawat, Piyanan; Chungpivat, Sudchit; Maleewong, Wanchai

2015-03-01

A real-time fluorescence resonance energy transfer polymerase chain reaction (qFRET PCR) coupled with melting curve analysis was developed for detection of Babesia canis vogeli and Hepatozoon canis infections in canine blood samples in a single tube assay. The target of the assay was a region within the 18S ribosomal RNA gene amplified in either species by a single pair of primers. Following amplification from the DNA of infected dog blood, a fluorescence melting curve analysis was done. The 2 species, B. canis vogeli and H. canis, could be detected and differentiated in infected dog blood samples (n = 37) with high sensitivity (100%). The detection limit for B. canis vogeli was 15 copies of a positive control plasmid, and for H. canis, it was 150 copies of a positive control plasmid. The assay could simultaneously distinguish the DNA of both parasites from the DNA of controls. Blood samples from 5 noninfected dogs were negative, indicating high specificity. Several samples can be run at the same time. The assay can reduce misdiagnosis and the time associated with microscopic examination, and is not prone to the carryover contamination associated with the agarose gel electrophoresis step of conventional PCR. In addition, this qFRET PCR method would be useful to accurately determine the range of endemic areas or to discover those areas where the 2 parasites co-circulate. © 2015 The Author(s).

A novel real time PCR assay using melt curve analysis for ivory identification.

PubMed

Kitpipit, Thitika; Penchart, Kitichaya; Ouithavon, Kanita; Satasook, Chutamas; Linacre, Adrian; Thanakiatkrai, Phuvadol

2016-10-01

Demand for ivory and expansion of human settlements have resulted in a rapid decline in the number of elephants. Enforcement of local and international laws and regulations requires identification of the species from which any ivory, or ivory products, originated. Further geographical assignment of the dead elephant from which the ivory was taken can assist in forensic investigations. In this study, a real-time PCR assay using melt curve analysis was developed and fully validated for forensic use. The presence or absence of three Elephantidae-specific and elephant species-specific melting peaks was used to identify the elephant species. Using 141 blood and ivory samples from the three extant elephant species, the assay demonstrated very high reproducibility and accuracy. The limit of detection was as low as 0.031ng of input DNA for conventional amplification and 0.002ng for nested amplification. Both DNA concentrations are typically encountered in forensic casework, especially for degraded samples. No cross-reactivity was observed for non-target species. Evaluation of direct amplification and nested amplification demonstrated the assay's flexibility and capability of analyzing low-template DNA samples and aged samples. Additionally, blind trial testing showed the assay's suitability application in real casework. In conclusion, wildlife forensic laboratories could use this novel, quick, and low-cost assay to help combat the continuing poaching crises leading to the collapse of elephant numbers in the wild. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Optimizing high-resolution melting analysis for the detection of mutations of GPR30/GPER-1 in breast cancer.

PubMed

Aihara, Masamune; Yamamoto, Shigeru; Nishioka, Hiroko; Inoue, Yutaro; Hamano, Kimikazu; Oka, Masaaki; Mizukami, Yoichi

2012-06-15

G protein-coupled receptor 30/G protein estrogen receptor-1 (GPR30/GPER-1) is a novel membrane receptor for estrogen whose mRNA is expressed at high levels in estrogen-dependent cells such as breast cancer cell lines. However, mutations in GRP30 related to diseases remain unreported. To detect unknown mutations in the GPR30 open reading frame (ORF) quickly, the experimental conditions for high-resolution melting (HRM) analysis were examined for PCR primers, Taq polymerases, saturation DNA binding dyes, Mg(2+) concentration, and normalized temperatures. Nine known SNPs and 13 artificial point mutations within the GPR30 ORF, as well as single nucleotide variants in DNA extracted from subjects with breast cancers were tested under the optimal experimental conditions. The combination of Expand High Fidelity(PLUS) and SYTO9 in the presence of 2.0 mM MgCl(2) produced the best separation in melting curves of mutations in all regions of the GPR30 ORF. Under these experimental conditions, the mutations were clearly detected in both heterozygotes and homozygotes. HRM analysis of GPR30 using genomic DNA from subjects with breast cancers showed a novel single nucleotide variant, 111C>T in GPR30 and 4 known SNPs. The experimental conditions determined in this study for HRM analysis are useful for high throughput assays to detect unknown mutations within the GPR30 ORF. Copyright © 2012 Elsevier B.V. All rights reserved.

Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases

DOE PAGES

Manthei, Kelly A.; Hill, Morgan C.; Burke, Jordan E.; ...

2015-03-23

RecQ helicases unwind remarkably diverse DNA structures as key components of many cellular processes. How RecQ enzymes accommodate different substrates in a unified mechanism that couples ATP hydrolysis to DNA unwinding is unknown. In this paper, the X-ray crystal structure of the Cronobacter sakazakii RecQ catalytic core domain bound to duplex DNA with a 3' single-stranded extension identifies two DNA-dependent conformational rearrangements: a winged-helix domain pivots ~90° to close onto duplex DNA, and a conserved aromatic-rich loop is remodeled to bind ssDNA. These changes coincide with a restructuring of the RecQ ATPase active site that positions catalytic residues for ATPmore » hydrolysis. Complex formation also induces a tight bend in the DNA and melts a portion of the duplex. Finally, this bending, coupled with translocation, could provide RecQ with a mechanism for unwinding duplex and other DNA structures.« less

EG-13GENOME-WIDE METHYLATION ANALYSIS IDENTIFIES GENOMIC DNA DEMETHYLATION DURING MALIGNANT PROGRESSION OF GLIOMAS

PubMed Central

Saito, Kuniaki; Mukasa, Akitake; Nagae, Genta; Aihara, Koki; Otani, Ryohei; Takayanagi, Shunsaku; Omata, Mayu; Tanaka, Shota; Shibahara, Junji; Takahashi, Miwako; Momose, Toshimitsu; Shimamura, Teppei; Miyano, Satoru; Narita, Yoshitaka; Ueki, Keisuke; Nishikawa, Ryo; Nagane, Motoo; Aburatani, Hiroyuki; Saito, Nobuhito

2014-01-01

Low-grade gliomas often undergo malignant progression, and these transformations are a leading cause of death in patients with low-grade gliomas. However, the molecular mechanisms underlying malignant tumor progression are still not well understood. Recent evidence indicates that epigenetic deregulation is an important cause of gliomagenesis; therefore, we examined the impact of epigenetic changes during malignant progression of low-grade gliomas. Specifically, we used the Illumina Infinium Human Methylation 450K BeadChip to perform genome-wide DNA methylation analysis of 120 gliomas and four normal brains. This study sample included 25 matched-pairs of initial low-grade gliomas and recurrent tumors (temporal heterogeneity) and 20 of the 25 recurring tumors recurred as malignant progressions, and one matched-pair of newly emerging malignant lesions and pre-existing lesions (spatial heterogeneity). Analyses of methylation profiles demonstrated that most low-grade gliomas in our sample (43/51; 84%) had a CpG island methylator phenotype (G-CIMP). Remarkably, approximately 50% of secondary glioblastomas that had progressed from low-grade tumors with the G-CIMP status exhibited a characteristic partial demethylation of genomic DNA during malignant progression, but other recurrent gliomas showed no apparent change in DNA methylation pattern. Interestingly, we found that most loci that were demethylated during malignant progression were located outside of CpG islands. The information of histone modifications patterns in normal human astrocytes and embryonal stem cells also showed that the ratio of active marks at the site corresponding to DNA demethylated loci in G-CIMP-demethylated tumors was significantly lower; this finding indicated that most demethylated loci in G-CIMP-demethylated tumors were likely transcriptionally inactive. A small number of the genes that were upregulated and had demethylated CpG islands were associated with cell cycle-related pathway. In summary, we demonstrated that characteristic DNA demethylation occurred during malignant progression of a subset of low-grade gliomas. The mechanisms underlying and consequences of such DNA demethylation should be studied further.

TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism

PubMed Central

Leitch, Andrea; Higgs, Martin R.; Bicknell, Louise S.; Yigit, Gökhan; Blackford, Andrew N.; Zlatanou, Anastasia; Mackenzie, Karen J.; Reddy, Kaalak; Halachev, Mihail; McGlasson, Sarah; Reijns, Martin A. M.; Fluteau, Adeline; Martin, Carol-Anne; Sabbioneda, Simone; Elcioglu, Nursel H.; Altmüller, Janine; Thiele, Holger; Greenhalgh, Lynn; Chessa, Luciana; Maghnie, Mohamad; Salim, Mahmoud; Bober, Michael B.; Nürnberg, Peter; Jackson, Stephen P.; Hurles, Matthew E.; Wollnik, Bernd; Stewart, Grant S.; Jackson, Andrew P.

2015-01-01

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism/Seckel syndrome. We establish that TRAIP relocalizes to sites of DNA damage where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to UV irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a novel component of the DNA damage response to replication-blocking DNA lesions. PMID:26595769

Conditions of High Resolution Melting Analysis on the Cobas z480 Instrument for the Genotyping of VKORC1 in the Clinical Routine Laboratory.

PubMed

Paar, Christian; Hammerl, Verena; Blessberger, Hermann; Stekel, Herbert; Steinwender, Clemens; Berg, Jörg

2016-12-01

High resolution melting (HRM) of amplicons is a simple method for genotyping of single nucleotide polymorphisms (SNPs). Albeit many applications reported, HRM seems to be rarely used in clinical laboratories. The suitability of HRM-PCR for the clinical laboratory was investigated for genotyping of SNPs of the vitamin K epoxide reductase complex unit 1 gene. About 100 DNA samples were analyzed by two different HRM-PCRs on the Cobas z480 instrument and compared with a PCR with fluorescently labeled probes (HybProbe-PCR) on the LightCycler 2.0 instrument as reference. Reliable genotyping with 100% matching results was obtained, when the amplicon size was small (63 bp) and DNA input was limited by e.g., sample dilution with salt-free water. DNA extracted by differing methods may be used for genotyping by HRM-PCR. Compared with HybProbe-PCR, HRM-PCR on the Cobas z480 instrument allows for higher through-put, however, at the cost of a higher degree of laboratory standardization and a slower turnaround.

Development of the Plastic Melt Waste Compactor- Design and Fabrication of the Half-Scale Prototype

NASA Technical Reports Server (NTRS)

Pace, Gregory S.; Fisher, John

2005-01-01

A half scale version of a device called the Plastic Melt Waste Compactor prototype has been developed at NASA Ames Research Center to deal with plastic based wastes that are expected to be encountered in future human space exploration scenarios such as Lunar or Martian Missions. The Plastic Melt Waste Compactor design was based on the types of wastes produced on the International Space Station, Space Shuttle, MIR and Skylab missions. The half scale prototype unit will lead to the development of a full scale Plastic Melt Waste Compactor prototype that is representative of flight hardware that would be used on near and far term space missions. This report details the progress of the Plastic Melt Waste Compactor Development effort by the Solid Waste Management group at NASA Ames Research Center.

Increased mantle heat flow with on-going rifting of the West Antarctic rift system inferred from characterisation of plagioclase peridotite in the shallow Antarctic mantle

NASA Astrophysics Data System (ADS)

Martin, A. P.; Cooper, A. F.; Price, R. C.

2014-03-01

The lithospheric, and shallow asthenospheric, mantle in Southern Victoria Land are known to record anomalously high heat flow but the cause remains imperfectly understood. To address this issue plagioclase peridotite xenoliths have been collected from Cenozoic alkalic igneous rocks at three localities along a 150 km transect across the western shoulder of the West Antarctic rift system in Southern Victoria Land, Antarctica. There is a geochemical, thermal and chronological progression across this section of the rift shoulder from relatively hot, young and thick lithosphere in the west to cooler, older and thinner lithosphere in the east. Overprinting this progression are relatively more recent mantle refertilising events. Melt depletion and refertilisation was relatively limited in the lithospheric mantle to the west but has been more extensive in the east. Thermometry obtained from orthopyroxene in these plagioclase peridotites indicates that those samples most recently affected by refertilising melts have attained the highest temperatures, above those predicted from idealised dynamic rift or Northern Victoria Land geotherms and higher than those prevailing in the equivalent East Antarctic mantle. Anomalously high heat flow can thus be attributed to entrapment of syn-rift melts in the lithosphere, probably since regional magmatism commenced at least 24 Myr ago. The chemistry and mineralogy of shallow plagioclase peridotite mantle can be explained by up to 8% melt extraction and a series of refertilisation events. These include: (a) up to 8% refertilisation by a N-MORB melt; (b) metasomatism involving up to 1% addition of a subduction-related component; and (c) addition of ~ 1.5% average calcio-carbonatite. A high MgO group of clinopyroxenes can be modelled by the addition of up to 1% alkalic melt. Melt extraction and refertilisation mainly occurred in the spinel stability field prior to decompression and uplift. In this region mantle plagioclase originates by a combination of subsolidus recrystallisation during decompression within the plagioclase stability field and refertilisation by basaltic melt.

Preliminary biogeochemical assessment of EPICA LGM and Holocene ice samples

NASA Astrophysics Data System (ADS)

Bulat, S.; Alekhina, I.; Marie, D.; Wagenbach, D.; Raynaud, D.; Petit, J. R.

2009-04-01

We are investigating the biological content (biomass and microbial diversity of Aeolian origin) of EPICA ice core within the frame of EPICA Microbiology consortium*. Two ice core sections were selected from EPICA Dome C and Droning Maud Land, both from LGM and Holocene. Preliminary measurements of DOC (dissolved organic content) and microbial cell concentrations have been performed. Both analyses showed the very low biomass and ultra low DOC content. Trace DNA analyses are in a progress. The ice sections were decontaminated in LGGE cold and clean room facilities benefiting the protocol developed for Vostok ice core studies. The melt water was then shared between two party laboratories for a complementary approach in studying microbial content. Prior to biology the melt water was tested for chemical contaminant ions and organic acids, DOC and dust contents. The biological methods included all the spectra of appropriate molecular techniques (gDNA extraction, PCR, clone libraries and sequencing). As preliminary results, both LGM (well identified by dust fallout) and Holocene ice samples (EDC99 and EDML) proved to be extremely clear (i.e. pristine) in terms of biomass (less then 4 cells per ml) and DOC contents (less then 5 ppbC). There was no obvious difference between LGM and Holocene in cell counts, while LGM showed a bit high organic carbon content. The latter in terms of biology means ultra-oligotrophic conditions (i.e., no possibility for heterotrophic life style). In fact no metabolizing microbial cells or propagating populations are expected at these depths at temperature -38oC and lower (limiting life temperature threshold is -20°C). Nevertheless some life seeds brought in Antarctica with precipitation could be well preserved because the age is rather young (21 kyr and less). Trying to identify these aliens and document their distribution during last climate cycle the meltwater was concentrated about 1000 times down. The genomic DNA was extracted and very weak signals were possible to generate which are now under cloning. The signals were hard to reproduce because of rather low volume of samples. More ice volume is needed to get the biosignal stronger and reproducible. Meantime we are adjusting PCR and in addition testing DNA repair-enzyme cocktail in case of DNA damage. As a preliminary conclusion we would like to highlight the following. Both Holocene and LGM ice samples (EDC99 and EDML) are very clean in terms of Ultra low biomass and Ultra low DOC content. The most basal ice of EDC and EDML ice cores could help in assessing microbial biomass and diversity if present under the glacier at the ice-bedrock boundary. * The present-day consortium includes S. Bulat, I. Alekhina, P. Normand, D. Prieur, J-R. Petit and D. Raynaud (France) and E. Willerslev and J.P. Steffensen (Denmark)

DNA-based dye lasers: progress in this half a decade

NASA Astrophysics Data System (ADS)

Kawabe, Yutaka

2016-09-01

After the invention of DNA-surfactant films and the proposal of dye doping into them by Ogata, many applications were demonstrated. Among them tunable thin film laser is one of the most attractive functional devices. Development and progress in DNA based lasers after the first observation of amplified spontaneous emission (ASE) by us has been reviewed in a former paper published in 2011.1 In this proceeding, progresses in the subsequent half a decade are described.

A novel mitochondrial DNA deletion in a patient with Pearson syndrome and neonatal diabetes mellitus provides insight into disease etiology, severity and progression.

PubMed

Chen, Xin-Yu; Zhao, Si-Yu; Wang, Yan; Wang, Dong; Dong, Chang-Hu; Yang, Ying; Wang, Zhi-Hua; Wu, Yuan-Ming

2016-07-01

Pearson syndrome (PS) is a rare, mitochondrial DNA (mtDNA) deletion disorder mainly affecting hematopoietic system and exocrine pancreas in early infancy, which is characterized by multi-organ involvement, variable manifestations and poor prognosis. Since the clinical complexity and uncertain outcome of PS, the ability to early diagnose and anticipate disease progression is of great clinical importance. We described a patient with severe anemia and hyperglycinemia at birth was diagnosed with neonatal diabetes mellitus, and later with PS. Genetic testing revealed that a novel mtDNA deletion existed in various non-invasive tissues from the patient. The disease course was monitored by mtDNA deletion heteroplasmy and mtDNA/nucleus DNA genome ratio in different tissues and at different time points, showing a potential genotype-phenotype correlation. Our findings suggest that for patient suspected for PS, it may be therapeutically important to first perform detailed mtDNA analysis on non-invasive tissues at the initial diagnosis and during disease progression.

In vitro DNA binding studies of therapeutic and prophylactic drug citral.

PubMed

Alam, Md Fazle; Varshney, Supriya; Khan, Masood Alam; Laskar, Amaj Ahmed; Younus, Hina

2018-07-01

The study of drug-DNA interactions is of great importance, as it paves the way towards the design of better therapeutic agents. Here, the interaction of DNA with a therapeutic and prophylactic drug citral has been studied. We have attempted to ascertain the mode of binding of citral with calf thymus DNA (Ct-DNA) through various biophysical techniques. Analysis of the UV-visible absorbance spectra and fluorescence spectra indicated the formation of a complex between citral and Ct-DNA. Competitive binding assays with ethidium bromide (EB), acridine orange (AO) and Hoechst 33258 reflected that citral possibly intercalates within the Ct-DNA. These observations were further confirmed by circular dichroism (CD) spectral analysis, viscosity measurements, DNA melting and molecular docking studies. This study is expected to contribute to a better understanding of molecular mechanisms of citral, and design of new drugs in the future. Copyright © 2018 Elsevier B.V. All rights reserved.

Lanthanum induced B-to-Z transition in self-assembled Y-shaped branched DNA structure

PubMed Central

Nayak, Ashok K.; Mishra, Aseem; Jena, Bhabani S.; Mishra, Barada K.; Subudhi, Umakanta

2016-01-01

Controlled conversion of right-handed B-DNA to left-handed Z-DNA is one of the greatest conformational transitions in biology. Recently, the B-Z transition has been explored from nanotechnological points of view and used as the driving machinery of many nanomechanical devices. Using a combination of CD spectroscopy, fluorescence spectroscopy, and PAGE, we demonstrate that low concentration of lanthanum chloride can mediate B-to-Z transition in self-assembled Y-shaped branched DNA (bDNA) structure. The transition is sensitive to the sequence and structure of the bDNA. Thermal melting and competitive dye binding experiments suggest that La3+ ions are loaded to the major and minor grooves of DNA and stabilize the Z-conformation. Our studies also show that EDTA and EtBr play an active role in reversing the transition from Z-to-B DNA. PMID:27241949

Lanthanum induced B-to-Z transition in self-assembled Y-shaped branched DNA structure

NASA Astrophysics Data System (ADS)

Nayak, Ashok K.; Mishra, Aseem; Jena, Bhabani S.; Mishra, Barada K.; Subudhi, Umakanta

2016-05-01

Controlled conversion of right-handed B-DNA to left-handed Z-DNA is one of the greatest conformational transitions in biology. Recently, the B-Z transition has been explored from nanotechnological points of view and used as the driving machinery of many nanomechanical devices. Using a combination of CD spectroscopy, fluorescence spectroscopy, and PAGE, we demonstrate that low concentration of lanthanum chloride can mediate B-to-Z transition in self-assembled Y-shaped branched DNA (bDNA) structure. The transition is sensitive to the sequence and structure of the bDNA. Thermal melting and competitive dye binding experiments suggest that La3+ ions are loaded to the major and minor grooves of DNA and stabilize the Z-conformation. Our studies also show that EDTA and EtBr play an active role in reversing the transition from Z-to-B DNA.

Eukaryotic Replicative Helicase Subunit Interaction with DNA and Its Role in DNA Replication

PubMed Central

Martinez, Matthew P.; Wacker, Amanda L.; Bruck, Irina; Kaplan, Daniel L.

2017-01-01

The replicative helicase unwinds parental double-stranded DNA at a replication fork to provide single-stranded DNA templates for the replicative polymerases. In eukaryotes, the replicative helicase is composed of the Cdc45 protein, the heterohexameric ring-shaped Mcm2-7 complex, and the tetrameric GINS complex (CMG). The CMG proteins bind directly to DNA, as demonstrated by experiments with purified proteins. The mechanism and function of these DNA-protein interactions are presently being investigated, and a number of important discoveries relating to how the helicase proteins interact with DNA have been reported recently. While some of the protein-DNA interactions directly relate to the unwinding function of the enzyme complex, other protein-DNA interactions may be important for minichromosome maintenance (MCM) loading, origin melting or replication stress. This review describes our current understanding of how the eukaryotic replicative helicase subunits interact with DNA structures in vitro, and proposed models for the in vivo functions of replicative helicase-DNA interactions are also described. PMID:28383499

An Mcm10 Mutant Defective in ssDNA Binding Shows Defects in DNA Replication Initiation.

PubMed

Perez-Arnaiz, Patricia; Kaplan, Daniel L

2016-11-20

Mcm10 is an essential protein that functions to initiate DNA replication after the formation of the replication fork helicase. In this manuscript, we identified a budding yeast Mcm10 mutant (Mcm10-m2,3,4) that is defective in DNA binding in vitro. Moreover, this Mcm10-m2,3,4 mutant does not stimulate the phosphorylation of Mcm2 by Dbf4-dependent kinase (DDK) in vitro. When we expressed wild-type levels of mcm10-m2,3,4 in budding yeast cells, we observed a severe growth defect and a substantially decreased DNA replication. We also observed a substantially reduced replication protein A- chromatin immunoprecipitation signal at origins of replication, reduced levels of DDK-phosphorylated Mcm2, and diminished Go, Ichi, Ni, and San (GINS) association with Mcm2-7 in vivo. mcm5-bob1 bypasses the growth defect conferred by DDK-phosphodead Mcm2 in budding yeast. However, the growth defect observed by expressing mcm10-m2,3,4 is not bypassed by the mcm5-bob1 mutation. Furthermore, origin melting and GINS association with Mcm2-7 are substantially decreased for cells expressing mcm10-m2,3,4 in the mcm5-bob1 background. Thus, the origin melting and GINS-Mcm2-7 interaction defects we observed for mcm10-m2,3,4 are not explained by decreased Mcm2 phosphorylation by DDK, since the defects persist in an mcm5-bob1 background. These data suggest that DNA binding by Mcm10 is essential for the initiation of DNA replication. Copyright © 2016 Elsevier Ltd. All rights reserved.

Manipulation of double-stranded DNA melting by force

NASA Astrophysics Data System (ADS)

Singh, Amit Raj; Granek, Rony

2017-09-01

By integrating elasticity—as described by the Gaussian network model—with bond binding energies that distinguish between different base-pair identities and stacking configurations, we study the force induced melting of a double-stranded DNA (dsDNA). Our approach is a generalization of our previous study of thermal dsDNA denaturation [J. Chem. Phys. 145, 144101 (2016), 10.1063/1.4964285] to that induced by force at finite temperatures. It allows us to obtain semimicroscopic information about the opening of the chain, such as whether the dsDNA opens from one of the ends or from the interior, forming an internal bubble. We study different types of force manipulation: (i) "end unzipping," with force acting at a single end base pair perpendicular to the helix, (ii) "midunzipping," with force acting at a middle base pair perpendicular to the helix, and (iii) "end shearing," where the force acts at opposite ends along the helix. By monitoring the free-energy landscape and probability distribution of intermediate denaturation states, we show that different dominant intermediate states are stabilized depending on the type of force manipulation used. In particular, the bubble state of the sequence L60B36, which we have previously found to be a stable state during thermal denaturation, is absent for end unzipping and end shearing, whereas very similar bubbles are stabilized by midunzipping, or when the force location is near the middle of the chain. Ours results offer a simple tool for stabilizing bubbles and loops using force manipulations at different temperatures, and may implicate on the mechanism in which DNA enzymes or motors open regions of the chain.

A Dual-Porosity, In Situ Crystallisation Model For Fast-Spreading Mid-Ocean Ridge Magma Chambers Based Upon Direct Observation From Hess Deep

NASA Astrophysics Data System (ADS)

MacLeod, C. J.; Lissenberg, C. J.

2014-12-01

We propose a revised magma chamber model for fast-spreading mid-ocean ridges based upon a synthesis of new data from a complete section of lower crust from the East Pacific Rise, reconstructed from samples collected from the Hess Deep rift valley during cruise JC21. Our investigation includes detailed sampling across critical transitions in the upper part of the plutonic section, including the inferred axial melt lens (AML) within the dyke-gabbro transition. We find that an overall petrological progression, from troctolite and primitive gabbro at the base up into evolved (oxide) gabbro and gabbronorite at the top of the lower crustal section, is mirrored by a progressive upward chemical fractionation as recorded in bulk rock and mineral compositions. Crystallographic preferred orientations measured using EBSD show that the downward increase in deformation of mush required in crystal subsidence models is not observed. Together these observations are consistent only with a model in which crystallisation of upward migrating evolving melts occurs in situ in the lower crust. Over-enrichment in incompatible trace element concentrations and ratios above that possible by fractional crystallisation is ubiquitous. This implies redistribution of incompatible trace elements in the lower crust by low porosity, near-pervasive reactive porous flow of interstitial melt moving continuously upward through the mush pile. Mass balance calculations reveal a significant proportion of this trace element enriched melt is trapped at mid-crustal levels. Mineral compositions in the upper third to half of the plutonic section are too evolved to represent the crystal residues of MORB. Erupted MORB therefore must be fed from melts sourced in the deeper part of the crystal mush pile, and which must ascend rapidly without significant modification in the upper plutonics or AML. From physical models of mush processes we posit that primitive melts are transported through transient, high porosity channels generated by gravitational instabilities that periodically overturn and drain crystallising melt bodies (sills) from deeper levels of the lower crustal mush. We conclude that magma chambers are characterised by melt delivery to the deep crust, followed by in situ crystallisation of melts transported upwards via a dual-porosity system.

DNA unzipping phase diagram calculated via replica theory.

PubMed

Roland, C Brian; Hatch, Kristi Adamson; Prentiss, Mara; Shakhnovich, Eugene I

2009-05-01

We show how single-molecule unzipping experiments can provide strong evidence that the zero-force melting transition of long molecules of natural dsDNA should be classified as a phase transition of the higher-order type (continuous). Toward this end, we study a statistical-mechanics model for the fluctuating structure of a long molecule of dsDNA, and compute the equilibrium phase diagram for the experiment in which the molecule is unzipped under applied force. We consider a perfect-matching dsDNA model, in which the loops are volume-excluding chains with arbitrary loop exponent c . We include stacking interactions, hydrogen bonds, and main-chain entropy. We include sequence heterogeneity at the level of random sequences; in particular, there is no correlation in the base-pairing (bp) energy from one sequence position to the next. We present heuristic arguments to demonstrate that the low-temperature macrostate does not exhibit degenerate ergodicity breaking. We use this claim to understand the results of our replica-theoretic calculation of the equilibrium properties of the system. As a function of temperature, we obtain the minimal force at which the molecule separates completely. This critical-force curve is a line in the temperature-force phase diagram that marks the regions where the molecule exists primarily as a double helix versus the region where the molecule exists as two separate strands. We compare our random-sequence model to magnetic tweezer experiments performed on the 48 502 bp genome of bacteriophage lambda . We find good agreement with the experimental data, which is restricted to temperatures between 24 and 50 degrees C . At higher temperatures, the critical-force curve of our random-sequence model is very different for that of the homogeneous-sequence version of our model. For both sequence models, the critical force falls to zero at the melting temperature T_{c} like |T-T_{c}|;{alpha} . For the homogeneous-sequence model, alpha=1/2 almost exactly, while for the random-sequence model, alpha approximately 0.9 . Importantly, the shape of the critical-force curve is connected, via our theory, to the manner in which the helix fraction falls to zero at T_{c} . The helix fraction is the property that is used to classify the melting transition as a type of phase transition. In our calculation, the shape of the critical-force curve holds strong evidence that the zero-force melting transition of long natural dsDNA should be classified as a higher-order (continuous) phase transition. Specifically, the order is 3rd or greater.

Analysis of Summer 2002 Melt Extent on the Greenland Ice Sheet using MODIS and SSM/I Data

NASA Technical Reports Server (NTRS)

Hall, Dorothy K.; Williams, Richard S., Jr.; Steffen, Konrad; Chien, Y. L.; Foster, James L.; Robinson, David A.; Riggs, George A.

2004-01-01

Previous work has shown that the summer of 2002 had the greatest area of snow melt extent on the Greenland ice sheet ever recorded using passive-microwave data. In this paper, we compare the 0 degree isotherm derived from the Moderate-Resolution Imaging Spectroradiometer (MODIS) instrument, with Special Sensor Microwave/Imager (SSM/I)-derived melt, at the time of the maximum melt extent in 2002. To validate the MODIS-derived land-surface temperatures (LSTs), we compared the MODIS LSTs with air temperatures from nine stations (using 11 different data points) and found that they agreed to within 2.3 plus or minus 2.09 C, with station temperatures consistently lower than the MODIS LSTs. According to the MODIS LST, the maximum surface melt extended to approximately 2300 m in southern Greenland; while the SSM/I measurements showed that the maximum melt extended to nearly 2700 m in southeastern Greenland. The MODIS and SSM/I data are complementary in providing detailed information about the progression of surface and near-surface melt on the Greenland ice sheet.

Analysis of Summer 2002 Melt Extent on the Greenland Ice Sheet using MODIS and SSM/I Data

NASA Technical Reports Server (NTRS)

Hall, Dorothy K.; Williams, Richard S.; Steffen, Konrad; Chien, Janet Y. L.

2004-01-01

Previous work has shown that the summer of 2002 had the greatest area of snow melt extent on the Greenland ice sheet ever recorded using passive-microwave data. In this paper, we compare the 0 deg. isotherm derived from the Moderate-Resolution Imaging Spectroradiometer (MODIS) instrument, with Special Sensor Microwave/Imager (SSM/I)-derived melt, at the time of the maximum melt extent in 2002. To validate the MODIS derived land-surface temperatures (LSTs), we compared the MODIS LSTs with air temperatures from nine stations (using 11 different data points) and found that they agreed to within 2.3 +/- 2.09 C, with station temperatures consistently lower than the MODIS LSTs. According to the MODIS LST, the maximum surface melt extended to approx. 2300 m in southern Greenland; while the SSM/I measurements showed that the maximum melt extended to nearly 2700 m in southeastern Greenland. The MODIS and SSM/I data are complementary in providing detailed information about the progression of surface and near- surface melt on the Greenland ice sheet.

Analysis of summer 2002 melt extent on the Greenland ice sheet using MODIS and SSM/I data

USGS Publications Warehouse

Hall, D.K.; Williams, R.S.; Steffen, K.; Chien, Janet Y.L.

2004-01-01

Previous work has shown that the summer of 2002 had the greatest area of snow melt extent on the Greenland ice sheet ever recorded using passive-microwave data. In this paper, we compare the 0?? isotherm derived from the Moderate-Resolution Imaging Spectroradiometer (MODIS) instrument, with Special Sensor Microwave/Imager (SSM/I)-derived melt, at the time of the maximum melt extent in 2002. To validate the MODIS-derived land-surface temperatures (LSTs), we compared the MODIS LSTs with air temperatures from nine stations (using 11 different data points) and found that they agreed to within 2.3??2.09??C, with station temperatures consistently lower than the MODIS LSTs. According to the MODIS LST, the maximum surface melt extended to ???2300 m in southern Greenland; while the SSM/I measurements showed that the maximum melt extended to nearly 2700 m in southeastern Greenland. The MODIS and SSM/I data are complementary in providing detailed information about the progression of surface and near-surface melt on the Greenland ice sheet.

Analysis of summer 2002 melt extent on the Greenland ice sheet using MODIS and SSM/I data

USGS Publications Warehouse

Hall, D. K.; Williams, R.S.; Steffen, K.; Chien, Janet Y.L.

2004-01-01

Previous work has shown that the summer of 2002 had the greatest area of snow melt extent on the Greenland ice sheet ever recorded using passive-microwave data. In this paper, we compare the 0deg isotherm derived from the Moderate-Resolution Imaging Spectroradiometer (MODIS) instrument, with Special Sensor Microwave/Imager (SSM/I)-derived melt, at the time of the maximum melt extent in 2002. To validate the MODIS-derived land-surface temperatures (LSTs), we compared the MODIS LSTs with air temperatures from nine stations (using 11 different data points) and found that they agreed to within 2.3 plusmn 2.09 degC, with station temperatures consistently lower than the MODIS LSTs. According to the MODIS LST, the maximum surface melt extended to ~2300 m in southern Greenland; while the SSM/I measurements showed that the maximum melt extended to nearly 2700 m in southeastern Greenland. The MODIS and SSM/I data are complementary in providing detailed information about the progression of surface and near-surface melt on the Greenland ice sheet.

Gaussian decomposition of high-resolution melt curve derivatives for measuring genome-editing efficiency

PubMed Central

Zaboikin, Michail; Freter, Carl

2018-01-01

We describe a method for measuring genome editing efficiency from in silico analysis of high-resolution melt curve data. The melt curve data derived from amplicons of genome-edited or unmodified target sites were processed to remove the background fluorescent signal emanating from free fluorophore and then corrected for temperature-dependent quenching of fluorescence of double-stranded DNA-bound fluorophore. Corrected data were normalized and numerically differentiated to obtain the first derivatives of the melt curves. These were then mathematically modeled as a sum or superposition of minimal number of Gaussian components. Using Gaussian parameters determined by modeling of melt curve derivatives of unedited samples, we were able to model melt curve derivatives from genetically altered target sites where the mutant population could be accommodated using an additional Gaussian component. From this, the proportion contributed by the mutant component in the target region amplicon could be accurately determined. Mutant component computations compared well with the mutant frequency determination from next generation sequencing data. The results were also consistent with our earlier studies that used difference curve areas from high-resolution melt curves for determining the efficiency of genome-editing reagents. The advantage of the described method is that it does not require calibration curves to estimate proportion of mutants in amplicons of genome-edited target sites. PMID:29300734

Analysis of Chromatin Organisation

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2011-01-01

Terms to be familiar with before you start to solve the test: chromatin, nucleases, sucrose density gradient centrifugation, melting point, gel electrophoresis, ethidium bromide, autoradiography, Southern blotting, Northern blotting, Sanger sequencing, restriction endonucleases, exonucleases, linker DNA, chloroform extraction, nucleosomes,…

Polymerase cross-linking spiral reaction (PCLSR) for detection of African swine fever virus (ASFV) in pigs and wild boars

PubMed Central

Woźniakowski, Grzegorz; Frączyk, Magdalena; Kowalczyk, Andrzej; Pomorska-Mól, Małgorzata; Niemczuk, Krzysztof; Pejsak, Zygmunt

2017-01-01

The study reports the development of a polymerase cross-linking spiral reaction (PCLSR) for the detection of African swine fever virus (ASFV) DNA in blood collected from infected pigs and wild boars. The method uses 3 specifically designed primers. Two outer-spiral primers comprising of 3′ sequences complementary to ASFV p72 gene sequence and 5′end sequences complementary to exogenous gene of black widow alpha-latrotoxin as well as additional ASFV specific cross-linking primer. The method is specific exclusively to ASFV DNA without cross-reactions with cDNA of classical swine fever virus (CSFV), porcine reproductive respiratory syndrome (PRRSV) or porcine epidemic diarrhea virus (PEDV). The sensitivity of this technique reached 7.2 × 102 copies per μl−1 of plasmid containing p72 gene. The PCLSR was conducted at 65 °C creating cross-linked complex structures. The results of PCLSR were visualized using SYBR Green I dye, gel electrophoresis while the reaction progress was traced using real-time PCR system that resulted in registration of fluorescent curves and melting peaks at 85.3 °C. The developed PCLSR was examined using blood or tissue samples collected from selected 17 ASF cases from infected wild boars and 3 outbreaks in pigs. Further tests have been also conducted using 55 tissue samples from 23 outbreaks and 22 cases. These results showed that PCLSR might be further used for preliminary and cost-effective detection and surveillance of ASFV. PMID:28198455

Polymerase cross-linking spiral reaction (PCLSR) for detection of African swine fever virus (ASFV) in pigs and wild boars.

PubMed

Woźniakowski, Grzegorz; Frączyk, Magdalena; Kowalczyk, Andrzej; Pomorska-Mól, Małgorzata; Niemczuk, Krzysztof; Pejsak, Zygmunt

2017-02-15

The study reports the development of a polymerase cross-linking spiral reaction (PCLSR) for the detection of African swine fever virus (ASFV) DNA in blood collected from infected pigs and wild boars. The method uses 3 specifically designed primers. Two outer-spiral primers comprising of 3' sequences complementary to ASFV p72 gene sequence and 5'end sequences complementary to exogenous gene of black widow alpha-latrotoxin as well as additional ASFV specific cross-linking primer. The method is specific exclusively to ASFV DNA without cross-reactions with cDNA of classical swine fever virus (CSFV), porcine reproductive respiratory syndrome (PRRSV) or porcine epidemic diarrhea virus (PEDV). The sensitivity of this technique reached 7.2 × 10 2 copies per μl -1 of plasmid containing p72 gene. The PCLSR was conducted at 65 °C creating cross-linked complex structures. The results of PCLSR were visualized using SYBR Green I dye, gel electrophoresis while the reaction progress was traced using real-time PCR system that resulted in registration of fluorescent curves and melting peaks at 85.3 °C. The developed PCLSR was examined using blood or tissue samples collected from selected 17 ASF cases from infected wild boars and 3 outbreaks in pigs. Further tests have been also conducted using 55 tissue samples from 23 outbreaks and 22 cases. These results showed that PCLSR might be further used for preliminary and cost-effective detection and surveillance of ASFV.

[GSTP1, APC and RASSF1 gene methylation in prostate cancer samples: comparative analysis of MS-HRM method and Infinium HumanMethylation450 BeadChip beadchiparray diagnostic value].

PubMed

Skorodumova, L O; Babalyan, K A; Sultanov, R; Vasiliev, A O; Govorov, A V; Pushkar, D Y; Prilepskaya, E A; Danilenko, S A; Generozov, E V; Larin, A K; Kostryukova, E S; Sharova, E I

2016-11-01

There is a clear need in molecular markers for prostate cancer (PC) risk stratification. Alteration of DNA methylation is one of processes that occur during ÐÑ progression. Methylation-sensitive PCR with high resolution melting curve analysis (MS-HRM) can be used for gene methylation analysis in routine laboratory practice. This method requires very small amounts of DNA for analysis. Numerous results have been accumulated on DNA methylation in PC samples analyzed by the Infinium HumanMethylation450 BeadChip (HM450). However, the consistency of MS-HRM results with chip hybridization results has not been examined yet. The aim of this study was to assess the consistency of results of GSTP1, APC and RASSF1 gene methylation analysis in ÐÑ biopsy samples obtained by MS-HRM and chip hybridization. The methylation levels of each gene determined by MS-HRM were statistically different in the group of PC tissue samples and the samples without signs of tumor growth. Chip hybridization data analysis confirmed the results obtained with the MS-HRM. Differences in methylation levels between tumor tissue and histologically intact tissue of each sample determined by MS-HRM and chip hybridization, were consistent with each other. Thus, we showed that the assessment of GSTP1, APC and RASSF1 gene methylation analysis using MS-HRM is suitable for the design of laboratory assays that will differentiate the PC tissue from the tissue without signs of tumor growth.

ITS2 barcoding DNA region combined with high resolution melting (HRM) analysis of Hyoscyami Semen, the mature seed of Hyoscyamus niger.

PubMed

Xiong, Chao; Hu, Zhi-Gang; Tu, Yuan; Liu, He-Gang; Wang, Ping; Zhao, Ming-Ming; SHIi, Yu-Hua; Wu, Lan; Sun, Wei; Chen, Shi-Lin

2016-12-01

Hyoscyami Semen, the mature dried seed of Hyoscyamus niger L., has long been used as a traditional Chinese medicine to treat human diseases. Hyoscyami Semen is found in local markets in China. In markets, sellers and buyers commonly inadvertently mix the seeds of H. niger with the seeds of related species such as Hygrophila salicifolia (Vahl) Nees, Astragalus complanatus R. Br., Cuscuta australis R. Br., Cuscuta chinensis Lam., and Impatiens balsamina L. because of their similar morphologies or similar names. Thus, developing a reliable method for discriminating H. niger seeds from its adulterants is necessary to reduce confusion and ensure the safe use of Hyoscyami Semen. The present study was designed to evaluate the efficiency of high-resolution melting analysis combined with DNA barcoding (Bar-HRM) with internal transcribed spacer 2 to discriminate H. niger. Our results show that Bar-HRM successfully identified the adulterants and detected the proportion of H. niger DNA extract within an admixture. In particular, HRM detected H. niger DNA extract in A. complanatus DNA extract at concentrations as low as 1%. In conclusion, the Bar-HRM method developed in the present study for authenticating H. niger is rapid and cost-effective. It can be used in the future to guarantee the purity of Hyoscyami Semen for the clinical use. Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Molecular mechanism of melting of a helical polymer crystal: Role of conformational order, packing and mobility of polymers

NASA Astrophysics Data System (ADS)

Cheerla, Ramesh; Krishnan, Marimuthu

2018-03-01

The molecular mechanism of melting of a superheated helical polymer crystal has been investigated using isothermal-isobaric molecular dynamics simulation that allows anisotropic deformation of the crystal lattice. A detailed microscopic analysis of the onset and progression of melting and accompanying changes in the polymer conformational order, translational, and orientation order of the solid along the melting pathway is presented. Upon gradual heating from room temperature to beyond the melting point at ambient pressure, the crystal exhibits signatures of premelting well below the solid-to-liquid melting transition at the melting point. The melting transition is manifested by abrupt changes in the crystal volume, lattice energy, polymer conformation, and dynamical properties. In the premelting stage, the crystal lattice structure and backbone orientation of the polymer chains are retained but with the onset of weakening of long-range helical order and interchain packing of polymers perpendicular to the fibre axis of the crystal. The premelting also marks the onset of conformational defects and anisotropic solid-state diffusion of polymers along the fibre axis. The present study underscores the importance of the interplay between intermolecular packing, interactions, and conformational dynamics at the atomic level in determining the macroscopic melting behavior of polymer crystals.

HIV-1 DNA predicts disease progression and post-treatment virological control

PubMed Central

Williams, James P; Hurst, Jacob; Stöhr, Wolfgang; Robinson, Nicola; Brown, Helen; Fisher, Martin; Kinloch, Sabine; Cooper, David; Schechter, Mauro; Tambussi, Giuseppe; Fidler, Sarah; Carrington, Mary; Babiker, Abdel; Weber, Jonathan

2014-01-01

In HIV-1 infection, a population of latently infected cells facilitates viral persistence despite antiretroviral therapy (ART). With the aim of identifying individuals in whom ART might induce a period of viraemic control on stopping therapy, we hypothesised that quantification of the pool of latently infected cells in primary HIV-1 infection (PHI) would predict clinical progression and viral replication following ART. We measured HIV-1 DNA in a highly characterised randomised population of individuals with PHI. We explored associations between HIV-1 DNA and immunological and virological markers of clinical progression, including viral rebound in those interrupting therapy. In multivariable analyses, HIV-1 DNA was more predictive of disease progression than plasma viral load and, at treatment interruption, predicted time to plasma virus rebound. HIV-1 DNA may help identify individuals who could safely interrupt ART in future HIV-1 eradication trials. Clinical trial registration: ISRCTN76742797 and EudraCT2004-000446-20 DOI: http://dx.doi.org/10.7554/eLife.03821.001 PMID:25217531

A novel typing method for Listeria monocytogenes using high-resolution melting analysis (HRMA) of tandem repeat regions.

PubMed

Ohshima, Chihiro; Takahashi, Hajime; Iwakawa, Ai; Kuda, Takashi; Kimura, Bon

2017-07-17

Listeria monocytogenes, which is responsible for causing food poisoning known as listeriosis, infects humans and animals. Widely distributed in the environment, this bacterium is known to contaminate food products after being transmitted to factories via raw materials. To minimize the contamination of products by food pathogens, it is critical to identify and eliminate factory entry routes and pathways for the causative bacteria. High resolution melting analysis (HRMA) is a method that takes advantage of differences in DNA sequences and PCR product lengths that are reflected by the disassociation temperature. Through our research, we have developed a multiple locus variable-number tandem repeat analysis (MLVA) using HRMA as a simple and rapid method to differentiate L. monocytogenes isolates. While evaluating our developed method, the ability of MLVA-HRMA, MLVA using capillary electrophoresis, and multilocus sequence typing (MLST) was compared for their ability to discriminate between strains. The MLVA-HRMA method displayed greater discriminatory ability than MLST and MLVA using capillary electrophoresis, suggesting that the variation in the number of repeat units, along with mutations within the DNA sequence, was accurately reflected by the melting curve of HRMA. Rather than relying on DNA sequence analysis or high-resolution electrophoresis, the MLVA-HRMA method employs the same process as PCR until the analysis step, suggesting a combination of speed and simplicity. The result of MLVA-HRMA method is able to be shared between different laboratories. There are high expectations that this method will be adopted for regular inspections at food processing facilities in the near future. Copyright © 2017. Published by Elsevier B.V.

On the origin of the decrease in stability of the DNA hairpin d(GCGAAGC) on complexation with aromatic drugs.

PubMed

Kostjukov, V V; Lantushenko, A O; Davies, D B; Evstigneev, M P

2007-08-01

Molecular dynamics simulations of drug-DNA complexes have been carried out in order to explain the experimentally observed decrease in thermal stability of the DNA hairpin d(GCGAAGC) on binding the aromatic drug molecules, daunomycin, ethidium bromide, novantrone and proflavine. This complexation behavior is in contrast to the stabilizing effect of the same aromatic drug molecules on DNA duplexes. Analysis of the energy parameters and the hydration properties of the complexes shows that the main factor correlating with the decrease in melting temperatures of the drug-hairpin complexes is the number of water bridges, with a reduction of at least 40% on ligand binding.

Mechanical control of Renilla luciferase.

PubMed

Tseng, Chiao-Yu; Zocchi, Giovanni

2013-08-14

We report experiments where the activity of the enzyme luciferase from Renilla reniformis is controlled through a DNA spring attached to the enzyme. In the wake of previous work on kinases, these results establish that mechanical stress applied through the DNA springs is indeed a general method for the artificial control of enzymes, and for the quantitative study of mechano-chemical coupling in these molecules. We also show proof of concept of the luciferase construct as a sensitive molecular probe, detecting a specific DNA target sequence in an easy, one-step, homogeneous assay, as well as SNP detection without melting curve analysis.

Crystallization of DNA-coated colloids

PubMed Central

Wang, Yu; Wang, Yufeng; Zheng, Xiaolong; Ducrot, Étienne; Yodh, Jeremy S.; Weck, Marcus; Pine, David J.

2015-01-01

DNA-coated colloids hold great promise for self-assembly of programmed heterogeneous microstructures, provided they not only bind when cooled below their melting temperature, but also rearrange so that aggregated particles can anneal into the structure that minimizes the free energy. Unfortunately, DNA-coated colloids generally collide and stick forming kinetically arrested random aggregates when the thickness of the DNA coating is much smaller than the particles. Here we report DNA-coated colloids that can rearrange and anneal, thus enabling the growth of large colloidal crystals from a wide range of micrometre-sized DNA-coated colloids for the first time. The kinetics of aggregation, crystallization and defect formation are followed in real time. The crystallization rate exhibits the familiar maximum for intermediate temperature quenches observed in metallic alloys, but over a temperature range smaller by two orders of magnitude, owing to the highly temperature-sensitive diffusion between aggregated DNA-coated colloids. PMID:26078020

DNA nanostructure-based fluorescence thermometer with silver nanoclusters

NASA Astrophysics Data System (ADS)

Bu, Congcong; Mu, Lixuan; Cao, Xingxing; Chen, Min; She, Guangwei; Shi, Wensheng

2018-07-01

DNA nanostructure-based fluorescence thermometers were fabricated by linking fluorescent silver nanoclusters (AgNCs) and guanine-rich（G-rich）DNA chains via a thermally sensitive DNA stem-loop at terminals 5‧ and 3‧. Variations of temperature alter the distance between the AgNCs and G-rich DNA chain, affecting the interaction between them. As a result, the intensity of fluorescence emission from the AgNCs at 636 nm can be sensitively modulated. It was found that the intensity of such red emission is more temperature sensitive than the equivalent green emission at 543 nm; sensitivity of ‑3.6%/°C was achieved. Through variation of the melting temperature of the DNA stem-loop, the response temperature range of the thermometers could be readily adjusted. Novel DNA nanostructure-based fluorescence thermometers as described in this work are anticipated to be able to measure the temperature of biological systems at small scales—even a single cell.

Tetrahelical monomolecular architecture of DNA: a new building block for nanotechnology.

PubMed

Kankia, Besik

2014-06-12

DNA nanotechnology typically relies on Watson-Crick base pairing as both a recognition and structural element. This limits structural versatility and introduces errors during self-assembly of DNA. Guanine (G) quartet motifs show promise as an alternative to DNA duplexes, but the synthesis of long, precisely defined molecules is a significant challenge. Here we demonstrate a continuous tetrahelical DNA architecture capable of programmed self-assembly. We report that the homopolymer consisting of (G3T)3G3 monomeric units has the capability to fold into a monomolecular DNA tetrahelix with unprecedented speed and stability. For instance, in the presence of 1 mM K(+) ions the dimer, (G3T)2, folds readily and melts above 100 °C. These findings have the potential to revolutionize DNA nanotechnology by introducing fast and error-free self-assembly of long and extraordinarily stable molecules.

DNA nanostructure-based fluorescence thermometer with silver nanoclusters.

PubMed

Bu, Congcong; Mu, Lixuan; Cao, XIngxing; Chen, Min; She, Guangwei; Shi, Wensheng

2018-04-27

Linking the fluorescent silver nanoclusters (AgNCs) and guanine-rich（G-rich）DNA chains by the thermal sensitive DNA stem-loop at teminal 5' and 3', DNA nanostructure-based fluorescence thermometers were fabricated. The variations of the temperature alter the distance between AgNCs and G-rich DNA chain, which could affect the interaction between them. As a result, the intensity of fluorescence emission from AgNCs at 636 nm can be sensitively modulated. It was found that such red emission is more sensitive to the temperature comparing with its intrinsic green emission at 543 nm, and sensitivity of -3.6%/℃ was achieved. Varying the melting temperature of the DNA stem-loop could readily adjust the response temperature range of thermometers. Novel DNA nanostructure-based fluorescence thermometers in this work could be anticipated to measure the temperature of biological system, even a single cell. © 2018 IOP Publishing Ltd.

Adakitic (tonalitic-trondhjemitic) magmas resulting from eclogite decompression and dehydration melting during exhumation in response to continental collision

NASA Astrophysics Data System (ADS)

Song, Shuguang; Niu, Yaoling; Su, Li; Wei, Chunjing; Zhang, Lifei

2014-04-01

Modern adakite or adakitic rocks are thought to result from partial melting of younger and thus warmer subducting ocean crust in subduction zones, with the melt interacting with or without mantle wedge peridotite during ascent, or from melting of thickened mafic lower crust. Here we show that adakitic (tonalitic-trondhjemitic) melts can also be produced by eclogite decompression during exhumation of subducted and metamorphosed oceanic/continental crust in response to continental collision, as exemplified by the adakitic rocks genetically associated with the early Paleozoic North Qaidam ultra-high pressure metamorphic (UHPM) belt on the northern margin of the Greater Tibetan Plateau. We present field evidence for partial melting of eclogite and its products, including adakitic melt, volumetrically significant plutons evolved from the melt, cumulate rocks precipitated from the melt, and associated granulitic residues. This “adakitic assemblage” records a clear progression from eclogite decompression and heating to partial melting, to melt fractionation and ascent/percolation in response to exhumation of the UHPM package. The garnetite and garnet-rich layers in the adakitic assemblage are of cumulate origin from the adakitic melt at high pressure, and accommodate much of the Nb-Ta-Ti. Zircon SHRIMP U-Pb dating shows that partial melting of the eclogite took place at ∼435-410 Ma, which postdates the seafloor subduction (>440 Ma) and temporally overlaps the UHPM (∼440-425 Ma). While the geological context and the timing of adakite melt formation we observe differ from the prevailing models, our observations and documentations demonstrate that eclogite melting during UHPM exhumation may be important in contributing to crustal growth.

Effect of Concentration on the Formation of Molecular Hybrids from T4 DNA

PubMed Central

Kozinski, Andrzej W.; Beer, Michael

1962-01-01

When the thymine of T4 DNA is replaced by 5-BU the melting temperature of T4 DNA is increased from about 83° to about 93°C. Heating and slow cooling of T4 DNA at concentrations of about 30 μg/ml leads to aggregates which consist of several polynucleotide chains which appear in the electron microscope as a branched structure. The aggregates have regions which are true hybrids. When the concentration of T4 DNA is lowered to less than 1 μg/ml the products of hybridization are not aggregates but have the morphology of native DNA molecules and the density labels are distributed as expected from the fusing of two chains of approximately equal length. ImagesFigure 6Figure 7Figure 8 PMID:14459098

DIVA V2.0

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

CHEN, JOANNA; SIMIRENKO, LISA; TAPASWI, MANJIRI

The DIVA software interfaces a process in which researchers design their DNA with a web-based graphical user interface, submit their designs to a central queue, and a few weeks later receive their sequence-verified clonal constructs. Each researcher independently designs the DNA to be constructed with a web-based BioCAD tool, and presses a button to submit their designs to a central queue. Researchers have web-based access to their DNA design queues, and can track the progress of their submitted designs as they progress from "evaluation", to "waiting for reagents", to "in progress", to "complete". Researchers access their completed constructs through themore » central DNA repository. Along the way, all DNA construction success/failure rates are captured in a central database. Once a design has been submitted to the queue, a small number of dedicated staff evaluate the design for feasibility and provide feedback to the responsible researcher if the design is either unreasonable (e.g., encompasses a combinatorial library of a billion constructs) or small design changes could significantly facilitate the downstream implementation process. The dedicated staff then use DNA assembly design automation software to optimize the DNA construction process for the design, leveraging existing parts from the DNA repository where possible and ordering synthetic DNA where necessary. SynTrack software manages the physical locations and availability of the various requisite reagents and process inputs (e.g., DNA templates). Once all requisite process inputs are available, the design progresses from "waiting for reagents" to "in progress" in the design queue. Human-readable and machine-parseable DNA construction protocols output by the DNA assembly design automation software are then executed by the dedicated staff exploiting lab automation devices wherever possible. Since the all employed DNA construction methods are sequence-agnostic, standardized (utilize the same enzymatic master mixes and reaction conditions), completely independent DNA construction tasks can be aggregated into the same multi-well plates and pursued in parallel. The resulting sets of cloned constructs can then be screened by high-throughput next-gen sequencing platforms for sequence correctness. A combination of long read-length (e.g., PacBio) and paired-end read platforms (e.g., Illumina) would be exploited depending the particular task at hand (e.g., PacBio might be sufficient to screen a set of pooled constructs with significant gene divergence). Post sequence verification, designs for which at least one correct clone was identified will progress to a "complete" status, while designs for which no correct clones wereidentified will progress to a "failure" status. Depending on the failure mode (e.g., no transformants), and how many prior attempts/variations of assembly protocol have been already made for a given design, subsequent attempts may be made or the design can progress to a "permanent failure" state. All success and failure rate information will be captured during the process, including at which stage a given clonal construction procedure failed (e.g., no PCR product) and what the exact failure was (e.g. assembly piece 2 missing). This success/failure rate data can be leveraged to refine the DNA assembly design process.« less

The mechanism of the emergence of distinct overstretched DNA states

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

Zhu, You-Liang; Sun, Zhao-Yan, E-mail: zysun@ciac.ac.cn; Lu, Zhong-Yuan

Although multiple overstretched DNA states were identified in experiments, the mechanism of the emergence of distinct states is still unclear. Molecular dynamics simulation is an ideal tool to clarify the mechanism, but the force loading rates in stretching achieved by conventional all-atom DNA models are much faster, which essentially affect overstretching states. We employed a modified coarse-grained DNA model with an unprecedented low loading rate in simulations to study the overstretching transitions of end-opened double-stranded DNA. We observed two-strand peeling off for DNA with low stability and the S-DNA with high stability under tension. By introducing a melting-forbidden model whichmore » prevents base-pair breaking, we still observed the overstretching transition induced by the formation of S-DNA due to the change of dihedral angle. Hence, we confirmed that the competition between the two strain-softening manners, i.e., base-pair breaking and dihedral angle variation, results in the emergence of distinct overstretched DNA states.« less

Real-time electrochemical LAMP: a rational comparative study of different DNA intercalating and non-intercalating redox probes.

PubMed

Martin, Alexandra; Bouffier, Laurent; Grant, Kathryn B; Limoges, Benoît; Marchal, Damien

2016-06-20

We present a comparative study of ten redox-active probes for use in real-time electrochemical loop-mediated isothermal amplification (LAMP). Our main objectives were to establish the criteria that need to be fulfilled for minimizing some of the current limitations of the technique and to provide future guidelines in the search for ideal redox reporters. To ensure a reliable comparative study, each redox probe was tested under similar conditions using the same LAMP reaction and the same entirely automatized custom-made real-time electrochemical device (designed for electrochemically monitoring in real-time and in parallel up to 48 LAMP samples). Electrochemical melt curve analyses were recorded immediately at the end of each LAMP reaction. Our results show that there are a number of intercalating and non-intercalating redox compounds suitable for real-time electrochemical LAMP and that the best candidates are those able to intercalate strongly into ds-DNA but not too much to avoid inhibition of the LAMP reaction. The strongest intercalating redox probes were finally shown to provide higher LAMP sensitivity, speed, greater signal amplitude, and cleaner-cut DNA melting curves than the non-intercalating molecules.

Recognition of RNA by amide modified backbone nucleic acids: molecular dynamics simulations of DNA-RNA hybrids in aqueous solution.

PubMed

Nina, Mafalda; Fonné-Pfister, Raymonde; Beaudegnies, Renaud; Chekatt, Habiba; Jung, Pierre M J; Murphy-Kessabi, Fiona; De Mesmaeker, Alain; Wendeborn, Sebastian

2005-04-27

Thermodynamic and structural properties of a chemically modified DNA-RNA hybrid in which a phosphodiester linkage is replaced by a neutral amide-3 linkage (3'-CH(2)-CONH-5') were investigated using UV melting experiments, molecular dynamics simulations in explicit water, and continuum solvent models. van't Hoff analysis of the experimental UV melting curves suggests that the significant increase of the thermodynamic stability of a 15-mer DNA-RNA with seven alternated amide-3 modifications (+11 degrees C) is mainly due to an increased binding enthalpy. To further evaluate the origin in the observed affinities differences, the electrostatic contribution to the binding free energy was calculated by solving the Poisson-Boltzmann equation numerically. The nonelectrostatic contribution was estimated as the product of a hydrophobic surface tension coefficient and the surface area that is buried upon double strand formation. Structures were taken from 10 ns molecular dynamics simulations computed in a consistent fashion using explicit solvent, counterions, and the particle-mesh Ewald procedure. The present preliminary thermodynamic study suggests that the favorable binding free energy of the amide-3 DNA single strand to the complementary RNA is equally driven by electrostatic and nonpolar contributions to the binding compared to their natural analogues. In addition, molecular dynamics simulations in explicit water were performed on an amide-3 DNA single strand and the corresponding natural DNA. Results from the conformations cluster analysis of the simulated amide-3 DNA single strand ensembles suggest that the 25% of the population sampled within 10 ns has a pre-organized conformation where the sugar C3' endo pucker is favored at the 3'-flanking nucleotides. These structural and thermodynamic features contribute to the understanding of the observed increased affinities of the amide-3 DNA-RNA hybrids at the microscopic level.

High resolution DNA melting analysis: an application for prenatal control of alpha-thalassemia.

PubMed

Sirichotiyakul, Supatra; Wanapirak, Chanane; Saetung, Rattika; Sanguansermsri, Torpong

2010-04-01

To report the use of real-time gap-PCR using SYTO9 with high-resolution melting analysis (HRMA) in prenatal diagnosis of alpha-thalassemia 1. Real-time gap-PCR using SYTO9 with HRMA was performed in 33 DNA samples from chorionic villi sampling (8 normal, 16 heterozygous, and 9 homozygous) to determine the alpha-thalassemia 1 gene [normal and Southeast Asia (-SEA) allele]. The dissociation curve analysis in normal and - SEA allele gave a peak of T(m) at 91.80 +/- 0.14 degrees C and 88.67 +/- 0.08 degrees C, respectively. Normal genotype and homozygous alpha-thalassemia 1 showed a single peak of T(m) that corresponded to their alleles. The heterozygotes gave both peaks with higher normal peak and smaller - SEA peak. Thirty one samples showed consistent results with the conventional gap-PCR. Two samples with ambiguous results were confirmed to be maternal DNA contamination on real-time quantitative PCR and microsatellite assay. HRMA from both samples showed similar pattern to that of heterozygotes. However, they showed much smaller normal peak compared with the - SEA peak, which is in contrast to those of heterozygotes and can readily be distinguished. HRMA with SYTO9 is feasible for prenatal diagnosis of alpha-thalassemia. It had potential advantage of prompt detection maternal DNA contamination. Copyright (c) 2010 John Wiley & Sons, Ltd.

Direct non transcriptional role of NF-Y in DNA replication.

PubMed

Benatti, Paolo; Belluti, Silvia; Miotto, Benoit; Neusiedler, Julia; Dolfini, Diletta; Drac, Marjorie; Basile, Valentina; Schwob, Etienne; Mantovani, Roberto; Blow, J Julian; Imbriano, Carol

2016-04-01

NF-Y is a heterotrimeric transcription factor, which plays a pioneer role in the transcriptional control of promoters containing the CCAAT-box, among which genes involved in cell cycle regulation, apoptosis and DNA damage response. The knock-down of the sequence-specific subunit NF-YA triggers defects in S-phase progression, which lead to apoptotic cell death. Here, we report that NF-Y has a critical function in DNA replication progression, independent from its transcriptional activity. NF-YA colocalizes with early DNA replication factories, its depletion affects the loading of replisome proteins to DNA, among which Cdc45, and delays the passage from early to middle-late S phase. Molecular combing experiments are consistent with a role for NF-Y in the control of fork progression. Finally, we unambiguously demonstrate a direct non-transcriptional role of NF-Y in the overall efficiency of DNA replication, specifically in the DNA elongation process, using a Xenopus cell-free system. Our findings broaden the activity of NF-Y on a DNA metabolism other than transcription, supporting the existence of specific TFs required for proper and efficient DNA replication. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Evaluation of a PCR melting profile method for intraspecies differentiation of Trichophyton rubrum and Trichophyton interdigitale.

PubMed

Leibner-Ciszak, Justyna; Dobrowolska, Anita; Krawczyk, Beata; Kaszuba, Aleksandra; Staczek, Paweł

2010-02-01

In order to identify the source of infections caused by dermatophytes, as well as the pathogen transmission pathway, there is a need to determine methods that allow detailed genetic differentiation of the strains within the dermatophyte genera. In this work, a PCR melting profile (PCR-MP) technique based on the ligation of adaptors and the difference in melting temperatures of DNA restriction fragments was used for the first time for intraspecies genotyping of dermatophytes. Clinical isolates and reference strains of dermatophytes isolated from skin, scalp, toenails and fingernails were used for this study. PCR-MP and random amplification of polymorphic DNA (RAPD) were used to type 11 isolates of Trichophyton rubrum, 40 isolates of Trichophyton interdigitale and 14 isolates of Microsporum canis. The results distinguished five types (containing one subtype) characteristic for T. rubrum and seven types characteristic for T. interdigitale using the PCR-MP technique. Analysis conducted using RAPD revealed five types for T. rubrum and four types for T. interdigitale isolates. No differentiation was observed for the M. canis isolates with either method. These results demonstrate that PCR-MP is a reliable method for the differentiation of T. rubrum and T. interdigitale strains and yields a discriminatory power that is at least equal to that of RAPD.

High-resolution melting analysis for detection of MYH9 mutations.

PubMed

Provaznikova, Dana; Kumstyrova, Tereza; Kotlin, Roman; Salaj, Peter; Matoska, Vaclav; Hrachovinova, Ingrid; Rittich, Simon

2008-09-01

May-Hegglin anomaly (MHA), Sebastian (SBS), Fechtner (FTNS) and Epstein (EPS) syndromes are rare autosomal dominant disorders with giant platelets and thrombocytopenia. Other manifestations of these disorders are combinations of the presence of granulocyte inclusions and deafness, cataracts and renal failure. Currently, MHA, SBS, FTNS and EPS are considered to be distinct clinical manifestation of a single illness caused by mutations of the MYH9 gene encoding the heavy chain of non-muscle myosin IIA (NMMHC-IIA). As the MYH9 gene has a high number of exons, it takes much time and material to use this method for the detection of MYH9 mutations. Recently, a new method has been introduced for scanning DNA mutations without the need for direct sequencing: high-resolution melting analysis (HRMA). Mutation detection with HRMA relies on the intercalation of the specific dye (LC Green plus) in double-strand DNA and fluorescence monitoring of PCR product melting profiles. In our study, we optimized the conditions and used HRMA for rapid screening of mutations in all MYH9 exons in seven affected individuals from four unrelated families with suspected MYH9 disorders. Samples identified by HRMA as positive for the mutation were analysed by direct sequencing. HRMA saved us over 85% of redundant sequencing.

Characterization of Microbial Communities Found in Bioreactor Effluent

NASA Technical Reports Server (NTRS)

Flowe, Candice

2013-01-01

The purpose of this investigation was to examine microbial communities of simulated wastewater effluent from hollow fiber membrane bioreactors collected from the Space Life Science Laboratory and Texas Technical University. Microbes were characterized using quantitative polymerase chain reaction where a total count of bacteria and fungi were determined. The primers that were used to determine the total count of bacteria and fungi were targeted for 16S rDNA genes and the internal transcribed spacer, respectively. PCR products were detected with SYBR Green I fluorescent dye and a melting curve analysis was performed to identify unique melt profiles resulting from DNA sequence variations from each species of the community. Results from both the total bacteria and total fungi count assays showed that distinct populations were present in isolates from these bioreactors. This was exhibited by variation in the number of peaks observed on the melting curve analysis graph. Further analysis of these results using species-specific primers will shed light on exactly which microbes are present in these effluents. Information gained from this study will enable the design of a system that can efficiently monitor microbes that play a role in the biogeochemical cycling of nitrogen in wastewater on the International Space Station to assist in the design of a sustainable system capable of converting this nutrient.

High Resolution Melting (HRM) for High-Throughput Genotyping-Limitations and Caveats in Practical Case Studies.

PubMed

Słomka, Marcin; Sobalska-Kwapis, Marta; Wachulec, Monika; Bartosz, Grzegorz; Strapagiel, Dominik

2017-11-03

High resolution melting (HRM) is a convenient method for gene scanning as well as genotyping of individual and multiple single nucleotide polymorphisms (SNPs). This rapid, simple, closed-tube, homogenous, and cost-efficient approach has the capacity for high specificity and sensitivity, while allowing easy transition to high-throughput scale. In this paper, we provide examples from our laboratory practice of some problematic issues which can affect the performance and data analysis of HRM results, especially with regard to reference curve-based targeted genotyping. We present those examples in order of the typical experimental workflow, and discuss the crucial significance of the respective experimental errors and limitations for the quality and analysis of results. The experimental details which have a decisive impact on correct execution of a HRM genotyping experiment include type and quality of DNA source material, reproducibility of isolation method and template DNA preparation, primer and amplicon design, automation-derived preparation and pipetting inconsistencies, as well as physical limitations in melting curve distinction for alternative variants and careful selection of samples for validation by sequencing. We provide a case-by-case analysis and discussion of actual problems we encountered and solutions that should be taken into account by researchers newly attempting HRM genotyping, especially in a high-throughput setup.

High Resolution Melting (HRM) for High-Throughput Genotyping—Limitations and Caveats in Practical Case Studies

PubMed Central

Słomka, Marcin; Sobalska-Kwapis, Marta; Wachulec, Monika; Bartosz, Grzegorz

2017-01-01

High resolution melting (HRM) is a convenient method for gene scanning as well as genotyping of individual and multiple single nucleotide polymorphisms (SNPs). This rapid, simple, closed-tube, homogenous, and cost-efficient approach has the capacity for high specificity and sensitivity, while allowing easy transition to high-throughput scale. In this paper, we provide examples from our laboratory practice of some problematic issues which can affect the performance and data analysis of HRM results, especially with regard to reference curve-based targeted genotyping. We present those examples in order of the typical experimental workflow, and discuss the crucial significance of the respective experimental errors and limitations for the quality and analysis of results. The experimental details which have a decisive impact on correct execution of a HRM genotyping experiment include type and quality of DNA source material, reproducibility of isolation method and template DNA preparation, primer and amplicon design, automation-derived preparation and pipetting inconsistencies, as well as physical limitations in melting curve distinction for alternative variants and careful selection of samples for validation by sequencing. We provide a case-by-case analysis and discussion of actual problems we encountered and solutions that should be taken into account by researchers newly attempting HRM genotyping, especially in a high-throughput setup. PMID:29099791

A novel HRM assay for the simultaneous detection and differentiation of eight poxviruses of medical and veterinary importance

PubMed Central

Gelaye, Esayas; Mach, Lukas; Kolodziejek, Jolanta; Grabherr, Reingard; Loitsch, Angelika; Achenbach, Jenna E.; Nowotny, Norbert; Diallo, Adama; Lamien, Charles Euloge

2017-01-01

Poxviruses belonging to the Orthopoxvirus, Capripoxvirus and Parapoxvirus genera share common host species and create a challenge for diagnosis. Here, we developed a novel multiplex PCR method for the simultaneous detection and differentiation of eight poxviruses, belonging to three genera: cowpox virus (CPXV) and camelpox virus (CMLV) [genus Orthopoxvirus]; goatpox virus (GTPV), sheeppox virus (SPPV) and lumpy skin disease virus (LSDV) [genus Capripoxvirus]; orf virus (ORFV), pseudocowpox virus (PCPV) and bovine papular stomatitis virus (BPSV) [genus Parapoxvirus]. The assay is based on high-resolution melting curve analysis (HRMCA) of PCR amplicons produced using genus specific primer pairs and dsDNA binding dye. Differences in fragment size and GC content were used as discriminating power. The assay generated three well separated melting regions for each genus and provided additional intra-genus genotyping allowing the differentiation of the eight poxviruses based on amplicon melting temperature. Out of 271 poxviral DNA samples tested: seven CPXV, 25 CMLV, 42 GTPV, 20 SPPV, 120 LSDV, 33 ORFV, 20 PCPV and two BPSV were detected; two samples presented co-infection with CMLV and PCPV. The assay provides a rapid, sensitive, specific and cost-effective method for the detection of pox diseases in a broad range of animal species and humans. PMID:28216667

Massively parallel digital high resolution melt for rapid and absolutely quantitative sequence profiling

NASA Astrophysics Data System (ADS)

Velez, Daniel Ortiz; Mack, Hannah; Jupe, Julietta; Hawker, Sinead; Kulkarni, Ninad; Hedayatnia, Behnam; Zhang, Yang; Lawrence, Shelley; Fraley, Stephanie I.

2017-02-01

In clinical diagnostics and pathogen detection, profiling of complex samples for low-level genotypes represents a significant challenge. Advances in speed, sensitivity, and extent of multiplexing of molecular pathogen detection assays are needed to improve patient care. We report the development of an integrated platform enabling the identification of bacterial pathogen DNA sequences in complex samples in less than four hours. The system incorporates a microfluidic chip and instrumentation to accomplish universal PCR amplification, High Resolution Melting (HRM), and machine learning within 20,000 picoliter scale reactions, simultaneously. Clinically relevant concentrations of bacterial DNA molecules are separated by digitization across 20,000 reactions and amplified with universal primers targeting the bacterial 16S gene. Amplification is followed by HRM sequence fingerprinting in all reactions, simultaneously. The resulting bacteria-specific melt curves are identified by Support Vector Machine learning, and individual pathogen loads are quantified. The platform reduces reaction volumes by 99.995% and achieves a greater than 200-fold increase in dynamic range of detection compared to traditional PCR HRM approaches. Type I and II error rates are reduced by 99% and 100% respectively, compared to intercalating dye-based digital PCR (dPCR) methods. This technology could impact a number of quantitative profiling applications, especially infectious disease diagnostics.

Fluorescence resonance energy transfer-based real-time polymerase chain reaction method without DNA extraction for the genotyping of F5, F2, F12, MTHFR, and HFE.

PubMed

Martinez-Serra, Jordi; Robles, Juan; Nicolàs, Antoni; Gutierrez, Antonio; Ros, Teresa; Amat, Juan Carlos; Alemany, Regina; Vögler, Oliver; Abelló, Aina; Noguera, Aina; Besalduch, Joan

2014-01-01

Blood samples are extensively used for the molecular diagnosis of many hematological diseases. The daily practice in a clinical laboratory of molecular diagnosis in hematology involves using a variety of techniques, based on the amplification of nucleic acids. Current methods for polymerase chain reaction (PCR) use purified genomic DNA, mostly isolated from total peripheral blood cells or white blood cells (WBC). In this paper we describe a real-time fluorescence resonance energy transfer-based method for genotyping directly from blood cells. Our strategy is based on an initial isolation of the WBCs, allowing the removal of PCR inhibitors, such as the heme group, present in the erythrocytes. Once the erythrocytes have been lysed, in the LightCycler(®) 2.0 Instrument, we perform a real-time PCR followed by a melting curve analysis for different genes (Factors 2, 5, 12, MTHFR, and HFE). After testing 34 samples comparing the real-time crossing point (CP) values between WBC (5×10(6) WBC/mL) and purified DNA (20 ng/μL), the results for F5 Leiden were as follows: CP mean value for WBC was 29.26±0.566 versus purified DNA 24.79±0.56. Thus, when PCR was performed from WBC (5×10(6) WBC/mL) instead of DNA (20 ng/μL), we observed a delay of about 4 cycles. These small differences in CP values were similar for all genes tested and did not significantly affect the subsequent analysis by melting curves. In both cases the fluorescence values were high enough, allowing a robust genotyping of all these genes without a previous DNA purification/extraction.

Development of a novel method to determine the concentration of heavy metal cations: application of the specific interaction between heavy metal cation and mismatch DNA base pair.

PubMed

Kozasa, Tetsuo; Miyakawa, Yukako; Fukushi, Miyako; Ono, Akira; Torigoe, Hidetaka

2009-01-01

We have already found that Hg(II) cation specifically binds to T:T mismatch base pair in heteroduplex DNA, which increases the melting temperature of heteroduplex DNA involving T:T mismatch base pair by about 4 degrees C. We have also found that Ag(I) cation specifically binds to C:C mismatch base pair in heteroduplex DNA, which increases the melting temperature of heteroduplex DNA involving C:C mismatch base pair by about 4 degrees C. Using the specific interaction, we developed a novel sensor to determine the concentration of each of Hg(II) and Ag(I) cation. The sensor is composed of a dye-labelled T-rich or C-rich DNA oligonucleotide, F2T6W2D: 5'-Fam-T(2)CT(2)CT(2)C(4)T(2)GT(2)GT(2)-Dabcyl-3' or F2C6W2D: 5'-Fam-C(2)TC(2)TC(2)T(4)C(2)AC(2)AC(2)-Dabcyl-3', where 6-carboxyfluorescein (Fam) is a fluorophore and Dabcyl is a quencher. The addition of Hg(II) cation decreased the intensity of Fam emission of F2T6W2D at 520 nm in a concentration-dependent manner. Also, the addition of Ag(I) cation decreased the intensity of Fam emission of F2C6W2D at 520 nm in a concentration-dependent manner. We conclude that, using the novel sensor developed in this study, the concentration of each of Hg(II) and Ag(I) cation can be determined from the intensity of Fam emission at 520 nm.

High-resolution melting PCR assay, applicable for diagnostics and screening studies, allowing detection and differentiation of several Babesia spp. infecting humans and animals.

PubMed

Rozej-Bielicka, Wioletta; Masny, Aleksander; Golab, Elzbieta

2017-10-01

The goal of the study was to design a single tube PCR test for detection and differentiation of Babesia species in DNA samples obtained from diverse biological materials. A multiplex, single tube PCR test was designed for amplification of approximately 400 bp region of the Babesia 18S rRNA gene. Universal primers were designed to match DNA of multiple Babesia spp. and to have low levels of similarity to DNA sequences of other intracellular protozoa and Babesia hosts. The PCR products amplified from Babesia DNA isolated from human, dog, rodent, deer, and tick samples were subjected to high-resolution melting analysis for Babesia species identification. The designed test allowed detection and differentiation of four Babesia species, three zoonotic (B. microti, B. divergens, B. venatorum) and one that is generally not considered zoonotic-Babesia canis. Both detection and identification of all four species were possible based on the HRM curves of the PCR products in samples obtained from the following: humans, dogs, rodents, and ticks. No cross-reactivity with DNA of Babesia hosts or Plasmodium falciparum and Toxoplasma gondii was observed. The lack of cross-reactivity with P. falciparum DNA might allow using the assay in endemic malaria areas. The designed assay is the first PCR-based test for detection and differentiation of several Babesia spp. of medical and veterinary importance, in a single tube reaction. The results of the study show that the designed assay for Babesia detection and identification could be a practical and inexpensive tool for diagnostics and screening studies of diverse biological materials.

Rapid detection of Wuchereria bancrofti and Brugia malayi in mosquito vectors (Diptera: Culicidae) using a real-time fluorescence resonance energy transfer multiplex PCR and melting curve analysis.

PubMed

Intapan, Pewpan M; Thanchomnang, Tongjit; Lulitanond, Viraphong; Maleewong, Wanchai

2009-01-01

We developed a single-step real-time fluorescence resonance energy transfer (FRET) multiplex polymerase chain reaction (PCR) merged with melting curve analysis for the detection of Wuchereria bancrofti and Brugia malayi DNA in blood-fed mosquitoes. Real-time FRET multiplex PCR is based on fluorescence melting curve analysis of a hybrid of amplicons generated from two families of repeated DNA elements: the 188 bp SspI repeated sequence, specific to W. bancrofti, and the 153-bp HhaI repeated sequence, specific to the genus Brugia and two pairs of specific fluorophore-labeled probes. Both W. bancrofti and B. malayi can be differentially detected in infected vectors by this process through their different fluorescence channel and melting temperatures. The assay could distinguish both human filarial DNAs in infected vectors from the DNAs of Dirofilaria immitis- and Plasmodium falciparum-infected human red blood cells and noninfected mosquitoes and human leukocytes. The technique showed 100% sensitivity and specificity and offers a rapid and reliable procedure for differentially identifying lymphatic filariasis. The introduced real-time FRET multiplex PCR can reduce labor time and reagent costs and is not prone to carry over contamination. The test can be used to screen mosquito vectors in endemic areas and therefore should be a useful diagnostic tool for the evaluation of infection rate of the mosquito populations and for xenomonitoring in the community after eradication programs such as the Global Program to Eliminate Lymphatic Filariasis.

Rapid detection of Echinococcus species by a high-resolution melting (HRM) approach.

PubMed

Santos, Guilherme Brzoskowski; Espínola, Sergio Martín; Ferreira, Henrique Bunselmeyer; Margis, Rogerio; Zaha, Arnaldo

2013-11-14

High-resolution melting (HRM) provides a low-cost, fast and sensitive scanning method that allows the detection of DNA sequence variations in a single step, which makes it appropriate for application in parasite identification and genotyping. The aim of this work was to implement an HRM-PCR assay targeting part of the mitochondrial cox1 gene to achieve an accurate and fast method for Echinococcus spp. differentiation. For melting analysis, a total of 107 samples from seven species were used in this study. The species analyzed included Echinococcus granulosus (n = 41) and Echinococcus ortleppi (n = 50) from bovine, Echinococcus vogeli (n = 2) from paca, Echinococcus oligarthra (n = 3) from agouti, Echinococcus multilocularis (n = 6) from monkey and Echinococcus canadensis (n = 2) and Taenia hydatigena (n = 3) from pig. DNA extraction was performed, and a 444-bp fragment of the cox1 gene was amplified. Two approaches were used, one based on HRM analysis, and a second using SYBR Green Tm-based. In the HRM analysis, a specific profile for each species was observed. Although some species exhibited almost the same melting temperature (Tm) value, the HRM profiles could be clearly discriminated. The SYBR Green Tm-based analysis showed differences between E. granulosus and E. ortleppi and between E. vogeli and E. oligarthra. In this work, we report the implementation of HRM analysis to differentiate species of the genus Echinococcus using part of the mitochondrial gene cox1. This method may be also potentially applied to identify other species belonging to the Taeniidae family.

Rapid detection of Echinococcus species by a high-resolution melting (HRM) approach

PubMed Central

2013-01-01

Background High-resolution melting (HRM) provides a low-cost, fast and sensitive scanning method that allows the detection of DNA sequence variations in a single step, which makes it appropriate for application in parasite identification and genotyping. The aim of this work was to implement an HRM-PCR assay targeting part of the mitochondrial cox1 gene to achieve an accurate and fast method for Echinococcus spp. differentiation. Findings For melting analysis, a total of 107 samples from seven species were used in this study. The species analyzed included Echinococcus granulosus (n = 41) and Echinococcus ortleppi (n = 50) from bovine, Echinococcus vogeli (n = 2) from paca, Echinococcus oligarthra (n = 3) from agouti, Echinococcus multilocularis (n = 6) from monkey and Echinococcus canadensis (n = 2) and Taenia hydatigena (n = 3) from pig. DNA extraction was performed, and a 444-bp fragment of the cox1 gene was amplified. Two approaches were used, one based on HRM analysis, and a second using SYBR Green Tm-based. In the HRM analysis, a specific profile for each species was observed. Although some species exhibited almost the same melting temperature (Tm) value, the HRM profiles could be clearly discriminated. The SYBR Green Tm-based analysis showed differences between E. granulosus and E. ortleppi and between E. vogeli and E. oligarthra. Conclusions In this work, we report the implementation of HRM analysis to differentiate species of the genus Echinococcus using part of the mitochondrial gene cox1. This method may be also potentially applied to identify other species belonging to the Taeniidae family. PMID:24517106

Studies of interaction of emodin and DNA in the presence of ethidium bromide by spectroscopic method

NASA Astrophysics Data System (ADS)

Bi, Shuyun; Zhang, Hanqi; Qiao, Chunyu; Sun, Ying; Liu, Chunming

2008-01-01

Emodin interacting with deoxyribonucleic acid (DNA) has been studied by different spectroscopic techniques, such as fluorescence, ultraviolet and visible (UV-vis), and fourier transform infared (FT-IR) spectroscopies, using ethidium bromide (EB) as a fluorescence probe of DNA. The decrease in the fluorescence of DNA-EB system on addition of emodin shows that the fluorescence quenching of DNA-EB complex by emodin occurs. The binding constants of emodin with DNA in the presence of EB are 6.02 × 10 4, 9.20 × 10 4 and 1.17 × 10 5 L mol -1 at 20, 35 and 50 °C, respectively. FT-IR spectrum further suggests that both the phosphate groups and the bases of DNA react with emodin. The reaction of DNA with emodin in the presence of EB is affected by ionic strength and temperature. The values of melting temperature ( Tm) of DNA-EB complex and emodin-DNA-EB complexes were determined, respectively. From the experiment evidences, the major binding mode of emodin with DNA should be the groove binding.

The mechanics of granitoid systems and maximum entropy production rates.

PubMed

Hobbs, Bruce E; Ord, Alison

2010-01-13

A model for the formation of granitoid systems is developed involving melt production spatially below a rising isotherm that defines melt initiation. Production of the melt volumes necessary to form granitoid complexes within 10(4)-10(7) years demands control of the isotherm velocity by melt advection. This velocity is one control on the melt flux generated spatially just above the melt isotherm, which is the control valve for the behaviour of the complete granitoid system. Melt transport occurs in conduits initiated as sheets or tubes comprising melt inclusions arising from Gurson-Tvergaard constitutive behaviour. Such conduits appear as leucosomes parallel to lineations and foliations, and ductile and brittle dykes. The melt flux generated at the melt isotherm controls the position of the melt solidus isotherm and hence the physical height of the Transport/Emplacement Zone. A conduit width-selection process, driven by changes in melt viscosity and constitutive behaviour, operates within the Transport Zone to progressively increase the width of apertures upwards. Melt can also be driven horizontally by gradients in topography; these horizontal fluxes can be similar in magnitude to vertical fluxes. Fluxes induced by deformation can compete with both buoyancy and topographic-driven flow over all length scales and results locally in transient 'ponds' of melt. Pluton emplacement is controlled by the transition in constitutive behaviour of the melt/magma from elastic-viscous at high temperatures to elastic-plastic-viscous approaching the melt solidus enabling finite thickness plutons to develop. The system involves coupled feedback processes that grow at the expense of heat supplied to the system and compete with melt advection. The result is that limits are placed on the size and time scale of the system. Optimal characteristics of the system coincide with a state of maximum entropy production rate. This journal is © 2010 The Royal Society

HYBRIDIZATION PROPERTIES OF DNA SEQUENCES DIRECTING THE SYNTHESIS OF MESSENGER RNA AND HETEROGENEOUS NUCLEAR RNA

PubMed Central

Greenberg, Jay R.; Perry, Robert P.

1971-01-01

The relationship of the DNA sequences from which polyribosomal messenger RNA (mRNA) and heterogeneous nuclear RNA (NRNA) of mouse L cells are transcribed was investigated by means of hybridization kinetics and thermal denaturation of the hybrids. Hybridization was performed in formamide solutions at DNA excess. Under these conditions most of the hybridizing mRNA and NRNA react at values of Dot (DNA concentration multiplied by time) expected for RNA transcribed from the nonrepeated or rarely repeated fraction of the genome. However, a fraction of both mRNA and NRNA hybridize at values of Dot about 10,000 times lower, and therefore must be transcribed from highly redundant DNA sequences. The fraction of NRNA hybridizing to highly repeated sequences is about 1.7 times greater than the corresponding fraction of mRNA. The hybrids formed by the rapidly reacting fractions of both NRNA and mRNA melt over a narrow temperature range with a midpoint about 11°C below that of native L cell DNA. This indicates that these hybrids consist of partially complementary sequences with approximately 11% mismatching of bases. Hybrids formed by the slowly reacting fraction of NRNA melt within 4°–6°C of native DNA, indicating very little, if any, mismatching of bases. Hybrids of the slowly reacting components of mRNA, formed under conditions of sufficiently low RNA input, have a high thermal stability, similar to that observed for hybrids of the slowly reacting NRNA component. However, when higher inputs of mRNA are used, hybrids are formed which have a strikingly lower thermal stability. This observation can be explained by assuming that there is sufficient similarity among the relatively rare DNA sequences coding for mRNA so that under hybridization conditions, in which these DNA sequences are not truly in excess, reversible hybrids exhibiting a considerable amount of mispairing are formed. The fact that a comparable phenomenon has not been observed for NRNA may mean that there is less similarity among the relatively rare DNA sequences coding for NRNA than there is among the rare sequences coding for mRNA. PMID:4999767

Mutant POLG2 Disrupts DNA Polymerase γ Subunits and Causes Progressive External Ophthalmoplegia

PubMed Central

Longley, Matthew J.; Clark, Susanna; Yu Wai Man, Cynthia; Hudson, Gavin; Durham, Steve E.; Taylor, Robert W.; Nightingale, Simon; Turnbull, Douglass M.; Copeland, William C.; Chinnery, Patrick F.

2006-01-01

DNA polymerase γ (pol γ) is required to maintain the genetic integrity of the 16,569-bp human mitochondrial genome (mtDNA). Mutation of the nuclear gene for the catalytic subunit of pol γ (POLG) has been linked to a wide range of mitochondrial diseases involving mutation, deletion, and depletion of mtDNA. We describe a heterozygous dominant mutation (c.1352G→A/p.G451E) in POLG2, the gene encoding the p55 accessory subunit of pol γ, that causes progressive external ophthalmoplegia with multiple mtDNA deletions and cytochrome c oxidase (COX)–deficient muscle fibers. Biochemical characterization of purified, recombinant G451E-substituted p55 protein in vitro revealed incomplete stimulation of the catalytic subunit due to compromised subunit interaction. Although G451E p55 retains a wild-type ability to bind DNA, it fails to enhance the DNA-binding strength of the p140-p55 complex. In vivo, the disease most likely arises through haplotype insufficiency or heterodimerization of the mutated and wild-type proteins, which promote mtDNA deletions by stalling the DNA replication fork. The progressive accumulation of mtDNA deletions causes COX deficiency in muscle fibers and results in the clinical phenotype. PMID:16685652

[Current status of DNA databases in the forensic field: new progress, new legal needs].

PubMed

Baeta, Miriam; Martínez-Jarreta, Begoña

2009-01-01

One of the most polemic issues regarding the use of deoxyribonucleic acid (DNA) in the legal sphere, refers to the creation of DNA databases. Until relatively recently, Spain did not have a law to support the establishment of a national DNA profile bank for forensic purposes, and preserve the fundamental rights of subjects whose data are archived therein. The regulatory law of police databases regarding identifiers obtained from DNA approved in 2007, covers this void in the Spanish legislation and responds to the incessant need to adapt the laws to continuous scientific and technological progress.

A comprehensive approach to ascertain the binding mode of curcumin with DNA

NASA Astrophysics Data System (ADS)

Haris, P.; Mary, Varughese; Aparna, P.; Dileep, K. V.; Sudarsanakumar, C.

2017-03-01

Curcumin is a natural phytochemical from the rhizoma of Curcuma longa, the popular Indian spice that exhibits a wide range of pharmacological properties like antioxidant, anticancer, anti-inflammatory, antitumor, and antiviral activities. In the published literatures we can see different studies and arguments on the interaction of curcumin with DNA. The intercalative binding, groove binding and no binding of curcumin with DNA were reported. In this context, we conducted a detailed study to understand the mechanism of recognition of dimethylsulfoxide-solubilized curcumin by DNA. The interaction of curcumin with calf thymus DNA (ctDNA) was confirmed by agarose gel electrophoresis. The nature of binding and energetics of interaction were studied by Isothermal Titration Calorimetry (ITC), Differential Scanning Calorimetry (DSC), UV-visible, fluorescence and melting temperature (Tm) analysis. The experimental data were compared with molecular modeling studies. Our investigation confirmed that dimethylsulfoxide-solubilized curcumin binds in the minor groove of the ctDNA without causing significant structural alteration to the DNA.

Deciphering the mechanism of interaction of edifenphos with calf thymus DNA

NASA Astrophysics Data System (ADS)

Ahmad, Ajaz; Ahmad, Masood

2018-01-01

Edifenphos is an important organophosphate pesticide with many antifungal and anti-insecticidal properties but it may cause potential hazards to human health. In this work, we have tried to explore the binding mode of action and mechanism of edifenphos to calf thymus DNA (CT-DNA). Several experiments such as ultraviolet-visible absorption spectra and emission spectroscopy showed complex formation between edifenphos and CT-DNA and low binding constant values supporting groove binding mode. These results were further confirmed by circular dichroism (CD), CT-DNA melting studies, viscosity measurements, density functional theory and molecular docking. CD study suggests that edifenphos does not alter native structure of CT-DNA. Isothermal calorimetry reveals that binding of edifenphos with CT-DNA is enthalpy driven process. Competitive binding assay and effect of ionic strength showed that edifenphos binds to CT-DNA via groove binding manner. Hence, edifenphos is a minor groove binder preferably interacting with A-T regions with docking score - 6.84 kJ/mol.

Pico-level DNA sensing by hetero-polymetalate, Na10{Dy2W10O30(µ-S)6}·80H2O, cluster

NASA Astrophysics Data System (ADS)

Dutta, Taposhree; Ganguly, Jhuma; Sarkar, Sabyasachi

2018-04-01

The polyoxometalate dysprosium cluster, (Dy-S-W POM) , Na10[Dy2W10O30(µ-S)6]·80H2O, shows remarkable dsDNA denaturation property. In the presence of 0.22 µmol of this Dy-S-W POM, the melting temperature (Tm) of calf-thymus (CT) dsDNA is decreased to 62.35 °C. Dy-S-W POM shows bleaching of methylene blue (MB). Addition of CT-DNA in the MB bleached solution of Dy-S-W POM apparently intercalates MB. Such trapped MB by CT-DNA responds to its re-oxidation by elemental sulfur formed in the bleaching process involving Dy-S-W POM. This reduction-oxidation property of MB with Dy-S-W POM led to the detection of pico (13.20 pmol) level of DNA even by naked eye, which will be helpful for rapid trace DNA detection in forensic science and DNA-related diagnostics, complimenting time-consuming sophisticated methodology.

High Resolution Melting (HRM) applied to wine authenticity.

PubMed

Pereira, Leonor; Gomes, Sónia; Castro, Cláudia; Eiras-Dias, José Eduardo; Brazão, João; Graça, António; Fernandes, José R; Martins-Lopes, Paula

2017-02-01

Wine authenticity methods are in increasing demand mainly in Denomination of Origin designations. The DNA-based methodologies are a reliable means of tracking food/wine varietal composition. The main aim of this work was the study of High Resolution Melting (HRM) application as a screening method for must and wine authenticity. Three sample types (leaf, must and wine) were used to validate the three developed HRM assays (Vv1-705bp; Vv2-375bp; and Vv3-119bp). The Vv1 HRM assay was only successful when applied to leaf and must samples. The Vv2 HRM assay successfully amplified all sample types, allowing genotype discrimination based on melting temperature values. The smallest amplicon, Vv3, produced a coincident melting curve shape in all sample types (leaf and wine) with corresponding genotypes. This study presents sensitive, rapid and efficient HRM assays applied for the first time to wine samples suitable for wine authenticity purposes. Copyright © 2016 Elsevier Ltd. All rights reserved.

An experimentally-informed coarse-grained 3-site-per-nucleotide model of DNA: Structure, thermodynamics, and dynamics of hybridization

PubMed Central

Hinckley, Daniel M.; Freeman, Gordon S.; Whitmer, Jonathan K.; de Pablo, Juan J.

2013-01-01

A new 3-Site-Per-Nucleotide coarse-grained model for DNA is presented. The model includes anisotropic potentials between bases involved in base stacking and base pair interactions that enable the description of relevant structural properties, including the major and minor grooves. In an improvement over available coarse-grained models, the correct persistence length is recovered for both ssDNA and dsDNA, allowing for simulation of non-canonical structures such as hairpins. DNA melting temperatures, measured for duplexes and hairpins by integrating over free energy surfaces generated using metadynamics simulations, are shown to be in quantitative agreement with experiment for a variety of sequences and conditions. Hybridization rate constants, calculated using forward-flux sampling, are also shown to be in good agreement with experiment. The coarse-grained model presented here is suitable for use in biological and engineering applications, including nucleosome positioning and DNA-templated engineering. PMID:24116642

Re-entrant DNA gels

PubMed Central

Bomboi, Francesca; Romano, Flavio; Leo, Manuela; Fernandez-Castanon, Javier; Cerbino, Roberto; Bellini, Tommaso; Bordi, Federico; Filetici, Patrizia; Sciortino, Francesco

2016-01-01

DNA is acquiring a primary role in material development, self-assembling by design into complex supramolecular aggregates, the building block of a new-materials world. Using DNA nanoconstructs to translate sophisticated theoretical intuitions into experimental realizations by closely matching idealized models of colloidal particles is a much less explored avenue. Here we experimentally show that an appropriate selection of competing interactions enciphered in multiple DNA sequences results into the successful design of a one-pot DNA hydrogel that melts both on heating and on cooling. The relaxation time, measured by light scattering, slows down dramatically in a limited window of temperatures. The phase diagram displays a peculiar re-entrant shape, the hallmark of the competition between different bonding patterns. Our study shows that it is possible to rationally design biocompatible bulk materials with unconventional phase diagrams and tuneable properties by encoding into DNA sequences both the particle shape and the physics of the collective response. PMID:27767029

Use and Limitations of a Climate-Quality Data Record to Study Temperature Trends on the Greenland Ice Sheet

NASA Technical Reports Server (NTRS)

Hall, Dorothy K.; Comiso, Josefino C.; Shuman, Christopher A.; Koenig, Lora S.; DiGirolamo, Nicolo E.

2011-01-01

Enhanced melting of the Greenland Ice Sheet has been documented in recent literature along with surface-temperature increases measured using infrared satellite data since 1981. Using a recently-developed climate-quality data record, 11- and 12-year trends in the clear-sky ice-surface temperature (IST) of the Greenland Ice Sheet have been studied using the Moderate-Resolution Imaging Spectroradiometer (MODIS) IST product. Daily and monthly MODIS ISTs of the Greenland Ice Sheet beginning on 1 March 2000 and continuing through 31 December 2010 are now available at 6.25-km spatial resolution on a polar stereographic grid as described in Hall et al. (submitted). This record will be elevated in status to a climate-data record (CDR) when more years of data become available either from the MODIS on the Terra or Aqua satellites, or from the Visible Infrared Imager Radiometer Suite (VIIRS) to be launched in October 2011. Maps showing the maximum extent of melt for the entire ice sheet and for the six major drainage basins have been developed from the MODIS IST dataset. Twelve-year trends of the duration of the melt season on the ice sheet vary in different drainage basins with some basins melting progressively earlier over the course of the study period. Some (but not all) of the basins also show a progressively-longer duration of melt. IST 12-year trends are compared with in-situ data, and climate data from the Modern Era Retrospective-Analysis for Research and Applications (MERRA) Reanalysis.

“Ultra-high resolution optical trap with single fluorophore sensitivity”

PubMed Central

Comstock, Matthew J; Ha, Taekjip; Chemla, Yann R

2013-01-01

We present a single-molecule instrument that combines a timeshared ultra-high resolution dual optical trap interlaced with a confocal fluorescence microscope. In a demonstration experiment, individual single-fluorophore labeled DNA oligonucleotides were observed to bind and unbind to complementary DNA suspended between two trapped beads. Simultaneous with the single-fluorophore detection, coincident angstrom-scale changes in tether extension could be clearly observed. Fluorescence readout allowed us to determine the duplex melting rate as a function of force. The new instrument will enable the simultaneous measurement of angstrom-scale mechanical motion of individual DNA-binding proteins (e.g., single base pair stepping of DNA translocases) along with the detection of fluorescently labeled protein properties (e.g., internal configuration). PMID:21336286

Targeting DNA repair pathways for cancer treatment: what's new?

PubMed Central

Kelley, Mark R; Logsdon, Derek; Fishel, Melissa L

2014-01-01

Disruptions in DNA repair pathways predispose cells to accumulating DNA damage. A growing body of evidence indicates that tumors accumulate progressively more mutations in DNA repair proteins as cancers progress. DNA repair mechanisms greatly affect the response to cytotoxic treatments, so understanding those mechanisms and finding ways to turn dysregulated repair processes against themselves to induce tumor death is the goal of all DNA repair inhibition efforts. Inhibition may be direct or indirect. This burgeoning field of research is replete with promise and challenge, as more intricacies of each repair pathway are discovered. In an era of increasing concern about healthcare costs, use of DNA repair inhibitors can prove to be highly effective stewardship of R&D resources and patient expenses. PMID:24947262

High-resolution DNA melting analysis in plant research

USDA-ARS?s Scientific Manuscript database

Genetic and genomic studies provide valuable insight into the inheritance, structure, organization, and function of genes. The knowledge gained from the analysis of plant genes is beneficial to all aspects of plant research, including crop improvement. New methods and tools are continually developed...

Molecular characterization of dissolved organic matter during the Arctic spring melt period

NASA Astrophysics Data System (ADS)

Gueguen, C.; Mangal, V.; Shi, Y. X.

2016-02-01

The application of high resolution electrospray ionization mass spectrometry has advanced our understanding of dissolved organic matter (DOM) at molecular level. The arctic spring melt period has been largely undersampled owing to logistical and safety issues, yet this period is extremely important to the overall flux of DOM and related contaminants including metals from high latitude rivers. In this study, we present high resolution molecular composition of 35 DOM samples collected in the Churchill River (Manitoba) during the 2015 spring melt period. As spring melt progresses, a significant change in the two most dominant carbon pools, protein and lignin, was observed. For example, the relative abundance of proteins detected in the river DOM samples increased from 19 to 44% during the spring flush, likely reflecting a change in DOM source. Similar patterns were found using fluorescence spectroscopy.

ATR-like kinase Mec1 facilitates both chromatin accessibility at DNA replication forks and replication fork progression during replication stress

PubMed Central

Rodriguez, Jairo; Tsukiyama, Toshio

2013-01-01

Faithful DNA replication is essential for normal cell division and differentiation. In eukaryotic cells, DNA replication takes place on chromatin. This poses the critical question as to how DNA replication can progress through chromatin, which is inhibitory to all DNA-dependent processes. Here, we developed a novel genome-wide method to measure chromatin accessibility to micrococcal nuclease (MNase) that is normalized for nucleosome density, the NCAM (normalized chromatin accessibility to MNase) assay. This method enabled us to discover that chromatin accessibility increases specifically at and ahead of DNA replication forks in normal S phase and during replication stress. We further found that Mec1, a key regulatory ATR-like kinase in the S-phase checkpoint, is required for both normal chromatin accessibility around replication forks and replication fork rate during replication stress, revealing novel functions for the kinase in replication stress response. These results suggest a possibility that Mec1 may facilitate DNA replication fork progression during replication stress by increasing chromatin accessibility around replication forks. PMID:23307868

Replication stress-induced chromosome breakage is correlated with replication fork progression and is preceded by single-stranded DNA formation.

PubMed

Feng, Wenyi; Di Rienzi, Sara C; Raghuraman, M K; Brewer, Bonita J

2011-10-01

Chromosome breakage as a result of replication stress has been hypothesized to be the direct consequence of defective replication fork progression, or "collapsed" replication forks. However, direct and genome-wide evidence that collapsed replication forks give rise to chromosome breakage is still lacking. Previously we showed that a yeast replication checkpoint mutant mec1-1, after transient exposure to replication impediment imposed by hydroxyurea (HU), failed to complete DNA replication, accumulated single-stranded DNA (ssDNA) at the replication forks, and fragmented its chromosomes. In this study, by following replication fork progression genome-wide via ssDNA detection and by direct mapping of chromosome breakage after HU exposure, we have tested the hypothesis that the chromosome breakage in mec1 cells occurs at collapsed replication forks. We demonstrate that sites of chromosome breakage indeed correlate with replication fork locations. Moreover, ssDNA can be detected prior to chromosome breakage, suggesting that ssDNA accumulation is the common precursor to double strand breaks at collapsed replication forks.

Melt Analysis of Mismatch Amplification Mutation Assays (Melt-MAMA): A Functional Study of a Cost-Effective SNP Genotyping Assay in Bacterial Models

PubMed Central

Birdsell, Dawn N.; Pearson, Talima; Price, Erin P.; Hornstra, Heidie M.; Nera, Roxanne D.; Stone, Nathan; Gruendike, Jeffrey; Kaufman, Emily L.; Pettus, Amanda H.; Hurbon, Audriana N.; Buchhagen, Jordan L.; Harms, N. Jane; Chanturia, Gvantsa; Gyuranecz, Miklos; Wagner, David M.; Keim, Paul S.

2012-01-01

Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and biologically informative markers extensively used across broad scientific disciplines. Newly identified SNP markers are publicly available at an ever-increasing rate due to advancements in sequencing technologies. Efficient, cost-effective SNP genotyping methods to screen sample populations are in great demand in well-equipped laboratories, but also in developing world situations. Dual Probe TaqMan assays are robust but can be cost-prohibitive and require specialized equipment. The Mismatch Amplification Mutation Assay, coupled with melt analysis (Melt-MAMA), is flexible, efficient and cost-effective. However, Melt-MAMA traditionally suffers from high rates of assay design failures and knowledge gaps on assay robustness and sensitivity. In this study, we identified strategies that improved the success of Melt-MAMA. We examined the performance of 185 Melt-MAMAs across eight different pathogens using various optimization parameters. We evaluated the effects of genome size and %GC content on assay development. When used collectively, specific strategies markedly improved the rate of successful assays at the first design attempt from ∼50% to ∼80%. We observed that Melt-MAMA accurately genotypes across a broad DNA range (∼100 ng to ∼0.1 pg). Genomic size and %GC content influence the rate of successful assay design in an independent manner. Finally, we demonstrated the versatility of these assays by the creation of a duplex Melt-MAMA real-time PCR (two SNPs) and conversion to a size-based genotyping system, which uses agarose gel electrophoresis. Melt-MAMA is comparable to Dual Probe TaqMan assays in terms of design success rate and accuracy. Although sensitivity is less robust than Dual Probe TaqMan assays, Melt-MAMA is superior in terms of cost-effectiveness, speed of development and versatility. We detail the parameters most important for the successful application of Melt-MAMA, which should prove useful to the wider scientific community. PMID:22438886

Rapid and accurate identification of Mycobacterium tuberculosis complex and common non-tuberculous mycobacteria by multiplex real-time PCR targeting different housekeeping genes.

PubMed

Nasr Esfahani, Bahram; Rezaei Yazdi, Hadi; Moghim, Sharareh; Ghasemian Safaei, Hajieh; Zarkesh Esfahani, Hamid

2012-11-01

Rapid and accurate identification of mycobacteria isolates from primary culture is important due to timely and appropriate antibiotic therapy. Conventional methods for identification of Mycobacterium species based on biochemical tests needs several weeks and may remain inconclusive. In this study, a novel multiplex real-time PCR was developed for rapid identification of Mycobacterium genus, Mycobacterium tuberculosis complex (MTC) and the most common non-tuberculosis mycobacteria species including M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and the M. gordonae in three reaction tubes but under same PCR condition. Genetic targets for primer designing included the 16S rDNA gene, the dnaJ gene, the gyrB gene and internal transcribed spacer (ITS). Multiplex real-time PCR was setup with reference Mycobacterium strains and was subsequently tested with 66 clinical isolates. Results of multiplex real-time PCR were analyzed with melting curves and melting temperature (T (m)) of Mycobacterium genus, MTC, and each of non-tuberculosis Mycobacterium species were determined. Multiplex real-time PCR results were compared with amplification and sequencing of 16S-23S rDNA ITS for identification of Mycobacterium species. Sensitivity and specificity of designed primers were each 100 % for MTC, M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and M. gordonae. Sensitivity and specificity of designed primer for genus Mycobacterium was 96 and 100 %, respectively. According to the obtained results, we conclude that this multiplex real-time PCR with melting curve analysis and these novel primers can be used for rapid and accurate identification of genus Mycobacterium, MTC, and the most common non-tuberculosis Mycobacterium species.

Diagnosis and identification of Leishmania spp. from Giemsa-stained slides, by real-time PCR and melting curve analysis in south-west of Iran.

PubMed

Khademvatan, S; Neisi, N; Maraghi, S; Saki, J

2011-12-01

The aim of present study was describing a real-time PCR assay for the diagnosis and direct identification of Leishmania species on Giemsa-stained slides in south-west of Iran. Altogether, 102 Giemsa-stained slides were collected from different part of south-west of Iran between 2008 and 2011. All the Giemsa-stained slides were examined under light microscope. After DNA extraction, real-time PCR amplification and detection were conducted with fluorescent SYBR Green I. For identification, PCR products were analysed with melting curve analysis. One hundred and two archived slides from suspected lesion examined by microscopy and real-time PCR. The sensitivity of the real-time PCR on Giemsa-stained slid was 98% (96/102). The melting curve analysis (T(m)) were 88·3±0·2°C for L. tropica (MHOM/IR/02/Mash10), 86·5±0·2°C for L. major (MHOM/IR/75/ER) and 89·4±0·3°C for L. infantum (MCAN/IR/97/LON 49), respectively. This study is first report in use of real-time PCR for diagnosis and identification of Leishmania spp. in Iran. Up to now, in Iran, the majority of identification of Leishmania species is restriction fragment length polymorphism (PCR-RFLP) of ITS1 and kinetoplast DNA. Our data showed that Giemsa-stained slides that were stored more than 3 years, can be use for Leishmania DNA extraction and amplification by real-time PCR. Compared to conventional PCR-based methods, the real-time PCR is extremely rapid with results and more samples can be processed at one time.

High-Throughput Genotyping of Single Nucleotide Polymorphisms in the Plasmodium falciparum dhfr Gene by Asymmetric PCR and Melt-Curve Analysis▿

PubMed Central

Cruz, Rochelle E.; Shokoples, Sandra E.; Manage, Dammika P.; Yanow, Stephanie K.

2010-01-01

Mutations within the Plasmodium falciparum dihydrofolate reductase gene (Pfdhfr) contribute to resistance to antimalarials such as sulfadoxine-pyrimethamine (SP). Of particular importance are the single nucleotide polymorphisms (SNPs) within codons 51, 59, 108, and 164 in the Pfdhfr gene that are associated with SP treatment failure. Given that traditional genotyping methods are time-consuming and laborious, we developed an assay that provides the rapid, high-throughput analysis of parasite DNA isolated from clinical samples. This assay is based on asymmetric real-time PCR and melt-curve analysis (MCA) performed on the LightCycler platform. Unlabeled probes specific to each SNP are included in the reaction mixture and hybridize differentially to the mutant and wild-type sequences within the amplicon, generating distinct melting curves. Since the probe is present throughout PCR and MCA, the assay proceeds seamlessly with no further addition of reagents. This assay was validated for analytical sensitivity and specificity using plasmids, purified genomic DNA from reference strains, and parasite cultures. For all four SNPs, correct genotypes were identified with 100 copies of the template. The performance of the assay was evaluated with a blind panel of clinical isolates from travelers with low-level parasitemia. The concordance between our assay and DNA sequencing ranged from 84 to 100% depending on the SNP. We also directly compared our MCA assay to a published TaqMan real-time PCR assay and identified major issues with the specificity of the TaqMan probes. Our assay provides a number of technical improvements that facilitate the high-throughput screening of patient samples to identify SP-resistant malaria. PMID:20631115

A singleplex real-time fluorescence resonance energy transfer PCR with melting curve analysis for the differential detection of Paragonimus heterotremus, Echinostoma malayanum and Fasciola gigantica eggs in faeces.

PubMed

Tantrawatpan, Chairat; Saijuntha, Weerachai; Manochantr, Sirikul; Kheolamai, Pakpoom; Thanchomnang, Tongjit; Sadaow, Lakkhana; Intapan, Pewpan M; Maleewong, Wanchai

2016-01-01

Because the eggs of Paragonimus, Echinostoma and Fasciola are very similar in size and shape, it is difficult to distinguish and accurately identify species by the morphology of their eggs, which is a standard diagnostic method. In this study, a novel assay combining a real-time fluorescence resonance energy transfer PCR and melting curve analysis using one set of primers and fluorophore-labelled hybridization probes specific for the 28S rDNA region was developed for the molecular detection of Paragonimus heterotremus, Echinostoma malayanum and Fasciola gigantica eggs. This assay could detect and distinguish P. heterotremus, E. malayanum and F. gigantica DNA with the distinct melting temperature (Tm) values of 57.99±0.08, 62.12±0.15 and 74.10±0.18, respectively. The assay can also be used to detect and distinguish DNA from P. bangkokensis, P. harinasutai, P. machorchis, E. revolutum, Hypodereum conoideum and F. hepatica, which have different Tm values. The sensitivity of this assay enabled the detection of one egg of P. heterotremus, E. malayanum or F. gigantica per 100 mg of faeces. In addition, the specificity testing showed no fluorescence signal for other parasites. Due to the sensitivity and specificity of our assay in detecting P. heterotremus, E. malayanum and F. gigantica, our method could be used to accurately diagnose these three medically important parasitic groups and has potential implications for molecular epidemiological investigations of human and/or animal infections. © The Author 2015. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Rapid Detection and Identification of Human Hookworm Infections through High Resolution Melting (HRM) Analysis

PubMed Central

Ngui, Romano; Lim, Yvonne A. L.; Chua, Kek Heng

2012-01-01

Background Hookworm infections are still endemic in low and middle income tropical countries with greater impact on the socioeconomic and public health of the bottom billion of the world's poorest people. In this study, a real-time polymerase chain reaction (PCR) coupled with high resolution melting-curve (HRM) analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on the five human hookworm species. Methods Real-time PCR coupled with HRM analysis targeting the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish hookworm species in human samples. Unique and distinct characteristics of HRM patterns were produced for each of the five hookworm species. The melting curves were characterized by peaks of 79.24±0.05°C and 83.00±0.04°C for Necator americanus, 79.12±0.10°C for Ancylostoma duodenale, 79.40±0.10°C for Ancylostoma ceylanicum, 79.63±0.05°C for Ancylostoma caninum and 79.70±0.14°C for Ancylostoma braziliense. An evaluation of the method's sensitivity and specificity revealed that this assay was able to detect as low as 0.01 ng/µl hookworm DNA and amplification was only recorded for hookworm positive samples. Conclusion The HRM assay developed in this study is a rapid and straightforward method for the diagnosis, identification and discrimination of five human hookworms. This assay is simple compared to other probe-based genotyping methods as it does not require multiplexing, DNA sequencing or post-PCR processing. Therefore, this method offers a new alternative for rapid detection of human hookworm species. PMID:22844538

DNA-based techniques for authentication of processed food and food supplements.

PubMed

Lo, Yat-Tung; Shaw, Pang-Chui

2018-02-01

Authentication of food or food supplements with medicinal values is important to avoid adverse toxic effects, provide consumer rights, as well as for certification purpose. Compared to morphological and spectrometric techniques, molecular authentication is found to be accurate, sensitive and reliable. However, DNA degradation and inclusion of inhibitors may lead to failure in PCR amplification. This paper reviews on the existing DNA extraction and PCR protocols, and the use of small size DNA markers with sufficient discriminative power for molecular authentication. Various emerging new molecular techniques such as isothermal amplification for on-site diagnosis, next-generation sequencing for high-throughput species identification, high resolution melting analysis for quick species differentiation, DNA array techniques for rapid detection and quantitative determination in food products are also discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Direct Determination of the Base-Pair Force Constant of DNA from the Acoustic Phonon Dispersion of the Double Helix

NASA Astrophysics Data System (ADS)

van Eijck, L.; Merzel, F.; Rols, S.; Ollivier, J.; Forsyth, V. T.; Johnson, M. R.

2011-08-01

Quantifying the molecular elasticity of DNA is fundamental to our understanding of its biological functions. Recently different groups, through experiments on tailored DNA samples and numerical models, have reported a range of stretching force constants (0.3 to 3N/m). However, the most direct, microscopic measurement of DNA stiffness is obtained from the dispersion of its vibrations. A new neutron scattering spectrometer and aligned, wet spun samples have enabled such measurements, which provide the first data of collective excitations of DNA and yield a force constant of 83N/m. Structural and dynamic order persists unchanged to within 15 K of the melting point of the sample, precluding the formation of bubbles. These findings are supported by large scale phonon and molecular dynamics calculations, which reconcile hard and soft force constants.

Ternary Magnesium-Lithium Base Constitution Diagrams and Magnesium Alloys of Low Alloy Additions

DTIC Science & Technology

1951-03-01

progress In eperimental development of mgmesiu-bease &alls with low alloy additions. The primry purpose of this investiptiU is to obtain alloys baving a...Casting Magnesium-Lithium Base Ternary Alloys Melting and Castirg Technigue The design , construction and operation of equipment for melting and...protection during heat treatment were: 1. Design and construction of a specimen container to hold a number of specimens in an inert atmosphere in order to WAC

Geodynamics of seafloor spreading extinction: Constraints from the South China Sea

NASA Astrophysics Data System (ADS)

Zhang, X.; Lin, J.; Behn, M. D.

2016-12-01

We investigate magmatism and mantle thermal structure beneath fossil spreading centers in the South China Sea (SCS), focusing on two aspects: (1) mantle thermal structure and melting, and (2) magmatism associated with seamounts. We carried out 3D geodynamic models to study thermal structure beneath the SCS during the process from initiation to cessation of seafloor spreading. Modeling results suggested that the overall mantle temperatures of the East Subbasin were significantly greater than that of the Southwest Subbasin when the seafloor spreading of both subbasins ceased at about 15-16 Ma. However, the differences in thermal structure between the two subbasins were calculated to have decreased with time. Work is in progress to couple geochemical and geophysical constraints with geodynamic modeling to investigate melt generation, fractional crystallization, and melt extraction at the fossil spreading centers in the SCS. Among the seamounts that can be identified on multi-beam bathymetry data, about half of them are located along the fossil spreading centers while the remaining located off axis. This is in contrast to fossil spreading ridges in the West Scotia Sea and Phoenix Ridge, where most seamounts are located off axis. The off-axis seamounts in the SCS also show strong asymmetry about the fossil spreading centers with most seamounts concentrated in the northern flank. Work is in progress to investigate the melting processes associated with seamounts.

Role of the heat capacity change in understanding and modeling melting thermodynamics of complementary duplexes containing standard and nucleobase-modified LNA.

PubMed

Hughesman, Curtis B; Turner, Robin F B; Haynes, Charles A

2011-06-14

Melting thermodynamic data obtained by differential scanning calorimetry (DSC) are reported for 43 duplexed oligonucleotides containing one or more locked nucleic acid (LNA) substitutions. The measured heat capacity change (ΔC(p)) for the helix-to-coil transition is used to compute the changes in enthalpy and entropy for melting of an LNA-bearing duplex at the T(m) of its corresponding isosequential unmodified DNA duplex to allow rigorous thermodynamic analysis of the stability enhancements provided by LNA substitutions. Contrary to previous studies, our analysis shows that the origin of the improved stability is almost exclusively a net reduction (ΔΔS° < 0) in the entropy gain accompanying the helix-to-coil transition, with the magnitude of the reduction dependent on the type of nucleobase and its base pairing properties. This knowledge and our average measured value for ΔC(p) of 42 ± 11 cal mol(-1) K(-1) bp(-1) are then used to derive a new model that accurately predicts melting thermodynamics and the increased melting temperature (ΔT(m)) of heteroduplexes formed between an unmodified DNA strand and a complementary strand containing any number and configuration of standard LNA nucleotides A, T, C, and G. This single-base thermodynamic (SBT) model requires only four entropy-related parameters in addition to ΔC(p). Finally, DSC data for 20 duplexes containing the nucleobase-modified LNAs 2-aminoadenine (D) and 2-thiothymine (H) are reported and used to determine SBT model parameters for D and H. The data and model suggest that along with the greater stability enhancement provided by D and H bases relative to their corresponding A and T analogues, the unique pseudocomplementary properties of D-H base pairs may make their use appealing for in vitro and in vivo applications.

DNA Methylation as an Epigenetic Factor in the Development and Progression of Polycythemia Vera

DTIC Science & Technology

2007-11-01

in the Development and Progression of Polycythemia Vera PRINCIPAL INVESTIGATOR: Jean-Pierre Issa, M.D. CONTRACTING...NUMBER DNA Methylation as an Epigenetic Factor in the Development and Progression of Polycythemia Vera 5b. GRANT NUMBER W81XWH-05-1-0535 5c...AVAILABILITY STATEMENT Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Polycythemia vera (PV

Candidate DNA Barcode Tags Combined With High Resolution Melting (Bar-HRM) Curve Analysis for Authentication of Senna alexandrina Mill. With Validation in Crude Drugs.

PubMed

Mishra, Priyanka; Shukla, Ashutosh K; Sundaresan, Velusamy

2018-01-01

Senna alexandrina (Fabaceae) is a globally recognized medicinal plant for its laxative properties as well as the only source of sennosides, and is highly exported bulk herb from India. Its major procurement is exclusively from limited cultivation, which leads to risks of deliberate or unintended adulteration. The market raw materials are in powdered or finished product form, which lead to difficulties in authentication. Here, DNA barcode tags based on chloroplast genes ( rbcL and matK ) and intergenic spacers ( psbA-trnH and ITS ) were developed for S. alexandrina along with the allied species. The ability and performance of the ITS1 region to discriminate among the Senna species resulted in the present proposal of the ITS1 tags as successful barcode. Further, these tags were coupled with high-resolution melting (HRM) curve analysis in a real-time PCR genotyping method to derive Bar-HRM (Barcoding-HRM) assays. Suitable HRM primer sets were designed through SNP detection and mutation scanning in genomic signatures of Senna species. The melting profiles of S. alexandrina and S . italica subsp. micrantha were almost identical and the remaining five species were clearly separated so that they can be differentiated by HRM method. The sensitivity of the method was utilized to authenticate market samples [Herbal Sample Assays (HSAs)]. HSA01 ( S. alexandrina crude drug sample from Bangalore) and HSA06 ( S. alexandrina crude drug sample from Tuticorin, Tamil Nadu, India) were found to be highly contaminated with S . italica subsp. micrantha . Species admixture samples mixed in varying percentage was identified sensitively with detection of contamination as low as 1%. The melting profiles of PCR amplicons are clearly distinct, which enables the authentic differentiation of species by the HRM method. This study reveals that DNA barcoding coupled with HRM is an efficient molecular tool to authenticate Senna herbal products in the market for quality control in the drug supply chain. CIMAP Communication Number: CIMAP/PUB/2017/31.

Fast and reliable detection of toxic Crotalaria spectabilis Roth. in Thunbergia laurifolia Lindl. herbal products using DNA barcoding coupled with HRM analysis.

PubMed

Singtonat, Sahachat; Osathanunkul, Maslin

2015-05-30

Nowadays, medicinal plants are used as a popular alternative to synthetic drugs. Many medicinal plant products have now been commercialized throughout various markets. These products are commonly sold in processed or modified forms such as powders, dried material and capsules, making it almost impossible to accurately identify the constituent species. The herbal plant known as 'Rang Chuet' in Thai has been widely used as remedies for various ailments. However, two medicinal plants species, Thunbergia laurifolia and Crotalaria spectabilis share this name. Duo to the similarity in nomenclature, the commercial products labeled as 'Rang Chuet' could be any of them. Recently, the evidence of hepatotoxic effects linked to use of C. spectabilis were reported and is now seriously concern. There is a need to find an approach that could help with species identification of these herbal products to ensure the safety and efficacy of the herbal drug. Here DNA barcoding was used in combination with High Resolution Melting analysis (Bar-HRM) to authenticate T. laurifolia species. Four DNA barcodes including matK, rbcL, rpoC and trnL were selected for use in primers design for HRM analysis to produce standard melting profiles of the selected species. Commercial products labeled as 'Rang Chuet' were purchased from Thai markets and authentication by HRM analyses. Melting data from the HRM assay using the designed primers showed that the two 'Rang Chuet' species could easily be distinguished from each other. The melting profiles of the all four region amplicons of each species are clearly separated in all three replicates. The method was then applied to authenticate products in powdered form. HRM curves of all ten test samples indicated that three of the tested products did not only contain the T. laurifolia species. The herbal drugs derived from different plants must be distinguished from each other even they share the same vernacular name. The Bar-HRM method developed here proved useful in the identification and authentication of herbal species in processed samples. In the future, species authentication through Bar-HRM could be used to promote consumer trust, as well as raising the quality of herbal products.

Candidate DNA Barcode Tags Combined With High Resolution Melting (Bar-HRM) Curve Analysis for Authentication of Senna alexandrina Mill. With Validation in Crude Drugs

PubMed Central

Mishra, Priyanka; Shukla, Ashutosh K.; Sundaresan, Velusamy

2018-01-01

Senna alexandrina (Fabaceae) is a globally recognized medicinal plant for its laxative properties as well as the only source of sennosides, and is highly exported bulk herb from India. Its major procurement is exclusively from limited cultivation, which leads to risks of deliberate or unintended adulteration. The market raw materials are in powdered or finished product form, which lead to difficulties in authentication. Here, DNA barcode tags based on chloroplast genes (rbcL and matK) and intergenic spacers (psbA-trnH and ITS) were developed for S. alexandrina along with the allied species. The ability and performance of the ITS1 region to discriminate among the Senna species resulted in the present proposal of the ITS1 tags as successful barcode. Further, these tags were coupled with high-resolution melting (HRM) curve analysis in a real-time PCR genotyping method to derive Bar-HRM (Barcoding-HRM) assays. Suitable HRM primer sets were designed through SNP detection and mutation scanning in genomic signatures of Senna species. The melting profiles of S. alexandrina and S. italica subsp. micrantha were almost identical and the remaining five species were clearly separated so that they can be differentiated by HRM method. The sensitivity of the method was utilized to authenticate market samples [Herbal Sample Assays (HSAs)]. HSA01 (S. alexandrina crude drug sample from Bangalore) and HSA06 (S. alexandrina crude drug sample from Tuticorin, Tamil Nadu, India) were found to be highly contaminated with S. italica subsp. micrantha. Species admixture samples mixed in varying percentage was identified sensitively with detection of contamination as low as 1%. The melting profiles of PCR amplicons are clearly distinct, which enables the authentic differentiation of species by the HRM method. This study reveals that DNA barcoding coupled with HRM is an efficient molecular tool to authenticate Senna herbal products in the market for quality control in the drug supply chain. CIMAP Communication Number: CIMAP/PUB/2017/31 PMID:29593755

Analysis of Apoptosis in Ultraviolet-Induced Sea Cucumber (Stichopus japonicus) Melting Using Terminal Deoxynucleotidyl-Transferase-Mediated dUTP Nick End-Labeling Assay and Cleaved Caspase-3 Immunohistochemistry.

PubMed

Yang, Jing-Feng; Gao, Rong-Chun; Wu, Hai-Tao; Li, Peng-Fei; Hu, Xian-Shu; Zhou, Da-Yong; Zhu, Bei-Wei; Su, Yi-Cheng

2015-11-04

The sea cucumber body wall melting phenomenon occurs under certain circumstances, and the mechanism of this phenomenon remains unclear. This study investigated the apoptosis in the ultraviolet (UV)-induced sea cucumber melting phenomenon. Fresh sea cucumbers (Stichopus japonicus) were exposed to UV radiation for half an hour at an intensity of 0.056 mW/cm(2) and then held at room temperature for melting development. The samples were histologically processed into formalin-fixed paraffin-embedded tissues. The apoptosis of samples was analyzed with the terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL) assay and cleaved caspase-3 immunohistochemistry. The emergence of TUNEL-positive cells speeds up between 0.5 and 2 h after UV irradiation. Cleaved caspase-3 positive cells were obviously detected in sample tissues immediately after the UV irradiation. These results demonstrated that sea cucumber melting induced by UV irradiation was triggered by the activation of caspase-3 followed by DNA fragmentation in sea cucumber tissue, which was attributed to apoptosis but was not a consequence of autolysis activity.

Simultaneous Identification of Four "Legal High" Plant Species in a Multiplex PCR High-Resolution Melt Assay.

PubMed

Elkins, Kelly M; Perez, Anjelica C U; Quinn, Alicia A

2017-05-01

The international prevalence of "legal high" drugs necessitates the development of a method for their detection and identification. Herein, we describe the development and validation of a tetraplex multiplex real-time polymerase chain reaction (PCR) assay used to simultaneously identify morning glory, jimson weed, Hawaiian woodrose, and marijuana detected by high-resolution melt using LCGreen Plus ® . The PCR assay was evaluated based on the following: (i) specificity and selectivity-primers were tested on DNA extracted from 30 species and simulated forensic samples, (ii) sensitivity-serial dilutions of the target DNA were prepared, and (iii) reproducibility and reliability-sample replicates were tested and remelted on different days. The assay is ideal for cases in which inexpensive assays are needed to quickly detect and identify trace biological material present on drug paraphernalia that is too compromised for botanical microscopic identification and for which analysts are unfamiliar with the morphology of the emerging "legal high" species. © 2016 American Academy of Forensic Sciences.

DNA replication depends on photosynthetic electron transport in cyanobacteria.

PubMed

Ohbayashi, Ryudo; Watanabe, Satoru; Kanesaki, Yu; Narikawa, Rei; Chibazakura, Taku; Ikeuchi, Masahiko; Yoshikawa, Hirofumi

2013-07-01

The freshwater cyanobacterium Synechococcus elongatus PCC 7942 exhibits light-dependent growth. Although it has been reported that DNA replication also depends on light irradiation in S. elongatus 7942, the involvement of the light in the regulation of DNA replication remains unclear. To elucidate the regulatory pathway of DNA replication by light, we studied the effect of several inhibitors, including two electron transport inhibitors, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), on DNA replication in S. elongatus 7942. DCMU inhibited only DNA replication initiation, whereas DBMIB blocked both the initiation and progression of DNA replication. These results suggest that DNA replication depends on the photosynthetic electron transport activity and initiation and progression of DNA replication are regulated in different ways. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Recent progress on DNA based walkers.

PubMed

Pan, Jing; Li, Feiran; Cha, Tae-Gon; Chen, Haorong; Choi, Jong Hyun

2015-08-01

DNA based synthetic molecular walkers are reminiscent of biological protein motors. They are powered by hybridization with fuel strands, environment induced conformational transitions, and covalent chemistry of oligonucleotides. Recent developments in experimental techniques enable direct observation of individual walkers with high temporal and spatial resolution. The functionalities of state-of-the-art DNA walker systems can thus be analyzed for various applications. Herein we review recent progress on DNA walker principles and characterization methods, and evaluate various aspects of their functions for future applications. Copyright © 2014 Elsevier Ltd. All rights reserved.

Understanding Melt-Memory of Commercial Polyolefins

NASA Astrophysics Data System (ADS)

Alamo, Rufina

Self-nucleation (SN) or controlling self-generated seeds in a polymer melt is an avenue to increase the rate of solidification of semicrystalline polymers of commercial relevance. Self-nuclei are remains in the melt of the segmental self-assembly to form polymer crystallites providing a path to enhance primary crystal nucleation. SN has been extensively studied in homopolymers such as iPP. Recently, a strong memory effect of crystallization has been observed in melts of random ethylene copolymers well above the equilibrium melting temperature. The melt memory is associated with clusters or seeds that remain in the melt from the copolymer's sequence length partitioning. Cooling from progressively lower self-seeded melt temperatures, ethylene copolymers with a broad inter-chain comonomer composition (1 - 15 mol%) display first the expected accelerated crystallization, followed by a decrease in the rate in a range of melt temperatures where narrow copolymers show a continuous acceleration of the rate. This unusual inversion of the crystallization rate was postulated to arise from the onset of liquid-liquid phase separation (LLPS) between comonomer-rich and comonomer-poor components of the broad copolymer. The UCST type phase diagram of these commercial copolymers has been documented via SANS using a blend of components, some deuterated, to reproduce the broad distribution. Furthermore, the components that contribute to LLPS have been identified by the crystallization behavior of molar mass fractions. The influence of long chain branching on the topology of copolymer melts has been analyzed using model 3-arm stars hydrogenated polybutadienes. The effect of melt viscosity on strength of melt memory is also evident when SN data of random ethylene copolymers are compared with those of propylene-ethylene copolymers. The strong dependence of melt viscosity on melt memory, and a critical threshold crystallinity level to observe the effect of melt memory on crystallization rate, support the kinetic nature of the SN phenomenon. Support from NSF, DMR-1105129 and DMR-1607786 is gratefully acknowledged.

Structures and functions in the crowded nucleus: new biophysical insights

NASA Astrophysics Data System (ADS)

Hancock, Ronald

2014-09-01

Concepts and methods from the physical sciences have catalysed remarkable progress in understanding the cell nucleus in recent years. To share this excitement with physicists and encourage their interest in this field, this review offers an overview of how the physics which underlies structures and functions in the nucleus is becoming more clear thanks to methods which have been developed to simulate and study macromolecules, polymers, and colloids. The environment in the nucleus is very crowded with macromolecules, making entropic (depletion) forces major determinants of interactions. Simulation and experiments are consistent with their key role in forming membraneless compartments such as nucleoli, PML and Cajal bodies, and discrete "territories" for chromosomes. The chromosomes, giant linear polyelectrolyte polymers, exist in vivo in a state like a polymer melt. Looped conformations are predicted in crowded conditions, and have been confirmed experimentally and are central to the regulation of gene expression. Polymer theory has revealed how the chromosomes are so highly compacted in the nucleus, forming a "crumpled globule" with fractal properties which avoids knots and entanglements in DNA while allowing facile accessibility for its replication and transcription. Entropic repulsion between looped polymers can explain the confinement of each chromosome to a discrete region of the nucleus. Crowding and looping are predicted to facilitate finding the specific targets of factors which modulate activities of DNA. Simulation shows that entropic effects contribute to finding and repairing potentially lethal double-strand breaks in DNA by increasing the mobility of the broken ends, favouring their juxtaposition for repair. Signaling pathways are strongly influenced by crowding, which favours a processive mode of response (consecutive reactions without releasing substrates). This new information contributes to understanding the sometimes counter-intuitive consequences.

A linear biopolymer in the vicinity of the triple point. The homopolymer case.

PubMed

Frank-Kamenetskii, M D; Chogovadze, G I

1984-06-01

This is a theoretical study of a situation where each residue of a linear biopolymer may adopt one of three conformational states. Such a situation exists in the case of DNA, since it may be in helical A, B, . . ., Z forms as well as the melted state. In the vicinity of the triple point in the phrase diagram three states, e.g. the A form, the B form and the denatured state, co-exist within a given molecule. We present an exact analytical solution of the simplest homopolymer model. Theory predicts that the presence of two helical states in one molecule should affect the helix-coil transition in two ways. The melting temperature experiences an upward shift and the melting range width is increased, by a factor of square root of two as a maximum.

Identification of feline polycystic kidney disease mutation using fret probes and melting curve analysis.

PubMed

Criado-Fornelio, A; Buling, A; Barba-Carretero, J C

2009-02-01

We developed and validated a real-time polymerase chain reaction (PCR) assay using fluorescent hybridization probes and melting curve analysis to identify the PKD1 exon 29 (C-->A) mutation, which is implicated in polycystic kidney disease of cats. DNA was isolated from peripheral blood of 20 Persian cats. The employ of the new real-time PCR and melting curve analysis in these samples indicated that 13 cats (65%) were wild type homozygotes and seven cats (35%) were heterozygotes. Both PCR-RFLP and sequencing procedures were in full agreement with real-time PCR test results. Sequence analysis showed that the mutant gene had the expected base change compared to the wild type gene. The new procedure is not only very reliable but also faster than the techniques currently applied for diagnosis of the mutation.

Recent progress on the mechanics of sharply bent DNA

NASA Astrophysics Data System (ADS)

Cong, PeiWen; Yan, Jie

2016-08-01

Despite extensive studies on the mechanics of DNA under external constrains, such as tension, torsion, and bending, several important aspects have remained poorly understood. One biologically important example is the mechanics of DNA under sharp bending conditions, which has been debated for a decade without thorough comprehension. The debate is about the interesting phenomenon raised from a series of different experiments: sharply bent DNA has a surprisingly high apparent bending flexibility that deviates from the canonical bending elasticity of DNA. This finding has motivated various theoretical models, which mainly incorporate the excitation of mechanical defects inside severely bent DNA molecules. Here, we review the recent progress on the understanding of the mechanics of sharply bent DNA and provide our view on this important question by interrogating the theoretical foundation of these experimental measurements.

Silver (I) as DNA glue: Ag+-mediated guanine pairing revealed by removing Watson-Crick constraints

PubMed Central

Swasey, Steven M.; Leal, Leonardo Espinosa; Lopez-Acevedo, Olga; Pavlovich, James; Gwinn, Elisabeth G.

2015-01-01

Metal ion interactions with DNA have far-reaching implications in biochemistry and DNA nanotechnology. Ag+ is uniquely interesting because it binds exclusively to the bases rather than the backbone of DNA, without the toxicity of Hg2+. In contrast to prior studies of Ag+ incorporation into double-stranded DNA, we remove the constraints of Watson-Crick pairing by focusing on homo-base DNA oligomers of the canonical bases. High resolution electro-spray ionization mass spectrometry reveals an unanticipated Ag+-mediated pairing of guanine homo-base strands, with higher stability than canonical guanine-cytosine pairing. By exploring unrestricted binding geometries, quantum chemical calculations find that Ag+ bridges between non-canonical sites on guanine bases. Circular dichroism spectroscopy shows that the Ag+-mediated structuring of guanine homobase strands persists to at least 90 °C under conditions for which canonical guanine-cytosine duplexes melt below 20 °C. These findings are promising for DNA nanotechnology and metal-ion based biomedical science. PMID:25973536

Silver (I) as DNA glue: Ag(+)-mediated guanine pairing revealed by removing Watson-Crick constraints.

PubMed

Swasey, Steven M; Leal, Leonardo Espinosa; Lopez-Acevedo, Olga; Pavlovich, James; Gwinn, Elisabeth G

2015-05-14

Metal ion interactions with DNA have far-reaching implications in biochemistry and DNA nanotechnology. Ag(+) is uniquely interesting because it binds exclusively to the bases rather than the backbone of DNA, without the toxicity of Hg(2+). In contrast to prior studies of Ag(+) incorporation into double-stranded DNA, we remove the constraints of Watson-Crick pairing by focusing on homo-base DNA oligomers of the canonical bases. High resolution electro-spray ionization mass spectrometry reveals an unanticipated Ag(+)-mediated pairing of guanine homo-base strands, with higher stability than canonical guanine-cytosine pairing. By exploring unrestricted binding geometries, quantum chemical calculations find that Ag(+) bridges between non-canonical sites on guanine bases. Circular dichroism spectroscopy shows that the Ag(+)-mediated structuring of guanine homobase strands persists to at least 90 °C under conditions for which canonical guanine-cytosine duplexes melt below 20 °C. These findings are promising for DNA nanotechnology and metal-ion based biomedical science.

Experimental studies on the nature of bonding of DNA/bipyridyl-(ethylenediamine)platinum(II) and DNA/netropsin complexes in solution and oriented wet-spun films

NASA Astrophysics Data System (ADS)

Marlowe, R. L.; Szabo, A.; Lee, S. A.; Rupprecht, A.

2002-03-01

The stability of complexes of NaDNA with bipyridyl-(ethylenediamine)platinum(II) (abbreviated [(bipy)Pt(en)]) and with netropsin has been studied using two techniques: (i) ultraviolet melting experiments were done on NaDNA/[(bipy)Pt(en)], showing that the [(bipy)Pt(en)] ligand stabilizes the DNA double helix structure; and (ii) swelling measurements (via optical microscopy) as a function of relative humidity were done on wet-spun oriented films of NaDNA/[(bipy)Pt(en)] and of NaDNA/netropsin. The swelling data shows that an irreversible transition of the films occurs at high relative humidity, first for the NaDNA/netropsin, then for pure NaDNA, and lastly for the NaDNA/[(bipy)Pt(en)]. These results are indicative that the [(bipy)Pt(en)] complex stabilizes the intermolecular bonds which mediate the film swelling characteristics. A model is suggested for the binding of [(bipy)Pt(en)] to DNA to explain why the swelling experiments show this ligand as increasing the intermolecular bond strength between the DNA double helices, while netropsin decreases this degree of stabilization.

Rapid quantification and sex determination of forensic evidence materials.

PubMed

Andréasson, Hanna; Allen, Marie

2003-11-01

DNA quantification of forensic evidence is very valuable for an optimal use of the available biological material. Moreover, sex determination is of great importance as additional information in criminal investigations as well as in identification of missing persons, no suspect cases, and ancient DNA studies. While routine forensic DNA analysis based on short tandem repeat markers includes a marker for sex determination, analysis of samples containing scarce amounts of DNA is often based on mitochondrial DNA, and sex determination is not performed. In order to allow quantification and simultaneous sex determination on minute amounts of DNA, an assay based on real-time PCR analysis of a marker within the human amelogenin gene has been developed. The sex determination is based on melting curve analysis, while an externally standardized kinetic analysis allows quantification of the nuclear DNA copy number in the sample. This real-time DNA quantification assay has proven to be highly sensitive, enabling quantification of single DNA copies. Although certain limitations were apparent, the system is a rapid, cost-effective, and flexible assay for analysis of forensic casework samples.

Characterization of the geometry and topology of DNA pictured as a discrete collection of atoms

PubMed Central

Olson, Wilma K.

2014-01-01

The structural and physical properties of DNA are closely related to its geometry and topology. The classical mathematical treatment of DNA geometry and topology in terms of ideal smooth space curves was not designed to characterize the spatial arrangements of atoms found in high-resolution and simulated double-helical structures. We present here new and rigorous numerical methods for the rapid and accurate assessment of the geometry and topology of double-helical DNA structures in terms of the constituent atoms. These methods are well designed for large DNA datasets obtained in detailed numerical simulations or determined experimentally at high-resolution. We illustrate the usefulness of our methodology by applying it to the analysis of three canonical double-helical DNA chains, a 65-bp minicircle obtained in recent molecular dynamics simulations, and a crystallographic array of protein-bound DNA duplexes. Although we focus on fully base-paired DNA structures, our methods can be extended to treat the geometry and topology of melted DNA structures as well as to characterize the folding of arbitrary molecules such as RNA and cyclic peptides. PMID:24791158

Towards Estimate of Present Day Ice Melting in Polar Regions From Altimetry, Gravity, Ocean Bottom Pressure and GPS Observations

NASA Astrophysics Data System (ADS)

Jiang, Y.; Wu, X.; van den Broeke, M. R.; Munneke, P. K.; Simonsen, S. B.; van der Wal, W.; Vermeersen, B. L.

2013-12-01

The ice sheet in Polar Regions stores the largest freshwater bodies on Earth, sufficient to elevate global sea level by more than 65 meters if melted. The earth may have entered an intensive ice-melting episode, possibly due to anthropogenic global warming rather than natural orbit variations. Determining present-day ice mass balance, however, is complicated by the fact that most observations contain both present day ice melting signal and residual signals from past glacier melting. Despite decades of progress in geodynamic modeling and new observations, significant uncertainties remain in both. The key to separate present-day ice mass change and signals from past melting is to include data of different physical characteristics. We conducted a new global kinematic inversion scheme to estimate both present-day ice melting and past glacier signatures simultaneously and assess their contribution to current and future global mean sea level change. Our approach is designed to invert and separate present-day melting signal in the spherical harmonic domain using a globally distributed interdisciplinary data with distinct physical information. Interesting results with unprecedented precisions have been achieved so far. We will present our results of the estimated present-day ice mass balance trend in both Greenland and Antarctica ice sheet as well as other regions where significant mass change occurs.

Ex-Vessel Core Melt Modeling Comparison between MELTSPREAD-CORQUENCH and MELCOR 2.1

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

Robb, Kevin R.; Farmer, Mitchell; Francis, Matthew W.

System-level code analyses by both United States and international researchers predict major core melting, bottom head failure, and corium-concrete interaction for Fukushima Daiichi Unit 1 (1F1). Although system codes such as MELCOR and MAAP are capable of capturing a wide range of accident phenomena, they currently do not contain detailed models for evaluating some ex-vessel core melt behavior. However, specialized codes containing more detailed modeling are available for melt spreading such as MELTSPREAD as well as long-term molten corium-concrete interaction (MCCI) and debris coolability such as CORQUENCH. In a preceding study, Enhanced Ex-Vessel Analysis for Fukushima Daiichi Unit 1: Meltmore » Spreading and Core-Concrete Interaction Analyses with MELTSPREAD and CORQUENCH, the MELTSPREAD-CORQUENCH codes predicted the 1F1 core melt readily cooled in contrast to predictions by MELCOR. The user community has taken notice and is in the process of updating their systems codes; specifically MAAP and MELCOR, to improve and reduce conservatism in their ex-vessel core melt models. This report investigates why the MELCOR v2.1 code, compared to the MELTSPREAD and CORQUENCH 3.03 codes, yield differing predictions of ex-vessel melt progression. To accomplish this, the differences in the treatment of the ex-vessel melt with respect to melt spreading and long-term coolability are examined. The differences in modeling approaches are summarized, and a comparison of example code predictions is provided.« less

Investigation of FANCA gene in Fanconi anaemia patients in Iran

PubMed Central

Saffar Moghadam, Ali Akbar; Mahjoubi, Frouzandeh; Reisi, Nahid; Vosough, Parvaneh

2016-01-01

Background & objectives: Fanconi anaemia (FA) is a syndrome with a predisposition to bone marrow failure, congenital anomalies and malignancies. It is characterized by cellular hypersensitivity to cross-linking agents such as mitomycin C (MMC). In the present study, a new approach was selected to investigate FANCA (Fanconi anaemia complementation group A) gene in patients clinically diagnosed with cellular hypersensitivity to DNA cross-linking agent MMC. Methods: Chromosomal breakage analysis was performed to prove the diagnosis of Fanconi anaemia in 318 families. Of these, 70 families had a positive result. Forty families agreed to molecular genetic testing. In total, there were 27 patients with unknown complementary types. Genomic DNA was extracted and total RNA was isolated from fresh whole blood of the patients. The first-strand cDNA was synthesized and the cDNA of each patient was then tested with 21 pairs of overlapping primers. High resolution melting curve analysis was used to screen FANCA, and LinReg software version 1.7 was utilized for analysis of expression. Results: In total, six sequence alterations were identified, which included two stop codons, two frames-shift mutations, one large deletion and one amino acid exchange. FANCA expression was downregulated in patients who had sequence alterations. Interpretation & conclusions: The results of the present study show that high resolution melting (HRM) curve analysis may be useful in the detection of sequence alteration. It is simpler and more costeffective than the multiplex ligation-dependent probe amplification (MLPA) procedure. PMID:27121516

Investigation of FANCA gene in Fanconi anaemia patients in Iran.

PubMed

Moghadam, Ali Akbar Saffar; Mahjoubi, Frouzandeh; Reisi, Nahid; Vosough, Parvaneh

2016-02-01

Fanconi anaemia (FA) is a syndrome with a predisposition to bone marrow failure, congenital anomalies and malignancies. It is characterized by cellular hypersensitivity to cross-linking agents such as mitomycin C (MMC). In the present study, a new approach was selected to investigate FANCA (Fanconi anaemia complementation group A) gene in patients clinically diagnosed with cellular hypersensitivity to DNA cross-linking agent MMC. Chromosomal breakage analysis was performed to prove the diagnosis of Fanconi anaemia in 318 families. Of these, 70 families had a positive result. Forty families agreed to molecular genetic testing. In total, there were 27 patients with unknown complementary types. Genomic DNA was extracted and total RNA was isolated from fresh whole blood of the patients. The first-strand cDNA was synthesized and the cDNA of each patient was then tested with 21 pairs of overlapping primers. High resolution melting curve analysis was used to screen FANCA, and LinReg software version 1.7 was utilized for analysis of expression. In total, six sequence alterations were identified, which included two stop codons, two frames-shift mutations, one large deletion and one amino acid exchange. FANCA expression was downregulated in patients who had sequence alterations. The results of the present study show that high resolution melting (HRM) curve analysis may be useful in the detection of sequence alteration. It is simpler and more cost-effective than the multiplex ligation-dependent probe amplification (MLPA) procedure.

Taxonomic Identification of Mediterranean Pines and Their Hybrids Based on the High Resolution Melting (HRM) and trnL Approaches: From Cytoplasmic Inheritance to Timber Tracing

PubMed Central

Ganopoulos, Ioannis; Aravanopoulos, Filippos; Madesis, Panagiotis; Pasentsis, Konstantinos; Bosmali, Irene; Ouzounis, Christos; Tsaftaris, Athanasios

2013-01-01

Fast and accurate detection of plant species and their hybrids using molecular tools will facilitate the assessment and monitoring of local biodiversity in an era of climate and environmental change. Herein, we evaluate the utility of the plastid trnL marker for species identification applied to Mediterranean pines (Pinus spp.). Our results indicate that trnL is a very sensitive marker for delimiting species biodiversity. Furthermore, High Resolution Melting (HRM) analysis was exploited as a molecular fingerprint for fast and accurate discrimination of Pinus spp. DNA sequence variants. The trnL approach and the HRM analyses were extended to wood samples of two species (Pinus nigra and Pinus sylvestris) with excellent results, congruent to those obtained using leaf tissue. Both analyses demonstrate that hybrids from the P. brutia (maternal parent) × P. halepensis (paternal parent) cross, exhibit the P. halepensis profile, confirming paternal plastid inheritance in Group Halepensis pines. Our study indicates that a single one-step reaction method and DNA marker are sufficient for the identification of Mediterranean pines, their hybrids and the origin of pine wood. Furthermore, our results underline the potential for certain DNA regions to be used as novel biological information markers combined with existing morphological characters and suggest a relatively reliable and open taxonomic system that can link DNA variation to phenotype-based species or hybrid assignment status and direct taxa identification from recalcitrant tissues such as wood samples. PMID:23577179

Taxonomic identification of mediterranean pines and their hybrids based on the high resolution melting (HRM) and trnL approaches: from cytoplasmic inheritance to timber tracing.

PubMed

Ganopoulos, Ioannis; Aravanopoulos, Filippos; Madesis, Panagiotis; Pasentsis, Konstantinos; Bosmali, Irene; Ouzounis, Christos; Tsaftaris, Athanasios

2013-01-01

Fast and accurate detection of plant species and their hybrids using molecular tools will facilitate the assessment and monitoring of local biodiversity in an era of climate and environmental change. Herein, we evaluate the utility of the plastid trnL marker for species identification applied to Mediterranean pines (Pinus spp.). Our results indicate that trnL is a very sensitive marker for delimiting species biodiversity. Furthermore, High Resolution Melting (HRM) analysis was exploited as a molecular fingerprint for fast and accurate discrimination of Pinus spp. DNA sequence variants. The trnL approach and the HRM analyses were extended to wood samples of two species (Pinus nigra and Pinus sylvestris) with excellent results, congruent to those obtained using leaf tissue. Both analyses demonstrate that hybrids from the P. brutia (maternal parent) × P. halepensis (paternal parent) cross, exhibit the P. halepensis profile, confirming paternal plastid inheritance in Group Halepensis pines. Our study indicates that a single one-step reaction method and DNA marker are sufficient for the identification of Mediterranean pines, their hybrids and the origin of pine wood. Furthermore, our results underline the potential for certain DNA regions to be used as novel biological information markers combined with existing morphological characters and suggest a relatively reliable and open taxonomic system that can link DNA variation to phenotype-based species or hybrid assignment status and direct taxa identification from recalcitrant tissues such as wood samples.

Silicon Web Process Development

NASA Technical Reports Server (NTRS)

Duncan, C. S.; Seidensticker, R. G.; Hopkins, R. H.; Mchugh, J. P.; Hill, F. E.; Heimlich, M. E.; Driggers, J. M.

1978-01-01

Progress in the development of techniques to grow silicon web at 25 wq cm/min output rate is reported. Feasibility of web growth with simultaneous melt replenishment is discussed. Other factors covered include: (1) tests of aftertrimmers to improve web width; (2) evaluation of growth lid designs to raise speed and output rate; (3) tests of melt replenishment hardware; and (4) investigation of directed gas flow systems to control unwanted oxide deposition in the system and to improve convective cooling of the web. Compatibility with sufficient solar cell performance is emphasized.

Lunar production of oxygen by electrolysis

NASA Technical Reports Server (NTRS)

Keller, Rudolf

1991-01-01

Two approaches to prepare oxygen from lunar resources by direct electrolysis are discussed. Silicates can be melted or dissolved in a fused salt and electrolyzed with oxygen evolved at the anode. Direct melting and electrolysis is potentially a very simple process, but high temperatures of 1400-1500 C are required, which aggravates materials problems. Operating temperatures can be lowered to about 1000 C by employing a molten salt flux. In this case, however, losses of electrolyte components must be avoided. Experimentation on both approaches is progressing.

Use and Limitations of a Climate-Quality Data Record to Study Temperature Trends on the Greenland Ice Sheet

NASA Astrophysics Data System (ADS)

Hall, D. K.; Comiso, J. C.; Shuman, C. A.; Koenig, L.; DiGirolamo, N. E.

2011-12-01

Enhanced melting of the Greenland Ice Sheet has been documented in recent literature along with surface-temperature increases measured using infrared satellite data since 1981. Using a recently-developed climate-quality data record, 11- and 12-year trends in the clear-sky ice-surface temperature (IST) of the Greenland Ice Sheet have been studied using the Moderate-Resolution Imaging Spectroradiometer (MODIS) IST product. Daily and monthly MODIS ISTs of the Greenland Ice Sheet beginning on 1 March 2000 and continuing through 31 December 2010 are now available at 6.25-km spatial resolution on a polar stereographic grid as described in Hall et al. (submitted). This record will be elevated in status to a climate-data record (CDR) when more years of data become available either from the MODIS on the Terra or Aqua satellites, or from the Visible Infrared Imager Radiometer Suite (VIIRS) to be launched in October 2011. Maps showing the maximum extent of melt for the entire ice sheet and for the six major drainage basins have been developed from the MODIS IST dataset. Twelve-year trends in the extent of melt and duration of the melt season on the ice sheet vary in different drainage basins with some basins melting progressively earlier over the course of the study period. Some (but not all) of the basins also show a progressively-longer duration of melt. Twelve-year trends in IST are compared with in-situ data, and climate data from the Modern Era Retrospective-Analysis for Research and Applications (MERRA) Reanalysis. Hall, D.K., J.C. Comiso, N.E. DiGirolamo, C.A. Shuman, J. Key and L.S. Koenig, submitted for journal publication: A Satellite-Derived Climate-Quality Data Record of the Clear-Sky Surface Temperature of the Greenland Ice Sheet.

Direct Determination of the Equilibrium Unbinding Potential Profile for a Short DNA Duplex from Force Spectroscopy Data

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

Noy, A

2004-05-04

Modern force microscopy techniques allow researchers to use mechanical forces to probe interactions between biomolecules. However, such measurements often happen in non-equilibrium regime, which precludes straightforward extraction of the equilibrium energy information. Here we use the work averaging method based on Jarzynski equality to reconstruct the equilibrium interaction potential from the unbinding of a complementary 14-mer DNA duplex from the results of non-equilibrium single-molecule measurements. The reconstructed potential reproduces most of the features of the DNA stretching transition, previously observed only in equilibrium stretching of long DNA sequences. We also compare the reconstructed potential with the thermodynamic parameters of DNAmore » duplex unbinding and show that the reconstruction accurately predicts duplex melting enthalpy.« less

Seasonal Change in Bacterial Flora and Biomass in Mountain Snow from the Tateyama Mountains, Japan, Analyzed by 16S rRNA Gene Sequencing and Real-Time PCR

PubMed Central

Segawa, Takahiro; Miyamoto, Koji; Ushida, Kazunari; Agata, Kiyokazu; Okada, Norihiro; Kohshima, Shiro

2005-01-01

The bacterial flora and biomass in mountain snow from the Tateyama Mountains, Toyama Prefecture, Japan, one of the heaviest snowfall regions in the world, were analyzed by amplified ribosomal DNA restriction analysis followed by 16S rRNA gene sequencing and DNA quantification by real-time PCR. Samples of surface snow collected in various months during the melting season contained a psychrophilic bacterium, Cryobacterium psychrophilum, and two psychrotrophic bacteria, Variovorax paradoxus and Janthinobacterium lividum. Bacterial colonies that developed in an in situ meltwater medium at 4°C were revealed to be V. paradoxus. The biomasses of C. psychrophilum, J. lividum, and V. paradoxus, as estimated by real-time PCR, showed large increases during the melting season from March to October (2.0 × 105-fold, 1.5 × 105-fold, and 1.0 × 104-fold increases, respectively), suggesting their rapid growth in the surface snow. The biomasses of C. psychrophilum and J. lividum increased significantly from March to April, reached a maximum in August, and dropped at the end of the melting season. In contrast, the biomass of V. paradoxus did not increase as rapidly during the early melting season but continued to increase from June until October. The differences in development observed among these bacterial species suggest that their growth was promoted by different nutrients and/or environmental conditions in the snow. Since these three types of bacteria have also been reported to be present in a glacier in Antarctica and a Greenland ice core, they seem to be specialized members of the snow biota that are distributed in snow and ice environments in various parts of the world. PMID:15640179

Seasonal change in bacterial flora and biomass in mountain snow from the Tateyama Mountains, Japan, analyzed by 16S rRNA gene sequencing and real-time PCR.

PubMed

Segawa, Takahiro; Miyamoto, Koji; Ushida, Kazunari; Agata, Kiyokazu; Okada, Norihiro; Kohshima, Shiro

2005-01-01

The bacterial flora and biomass in mountain snow from the Tateyama Mountains, Toyama Prefecture, Japan, one of the heaviest snowfall regions in the world, were analyzed by amplified ribosomal DNA restriction analysis followed by 16S rRNA gene sequencing and DNA quantification by real-time PCR. Samples of surface snow collected in various months during the melting season contained a psychrophilic bacterium, Cryobacterium psychrophilum, and two psychrotrophic bacteria, Variovorax paradoxus and Janthinobacterium lividum. Bacterial colonies that developed in an in situ meltwater medium at 4 degrees C were revealed to be V. paradoxus. The biomasses of C. psychrophilum, J. lividum, and V. paradoxus, as estimated by real-time PCR, showed large increases during the melting season from March to October (2.0 x 10(5)-fold, 1.5 x 10(5)-fold, and 1.0 x 10(4)-fold increases, respectively), suggesting their rapid growth in the surface snow. The biomasses of C. psychrophilum and J. lividum increased significantly from March to April, reached a maximum in August, and dropped at the end of the melting season. In contrast, the biomass of V. paradoxus did not increase as rapidly during the early melting season but continued to increase from June until October. The differences in development observed among these bacterial species suggest that their growth was promoted by different nutrients and/or environmental conditions in the snow. Since these three types of bacteria have also been reported to be present in a glacier in Antarctica and a Greenland ice core, they seem to be specialized members of the snow biota that are distributed in snow and ice environments in various parts of the world.

High-resolution melt PCR analysis for genotyping of Ureaplasma parvum isolates directly from clinical samples.

PubMed

Payne, Matthew S; Tabone, Tania; Kemp, Matthew W; Keelan, Jeffrey A; Spiller, O Brad; Newnham, John P

2014-02-01

Ureaplasma sp. infection in neonates and adults underlies a variety of disease pathologies. Of the two human Ureaplasma spp., Ureaplasma parvum is clinically the most common. We have developed a high-resolution melt (HRM) PCR assay for the differentiation of the four serovars of U. parvum in a single step. Currently U. parvum strains are separated into four serovars by sequencing the promoter and coding region of the multiple-banded antigen (MBA) gene. We designed primers to conserved sequences within this region for PCR amplification and HRM analysis to generate reproducible and distinct melt profiles that distinguish clonal representatives of serovars 1, 3, 6, and 14. Furthermore, our HRM PCR assay could classify DNA extracted from 74 known (MBA-sequenced) test strains with 100% accuracy. Importantly, HRM PCR was also able to identify U. parvum serovars directly from 16 clinical swabs. HRM PCR performed with DNA consisting of mixtures of combined known serovars yielded profiles that were easily distinguished from those for single-serovar controls. These profiles mirrored clinical samples that contained mixed serovars. Unfortunately, melt curve analysis software is not yet robust enough to identify the composition of mixed serovar samples, only that more than one serovar is present. HRM PCR provides a single-step, rapid, cost-effective means to differentiate the four serovars of U. parvum that did not amplify any of the known 10 serovars of Ureaplasma urealyticum tested in parallel. Choice of reaction reagents was found to be crucial to allow sufficient sensitivity to differentiate U. parvum serovars directly from clinical swabs rather than requiring cell enrichment using microbial culture techniques.

High-Resolution Melting Analysis for Rapid Detection of Sequence Type 131 Escherichia coli.

PubMed

Harrison, Lucas B; Hanson, Nancy D

2017-06-01

Escherichia coli isolates belonging to the sequence type 131 (ST131) clonal complex have been associated with the global distribution of fluoroquinolone and β-lactam resistance. Whole-genome sequencing and multilocus sequence typing identify sequence type but are expensive when evaluating large numbers of samples. This study was designed to develop a cost-effective screening tool using high-resolution melting (HRM) analysis to differentiate ST131 from non-ST131 E. coli in large sample populations in the absence of sequence analysis. The method was optimized using DNA from 12 E. coli isolates. Singleplex PCR was performed using 10 ng of DNA, Type-it HRM buffer, and multilocus sequence typing primers and was followed by multiplex PCR. The amplicon sizes ranged from 630 to 737 bp. Melt temperature peaks were determined by performing HRM analysis at 0.1°C resolution from 50 to 95°C on a Rotor-Gene Q 5-plex HRM system. Derivative melt curves were compared between sequence types and analyzed by principal component analysis. A blinded study of 191 E. coli isolates of ST131 and unknown sequence types validated this methodology. This methodology returned 99.2% specificity (124 true negatives and 1 false positive) and 100% sensitivity (66 true positives and 0 false negatives). This HRM methodology distinguishes ST131 from non-ST131 E. coli without sequence analysis. The analysis can be accomplished in about 3 h in any laboratory with an HRM-capable instrument and principal component analysis software. Therefore, this assay is a fast and cost-effective alternative to sequencing-based ST131 identification. Copyright © 2017 Harrison and Hanson.

Applying high-resolution melting (HRM) technology to olive oil and wine authenticity.

PubMed

Pereira, Leonor; Gomes, Sónia; Barrias, Sara; Fernandes, José Ramiro; Martins-Lopes, Paula

2018-01-01

Olive oil and wine production have a worldwide economic impact. Their market reliability is under great concern because of the increasing number of fraud and adulteration attempts. The need for a traceability system in all its extension is crucial particularly for the cases of olive oils and wines with certified labels, in which only a limited number of olives and grapevine varieties, respectively, are allowed in a restricted well-defined geographical area. Molecular markers have been vastly applied to the food sector, and in particular High-Resolution DNA Melting technology has been successfully applied for olive oil and wine authentication, as part of the traceability system. In this review, the applications of HRM and their usefulness for this sector considering, Safety, Security and Authenticity will be reviewed. A broad overview of the HRM technique will be presented, focusing on the aspects that are crucial for its success, in particular the new generation of fluorescent dsDNA dyes used for amplicon detection and quantification, and the data analysis. A brief outlook on the olive oil and wine authenticity procedures, based on new DNA technology advances, and in which way this may influence the future establishment of a traceability system will be discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Classification of Rhizomonas suberifaciens, an unnamed Rhizomonas species, and Sphingomonas spp. in rRNA superfamily IV.

PubMed

van Bruggen, A H; Jochimsen, K N; Steinberger, E M; Segers, P; Gillis, M

1993-01-01

Thermal melting profiles of hybrids between 3H-labeled rRNA of Rhizomonas suberifaciens, the causal agent of corky root of lettuce, and chromosomal DNAs from 27 species of gram-negative bacteria indicated that the genus Rhizomonas belongs to superfamily IV of De Ley. On the basis of the melting temperatures of DNA hybrids with rRNAs from the type strains of R. suberifaciens, Sphingomonas paucimobilis, and Sphingomonas capsulata, Rhizomonas strains constitute a separate branch in superfamily IV, which is closely related to but separate from branches containing Zymomonas mobilis, Sphingomonas spp., and S. capsulata. Sphingomonas yanoikuyae and Rhizomonas sp. strain WI4 are located toward the base of the Rhizomonas rRNA branch. DNA-DNA hybridization indicated that S. yanoikuyae is equidistant from Rhizomonas sp. strain WI4 and S. paucimobilis. Sequences of 270 bp of 16S ribosomal DNAs from eight strains of Rhizomonas spp., eight strains of Sphingomonas spp., and Agrobacterium tumefaciens indicated that S. yanoikuyae and Rhizomonas sp. strains WI4 and CA16 are genetically more closely related to R. suberifaciens than to Sphingomonas spp. Thus, S. yanoikuyae may need to be transferred to the genus Rhizomonas on the basis of the results of further study.

Cell-free DNA as a molecular tool for monitoring disease progression and response to therapy in breast cancer patients.

PubMed

Liang, Diana H; Ensor, Joe E; Liu, Zhe-Bin; Patel, Asmita; Patel, Tejal A; Chang, Jenny C; Rodriguez, Angel A

2016-01-01

Due to the spatial and temporal genomic heterogeneity of breast cancer, genomic sequencing obtained from a single biopsy may not capture the complete genomic profile of tumors. Thus, we propose that cell-free DNA (cfDNA) in plasma may be an alternate source of genomic information to provide comprehensive data throughout a patient's clinical course. We performed a retrospective chart review of 100 patients with stage 4 or high-risk stage 3 breast cancer. The degree of agreement between genomic alterations found in tumor DNA (tDNA) and cfDNA was determined by Cohen's Kappa. Clinical disease progression was compared to mutant allele frequency using a two-sided Fisher's exact test. The presence of mutations and mutant allele frequency was correlated with progression-free survival (PFS) using a Cox proportional hazards model and a log-rank test. The most commonly found genomic alterations were mutations in TP53 and PIK3CA, and amplification of EGFR and ERBB2. PIK3CA mutation and ERBB2 amplification demonstrated robust agreement between tDNA and cfDNA (Cohen's kappa = 0.64 and 0.77, respectively). TP53 mutation and EGFR amplification demonstrated poor agreement between tDNA and cfDNA (Cohen's kappa = 0.18 and 0.33, respectively). The directional changes of TP53 and PIK3CA mutant allele frequency were closely associated with response to therapy (p = 0.002). The presence of TP53 mutation (p = 0.0004) and PIK3CA mutant allele frequency [p = 0.01, HR 1.074 (95 % CI 1.018-1.134)] was excellent predictors of PFS. Identification of selected cancer-specific genomic alterations from cfDNA may be a noninvasive way to monitor disease progression, predict PFS, and offer targeted therapy.

Barcoding Melting Curve Analysis for Rapid, Sensitive, and Discriminating Authentication of Saffron (Crocus sativus L.) from Its Adulterants

PubMed Central

Cao, Liang; Yuan, Yuan; Chen, Min; Jin, Yan; Huang, Luqi

2014-01-01

Saffron (Crocus sativus L.) is one of the most important and expensive medicinal spice products in the world. Because of its high market value and premium price, saffron is often adulterated through the incorporation of other materials, such as Carthamus tinctorius L. and Calendula officinalis L. flowers, Hemerocallis L. petals, Daucus carota L. fleshy root, Curcuma longa L. rhizomes, Zea may L., and Nelumbo nucifera Gaertn. stigmas. To develop a straightforward, nonsequencing method for rapid, sensitive, and discriminating detection of these adulterants in traded saffron, we report here the application of a barcoding melting curve analysis method (Bar-MCA) that uses the universal chloroplast plant DNA barcoding region trnH-psbA to identify adulterants. When amplified at DNA concentrations and annealing temperatures optimized for the curve analysis, peaks were formed at specific locations for saffron (81.92°C) and the adulterants: D. carota (81.60°C), C. tinctorius (80.10°C), C. officinalis (79.92°C), Dendranthema morifolium (Ramat.) Tzvel. (79.62°C), N. nucifera (80.58°C), Hemerocallis fulva (L.) L. (84.78°C), and Z. mays (84.33°C). The constructed melting curves for saffron and its adulterants have significantly different peak locations or shapes. In conclusion, Bar-MCA could be a faster and more cost-effective method to authenticate saffron and detect its adulterants. PMID:25548775

Barcoding melting curve analysis for rapid, sensitive, and discriminating authentication of saffron (Crocus sativus L.) from its adulterants.

PubMed

Jiang, Chao; Cao, Liang; Yuan, Yuan; Chen, Min; Jin, Yan; Huang, Luqi

2014-01-01

Saffron (Crocus sativus L.) is one of the most important and expensive medicinal spice products in the world. Because of its high market value and premium price, saffron is often adulterated through the incorporation of other materials, such as Carthamus tinctorius L. and Calendula officinalis L. flowers, Hemerocallis L. petals, Daucus carota L. fleshy root, Curcuma longa L. rhizomes, Zea may L., and Nelumbo nucifera Gaertn. stigmas. To develop a straightforward, nonsequencing method for rapid, sensitive, and discriminating detection of these adulterants in traded saffron, we report here the application of a barcoding melting curve analysis method (Bar-MCA) that uses the universal chloroplast plant DNA barcoding region trnH-psbA to identify adulterants. When amplified at DNA concentrations and annealing temperatures optimized for the curve analysis, peaks were formed at specific locations for saffron (81.92°C) and the adulterants: D. carota (81.60°C), C. tinctorius (80.10°C), C. officinalis (79.92°C), Dendranthema morifolium (Ramat.) Tzvel. (79.62°C), N. nucifera (80.58°C), Hemerocallis fulva (L.) L. (84.78°C), and Z. mays (84.33°C). The constructed melting curves for saffron and its adulterants have significantly different peak locations or shapes. In conclusion, Bar-MCA could be a faster and more cost-effective method to authenticate saffron and detect its adulterants.

Binding of DNA-binding alkaloids berberine and palmatine to tRNA and comparison to ethidium: Spectroscopic and molecular modeling studies

NASA Astrophysics Data System (ADS)

Islam, Md. Maidul; Pandya, Prateek; Chowdhury, Sebanti Roy; Kumar, Surat; Kumar, Gopinatha Suresh

2008-11-01

The interaction of two natural protoberberine plant alkaloids berberine and palmatine with tRNA phe was studied using various biophysical techniques and molecular modeling and the data were compared with the binding of the classical DNA intercalator, ethidium. Circular dichroic studies revealed that the tRNA conformation was moderately perturbed on binding of the alkaloids. The cooperative binding of both the alkaloids and ethidium to tRNA was revealed from absorbance and fluorescence studies. Fluorescence quenching studies advanced a conclusion that while berberine and palmatine are partially intercalated, ethidium is fully intercalated on the tRNA molecule. The binding of the alkaloids as well as ethidium stabilized the tRNA melting, and the binding constant evaluated from the averaged optical melting temperature data was in agreement with fluorescence spectral-binding data. Differential scanning calorimetry revealed that the tRNA melting showed three close transitions that were affected on binding of these small molecules. Molecular docking calculations performed showed the preferred regions of binding of these small molecules on the tRNA. Taken together, the results suggest that the binding of the alkaloids berberine and palmatine on the tRNA structure appears to be mostly by partial intercalation while ethidium intercalates fully on the tRNA. These results further advance our knowledge on the molecular aspects on the interaction of these alkaloids to tRNA.

Dynamics of EGFR Mutation Load in Plasma for Prediction of Treatment Response and Disease Progression in Patients With EGFR-Mutant Lung Adenocarcinoma.

PubMed

Taus, Álvaro; Camacho, Laura; Rocha, Pedro; Hardy-Werbin, Max; Pijuan, Lara; Piquer, Gabriel; López, Eva; Dalmases, Alba; Longarón, Raquel; Clavé, Sergi; Salido, Marta; Albanell, Joan; Bellosillo, Beatriz; Arriola, Edurne

2018-03-23

The assessment of epidermal growth factor receptor (EGFR) mutations is crucial for the management of patients with lung adenocarcinoma. Circulating tumor DNA (ctDNA)-based assessment offers advantages over tumor as a minimally invasive method able to capture tumor heterogeneity. Consecutive patients diagnosed with EGFR-mutant lung adenocarcinoma in tumor biopsy were included in this study. Plasma samples were obtained at different time points during the course of the disease. EGFR mutations in plasma were quantified using BEAMing (beads, emulsions, amplification, and magnetics) or digital PCR and were correlated with mutations in tumor and with radiologic response and progression. Two hundred twenty-one plasma samples from 33 patients were analyzed. EGFR mutations in plasma were detected in 83% of all patients and 100% of those with extrathoracic metastases. The dynamics of the EGFR mutation load predicted response in 93% and progression in 89% of cases well in advance of radiologic evaluation. Progression-free survival for patients in whom ctDNA was not detected in plasma during treatment was significantly longer than for those in whom ctDNA remained detectable (295 vs. 55 days; hazard ratio, 17.1; P < .001). The detection of EGFR mutations in ctDNA showed good correlation with that in tumor biopsy and predicted tumor response and progression in most patients. The liquid biopsy for ctDNA-based assessment of EGFR mutations is a reliable technique for diagnosis and follow-up in patients with EGFR-mutant lung adenocarcinoma in routine clinical practice. Copyright © 2018 Elsevier Inc. All rights reserved.

Scallop-Inspired DNA Nanomachine: A Ratiometric Nanothermometer for Intracellular Temperature Sensing.

PubMed

Xie, Nuli; Huang, Jin; Yang, Xiaohai; He, Xiaoxiao; Liu, Jianbo; Huang, Jiaqi; Fang, Hongmei; Wang, Kemin

2017-11-21

Accurate measurement of intracellular temperature is of great significance in biology and medicine. With use of DNA nanotechnology and inspiration by nature's examples of "protective and reversible responses" exoskeletons, a scallop-inspired DNA nanomachine (SDN) is desgined as a ratiometric nanothermometer for intracellular temperature sensing. The SDN is composed of a rigid DNA tetrahedron, where a thermal-sensitive molecular beacon (MB) is embedded in one edge of the DNA tetrahedron. Relying on the thermal-sensitive MB and fluorescence resonance energy transfer (FRET) signaling mechanism, the "On" to "Off" signal is reversibly responding to "below" and "over" the melting temperature. Mimicking the functional anatomy of a scallop, the SDN exhibits high cellular permeability and resistance to enzymatic degradation, good reversibility, and tunable response range. Furthermore, FRET ratiometric signal that allows the simultaneous recording of two emission intensities at different wavelengths can provide a feasible approach for precise detection, minimizing the effect of system fluctuations.

Are terrestrial plumes from motionless plates analogues to Martian plumes feeding the giant shield volcanoes?

NASA Astrophysics Data System (ADS)

Meyzen, Christine; Massironi, Matteo; Pozzobon, Riccardo; Dal Zilio, Luca

2014-05-01

The near "one-plate" planet evolution of Mars has led to the edification of long-lasting giant shied volcanoes. Unlike the Earth, Mars would have been a transient convecting planet, where plate tectonic would have possibly acted only during the first hundreds of million years of its history. On Earth, where plate tectonic is active, most of them are regenerated and recycled through convection. However, the Nubian and Antarctic plates could be considered as poorly mobile surfaces of various thicknesses that are acting as conductive lids on top of Earth's deeper convective system. In these environments, volcanoes do not show any linear age progression at least for the last 30 Ma, but constitute the sites of persistent, focused long-term magmatic activity, rather than a chain of volcanoes as observed in fast-moving plate plume environments. Here, the near stationary absolute plate motion probably exerts a primary control on volcanic processes, and more specifically, on the melting ones. The residual depleted mantle, that is left behind by the melting processes, cannot be swept away from the melting locus. Over time, the thickening of this near-stationary depleted layer progressively forces the termination of melting to higher depths, reducing the melt production rate. Such a process gradually leads both to decreasing efficient melt extraction and increasing mantle lithospheric-melt interactions. The accumulation of this refractory material also causes long-term fluctuations of the volcanic activity, in generating long periods of quiescence. The presence of this residual mantle keel induces over time a lateral flow deflection, which translates into a shift of future melting sites around it. This process gives rise to the horseshoe-like shape of some volcanic islands on slow-moving plates (e.g. Cape Verde, Crozet). Finally, the pronounced topographic swells/bulges observed in this environments may also be supported both by large scale mantle upwelling and their residual mantle roots. Most of these processes are likely similar to those observed on Martian giant shield volcanoes. The goal of this presentation will be to describe the essential characteristics of intra-oceanic plumes on slow moving plates on the Earth and to point out their similarities with those of the large shield volcanoes from the Tharsis region.

In-gel multiple displacement amplification of long DNA fragments diluted to the single molecule level.

PubMed

Michikawa, Yuichi; Sugahara, Keisuke; Suga, Tomo; Ohtsuka, Yoshimi; Ishikawa, Kenichi; Ishikawa, Atsuko; Shiomi, Naoko; Shiomi, Tadahiro; Iwakawa, Mayumi; Imai, Takashi

2008-12-15

The isolation and multiple genotyping of long individual DNA fragments are needed to obtain haplotype information for diploid organisms. Limiting dilution of sample DNA followed by multiple displacement amplification is a useful technique but is restricted to short (<5 kb) DNA fragments. In the current study, a novel modification was applied to overcome these problems. A limited amount of cellular DNA was carefully released from intact cells into a mildly heated alkaline agarose solution and mixed thoroughly. The solution was then gently aliquoted and allowed to solidify while maintaining the integrity of the diluted DNA. Exogenously provided Phi29 DNA polymerase was used to perform consistent genomic amplification with random hexameric oligonucleotides within the agarose gels. Simple heat melting of the gel allowed recovery of the amplified materials in a solution of the polymerase chain reaction (PCR)-ready form. The haplotypes of seven SNPs spanning 240 kb of the DNA surrounding the human ATM gene region on chromosome 11 were determined for 10 individuals, demonstrating the feasibility of this new method.

Studies on interaction of norbixin with DNA: Multispectroscopic and in silico analysis

NASA Astrophysics Data System (ADS)

Anantharaman, Amrita; Priya, Rajendra Rao; Hemachandran, Hridya; Sivaramakrishna, Akella; Babu, Subramanian; Siva, Ramamoorthy

2015-06-01

The interaction of food colorant norbixin with calf thymus DNA (CTDNA) was investigated through UV-Visible spectroscopy, Fourier Transform Infrared (FTIR), Circular Dichroism (CD), Nuclear Magnetic Resonance (NMR), DNA melting studies, electrophoretic analysis, histological staining technique and molecular docking studies. The results indicated that norbixin interacted with CTDNA by partial intercalation mode. The binding constant (K) of norbixin with CTDNA was calculated to be 5.08 × 105 Mol-1 L. FTIR and CD studies were coupled with 1H NMR spectra revealed that norbixin intercalates partially and binds to the groove's, phosphate group, deoxyribose sugar of DNA and also induces conformational transition of B-form to A-form DNA. Agarose gel electrophoretic and histological staining technique results further prove that, norbixin specifically binds to the DNA in the cell. Moreover, molecular docking studies on the specific binding of norbixin with CTDNA have exhibited lowest conformation energy score of -3.2. Therefore, this food colorant has the ability to interact with DNA and it could emerge as a promising class of natural DNA targeted therapeutic.

Molecular structure of bottlebrush polymers in melts

PubMed Central

Paturej, Jarosław; Sheiko, Sergei S.; Panyukov, Sergey; Rubinstein, Michael

2016-01-01

Bottlebrushes are fascinating macromolecules that display an intriguing combination of molecular and particulate features having vital implications in both living and synthetic systems, such as cartilage and ultrasoft elastomers. However, the progress in practical applications is impeded by the lack of knowledge about the hierarchic organization of both individual bottlebrushes and their assemblies. We delineate fundamental correlations between molecular architecture, mesoscopic conformation, and macroscopic properties of polymer melts. Numerical simulations corroborate theoretical predictions for the effect of grafting density and side-chain length on the dimensions and rigidity of bottlebrushes, which effectively behave as a melt of flexible filaments. These findings provide quantitative guidelines for the design of novel materials that allow architectural tuning of their properties in a broad range without changing chemical composition. PMID:28861466

Peripheral neuropathy predicts nuclear gene defect in patients with mitochondrial ophthalmoplegia.

PubMed

Horga, Alejandro; Pitceathly, Robert D S; Blake, Julian C; Woodward, Catherine E; Zapater, Pedro; Fratter, Carl; Mudanohwo, Ese E; Plant, Gordon T; Houlden, Henry; Sweeney, Mary G; Hanna, Michael G; Reilly, Mary M

2014-12-01

Progressive external ophthalmoplegia is a common clinical feature in mitochondrial disease caused by nuclear DNA defects and single, large-scale mitochondrial DNA deletions and is less frequently associated with point mutations of mitochondrial DNA. Peripheral neuropathy is also a frequent manifestation of mitochondrial disease, although its prevalence and characteristics varies considerably among the different syndromes and genetic aetiologies. Based on clinical observations, we systematically investigated whether the presence of peripheral neuropathy could predict the underlying genetic defect in patients with progressive external ophthalmoplegia. We analysed detailed demographic, clinical and neurophysiological data from 116 patients with genetically-defined mitochondrial disease and progressive external ophthalmoplegia. Seventy-eight patients (67%) had a single mitochondrial DNA deletion, 12 (10%) had a point mutation of mitochondrial DNA and 26 (22%) had mutations in either POLG, C10orf2 or RRM2B, or had multiple mitochondrial DNA deletions in muscle without an identified nuclear gene defect. Seventy-seven patients had neurophysiological studies; of these, 16 patients (21%) had a large-fibre peripheral neuropathy. The prevalence of peripheral neuropathy was significantly lower in patients with a single mitochondrial DNA deletion (2%) as compared to those with a point mutation of mitochondrial DNA or with a nuclear DNA defect (44% and 52%, respectively; P<0.001). Univariate analyses revealed significant differences in the distribution of other clinical features between genotypes, including age at disease onset, gender, family history, progressive external ophthalmoplegia at clinical presentation, hearing loss, pigmentary retinopathy and extrapyramidal features. However, binomial logistic regression analysis identified peripheral neuropathy as the only independent predictor associated with a nuclear DNA defect (P=0.002; odds ratio 8.43, 95% confidence interval 2.24-31.76). Multinomial logistic regression analysis identified peripheral neuropathy, family history and hearing loss as significant predictors of the genotype, and the same three variables showed the highest performance in genotype classification in a decision tree analysis. Of these variables, peripheral neuropathy had the highest specificity (91%), negative predictive value (83%) and positive likelihood ratio (5.87) for the diagnosis of a nuclear DNA defect. These results indicate that peripheral neuropathy is a rare finding in patients with single mitochondrial DNA deletions but that it is highly predictive of an underlying nuclear DNA defect. This observation may facilitate the development of diagnostic algorithms. We suggest that nuclear gene testing may enable a more rapid diagnosis and avoid muscle biopsy in patients with progressive external ophthalmoplegia and peripheral neuropathy. © The Author (2014). Published by Oxford University Press on behalf of the Guarantors of Brain.

Peripheral neuropathy predicts nuclear gene defect in patients with mitochondrial ophthalmoplegia

PubMed Central

Pitceathly, Robert D. S.; Blake, Julian C.; Woodward, Catherine E.; Zapater, Pedro; Fratter, Carl; Mudanohwo, Ese E.; Plant, Gordon T.; Houlden, Henry; Sweeney, Mary G.; Hanna, Michael G.; Reilly, Mary M.

2014-01-01

Progressive external ophthalmoplegia is a common clinical feature in mitochondrial disease caused by nuclear DNA defects and single, large-scale mitochondrial DNA deletions and is less frequently associated with point mutations of mitochondrial DNA. Peripheral neuropathy is also a frequent manifestation of mitochondrial disease, although its prevalence and characteristics varies considerably among the different syndromes and genetic aetiologies. Based on clinical observations, we systematically investigated whether the presence of peripheral neuropathy could predict the underlying genetic defect in patients with progressive external ophthalmoplegia. We analysed detailed demographic, clinical and neurophysiological data from 116 patients with genetically-defined mitochondrial disease and progressive external ophthalmoplegia. Seventy-eight patients (67%) had a single mitochondrial DNA deletion, 12 (10%) had a point mutation of mitochondrial DNA and 26 (22%) had mutations in either POLG, C10orf2 or RRM2B, or had multiple mitochondrial DNA deletions in muscle without an identified nuclear gene defect. Seventy-seven patients had neurophysiological studies; of these, 16 patients (21%) had a large-fibre peripheral neuropathy. The prevalence of peripheral neuropathy was significantly lower in patients with a single mitochondrial DNA deletion (2%) as compared to those with a point mutation of mitochondrial DNA or with a nuclear DNA defect (44% and 52%, respectively; P < 0.001). Univariate analyses revealed significant differences in the distribution of other clinical features between genotypes, including age at disease onset, gender, family history, progressive external ophthalmoplegia at clinical presentation, hearing loss, pigmentary retinopathy and extrapyramidal features. However, binomial logistic regression analysis identified peripheral neuropathy as the only independent predictor associated with a nuclear DNA defect (P = 0.002; odds ratio 8.43, 95% confidence interval 2.24–31.76). Multinomial logistic regression analysis identified peripheral neuropathy, family history and hearing loss as significant predictors of the genotype, and the same three variables showed the highest performance in genotype classification in a decision tree analysis. Of these variables, peripheral neuropathy had the highest specificity (91%), negative predictive value (83%) and positive likelihood ratio (5.87) for the diagnosis of a nuclear DNA defect. These results indicate that peripheral neuropathy is a rare finding in patients with single mitochondrial DNA deletions but that it is highly predictive of an underlying nuclear DNA defect. This observation may facilitate the development of diagnostic algorithms. We suggest that nuclear gene testing may enable a more rapid diagnosis and avoid muscle biopsy in patients with progressive external ophthalmoplegia and peripheral neuropathy. PMID:25281868

Development of a two-step high-resolution melting (HRM) analysis for screening sequence variants associated with resistance to the QoIs, benzimidazoles and dicarboximides in airborne inoculum of Botrytis cinerea.

PubMed

Chatzidimopoulos, Michael; Ganopoulos, Ioannis; Vellios, Evangelos; Madesis, Panagiotis; Tsaftaris, Athanasios; Pappas, Athanassios C

2014-11-01

A rapid, high-resolution melting (HRM) analysis protocol was developed to detect sequence variations associated with resistance to the QoIs, benzimidazoles and dicarboximides in Botrytis cinerea airborne inoculum. HRM analysis was applied directly in fungal DNA collected from air samplers with selective medium. Three and five different genotypes were detected and classified according to their melting profiles in BenA and bos1 genes associated with resistance to benzimidazoles and dicarboximides, respectively. The sensitivity of the methodology was evident in the case of the QoIs, where genotypes varying either by a single nucleotide polymorphism or an additional 1205-bp intron were separated accurately with a single pair of primers. The developed two-step protocol was completed in 82 min and showed reduced variation in the melting curves' formation. HRM analysis rapidly detected the major mutations found in greenhouse strains providing accurate data for successfully controlling grey mould. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Force regulated dynamics of RPA on a DNA fork

PubMed Central

Kemmerich, Felix E.; Daldrop, Peter; Pinto, Cosimo; Levikova, Maryna; Cejka, Petr; Seidel, Ralf

2016-01-01

Replication protein A (RPA) is a single-stranded DNA binding protein, involved in most aspects of eukaryotic DNA metabolism. Here, we study the behavior of RPA on a DNA substrate that mimics a replication fork. Using magnetic tweezers we show that both yeast and human RPA can open forked DNA when sufficient external tension is applied. In contrast, at low force, RPA becomes rapidly displaced by the rehybridization of the DNA fork. This process appears to be governed by the binding or the release of an RPA microdomain (toehold) of only few base-pairs length. This gives rise to an extremely rapid exchange dynamics of RPA at the fork. Fork rezipping rates reach up to hundreds of base-pairs per second, being orders of magnitude faster than RPA dissociation from ssDNA alone. Additionally, we show that RPA undergoes diffusive motion on ssDNA, such that it can be pushed over long distances by a rezipping fork. Generally the behavior of both human and yeast RPA homologs is very similar. However, in contrast to yeast RPA, the dissociation of human RPA from ssDNA is greatly reduced at low Mg2+ concentrations, such that human RPA can melt DNA in absence of force. PMID:27016742

Single molecule analysis of Thermus thermophilus SSB protein dynamics on single-stranded DNA.

PubMed

Zhang, Jichuan; Zhou, Ruobo; Inoue, Jin; Mikawa, Tsutomu; Ha, Taekjip

2014-04-01

Single-stranded (ss) DNA binding (SSB) proteins play central roles in DNA replication, recombination and repair in all organisms. We previously showed that Escherichia coli (Eco) SSB, a homotetrameric bacterial SSB, undergoes not only rapid ssDNA-binding mode transitions but also one-dimensional diffusion (or migration) while remaining bound to ssDNA. Whereas the majority of bacterial SSB family members function as homotetramers, dimeric SSB proteins were recently discovered in a distinct bacterial lineage of extremophiles, the Thermus-Deinococcus group. Here we show, using single-molecule fluorescence resonance energy transfer (FRET), that homodimeric bacterial SSB from Thermus thermophilus (Tth) is able to diffuse spontaneously along ssDNA over a wide range of salt concentrations (20-500 mM NaCl), and that TthSSB diffusion can help transiently melt the DNA hairpin structures. Furthermore, we show that two TthSSB molecules undergo transitions among different DNA-binding modes while remaining bound to ssDNA. Our results extend our previous observations on homotetrameric SSBs to homodimeric SSBs, indicating that the dynamic features may be shared among different types of SSB proteins. These dynamic features of SSBs may facilitate SSB redistribution and removal on/from ssDNA, and help recruit other SSB-interacting proteins onto ssDNA for subsequent DNA processing in DNA replication, recombination and repair.

Thermodynamics for the Formation of Double-Stranded DNA-Single-Walled Carbon Nanotube Hybrids.

PubMed

Shiraki, Tomohiro; Tsuzuki, Akiko; Toshimitsu, Fumiyuki; Nakashima, Naotoshi

2016-03-24

For the first time, the thermodynamics are described for the formation of double-stranded DNA (ds-DNA)-single-walled carbon nanotube (SWNT) hybrids. This treatment is applied to the exchange reaction of sodium cholate (SC) molecules on SWNTs and the ds-DNAs d(A)20 -d(T)20 and nuclear factor (NF)-κB decoy. UV/Vis/near-IR spectroscopy with temperature variations was used for analyzing the exchange reaction on the SWNTs with four different chiralities: (n,m)=(8,3), (6,5), (7,5), and (8,6). Single-stranded DNAs (ss-DNAs), including d(A)20 and d(T)20, are also used for comparison. The d(A)20-d(T)20 shows a drastic change in its thermodynamic parameters around the melting temperature (Tm ) of the DNA oligomer. No such Tm dependency was measured, owing to high Tm in the NF-κB decoy DNA and no Tm in the ss-DNA. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PrecisePrimer: an easy-to-use web server for designing PCR primers for DNA library cloning and DNA shuffling.

PubMed

Pauthenier, Cyrille; Faulon, Jean-Loup

2014-07-01

PrecisePrimer is a web-based primer design software made to assist experimentalists in any repetitive primer design task such as preparing, cloning and shuffling DNA libraries. Unlike other popular primer design tools, it is conceived to generate primer libraries with popular PCR polymerase buffers proposed as pre-set options. PrecisePrimer is also meant to design primers in batches, such as for DNA libraries creation of DNA shuffling experiments and to have the simplest interface possible. It integrates the most up-to-date melting temperature algorithms validated with experimental data, and cross validated with other computational tools. We generated a library of primers for the extraction and cloning of 61 genes from yeast DNA genomic extract using default parameters. All primer pairs efficiently amplified their target without any optimization of the PCR conditions. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

An experimentally-informed coarse-grained 3-site-per-nucleotide model of DNA: Structure, thermodynamics, and dynamics of hybridization

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

Hinckley, Daniel M.; Freeman, Gordon S.; Whitmer, Jonathan K.

2013-10-14

A new 3-Site-Per-Nucleotide coarse-grained model for DNA is presented. The model includes anisotropic potentials between bases involved in base stacking and base pair interactions that enable the description of relevant structural properties, including the major and minor grooves. In an improvement over available coarse-grained models, the correct persistence length is recovered for both ssDNA and dsDNA, allowing for simulation of non-canonical structures such as hairpins. DNA melting temperatures, measured for duplexes and hairpins by integrating over free energy surfaces generated using metadynamics simulations, are shown to be in quantitative agreement with experiment for a variety of sequences and conditions. Hybridizationmore » rate constants, calculated using forward-flux sampling, are also shown to be in good agreement with experiment. The coarse-grained model presented here is suitable for use in biological and engineering applications, including nucleosome positioning and DNA-templated engineering.« less

Theory and modeling of particles with DNA-mediated interactions

NASA Astrophysics Data System (ADS)

Licata, Nicholas A.

In recent years significant attention has been attracted to proposals which utilize DNA for nanotechnological applications. Potential applications of these ideas range from the programmable self-assembly of colloidal crystals, to biosensors and nanoparticle based drug delivery platforms. In Chapter I we introduce the system, which generically consists of colloidal particles functionalized with specially designed DNA markers. The sequence of bases on the DNA markers determines the particle type. Due to the hybridization between complementary single-stranded DNA, specific, type-dependent interactions can be introduced between particles by choosing the appropriate DNA marker sequences. In Chapter II we develop a statistical mechanical description of the aggregation and melting behavior of particles with DNA-mediated interactions. A quantitative comparison between the theory and experiments is made by calculating the experimentally observed melting profile. In Chapter III a model is proposed to describe the dynamical departure and diffusion of particles which form reversible key-lock connections. The model predicts a crossover from localized to diffusive behavior. The random walk statistics for the particles' in plane diffusion is discussed. The lateral motion is analogous to dispersive transport in disordered semiconductors, ranging from standard diffusion with a renormalized diffusion coefficient to anomalous, subdiffusive behavior. In Chapter IV we propose a method to self-assemble nanoparticle clusters using DNA scaffolds. An optimal concentration ratio is determined for the experimental implementation of our self-assembly proposal. A natural extension is discussed in Chapter V, the programmable self-assembly of nanoparticle clusters where the desired cluster geometry is encoded using DNA-mediated interactions. We determine the probability that the system self-assembles the desired cluster geometry, and discuss the connections to jamming in granular and colloidal systems. In Chapter VI we consider a nanoparticle based drug delivery platform for targeted, cell specific chemotherapy. A key-lock model is proposed to describe the results of in-vitro experiments, and the situation in-vivo is discussed. The cooperative binding, and hence the specificity to cancerous cells, is kinetically limited. The implications for optimizing the design of nanoparticle based drug delivery platforms is discussed. In Chapter VII we present prospects for future research: the connection between DNA-mediated colloidal crystallization and jamming, and the inverse problem in self-assembly.

"Isogaba Maware": quality control of genome DNA by checkpoints.

PubMed

Kitazono, A; Matsumoto, T

1998-05-01

Checkpoints maintain the interdependency of cell cycle events by permitting the onset of an event only after the completion of the preceding event. The DNA replication checkpoint induces a cell cycle arrest until the completion of the DNA replication. Similarly, the DNA damage checkpoint arrests cell cycle progression if DNA repair is incomplete. A number of genes that play a role in the two checkpoints have been identified through genetic studies in yeasts, and their homologues have been found in fly, mouse, and human. They form signaling cascades activated by a DNA replication block or DNA damage and subsequently generate the negative constraints on cell cycle regulators. The failure of these signaling cascades results in producing offspring that carry mutations or that lack a portion of the genome. In humans, defects in the checkpoints are often associated with cancer-prone diseases. Focusing mainly on the studies in budding and fission yeasts, we summarize the recent progress.

Modelling the seismic properties of fast-spreading ridge crustal Low-Velocity Zones: insights from Oman gabbro textures

NASA Astrophysics Data System (ADS)

Lamoureux, Gwenaëlle; Ildefonse, Benoı̂t; Mainprice, David

1999-11-01

Although considerable progress has been made in the study of fast-spreading, mid-ocean ridge magma chambers over the past fifteen years, the fraction of melt present in the chamber remains poorly constrained and controversial. We present new constraints obtained by modelling the seismic properties of partially molten gabbros at the ridge axis. P-wave velocities at low frequencies are calculated in the foliation/lineation reference frame using a differential effective medium technique. The model takes into account the lattice preferred orientation of the crystalline phase and the average shape of the melt phase. The structural parameters are obtained from the Oman ophiolite. The structural reference frame is given by the general trend of the gabbro foliation and the melt fraction and shape are estimated using the textures of nine upper gabbro samples. The estimated melt fraction and shape depend on the assumptions regarding which part of the observed textures represent the melt in the gabbroic mush of the magma chamber. However, we can put limits on the reasonable values for the melt fraction and shape. Our results are consistent with a melt fraction of the order of 10 to 20% in the Low-Velocity Zone (i.e. the magma chamber), which is anisotropically distributed with the melt pockets preferentially aligned parallel to the foliation and approximated by oblate ellipsoids with approximate dimensions of 4 : 4 : 1. These results are also consistent with the seismic structure of the East Pacific rise at 9°30'. The anisotropic melt distribution can, at least partially, explain the vertical velocity gradient described in the LVZ.

A polycomb-mediated epigenetic field defect precedes invasive cervical carcinoma

PubMed Central

Wijetunga, Neil Ari; Ben-Dayan, Miriam; Tozour, Jessica; Burk, Robert D.; Schlecht, Nicolas F.; Einstein, Mark H.; Greally, John M.

2016-01-01

Human papillomavirus (HPV)-associated cervical carcinoma is preceded by stages of cervical intra-epithelial neoplasia (CIN) that can variably progress to malignancy. Understanding the different molecular processes involved in the progression of pre-malignant CIN is critical to the development of improved predictive and interventional capabilities. We tested the role of regulators of transcription in both the development and the progression of HPV-associated CIN, performing the most comprehensive genomic survey to date of DNA methylation in HPV-associated cervical neoplasia, testing ~2 million loci throughout the human genome in biopsies from 78 HPV+ women, identifying changes starting in early CIN and maintained through carcinogenesis. We identified loci at which DNA methylation is consistently altered, beginning early in the course of neoplastic disease and progressing with disease advancement. While the loss of DNA methylation occurs mostly at intergenic regions, acquisition of DNA methylation is at sites involved in transcriptional regulation, with strong enrichment for targets of polycomb repression. Using an independent cohort from The Cancer Genome Atlas, we validated the loci with increased DNA methylation and found that these regulatory changes were associated with locally decreased gene expression. Secondary validation using immunohistochemistry showed that the progression of neoplasia was associated with increasing polycomb protein expression specifically in the cervical epithelium. We find that perturbations of genomic regulatory processes occur early and persist in cervical carcinoma. The results indicate a polycomb-mediated epigenetic field defect in cervical neoplasia that may represent a target for early, topical interventions using polycomb inhibitors. PMID:27557505

A polycomb-mediated epigenetic field defect precedes invasive cervical carcinoma.

PubMed

Wijetunga, Neil Ari; Ben-Dayan, Miriam; Tozour, Jessica; Burk, Robert D; Schlecht, Nicolas F; Einstein, Mark H; Greally, John M

2016-09-20

Human papillomavirus (HPV)-associated cervical carcinoma is preceded by stages of cervical intra-epithelial neoplasia (CIN) that can variably progress to malignancy. Understanding the different molecular processes involved in the progression of pre-malignant CIN is critical to the development of improved predictive and interventional capabilities. We tested the role of regulators of transcription in both the development and the progression of HPV-associated CIN, performing the most comprehensive genomic survey to date of DNA methylation in HPV-associated cervical neoplasia, testing ~2 million loci throughout the human genome in biopsies from 78 HPV+ women, identifying changes starting in early CIN and maintained through carcinogenesis. We identified loci at which DNA methylation is consistently altered, beginning early in the course of neoplastic disease and progressing with disease advancement. While the loss of DNA methylation occurs mostly at intergenic regions, acquisition of DNA methylation is at sites involved in transcriptional regulation, with strong enrichment for targets of polycomb repression. Using an independent cohort from The Cancer Genome Atlas, we validated the loci with increased DNA methylation and found that these regulatory changes were associated with locally decreased gene expression. Secondary validation using immunohistochemistry showed that the progression of neoplasia was associated with increasing polycomb protein expression specifically in the cervical epithelium. We find that perturbations of genomic regulatory processes occur early and persist in cervical carcinoma. The results indicate a polycomb-mediated epigenetic field defect in cervical neoplasia that may represent a target for early, topical interventions using polycomb inhibitors.

Time lapse microscopy of temperature control during self-assembly of 3D DNA crystals

NASA Astrophysics Data System (ADS)

Conn, Fiona W.; Jong, Michael Alexander; Tan, Andre; Tseng, Robert; Park, Eunice; Ohayon, Yoel P.; Sha, Ruojie; Mao, Chengde; Seeman, Nadrian C.

2017-10-01

DNA nanostructures are created by exploiting the high fidelity base-pairing interactions of double-stranded branched DNA molecules. These structures present a convenient medium for the self-assembly of macroscopic 3D crystals. In some self-assemblies in this system, crystals can be formed by lowering the temperature, and they can be dissolved by raising it. The ability to monitor the formation and melting of these crystals yields information that can be used to monitor crystal formation and growth. Here, we describe the development of an inexpensive tool that enables direct observation of the crystal growth process as a function of both time and temperature. Using the hanging-drop crystallization of the well-characterized 2-turn DNA tensegrity triangle motif for our model system, its response to temperature has been characterized visually.

Cell cycle in egg cell and its progression during zygotic development in rice.

PubMed

Sukawa, Yumiko; Okamoto, Takashi

2018-03-01

Rice egg is arrested at G1 phase probably by OsKRP2. After fusion with sperm, karyogamy, OsWEE1-mediated parental DNA integrity in zygote nucleus, zygote progresses cell cycle to produce two-celled embryo. In angiosperms, female and male gametes exist in gametophytes after the complementation of meiosis and the progression of nuclear/cell division of the haploid cell. Within the embryo sac, the egg cell is specially differentiated for fertilization and subsequent embryogenesis, and cellular programs for embryonic development, such as restarting the cell cycle and de novo gene expression, are halted. There is only limited knowledge about how the cell cycle in egg cells restarts toward zygotic division, although the conversion of the cell cycle from a quiescent and arrested state to an active state is the most evident transition of cell status from egg cell to zygote. This is partly due to the difficulty in direct access and analysis of egg cells, zygotes and early embryos, which are deeply embedded in ovaries. In this study, precise relative DNA amounts in the nuclei of egg cells, developing zygotes and cells of early embryos were measured, and the cell cycle of a rice egg cell was estimated as the G1 phase with a 1C DNA level. In addition, increases in DNA content in zygote nuclei via karyogamy and DNA replication were also detectable according to progression of the cell cycle. In addition, expression profiles for cell cycle-related genes in egg cells and zygotes were also addressed, and it was suggested that OsKRP2 and OsWEE1 function in the inhibition of cell cycle progression in egg cells and in checkpoint of parental DNA integrity in zygote nucleus, respectively.

Toward Fully in Silico Melting Point Prediction Using Molecular Simulations

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

Zhang, Y; Maginn, EJ

2013-03-01

Melting point is one of the most fundamental and practically important properties of a compound. Molecular computation of melting points. However, all of these methods simulation methods have been developed for the accurate need an experimental crystal structure as input, which means that such calculations are not really predictive since the melting point can be measured easily in experiments once a crystal structure is known. On the other hand, crystal structure prediction (CSP) has become an active field and significant progress has been made, although challenges still exist. One of the main challenges is the existence of many crystal structuresmore » (polymorphs) that are very close in energy. Thermal effects and kinetic factors make the situation even more complicated, such that it is still not trivial to predict experimental crystal structures. In this work, we exploit the fact that free energy differences are often small between crystal structures. We show that accurate melting point predictions can be made by using a reasonable crystal structure from CSP as a starting point for a free energy-based melting point calculation. The key is that most crystal structures predicted by CSP have free energies that are close to that of the experimental structure. The proposed method was tested on two rigid molecules and the results suggest that a fully in silico melting point prediction method is possible.« less

Recombinase and translesion DNA polymerase decrease the speed of replication fork progression during the DNA damage response in Escherichia coli cells

PubMed Central

Tan, Kang Wei; Pham, Tuan Minh; Furukohri, Asako; Maki, Hisaji; Akiyama, Masahiro Tatsumi

2015-01-01

The SOS response is a DNA damage response pathway that serves as a general safeguard of genome integrity in bacteria. Extensive studies of the SOS response in Escherichia coli have contributed to establishing the key concepts of cellular responses to DNA damage. However, how the SOS response impacts on the dynamics of DNA replication fork movement remains unknown. We found that inducing the SOS response decreases the mean speed of individual replication forks by 30–50% in E. coli cells, leading to a 20–30% reduction in overall DNA synthesis. dinB and recA belong to a group of genes that are upregulated during the SOS response, and encode the highly conserved proteins DinB (also known as DNA polymerase IV) and RecA, which, respectively, specializes in translesion DNA synthesis and functions as the central recombination protein. Both genes were independently responsible for the SOS-dependent slowdown of replication fork progression. Furthermore, fork speed was reduced when each gene was ectopically expressed in SOS-uninduced cells to the levels at which they are expressed in SOS-induced cells. These results clearly indicate that the increased expression of dinB and recA performs a novel role in restraining the progression of an unperturbed replication fork during the SOS response. PMID:25628359

The Temporal Regulation of S Phase Proteins During G1

PubMed Central

Grant, Gavin D.; Cook, Jeanette G.

2018-01-01

Successful DNA replication requires intimate coordination with cell cycle progression. Prior to DNA replication initiation in S phase, a series of essential preparatory events in G1 phase ensures timely, complete, and precise genome duplication. Among the essential molecular processes are regulated transcriptional upregulation of genes that encode replication proteins, appropriate post-transcriptional control of replication factor abundance and activity, and the assembly of DNA-loaded protein complexes to license replication origins. In this chapter we describe these critical G1 events necessary for DNA replication and their regulation in the context of both cell cycle entry and cell cycle progression. PMID:29357066

Selective muscle fiber loss and molecular compensation in mitochondrial myopathy due to TK2 deficiency.

PubMed

Vilà, Maya R; Villarroya, Joan; García-Arumí, Elena; Castellote, Amparo; Meseguer, Anna; Hirano, Michio; Roig, Manuel

2008-04-15

A 12-year-old patient with mitochondrial DNA (mtDNA) depletion syndrome due to TK2 gene mutations has been evaluated serially over the last 10 years. We observed progressive muscle atrophy with selective loss of type 2 muscle fibers and, despite severe depletion of mtDNA, normal activities of respiratory chain (RC) complexes and levels of COX II mitochondrial protein in the remaining muscle fibers. These results indicate that compensatory mechanisms account for the slow progression of the disease. Identification of factors that ameliorate mtDNA depletion may reveal new therapeutic targets for these devastating disorders.

Some physical aspects of fluid-fluxed melting

NASA Astrophysics Data System (ADS)

Patiño Douce, A.

2012-04-01

Fluid-fluxed melting is thought to play a crucial role in the origin of many terrestrial magmas. We can visualize the fundamental physics of the process as follows. An infinitesimal amount of fluid infiltrates dry rock at the temperature of its dry solidus. In order to restore equilibrium the temperature must drop, so that enthalpy is released and immediately reabsorbed as enthalpy of melting. The amount of melt produced must be such that the energy balance and thermodynamic equilibrium conditions are simultaneously satisfied. We wish to understand how an initially dry rock melts in response to progressive fluid infiltration, under both batch and fractional melting constraints. The simplest physical model for this process is a binary system in which one of the components makes up a pure solid phase and the other component a pure fluid phase, and in which a binary melt phase exists over certain temperature range. Melting point depression is calculated under the assumption of ideal mixing. The equations of energy balance and thermodynamic equilibrium are solved simultaneously for temperature and melt fraction, using an iterative procedure that allows addition of fluid in infinitesimal increments. Batch melting and fractional melting are simulated by allowing successive melt increments to remain in the system (batch) or not (fractional). Despite their simplified nature, these calculations reveal some important aspects of fluid-fluxed melting. The model confirms that, if the solubility of the fluid in the melt is sufficiently high, fluid fluxed melting is an efficient mechanism of magma generation. One might expect that the temperature of the infiltrating fluid would have a significant effect on melt productivity, but the results of the calculations show this not to be the case, because a relatively small mass of low molecular weight fluid has a strong effect on the melting point of minerals with much higher molecular weights. The calculations reveal the somewhat surprising result that fluid infiltration produces more melt during fractional melting than during batch melting. This behavior, which is opposite to that of decompression melting of a dry solid, arises because the melting point depression effect of the added fluid is greater during fractional melting than during batch melting, which results in a greater release of enthalpy and, therefore, greater melt production for fractional melting than for batch melting, for the same total amount of fluid added. The difference may be considerable. As an example, suppose that 0.1 mols of H2O infiltrate 1 mol or silicate rock. Depending on the rock composition this may corresponds to ˜ 1 wt% H2O. For a given choice of model parameters (initial temperature, heat capacity and entropy of fusion), about 28% of the rock melts during fractional melting, versus some 23 % during batch melting. Fluid fluxing is a robust process of melt generation, without which magmatism at Earth's convergent plate margins would be impossible.

Recent progress in DNA origami technology.

PubMed

Endo, Masayuki; Sugiyama, Hiroshi

2011-06-01

DNA origami is an emerging technology for designing defined two-dimensional DNA nanostructures. In this review, we focus on and describe several types of DNA origami-related studies, as follows: (1) programmed DNA origami assembly, (2) DNA origami-templated molecular assembly, (3) design and construction of various three-dimensional DNA origami structures, (4) programmed functionalization of DNA origami and combination with top-down nanotechnology, (5) single molecular observation on a designed DNA origami, and (6) DNA nanomachines working on a DNA origami. © 2011 by John Wiley & Sons, Inc.

DNA nanostructure-based drug delivery nanosystems in cancer therapy.

PubMed

Wu, Dandan; Wang, Lei; Li, Wei; Xu, Xiaowen; Jiang, Wei

2017-11-25

DNA as a novel biomaterial can be used to fabricate different kinds of DNA nanostructures based on its principle of GC/AT complementary base pairing. Studies have shown that DNA nanostructure is a nice drug carrier to overcome big obstacles existing in cancer therapy such as systemic toxicity and unsatisfied drug efficacy. Thus, different types of DNA nanostructure-based drug delivery nanosystems have been designed in cancer therapy. To improve treating efficacy, they are also developed into more functional drug delivery nanosystems. In recent years, some important progresses have been made. The objective of this review is to make a retrospect and summary about these different kinds of DNA nanostructure-based drug delivery nanosystems and their latest progresses: (1) active targeting; (2) mutidrug co-delivery; (3) construction of stimuli-responsive/intelligent nanosystems. Copyright © 2017 Elsevier B.V. All rights reserved.

A process for developing and revising a learning progression on sea level rise using learners' explanations

NASA Astrophysics Data System (ADS)

McDonald, Robert Christopher

The purpose of this study was to explore the process of developing a learning progression (LP) on constructing explanations about sea level rise. I used a learning progressions theoretical framework informed by the situated cognition learning theory. During this exploration, I explicitly described my decision-making process as I developed and revised a hypothetical learning progression. Correspondingly, my research question was: What is a process by which a hypothetical learning progression on sea level rise is developed into an empirical learning progression using learners' explanations? To answer this question, I used a qualitative descriptive single case study with multiple embedded cases (Yin, 2014) that employed analytic induction (Denzin, 1970) to analyze data collected on middle school learners (grades 6-8). Data sources included written artifacts, classroom observations, and semi-structured interviews. Additionally, I kept a researcher journal to track my thinking about the learning progression throughout the research study. Using analytic induction to analyze collected data, I developed eight analytic concepts: participant explanation structures varied widely, global warming and ice melt cause sea level rise, participants held alternative conceptions about sea level rise, participants learned about thermal expansion as a fundamental aspect of sea level rise, participants learned to incorporate authentic scientific data, participants' mental models of the ocean varied widely, sea ice melt contributes to sea level rise, and participants held vague and alternative conceptions about how pollution impacts the ocean. I started with a hypothetical learning progression, gathered empirical data via various sources (especially semi-structured interviews), revised the hypothetical learning progression in response to those data, and ended with an empirical learning progression comprising six levels of learner thinking. As a result of developing an empirically based LP, I was able to compare two learning progressions on the same topic. By comparing my learning progression with the LP in Breslyn, McGinnis, McDonald, and Hestness (2016), I was able to confirm portions of the two learning progressions and explore different possible pathways for learners to achieve progress towards upper anchors of the LPs through targeted instruction. Implications for future LP research, curriculum, instruction, assessment, and policy related to learning progressions are presented.

Fukushima Daiichi unit 1 uncertainty analysis--Preliminary selection of uncertain parameters and analysis methodology

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

Cardoni, Jeffrey N.; Kalinich, Donald A.

2014-02-01

Sandia National Laboratories (SNL) plans to conduct uncertainty analyses (UA) on the Fukushima Daiichi unit (1F1) plant with the MELCOR code. The model to be used was developed for a previous accident reconstruction investigation jointly sponsored by the US Department of Energy (DOE) and Nuclear Regulatory Commission (NRC). However, that study only examined a handful of various model inputs and boundary conditions, and the predictions yielded only fair agreement with plant data and current release estimates. The goal of this uncertainty study is to perform a focused evaluation of uncertainty in core melt progression behavior and its effect on keymore » figures-of-merit (e.g., hydrogen production, vessel lower head failure, etc.). In preparation for the SNL Fukushima UA work, a scoping study has been completed to identify important core melt progression parameters for the uncertainty analysis. The study also lays out a preliminary UA methodology.« less

Liquid Biopsy Analysis of FGFR3 and PIK3CA Hotspot Mutations for Disease Surveillance in Bladder Cancer.

PubMed

Christensen, Emil; Birkenkamp-Demtröder, Karin; Nordentoft, Iver; Høyer, Søren; van der Keur, Kirstin; van Kessel, Kim; Zwarthoff, Ellen; Agerbæk, Mads; Ørntoft, Torben Falck; Jensen, Jørgen Bjerggaard; Dyrskjøt, Lars

2017-06-01

Disease surveillance in patients with bladder cancer is important for early diagnosis of progression and metastasis and for optimised treatment. To develop urine and plasma assays for disease surveillance for patients with FGFR3 and PIK3CA tumour mutations. Droplet digital polymerase chain reaction (ddPCR) assays were developed and tumour DNA from two patient cohorts was screened for FGFR3 and PIK3CA hotspot mutations. One cohort included 363 patients with non-muscle-invasive bladder cancer (NMIBC). The other cohort included 468 patients with bladder cancer undergoing radical cystectomy (Cx). Urine supernatants (NMIBC n=216, Cx n=27) and plasma samples (NMIBC n=39, Cx n=27) from patients harbouring mutations were subsequently screened using ddPCR assays. Progression-free survival, recurrence-free survival, and overall survival were measured. Fisher's exact test, the Wilcoxon rank-sum test and Cox regression analysis were applied. In total, 36% of the NMIBC patients (129/363) and 11% of the Cx patients (44/403) harboured at least one FGFR3 or PIK3CA mutation. Screening of DNA from serial urine supernatants from the NMIBC cohort revealed that high levels of tumour DNA (tDNA) were associated with later disease progression in NMIBC (p=0.003). Furthermore, high levels of tDNA in plasma samples were associated with recurrence in the Cx cohort (p=0.016). A positive correlation between tDNA levels in urine and plasma was observed (correlation coefficient 0.6). The retrospective study design and low volumes of plasma available for analysis were limitations of the study. Increased levels of FGFR3 and PIK3CA mutated DNA in urine and plasma are indicative of later progression and metastasis in bladder cancer. Urine and plasma from patients with bladder cancer may be monitored for diagnosis of progression and metastasis using mutation assays. Copyright © 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Cation radius effects on the helix-coil transition of DNA. Cryptates and other large cations.

PubMed Central

Trend, B L; Knoll, D A; Ueno, M; Evans, D F; Bloomfield, V A

1990-01-01

Most polyelectrolyte theories of the effect of ions on the thermal melting of DNA assume that the predominant influence of the cations comes through their charge. Ion size and structure are treated, for analytic convenience, as negligible variables. We have examined the validity of this assumption by measuring the melting temperature of calf thymus DNA as a function of salt concentration with four univalent cations of different hydrated radii. These are K+ (3.3 A), (n-Pr)4N+ (4.5 A), (EtOH)4N+ (4.5 A), and C222-K+ (5 A). C222-K+ is a complex of cryptand C222 with K+. With K+ as the sole cation, Tm varies linearly with the log of ionic strength over the range 0.001-0.1 M. With all the K+ sequestered by an equimolar amount of C222, Tm is depressed by 10-20 degrees C and the slope of Tm vs. ionic strength is lower. At low ionic strength, an even greater reduction in Tm is achieved with (n-Pr)4N+; but the similar-sized (EtOH)4N+ gives a curve more similar to K+. Theoretical modeling, taking into account cation size through the Poisson-Boltzmann equation for cylindrical polyelectrolytes, predicts that larger cations should be less effective in stabilizing the double helix; but the calculated effect is less than observed experimentally. These results show that valence, cation size, and specific solvation effects are all important in determining the stability of the double-helical form of DNA. PMID:2344467

Overcoming a nucleosomal barrier to replication

PubMed Central

Chang, Han-Wen; Pandey, Manjula; Kulaeva, Olga I.; Patel, Smita S.; Studitsky, Vasily M.

2016-01-01

Efficient overcoming and accurate maintenance of chromatin structure and associated histone marks during DNA replication are essential for normal functioning of the daughter cells. However, the molecular mechanisms of replication through chromatin are unknown. We have studied traversal of uniquely positioned mononucleosomes by T7 replisome in vitro. Nucleosomes present a strong, sequence-dependent barrier for replication, with particularly strong pausing of DNA polymerase at the +(31–40) and +(41–65) regions of the nucleosomal DNA. The exonuclease activity of T7 DNA polymerase increases the overall rate of progression of the replisome through a nucleosome, likely by resolving nonproductive complexes. The presence of nucleosome-free DNA upstream of the replication fork facilitates the progression of DNA polymerase through the nucleosome. After replication, at least 50% of the nucleosomes assume an alternative conformation, maintaining their original positions on the DNA. Our data suggest a previously unpublished mechanism for nucleosome maintenance during replication, likely involving transient formation of an intranucleosomal DNA loop. PMID:27847876

DNA polymerase η modulates replication fork progression and DNA damage responses in platinum-treated human cells

NASA Astrophysics Data System (ADS)

Sokol, Anna M.; Cruet-Hennequart, Séverine; Pasero, Philippe; Carty, Michael P.

2013-11-01

Human cells lacking DNA polymerase η (polη) are sensitive to platinum-based cancer chemotherapeutic agents. Using DNA combing to directly investigate the role of polη in bypass of platinum-induced DNA lesions in vivo, we demonstrate that nascent DNA strands are up to 39% shorter in human cells lacking polη than in cells expressing polη. This provides the first direct evidence that polη modulates replication fork progression in vivo following cisplatin and carboplatin treatment. Severe replication inhibition in individual platinum-treated polη-deficient cells correlates with enhanced phosphorylation of the RPA2 subunit of replication protein A on serines 4 and 8, as determined using EdU labelling and immunofluorescence, consistent with formation of DNA strand breaks at arrested forks in the absence of polη. Polη-mediated bypass of platinum-induced DNA lesions may therefore represent one mechanism by which cancer cells can tolerate platinum-based chemotherapy.

Silencing of human DNA polymerase λ causes replication stress and is synthetically lethal with an impaired S phase checkpoint

PubMed Central

Zucca, Elisa; Bertoletti, Federica; Wimmer, Ursula; Ferrari, Elena; Mazzini, Giuliano; Khoronenkova, Svetlana; Grosse, Nicole; van Loon, Barbara; Dianov, Grigory; Hübscher, Ulrich; Maga, Giovanni

2013-01-01

Human DNA polymerase (pol) λ functions in base excision repair and non-homologous end joining. We have previously shown that DNA pol λ is involved in accurate bypass of the two frequent oxidative lesions, 7,8-dihydro-8-oxoguanine and 1,2-dihydro-2-oxoadenine during the S phase. However, nothing is known so far about the relationship of DNA pol λ with the S phase DNA damage response checkpoint. Here, we show that a knockdown of DNA pol λ, but not of its close homologue DNA pol β, results in replication fork stress and activates the S phase checkpoint, slowing S phase progression in different human cancer cell lines. We furthermore show that DNA pol λ protects cells from oxidative DNA damage and also functions in rescuing stalled replication forks. Its absence becomes lethal for a cell when a functional checkpoint is missing, suggesting a DNA synthesis deficiency. Our results provide the first evidence, to our knowledge, that DNA pol λ is required for cell cycle progression and is functionally connected to the S phase DNA damage response machinery in cancer cells. PMID:23118481

Elongator complex is critical for cell cycle progression and leaf patterning in Arabidopsis.

PubMed

Xu, Deyang; Huang, Weihua; Li, Yang; Wang, Hua; Huang, Hai; Cui, Xiaofeng

2012-03-01

The mitotic cell cycle in higher eukaryotes is of pivotal importance for organ growth and development. Here, we report that Elongator, an evolutionarily conserved histone acetyltransferase complex, acts as an important regulator of mitotic cell cycle to promote leaf patterning in Arabidopsis. Mutations in genes encoding Elongator subunits resulted in aberrant cell cycle progression, and the altered cell division affects leaf polarity formation. The defective cell cycle progression is caused by aberrant DNA replication and increased DNA damage, which activate the DNA replication checkpoint to arrest the cell cycle. Elongator interacts with proliferating cell nuclear antigen (PCNA) and is required for efficient histone 3 (H3) and H4 acetylation coupled with DNA replication. Levels of chromatin-bound H3K56Ac and H4K5Ac known to associate with replicons during DNA replication were reduced in the mutants of both Elongator and chromatin assembly factor 1 (CAF-1), another protein complex that physically interacts with PCNA for DNA replication-coupled chromatin assembly. Disruptions of CAF-1 also led to severe leaf polarity defects, which indicated that Elongator and CAF-1 act, at least partially, in the same pathway to promote cell cycle progression. Collectively, our results demonstrate that Elongator is an important regulator of mitotic cell cycle, and the Elongator pathway plays critical roles in promoting leaf polarity formation. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

A novel technology for the detection, enrichment, and separation of trace amounts of target DNA based on amino-modified fluorescent magnetic composite nanoparticles.

PubMed

Wang, Guannan; Su, Xingguang

2010-06-01

A novel, highly sensitive technology for the detection, enrichment, and separation of trace amounts of target DNA was developed on the basis of amino-modified fluorescent magnetic composite nanoparticles (AFMN). In this study, the positively charged amino-modified composite nanoparticles conjugate with the negatively charged capture DNA through electrostatic binding. The optimal combination of AFMN and capture DNA was measured by dynamic light scattering (DLS) and UV-vis absorption spectroscopy. The highly sensitive detection of trace amounts of target DNA was achieved through enrichment by means of AFMN. The detection limit for target DNA is 0.4 pM, which could be further improved by using a more powerful magnet. Because of their different melting temperatures, single-base mismatched target DNA could be separated from perfectly complementary target DNA. In addition, the photoluminescence (PL) signals of perfectly complementary target DNA and single-base mismatched DNA as well as the hybridization kinetics of different concentrations of target DNA at different reaction times have also been studied. Most importantly, the detection, enrichment, and separation ability of AFMN was further verified with milk. Simple and satisfactory results were obtained, which show the great potential in the fields of mutation identification and clinical diagnosis.

Spectroscopic and electrochemical studies of the interaction between oleuropein, the major bio-phenol in olives, and salmon sperm DNA

NASA Astrophysics Data System (ADS)

Mohamadi, Maryam; Afzali, Daryoush; Esmaeili-Mahani, Saeed; Mostafavi, Ali; Torkzadeh-Mahani, Masoud

2015-09-01

Interaction of oleuropein, the major bio-phenol in olive leaf and fruit, with salmon sperm double-stranded DNA was investigated by employing electronic absorption titrations, fluorescence quenching spectroscopy, competitive fluorescence spectroscopy, thermal denaturation and voltammetric studies. Titration of oleuropein with the DNA caused a hypochromism accompanied with a red shift indicating an intercalative mode of interaction. Binding constant of 1.4 × 104 M-1 was obtained for this interaction. From the curves of fluorescence titration of oleuropein with the DNA, binding constant and binding sites were calculated to be 8.61 × 103 M-1 and 1.05, respectively. Competitive studies with ethidium bromide (a well-known DNA intercalator) showed that the bio-phenol could take the place of ethidium bromide in the DNA intercalation sites. The interaction of oleuropein with DNA was also studied electrochemically. In the presence of the DNA, the anodic and cathodic peak currents of oleuropein decreased accompanied with increases in peak-to-peak potential separation and formal potential, indicating the intercalation of oleuropein into the DNA double helix. Moreover, melting temperature of the DNA was found to increase in the presence of oleuropein, indicating the stabilization of the DNA double helix due to an intercalative interaction.

Thermal stability of G-rich anti-parallel DNA triplexes upon insertion of LNA and α-L-LNA.

PubMed

Kosbar, Tamer R; Sofan, Mamdouh A; Abou-Zeid, Laila; Pedersen, Erik B

2015-05-14

G-rich anti-parallel DNA triplexes were modified with LNA or α-L-LNA in their Watson-Crick and TFO strands. The triplexes were formed by targeting a pyrimidine strand to a putative hairpin formed by Hoogsteen base pairing in order to use the UV melting method to evaluate the stability of the triplexes. Their thermal stability was reduced when the TFO strand was modified with LNA or α-L-LNA. The same trend was observed when the TFO strand and the purine Watson-Crick strand both were modified with LNA. When all triad components were modified with α-L-LNA and LNA in the middle of the triplex, the thermal melting was increased. When the pyrimidine sequence was modified with a single insertion of LNA or α-L-LNA the ΔTm increased. Moreover, increasing the number of α-L-LNA in the pyrimidine target sequence to six insertions, leads to a high increase in the thermal stability. The conformational S-type structure of α-L-LNA in anti-parallel triplexes is preferable for triplex stability.

Transcription and replication: breaking the rules of the road causes genomic instability.

PubMed

Poveda, Ana Maria; Le Clech, Mikael; Pasero, Philippe

2010-01-01

Replication and transcription machineries progress at high speed on the same DNA template, which inevitably causes traffic accidents. Problems are not only caused by frontal collisions between polymerases, but also by cotranscriptional R-loops. These RNA-DNA hybrids induce genomic instability by blocking fork progression and could be implicated in the development of cancer.

Stacked-unstacked equilibrium at the nick site of DNA.

PubMed

Protozanova, Ekaterina; Yakovchuk, Peter; Frank-Kamenetskii, Maxim D

2004-09-17

Stability of duplex DNA with respect to separation of complementary strands is crucial for DNA executing its major functions in the cell and it also plays a central role in major biotechnology applications of DNA: DNA sequencing, polymerase chain reaction, and DNA microarrays. Two types of interaction are well known to contribute to DNA stability: stacking between adjacent base-pairs and pairing between complementary bases. However, their contribution into the duplex stability is yet to be determined. Now we fill this fundamental gap in our knowledge of the DNA double helix. We have prepared a series of 32, 300 bp-long DNA fragments with solitary nicks in the same position differing only in base-pairs flanking the nick. Electrophoretic mobility of these fragments in the gel has been studied. Assuming the equilibrium between stacked and unstacked conformations at the nick site, all 32 stacking free energy parameters have been obtained. Only ten of them are essential and they govern the stacking interactions between adjacent base-pairs in intact DNA double helix. A full set of DNA stacking parameters has been determined for the first time. From these data and from a well-known dependence of DNA melting temperature on G.C content, the contribution of base-pairing into duplex stability has been estimated. The obtained energy parameters of the DNA double helix are of paramount importance for understanding sequence-dependent DNA flexibility and for numerous biotechnology applications.

The Deep Crust Magmatic Refinery, Part 2 : The Magmatic Output of Numerical Models.

NASA Astrophysics Data System (ADS)

Bouilhol, P.; Riel, N., Jr.; Van Hunen, J.

2016-12-01

Metamorphic and magmatic processes occurring in the deep crust ultimately control the chemical and physical characteristic of the continental crust. A complex interplay between magma intrusion, crystallization, and reaction with the pre-existing crust provide a wide range of differentiated magma and cumulates (and / or restites) that will feed the upper crustal levels with evolved melt while constructing the lower crust. With growing evidence from field and experimental studies, it becomes clearer that crystallization and melting processes are non-exclusive but should be considered together. Incoming H2O bearing mantle melts will start to fractionate to a certain extent, forming cumulates but also releasing heat and H2O to the intruded host-rock allowing it to melt in saturated conditions. The end-result of such dynamic system is a function of the amount and composition of melt input, and extent of reaction with the host which is itself dependent on the migration mode of the melts. To better constrain lower crust processes, we have built up a numerical model [see Riel et al. associated abstract for methods] to explore different parameters, unravelling the complex interplay between melt percolation / crystallization and degassing / re-melting in a so called "hot zone" model. We simulated the intrusion of water bearing mantle melts at the base of an amphibolitized lower crust during a magmatic event that lasts 5 Ma. We varied several parameters such as Moho depth and melt rock ratio to better constrain what controls the final melt / lower crust composition.. We show the evolution of the chemical characteristics of the melt that escape the system during this magmatic event, as well as the resulting lower crust characteristics. We illustrate how the evolution of melt major elements composition reflects the progressive replacement of the crust towards compositions that are dominated by the mantle melt input. The resulting magmas cover a wide range of composition from tonalite to granite, and the modelled lower crust shows all the petrological characteristic of observed lower arc-crust.

Rapid detection and identification of four major Schistosoma species by high-resolution melt (HRM) analysis.

PubMed

Li, Juan; Zhao, Guang-Hui; Lin, RuiQing; Blair, David; Sugiyama, Hiromu; Zhu, Xing-Quan

2015-11-01

Schistosomiasis, caused by blood flukes belonging to several species of the genus Schistosoma, is a serious and widespread parasitic disease. Accurate and rapid differentiation of these etiological agents of animal and human schistosomiasis to species level can be difficult. We report a real-time PCR assay coupled with a high-resolution melt (HRM) assay targeting a portion of the nuclear 18S rDNA to detect, identify, and distinguish between four major blood fluke species (Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and Schistosoma mekongi). Using this system, the Schistosoma spp. was accurately identified and could also be distinguished from all other trematode species with which they were compared. As little as 10(-5) ng genomic DNA from a Schistosoma sp. could be detected. This process is inexpensive, easy, and can be completed within 3 h. Examination of 21 representative Schistosoma samples from 15 geographical localities in seven endemic countries validated the value of the HRM detection assay and proved its reliability. The melting curves were characterized by peaks of 83.65 °C for S. japonicum and S. mekongi, 85.65 °C for S. mansoni, and 85.85 °C for S. haematobium. The present study developed a real-time PCR coupled with HRM analysis assay for detection and differential identification of S. mansoni, S. haematobium, S. japonicum, and S. mekongi. This method is rapid, sensitive, and inexpensive. It has important implications for epidemiological studies of Schistosoma.

Binding of the Biogenic Polyamines to Deoxyribonucleic Acids of Varying Base Composition: Base Specificity and the Associated Energetics of the Interaction

PubMed Central

Kabir, Ayesha; Suresh Kumar, Gopinatha

2013-01-01

Background The thermodynamics of the base pair specificity of the binding of the polyamines spermine, spermidine, putrescine, and cadaverine with three genomic DNAs Clostridium perfringens, 27% GC, Escherichia coli, 50% GC and Micrococcus lysodeikticus, 72% GC have been studied using titration calorimetry and the data supplemented with melting studies, ethidium displacement and circular dichroism spectroscopy results. Methodology/Principal Findings Isothermal titration calorimetry, differential scanning calorimetry, optical melting studies, ethidium displacement, circular dichroism spectroscopy are the various techniques employed to characterize the interaction of four polyamines, spermine, spermidine, putersine and cadaverine with the DNAs. Polyamines bound stronger with AT rich DNA compared to the GC rich DNA and the binding varied depending on the charge on the polyamine as spermine>spermidine >putrescine>cadaverine. Thermodynamics of the interaction revealed that the binding was entropy driven with small enthalpy contribution. The binding was influenced by salt concentration suggesting the contribution from electrostatic forces to the Gibbs energy of binding to be the dominant contributor. Each system studied exhibited enthalpy-entropy compensation. The negative heat capacity changes suggested a role for hydrophobic interactions which may arise due to the non polar interactions between DNA and polyamines. Conclusion/Significance From a thermodynamic analysis, the AT base specificity of polyamines to DNAs has been elucidated for the first time and supplemented by structural studies. PMID:23894663

High Resolution Melting Analysis Targeting hsp70 as a Fast and Efficient Method for the Discrimination of Leishmania Species.

PubMed

Zampieri, Ricardo Andrade; Laranjeira-Silva, Maria Fernanda; Muxel, Sandra Marcia; Stocco de Lima, Ana Carolina; Shaw, Jeffrey Jon; Floeter-Winter, Lucile Maria

2016-02-01

Protozoan parasites of the genus Leishmania cause a large spectrum of clinical manifestations known as Leishmaniases. These diseases are increasingly important public health problems in many countries both within and outside endemic regions. Thus, an accurate differential diagnosis is extremely relevant for understanding epidemiological profiles and for the administration of the best therapeutic protocol. Exploring the High Resolution Melting (HRM) dissociation profiles of two amplicons using real time polymerase chain reaction (real-time PCR) targeting heat-shock protein 70 coding gene (hsp70) revealed differences that allowed the discrimination of genomic DNA samples of eight Leishmania species found in the Americas, including Leishmania (Leishmania) infantum chagasi, L. (L.) amazonensis, L. (L.) mexicana, L. (Viannia) lainsoni, L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) naiffi and L. (V.) shawi, and three species found in Eurasia and Africa, including L. (L.) tropica, L. (L.) donovani and L. (L.) major. In addition, we tested DNA samples obtained from standard promastigote culture, naturally infected phlebotomines, experimentally infected mice and clinical human samples to validate the proposed protocol. HRM analysis of hsp70 amplicons is a fast and robust strategy that allowed for the detection and discrimination of all Leishmania species responsible for the Leishmaniases in Brazil and Eurasia/Africa with high sensitivity and accuracy. This method could detect less than one parasite per reaction, even in the presence of host DNA.

A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression.

PubMed

Syed, Salahuddin; Desler, Claus; Rasmussen, Lene J; Schmidt, Kristina H

2016-12-01

In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186-212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks.

A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression

PubMed Central

Syed, Salahuddin; Desler, Claus; Rasmussen, Lene J.; Schmidt, Kristina H.

2016-01-01

In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186–212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks. PMID:27923055

Dynamic investigation of DNA bending and wrapping by type II topoisomerases

NASA Astrophysics Data System (ADS)

Shao, Qing; Finzi, Laura; Dunlap, David

2009-11-01

Type II topoisomerases catalyze DNA decatenation and unwinding which is crucial for cell division, and therefore type II topoisomerases are some of the main targets of anti-cancer drugs. A recent crystal structure shows that, during the catalytic cycle, a yeast type II topoimerase can bend a 10 base pair DNA segment by up to 150 degrees. Bacterial gyrase, another type II topoisomerase, can wrap DNA into a tight 180 degree turn. Bending a stiff polymer like DNA requires considerable energy and could represent the rate limiting step in the catalytic (topological) cycle. Using modified deoxyribonucleotides in PCR reactions, stiffer DNA fragments have been produced and used as substrates for topoisomerase II-mediated relaxation of plectonemes introduced in single molecules using magnetic tweezers. The wrapping ability of gyrase decreases for diamino-purine-substituted DNA in which every base pair has three hydrogen-bonds. The overall rate of relaxation of plectonemes by recombinant human topoisomerase II alpha also decreases. These results reveal the dynamic properties of DNA bending and wrapping by type II topisomerases and suggest that A:T base pair melting is a rate determining step for bending and wrapping.

Microscale models of partially molten rocks and their macroscale physical properties

NASA Astrophysics Data System (ADS)

Rudge, J. F.

2017-12-01

Any geodynamical model of melt transport in the Earth's mantle requires constitutive laws for the rheology of partially molten rock. These constitutive laws are poorly known, and one way to make progress in our understanding is through the upscaling of microscale models which describe physics at the scale of individual mineral grains. Crucially, many upscaled physical properties (such as permeability) depend not only on how much melt is present, but on how that melt is arranged at the microscale; i.e. on the geometry of the melt network. Here I will present some new calculations of equilibrium melt network geometries around idealised tetrakaidecahedral grains. In contrast to several previous calculations of textural equilibrium, these calculations allow for a both a liquid-phase and a solid-phase topology that can tile 3D space. The calculations are based on a simple minimisation of surface energy using the finite element method. In these simple models just two parameters control the topology of the melt network: the porosity (volume fraction of melt), and the dihedral angle. The consquences of these melt geometries for upscaled properties such as permeability; electrical conductivity; and importantly, effective viscosity will be explored. Recent theoretical work [1,2] has suggested that in diffusion creep a small amount of melt may dramatically reduce the effective shear viscosity of a partially molten rock, with profound consequences for the nature of the asthenosphere. This contribution will show that this reduction in viscosity may have been significantly overestimated, so that the drop in the effective viscosity at onset of melting is more modest. [1] Takei, Y., and B. K. Holtzman (2009), Viscous constitutive relations of solid-liquid composites in terms of grain boundary contiguity: 1. Grain boundary diffusion control model, J. Geophys. Res., 114, B06205.[2] Holtzmann B. K. (2016) Questions on the existence, persistence, and mechanical effects of a very small melt fraction in the asthenosphere, Geophys. Geochem. Geosyst. 17, 470-484.

A new structural framework for integrating replication protein A into DNA processing machinery

PubMed Central

Brosey, Chris A.; Yan, Chunli; Tsutakawa, Susan E.; Heller, William T.; Rambo, Robert P.; Tainer, John A.; Ivanov, Ivaylo; Chazin, Walter J.

2013-01-01

By coupling the protection and organization of single-stranded DNA (ssDNA) with recruitment and alignment of DNA processing factors, replication protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA coordinates biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA’s DNA-binding activity, combining small-angle X-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA’s DNA-binding core. The scattering data reveal compaction promoted by DNA binding; DNA-free RPA exists in an ensemble of states with inter-domain mobility and becomes progressively more condensed and less dynamic on binding ssDNA. Our results contrast with previous models proposing RPA initially binds ssDNA in a condensed state and becomes more extended as it fully engages the substrate. Moreover, the consensus view that RPA engages ssDNA in initial, intermediate and final stages conflicts with our data revealing that RPA undergoes two (not three) transitions as it binds ssDNA with no evidence for a discrete intermediate state. These results form a framework for understanding how RPA integrates the ssDNA substrate into DNA processing machinery, provides substrate access to its binding partners and promotes the progression and selection of DNA processing pathways. PMID:23303776

A new structural framework for integrating replication protein A into DNA processing machinery

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

Brosey, Chris; Yan, Chunli; Tsutakawa, Susan

2013-01-17

By coupling the protection and organization of single-stranded DNA (ssDNA) with recruitment and alignment of DNA processing factors, replication protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA coordinates biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA's DNA-binding activity, combining small-angle X-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA's DNA-binding core. The scattering data reveal compaction promoted by DNA binding; DNA-free RPA exists in an ensemble of states with inter-domain mobility and becomes progressively more condensed and less dynamicmore » on binding ssDNA. Our results contrast with previous models proposing RPA initially binds ssDNA in a condensed state and becomes more extended as it fully engages the substrate. Moreover, the consensus view that RPA engages ssDNA in initial, intermediate and final stages conflicts with our data revealing that RPA undergoes two (not three) transitions as it binds ssDNA with no evidence for a discrete intermediate state. These results form a framework for understanding how RPA integrates the ssDNA substrate into DNA processing machinery, provides substrate access to its binding partners and promotes the progression and selection of DNA processing pathways.« less

Theory and modeling of particles with DNA-mediated interactions

NASA Astrophysics Data System (ADS)

Licata, Nicholas A.

2008-05-01

In recent years significant attention has been attracted to proposals which utilize DNA for nanotechnological applications. Potential applications of these ideas range from the programmable self-assembly of colloidal crystals, to biosensors and nanoparticle based drug delivery platforms. In Chapter I we introduce the system, which generically consists of colloidal particles functionalized with specially designed DNA markers. The sequence of bases on the DNA markers determines the particle type. Due to the hybridization between complementary single-stranded DNA, specific, type-dependent interactions can be introduced between particles by choosing the appropriate DNA marker sequences. In Chapter II we develop a statistical mechanical description of the aggregation and melting behavior of particles with DNA-mediated interactions. In Chapter III a model is proposed to describe the dynamical departure and diffusion of particles which form reversible key-lock connections. In Chapter IV we propose a method to self-assemble nanoparticle clusters using DNA scaffolds. A natural extension is discussed in Chapter V, the programmable self-assembly of nanoparticle clusters where the desired cluster geometry is encoded using DNA-mediated interactions. In Chapter VI we consider a nanoparticle based drug delivery platform for targeted, cell specific chemotherapy. In Chapter VII we present prospects for future research: the connection between DNA-mediated colloidal crystallization and jamming, and the inverse problem in self-assembly.

Force regulated dynamics of RPA on a DNA fork.

PubMed

Kemmerich, Felix E; Daldrop, Peter; Pinto, Cosimo; Levikova, Maryna; Cejka, Petr; Seidel, Ralf

2016-07-08

Replication protein A (RPA) is a single-stranded DNA binding protein, involved in most aspects of eukaryotic DNA metabolism. Here, we study the behavior of RPA on a DNA substrate that mimics a replication fork. Using magnetic tweezers we show that both yeast and human RPA can open forked DNA when sufficient external tension is applied. In contrast, at low force, RPA becomes rapidly displaced by the rehybridization of the DNA fork. This process appears to be governed by the binding or the release of an RPA microdomain (toehold) of only few base-pairs length. This gives rise to an extremely rapid exchange dynamics of RPA at the fork. Fork rezipping rates reach up to hundreds of base-pairs per second, being orders of magnitude faster than RPA dissociation from ssDNA alone. Additionally, we show that RPA undergoes diffusive motion on ssDNA, such that it can be pushed over long distances by a rezipping fork. Generally the behavior of both human and yeast RPA homologs is very similar. However, in contrast to yeast RPA, the dissociation of human RPA from ssDNA is greatly reduced at low Mg(2+) concentrations, such that human RPA can melt DNA in absence of force. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

RNA-DNA and DNA-DNA base-pairing at the upstream edge of the transcription bubble regulate translocation of RNA polymerase and transcription rate.

PubMed

KIreeva, Maria; Trang, Cyndi; Matevosyan, Gayane; Turek-Herman, Joshua; Chasov, Vitaly; Lubkowska, Lucyna; Kashlev, Mikhail

2018-06-20

Translocation of RNA polymerase (RNAP) along DNA may be rate-limiting for transcription elongation. The Brownian ratchet model posits that RNAP rapidly translocates back and forth until the post-translocated state is stabilized by NTP binding. An alternative model suggests that RNAP translocation is slow and poorly reversible. To distinguish between these two models, we take advantage of an observation that pyrophosphorolysis rates directly correlate with the abundance of the pre-translocated fraction. Pyrophosphorolysis by RNAP stabilized in the pre-translocated state by bacteriophage HK022 protein Nun was used as a reference point to determine the pre-translocated fraction in the absence of Nun. The stalled RNAP preferentially occupies the post-translocated state. The forward translocation rate depends, among other factors, on melting of the RNA-DNA base pair at the upstream edge of the transcription bubble. DNA-DNA base pairing immediately upstream from the RNA-DNA hybrid stabilizes the post-translocated state. This mechanism is conserved between E. coli RNAP and S. cerevisiae RNA polymerase II and is partially dependent on the lid domain of the catalytic subunit. Thus, the RNA-DNA hybrid and DNA reannealing at the upstream edge of the transcription bubble emerge as targets for regulation of the transcription elongation rate.

Particle Image Velocimetry During Injection Molding

NASA Astrophysics Data System (ADS)

Bress, Thomas; Dowling, David

2012-11-01

Injection molding involves the unsteady non-isothermal flow of a non-Newtonian polymer melt. An optical-access mold has been used to perform particle image velocimetry (PIV) on molten polystyrene during injection molding. Velocimetry data of the mold-filling flow will be presented. Statistical assessments of the velocimetry data and scaled residuals of the continuity equation suggest that PIV can be conducted in molten plastics with an uncertainty of +/-2 percent. Simulations are often used to model polymer flow during injection molding to design molds and select processing parameters but it is difficult to determine the accuracy of these simulations due to a lack of in-mold velocimetry and melt-front progression data. Moldflow was used to simulate the filling of the optical-access mold, and these simulated results are compared to the appropriately-averaged time-varying velocity field measurements. Simulated results for melt-front progression are also compared with the experimentally observed flow fronts. The ratio of the experimentally measured average velocity magnitudes to the simulation magnitudes was found on average to be 0.99 with a standard deviation of 0.25, and the difference in velocity orientations was found to be 0.9 degree with a standard deviation of 3.2 degrees. formerly at the University of Michigan.

The Generation of Barriers to Melt Ascent in the Martian Lithosphere

NASA Astrophysics Data System (ADS)

Schools, Joe W.; Montési, Laurent G. J.

2018-01-01

Planetary mantles can be regarded as an aggregate of two phases: a solid, porous matrix and a liquid melt. Melt travels rapidly upward through the matrix due to its buoyancy. When this melt enters the colder lithosphere, it begins to crystallize. If crystallization happens at a high rate, the newly formed crystals can clog the pore space, reducing its permeability to essentially zero. This zone of zero permeability is the permeability barrier. We use the MELTS family of thermodynamic calculators to determine melt compositions and the crystallization sequence of ascending melt throughout Martian history and simulate the formation of permeability barriers. At lower strain rates (10-17-10-15 s-1) permeability barriers form deep in the lithosphere, possibly contributing to the formation of localized volcanic edifices on the Martian surface once fracturing or thermal erosion enables melt to traverse the lithosphere. Higher strain rates (10-13 s-1) yield shallower permeability barriers, perhaps producing extensive lava flows. Permeability barrier formation is investigated using an anhydrous mantle source or mantle sources that include up to 1,000 ppm H2O. Introducing even small amounts of water (25 ppm H2O) reduces mantle viscosity in a manner similar to increasing the strain rate and results in a shallower barrier than in the anhydrous case. Large amounts of water (1,000 ppm H2O) yield very shallow weak barriers or no barriers at all. The depth of the permeability barrier has evolved through time, likely resulting in a progression in the style of surface volcanism from widespread flows to massive, singular volcanoes.

The transition from granite to banded aplite-pegmatite sheet complexes: An example from Megiliggar Rocks, Tregonning topaz granite, Cornwall

NASA Astrophysics Data System (ADS)

Breiter, K.; Ďurišová, J.; Hrstka, T.; Korbelová, Z.; Vašinová Galiová, M.; Müller, A.; Simons, B.; Shail, R. K.; Williamson, B. J.; Davies, J. A.

2018-03-01

The genetic relationship between a granite pluton and adjacent complex of rare-metal pegmatite-aplite-banded sheets (Megiliggar Sheet Complex - MSC) has been studied at the border of the Tregonning topaz granite at Megiliggar Rocks, Cornwall, SW England. Similarities in whole-rock chemical and mineralogical compositions, together with a gradual change in textures away from the granite margin, provide strong evidence for a genetic link between the Tregonning Granite and MSC. The sheets are likely to represent apophyses of residual melt which escaped from the largely crystallized roof of the granite pluton. The escaping melt was peraluminous, had a composition near the F, B, Li slightly enriched granite minimum, and, in comparison with other Cornish granites, was enriched in F, Li, Rb, Cs, Sn, W, Nb, Ta, and U, and depleted in Fe, Mg, Ca, Sr, Th, Zr, and REE. With increasing distance from the Tregonning Granite, the silicate melt crystallized as homogeneous leucogranite sheets and banded complex sheets (i.e. combinations of bands with granitic, aplitic and pegmatitic textures), then layered aplite-pegmatites; this sequence becoming progressively more depleted in the fluxing and volatile elements F, Li, Rb, and Cs, but showing no change in Zr/Hf ratios. The fixed Zr/Hf ratio is interpreted as indicating a direct genetic link (parental melt) between all rock types, however the melt progressively lost fluxing and volatile elements with distance from the granite pluton, probably due to wall-rock reaction or fluid exsolution and migration via fractures. Differentiation of the primary melt into Na-Li-F-rich and separate K-B-rich domains was the dominant chemical process responsible for the textural and mineral diversity of the MSC. On a large (cliff-section) scale, the proximal Na-Li-F-rich leucogranite passes through complex sheets into K-B-rich aplite-pegmatites, whilst at a smaller (<1 m) scale, the K-B-rich bands are interspersed (largely overlain) by Na-Li-F-rich segregations. The grain size differences between the aplite and pegmatite could be related to pressure fluctuations and/or undercooling.

Correlation of DNA Ploidy with Progression of Cervical Cancer

PubMed Central

Singh, M.; Mehrotra, S.; Kalra, N.; Singh, U.; Shukla, Y.

2008-01-01

The majority of squamous cell carcinomas of cervix are preceded by visible changes in the cervix, most often detected by cervical smear. As cervical cancer is preceded by long precancerous stages, identification of the high-risk population through detection of DNA ploidy may be of importance in effective management of this disease. Here we attempted to correlate aneuploid DNA patterns and their influence on biological behavior of flow-cytometry analysis of DNA ploidy which was carried out in cytologically diagnosed cases of mild (79), moderate (36), and severe (12) dysplasia, as well as “atypical squamous cells of unknown significance (ASCUS)” (57) along with controls (69), in order to understand its importance in malignant progression of disease. Cytologically diagnosed dysplasias, which were employed for DNA ploidy studies, 39 mild, 28 moderate, and 11 severe dysplasia cases were found to be aneuploid. Out of the 69 control subjects, 6 cases showed aneuploidy pattern and the rest 63 subjects were diploid. An aneuploidy pattern was observed in 8 out of 57 cases of cytologically evaluated ASCUS. The results of the followup studies showed that aberrant DNA content reliably predicts the occurrence of squamous cell carcinoma in cervical smear. Flow cytometric analysis of DNA ploidy may provide a strategic diagnostic tool for early detection of carcinoma cervix. Therefore, it is a concept of an HPV screening with reflex cytology in combination with DNA flow cytometry to detect progressive lesions with the greatest possible sensitivity and specificity. PMID:20445775

Anomeric 2'-Deoxycytidines and Silver Ions: Hybrid Base Pairs with Greatly Enhanced Stability and Efficient DNA Mismatch Detection with α-dC.

PubMed

Guo, Xiurong; Seela, Frank

2017-09-04

α-d-Nucleosides are rare in nature but can develop fascinating properties when incorporated into DNA. This work reports on the first silver-mediated base pair constructed from two anomeric nucleosides: α-dC and β-dC. The hybrid base pair was integrated into the DNA and DNA/RNA double helix. A 12-mer duplex with α-dC and β-dC pair exhibits a higher thermal stability (T m =43 °C) than that incorporating the β-dC-Ag + -β-dC homo pair (T m =34 °C). Furthermore, α-dC shows excellent mismatch discrimination for DNA single nucleotide polymorphism (SNP). All four SNPs were identified on the basis of large T m value differences measured in the presence of silver ions. High resolution melting was not required. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mechanisms and regulation of DNA replication initiation in eukaryotes

PubMed Central

Parker, Matthew W.; Botchan, Michael R.; Berger, James M.

2017-01-01

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a given cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the Origin Recognition Complex (ORC), and subsequent activation of the helicase by incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here we review the molecular mechanisms that underpin eukaryotic DNA replication initiation – from selecting replication start sites to replicative helicase loading and activation – and describe how these events are often distinctly regulated across different eukaryotic model organisms. PMID:28094588

Mechanisms and regulation of DNA replication initiation in eukaryotes.

PubMed

Parker, Matthew W; Botchan, Michael R; Berger, James M

2017-04-01

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation - from selecting replication start sites to replicative helicase loading and activation - and describe how these events are often distinctly regulated across different eukaryotic model organisms.

Evaluation of suitable DNA regions for molecular identification of high value medicinal plants in genus Kaempferia.

PubMed

Osathanunkul, Maslin; Dheeranupattana, Srisulak; Rotarayanont, Siriphron; Sookkhee, Siriwoot; Osathanunkul, Khukrit; Madesis, Panagiotis

2017-12-02

DNA barcoding coupled high resolution melting (Bar-HRM) is an emerging method for species discrimination based on DNA dissociation kinetics. The aim of this work was to evaluate the suitability of different primer sets, derived from selected DNA regions, for Bar-HRM analysis of species in Kaempferia (Zingiberaceae). Four primer pairs were evaluated (rbcL, rpoC, trnL and ITS1). It was observed that the ITS1 barcode was the most useful DNA barcoding region overall for species discrimination out of all of the regions and primers assessed. Thus, the primer pair derived from the ITS1 region was the single most effective region for the identification of the tested species, whereas the rbcL primer pair gave the lowest resolution. Our Bar-HRM developed here would not only be useful for identification of Kaempferia plant specimens lacking essential parts for morphological identification but will be useful for authenticating products in powdered form of a high value medicinal species Kaempferia parviflora, in particular.

Antibacterial Drug Leads: DNA and Enzyme Multitargeting

DOE PAGES

Zhu, Wei; Wang, Yang; Li, Kai; ...

2015-01-09

Here, we report the results of an investigation of the activity of a series of amidine and bisamidine compounds against Staphylococcus aureus and Escherichia coli. The most active compounds bound to an AT-rich DNA dodecamer (CGCGAATTCGCG) 2 and using DSC were found to increase the melting transition by up to 24 °C. Several compounds also inhibited undecaprenyl diphosphate synthase (UPPS) with IC 50 values of 100–500 nM, and we found good correlations (R 2 = 0.89, S. aureus; R 2 = 0.79, E. coli) between experimental and predicted cell growth inhibition by using DNA ΔT m and UPPS IC 50more » experimental results together with one computed descriptor. Finally, we also solved the structures of three bisamidines binding to DNA as well as three UPPS structures. Overall, the results are of general interest in the context of the development of resistance-resistant antibiotics that involve multitargeting.« less

DNA methylation and the potential role of demethylating agents in prevention of progressive chronic kidney disease.

PubMed

Larkin, Benjamin P; Glastras, Sarah J; Chen, Hui; Pollock, Carol A; Saad, Sonia

2018-04-24

Chronic kidney disease (CKD) is a global epidemic, and its major risk factors include obesity and type 2 diabetes. Obesity not only promotes metabolic dysregulation and the development of diabetic kidney disease but also may independently lead to CKD by a variety of mechanisms, including endocrine and metabolic dysfunction, inflammation, oxidative stress, altered renal hemodynamics, and lipotoxicity. Deleterious renal effects of obesity can also be transmitted from one generation to the next, and it is increasingly recognized that offspring of obese mothers are predisposed to CKD. Epigenetic modifications are changes that regulate gene expression without altering the DNA sequence. Of these, DNA methylation is the most studied. Epigenetic imprints, particularly DNA methylation, are laid down during critical periods of fetal development, and they may provide a mechanism by which maternal-fetal transmission of chronic disease occurs. Our current review explores the evidence for the role of DNA methylation in the development of CKD, diabetic kidney disease, diabetes, and obesity. DNA methylation has been implicated in renal fibrosis-the final pathophysiologic pathway in the development of end-stage kidney disease-which supports the notion that demethylating agents may play a potential therapeutic role in preventing development and progression of CKD.-Larkin, B. P., Glastras, S. J., Chen, H., Pollock, C. A., Saad, S. DNA methylation and the potential role of demethylating agents in prevention of progressive chronic kidney disease.

Estimation of snow and glacier melt contribution to Liddar stream in a mountainous catchment, western Himalaya: an isotopic approach.

PubMed

Jeelani, Gh; Shah, Rouf A; Jacob, Noble; Deshpande, Rajendrakumar D

2017-03-01

Snow- and glacier-dominated catchments in the Himalayas are important sources of fresh water to more than one billion people. However, the contribution of snowmelt and glacier melt to stream flow remains largely unquantified in most parts of the Himalayas. We used environmental isotopes and geochemical tracers to determine the source water and flow paths of stream flow draining the snow- and glacier-dominated mountainous catchment of the western Himalaya. The study suggested that the stream flow in the spring season is dominated by the snowmelt released from low altitudes and becomes isotopically depleted as the melt season progressed. The tracer-based mixing models suggested that snowmelt contributed a significant proportion (5-66 %) to stream flow throughout the year with the maximum contribution in spring and summer seasons (from March to July). In 2013 a large and persistent snowpack contributed significantly (∼51 %) to stream flow in autumn (September and October) as well. The average annual contribution of glacier melt to stream flow is little (5 %). However, the monthly contribution of glacier melt to stream flow reaches up to 19 % in September during years of less persistent snow pack.

Nicotinamide Riboside and Mitochondrial Biogenesis

ClinicalTrials.gov

2018-03-15

Mitochondrial Diseases; Mitochondrial Myopathies; Progressive External Ophthalmoplegia; Progressive Ophthalmoplegia; Progressive; Ophthalmoplegia, External; Mitochondria DNA Deletion; MELAS; Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-Like Episodes; Mitochondrial Encephalopathy, Lactic Acidosis and Stroke-Like Episodes (MELAS Syndrome)

Separation/extraction, detection, and interpretation of DNA mixtures in forensic science (review).

PubMed

Tao, Ruiyang; Wang, Shouyu; Zhang, Jiashuo; Zhang, Jingyi; Yang, Zihao; Sheng, Xiang; Hou, Yiping; Zhang, Suhua; Li, Chengtao

2018-05-25

Interpreting mixed DNA samples containing material from multiple contributors has long been considered a major challenge in forensic casework, especially when encountering low-template DNA (LT-DNA) or high-order mixtures that may involve missing alleles (dropout) and unrelated alleles (drop-in), among others. In the last decades, extraordinary progress has been made in the analysis of mixed DNA samples, which has led to increasing attention to this research field. The advent of new methods for the separation and extraction of DNA from mixtures, novel or jointly applied genetic markers for detection and reliable interpretation approaches for estimating the weight of evidence, as well as the powerful massively parallel sequencing (MPS) technology, has greatly extended the range of mixed samples that can be correctly analyzed. Here, we summarized the investigative approaches and progress in the field of forensic DNA mixture analysis, hoping to provide some assistance to forensic practitioners and to promote further development involving this issue.

CDK1 promotes nascent DNA synthesis and induces resistance of cancer cells to DNA-damaging therapeutic agents

PubMed Central

Liao, Hongwei; Ji, Fang; Geng, Xinwei; Xing, Meichun; Li, Wen; Chen, Zhihua; Shen, Huahao; Ying, Songmin

2017-01-01

Cyclin dependent kinase 1 (CDK1) is essential for cell viability and plays a vital role in many biological events including cell cycle control, DNA damage repair, and checkpoint activation. Here, we identify an unanticipated role for CDK1 in promoting nascent DNA synthesis during S-phase. We report that a short duration of CDK1 inhibition, which does not perturb cell cycle progression, triggers a replication-associated DNA damage response (DDR). This DDR is associated with a disruption of replication fork progression and leads to genome instability. Moreover, we show that compromised CDK1 activity dramatically increases the efficacy of chemotherapeutic agents that kill cancer cells through perturbing DNA replication, including Olaparib, an FDA approved PARP inhibitor. Our study has revealed an important role for CDK1 in the DNA replication program, and suggests that the therapeutic targeting CDK1 may be a novel approach for combination chemotherapy. PMID:29207595

A DNA methylation map of human cancer at single base-pair resolution.

PubMed

Vidal, E; Sayols, S; Moran, S; Guillaumet-Adkins, A; Schroeder, M P; Royo, R; Orozco, M; Gut, M; Gut, I; Lopez-Bigas, N; Heyn, H; Esteller, M

2017-10-05

Although single base-pair resolution DNA methylation landscapes for embryonic and different somatic cell types provided important insights into epigenetic dynamics and cell-type specificity, such comprehensive profiling is incomplete across human cancer types. This prompted us to perform genome-wide DNA methylation profiling of 22 samples derived from normal tissues and associated neoplasms, including primary tumors and cancer cell lines. Unlike their invariant normal counterparts, cancer samples exhibited highly variable CpG methylation levels in a large proportion of the genome, involving progressive changes during tumor evolution. The whole-genome sequencing results from selected samples were replicated in a large cohort of 1112 primary tumors of various cancer types using genome-scale DNA methylation analysis. Specifically, we determined DNA hypermethylation of promoters and enhancers regulating tumor-suppressor genes, with potential cancer-driving effects. DNA hypermethylation events showed evidence of positive selection, mutual exclusivity and tissue specificity, suggesting their active participation in neoplastic transformation. Our data highlight the extensive changes in DNA methylation that occur in cancer onset, progression and dissemination.

Muscovite-Dehydration Melting: A Textural Study of a Key Reaction in Transforming Continental Margin Strata Into a Migmatitic Orogenic Core

NASA Astrophysics Data System (ADS)

Dyck, B. J.; St Onge, M. R.; Waters, D. J.; Searle, M. P.

2015-12-01

Metamorphosed continental margin sedimentary sequences, which comprise the dominant tectonostratigraphic assemblage exposed in orogenic hinterlands, are crucial to understanding the architecture and evolution of collisional mountain belts. This study explores the textural effect of anatexis in amphibolite-grade conditions and documents the mineral growth mechanisms that control nucleation and growth of K-feldspar, sillimanite and silicate melt. The constrained textural evolution follows four stages: 1) Nucleation - K-feldspar is documented to nucleate epitaxially on isomorphic plagioclase in quartzofeldspathic (psammitic) domains, whereas sillimanite nucleates in the Al-rich (pelitic) domain, initially on [001] mica planes. The first melt forms at the site of muscovite breakdown. 2) Chemically driven growth - In the quartzofeldspathic domain, K-feldspar progressively replaces plagioclase by a K+ - Na+ cation transfer reaction, driven by the freeing of muscovite-bound K+ during breakdown of the mica. Sillimanite forms intergrowths with the remaining hydrous melt components, contained initially in ovoid clots. 3) Merge and coarsening - With an increase in pressure, melt and sillimanite migrate away from clots along grain boundaries. A melt threshold is reached once the grain-boundary network is wetted by melt, increasing the length-scale of diffusion, resulting in grain boundary migration and grain-size coarsening. The melt threshold denotes the transition to an open-system on the lithology scale, where melt is a transient phase. 4) Residual melt crystallization - Residual melt crystallizes preferentially on existing peritectic grains as anatectic quartz, plagioclase, and K-feldspar. As the system cools and closes, grain growth forces melt into the intersections of grain-boundaries, recognized as irregular shaped melt films, or as intergrowths of the volatile-rich phases (i.e. Tur-Ms-Ap). In the Himalayan metamorphic core these processes result in the formation of: pelitic K-feldspar augen gneiss, stockwork leucogranites, and an effective strengthening of the hinterland, as evidenced by a switch in tectonic deformation style, from thin-skinned cover sequence thrust imbrication and folding to out-of-sequence basement-involved thick-skinned thrusting and folding.

From mantle peridotites to hybrid troctolites: Textural, structural and geochemical evolution during multi-stage melt-rock interaction history

NASA Astrophysics Data System (ADS)

Basch, V.; Rampone, E.; Crispini, L.; Ferrando, C.; Ildefonse, B.; Godard, M.

2017-12-01

Recent studies investigate the replacive formation of hybrid troctolites from mantle peridotites after multiple stages of melt-rock reactions. However, none of these studies are conducted in a field-controlled geological setting displaying the clear evolution from peridotite to dunite to troctolite. We investigated the Mt.Maggiore and Erro Tobbio ophiolitic peridotites. They both preserve structural and chemical records of two distinct melt-rock interaction stages, from a reactive melt percolation at spinel facies to plagioclase-bearing melt impregnation at shallower lithospheric depths. We performed EBSD and in situ geochemical analyses to document the textural, structural and geochemical variations of the olivine matrix during melt-rock interactions and the associated evolution from peridotite to dunite to troctolite. The olivine-saturated reactive melt percolation leads to the dissolution of mantle pyroxenes in peridotite, and to the formation of replacive dunite. At shallower level, melt impregnation leads to the crystallization of plagioclase in the dunite, and to the formation of hybrid troctolite. The latter is characterized by textural, structural and geochemical features acquired during dunitization and impregnation processes. We documented a textural evolution of the olivine matrix (decrease in grain area, tortuosity and aspect ratio) during impregnation, with a progressive corrosion of mantle olivines by a reactive melt. As a result, olivine in the hybrid troctolites occurs both as coarse deformed relicts and disrupted undeformed grains. During melt-rock interactions, the variation in olivine Crystallographic Preferred Orientation is related to the local melt/rock ratio involved in the percolation process. At high melt/rock ratio, a change from axial-[100] to axial-[010] is observed, with the disaggregation of the solid matrix. REE-enriched compositions are observed in olivine of dunites and troctolites. A geochemical modeling of melt-rock interactions (Plate Model) fits the observed evolution of modal composition with the measured trace element composition variability. The combined field, structural, and geochemical investigation of the evolution from a mantle protolith to the product of the reactions truly supports the hybrid origin of an olivine-rich troctolite.

Ordered Conformational Changes in Damaged DNA Induced by Nucleotide Excision Repair Factors*

PubMed Central

Tapias, Angels; Auriol, Jerome; Forget, Diane; Enzlin, Jacqueline H.; Schärer, Orlando D; Coin, Frederic; Coulombe, Benoit; Egly, Jean-Marc

2015-01-01

In response to genotoxic attacks, cells activate sophisticated DNA repair pathways such as nucleotide excision repair (NER), which consists of damage removal via dual incision and DNA resynthesis. Using permanganate footprinting as well as highly purified factors, we show that NER is a dynamic process that takes place in a number of successive steps during which the DNA is remodeled around the lesion in response to the various NER factors. XPC/HR23B first recognizes the damaged structure and initiates the opening of the helix from position −3 to +6. TFIIH is then recruited and, in the presence of ATP, extends the opening from position −6 to +6; it also displaces XPC downstream from the lesion, thereby providing the topological structure for recruiting XPA and RPA, which will enlarge the opening. Once targeted by XPG, the damaged DNA is further melted from position −19 to +8. XPG and XPF/ERCC1 endo-nucleases then cut the damaged DNA at the limit of the opened structure that was previously “labeled” by the positioning of XPC/HR23B and TFIIH. PMID:14981083

[Oligonucleotide derivatives in the nucleic acid hybridization analysis. II. Isothermal signal amplification in process of DNA analysis by minisequencing].

PubMed

Dmitrienko, E V; Khomiakova, E A; Pyshnaia; Bragin, A G; Vedernikov, V E; Pyshnyĭ, D V

2010-01-01

The isothermal amplification of reporter signal via limited probe extension (minisequencing) upon hybridization of nucleic acids has been studied. The intensity of reporter signal has been shown to increase due to enzymatic labeling of multiple probes upon consecutive hybridization with one DNA template both in homophase and heterophase assays using various kinds of detection signal: radioisotope label, fluorescent label, and enzyme-linked assay. The kinetic scheme of the process has been proposed and kinetic parameters for each step have been determined. The signal intensity has been shown to correlate with physicochemical characteristics of both complexes: probe/DNA and product/DNA. The maximum intensity has been observed at minimal difference between the thermodynamic stability of these complexes, provided the reaction temperature has been adjusted near their melting temperature values; rising or lowering the reaction temperature reduces the amount of reporting product. The signal intensity has been shown to decrease significantly upon hybridization with the DNA template containing single-nucleotide mismatches. Limited probe extension assay is useful not only for detection of DNA template but also for its quantitative characterization.

Investigation of irradiated rats DNA in the presence of Cu(II) chelates of amino acids Schiff bases.

PubMed

Karapetyan, N H; Torosyan, A L; Malakyan, M; Bajinyan, S A; Haroutiunian, S G

2016-01-01

The new synthesized Cu(II) chelates of amino acids Schiff bases were studied as a potential radioprotectors. Male albino rats of Wistar strain were exposed to X-ray whole-body irradiation at 4.8 Gy. This dose caused 30% mortality of the animals (LD30). The survival of animals exposed to radiation after preliminary administration of 10 mg/kg Cu(II)(Nicotinyl-L-Tyrosinate)2 or Cu(II)(Nicotinyl-L-Tryptophanate)2 prior to irradiation was registered about 80 and 100% correspondingly. Using spectrophotometric melting and agarose gel electrophoresis methods, the differences between the DNA isolated from irradiated rats and rats pretreated with Cu(II) chelates were studied. The fragments of DNA with different breaks were revealed in DNA samples isolated from irradiated animals. While, the repair of the DNA structure was observed for animals pretreated with the Cu(II) chelates. The results suggested that pretreatment of the irradiated rats with Cu(II)(Nicotinyl-L-Tyrosinate)2 and Cu(II)(Nicotinyl-L-Tryptophanate)2 compounds improves the liver DNA characteristics.

Experimental Evidence for Fast Lithium Diffusion and Isotope Fractionation in Water-bearing Rhyolitic Melts at Magmatic Conditions

NASA Astrophysics Data System (ADS)

Cichy, S. B.; Till, C. B.; Roggensack, K.; Hervig, R. L.; Clarke, A. B.

2015-12-01

The aim of this work is to extend the existing database of experimentally-determined lithium diffusion coefficients to more natural cases of water-bearing melts at the pressure-temperature range of the upper crust. In particular, we are investigating Li intra-melt and melt-vapor diffusion and Li isotope fractionation, which have the potential to record short-lived magmatic processes (seconds to hours) in the shallow crust, especially during decompression-induced magma degassing. Hydrated intra-melt Li diffusion-couple experiments on Los Posos rhyolite glass [1] were performed in a piston cylinder at 300 MPa and 1050 °C. The polished interfaces between the diffusion couples were marked by addition of Pt powder for post-run detection. Secondary ion mass spectrometry analyses indicate that lithium diffuses extremely fast in the presence of water. Re-equilibration of a hydrated ~2.5 mm long diffusion-couple experiment was observed during the heating period from room temperature to the final temperature of 1050 °C at a rate of ~32 °C/min. Fractionation of ~40‰ δ7Li was also detected in this zero-time experiment. The 0.5h and 3h runs show progressively higher degrees of re-equilibration, while the isotope fractionation becomes imperceptible. Li contamination was observed in some experiments when flakes filed off Pt tubing were used to mark the diffusion couple boundary, while the use of high purity Pt powder produced better results and allowed easier detection of the diffusion-couple boundary. The preliminary lithium isotope fractionation results (δ7Li vs. distance) support findings from [2] that 6Li diffuses substantially faster than 7Li. Further experimental sets are in progress, including lower run temperatures (e.g. 900 °C), faster heating procedure (~100 °C/min), shorter run durations and the extension to mafic systems. [1] Stanton (1990) Ph.D. thesis, Arizona State Univ., [2] Richter et al. (2003) GCA 67, 3905-3923.

Detection of Prostate Cancer Progression by Serum DNA Integrity

DTIC Science & Technology

2010-04-01

qRT) Alu and direct qRT LINE1 is being optimized. We will also continue to develop circulating DNA methylated GSTP1 assay to complement the DNA...developed the LINE1 assay, assembled the manuscript on uLINE1, and performed preliminary analysis of circulating DNA GSTP1 methylation. The goal is to

Dynamic and Progressive Control of DNA Origami Conformation by Modulating DNA Helicity with Chemical Adducts.

PubMed

Chen, Haorong; Zhang, Hanyu; Pan, Jing; Cha, Tae-Gon; Li, Shiming; Andréasson, Joakim; Choi, Jong Hyun

2016-05-24

DNA origami has received enormous attention for its ability to program complex nanostructures with a few nanometer precision. Dynamic origami structures that change conformation in response to environmental cues or external signals hold great promises in sensing and actuation at the nanoscale. The reconfiguration mechanism of existing dynamic origami structures is mostly limited to single-stranded hinges and relies almost exclusively on DNA hybridization or strand displacement. Here, we show an alternative approach by demonstrating on-demand conformation changes with DNA-binding molecules, which intercalate between base pairs and unwind DNA double helices. The unwinding effect modulates the helicity mismatch in DNA origami, which significantly influences the internal stress and the global conformation of the origami structure. We demonstrate the switching of a polymerized origami nanoribbon between different twisting states and a well-constrained torsional deformation in a monomeric origami shaft. The structural transformation is shown to be reversible, and binding isotherms confirm the reconfiguration mechanism. This approach provides a rapid and reversible means to change DNA origami conformation, which can be used for dynamic and progressive control at the nanoscale.

Replication protein A: directing traffic at the intersection of replication and repair.

PubMed

Oakley, Greg G; Patrick, Steve M

2010-06-01

Since the initial discovery of replication protein A (RPA) as a DNA replication factor, much progress has been made on elucidating critical roles for RPA in other DNA metabolic pathways. RPA has been shown to be required for DNA replication, DNA repair, DNA recombination, and the DNA damage response pathway with roles in checkpoint activation. This review summarizes the current understanding of RPA structure, phosphorylation and protein-protein interactions in mediating these DNA metabolic processes.

Stability of non-Watson-Crick G-A/A-G base pair in synthetic DNA and RNA oligonucleotides.

PubMed

Ito, Yuko; Sone, Yumiko; Mizutani, Takaharu

2004-03-01

A non-Watson-Crick G-A/A-G base pair is found in SECIS (selenocysteine-insertion sequence) element in the 3'-untranslated region of Se-protein mRNAs and in the functional site of the hammerhead ribozyme. We studied the stability of G-A/A-G base pair (bold) in 17mer GT(U)GACGGAAACCGGAAC synthetic DNA and RNA oligonucleotides by thermal melting experiments and gel electrophoresis. The measured Tm value of DNA oligonucleotide having G-A/A-G pair showed an intermediate value (58 degrees C) between that of Watson-Crick G-C/C-G base pair (75 degrees C) and that of G-G/A-A of non-base-pair (40 degrees C). Similar thermal melting patterns were obtained with RNA oligonucleotides. This result indicates that the secondary structure of oligonucleotide having G-A/A-G base pair is looser than that of the G-C type Watson-Crick base pair. In the comparison between RNA and DNA having G-A/A-G base pair, the Tm value of the RNA oligonucleotide was 11 degrees C lower than that of DNA, indicating that DNA has a more rigid structure than RNA. The stained pattern of oligonucleotide on polyacrylamide gel clarified that the mobility of the DNA oligonucleotide G-A/A-G base pair changed according to the urea concentration from the rigid state (near the mobility of G-C/C-G oligonucleotide) in the absence of urea to the random state (near the mobility of G-G/A-A oligonucleotide) in 7 M urea. However, the RNA oligonucleotide with G-A/A-G pair moved at an intermediate mobility between that of oligonucleotide with G-C/C-G and of the oligonucleotide with G-G/A-A, and the mobility pattern did not depend on urea concentration. Thus, DNA and RNA oligonucleotides with the G-A/A-G base pair showed a pattern indicating an intermediate structure between the rigid Watson-Crick base pair and the random structure of non-base pair. RNA with G-A/A-G base pair has the intermediate structure not influenced by urea concentration. Finally, this study indicated that the intermediate rigidity imparted by Non-Watson-Crick base pair in SECIS element plays an important role in the selenocysteine expression by UGA codon.

The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis.

PubMed

Ceccarelli, Marcello; Galluzzi, Luca; Diotallevi, Aurora; Andreoni, Francesca; Fowler, Hailie; Petersen, Christine; Vitale, Fabrizio; Magnani, Mauro

2017-05-16

Leishmaniasis is a neglected disease caused by many Leishmania species, belonging to subgenera Leishmania (Leishmania) and Leishmania (Viannia). Several qPCR-based molecular diagnostic approaches have been reported for detection and quantification of Leishmania species. Many of these approaches use the kinetoplast DNA (kDNA) minicircles as the target sequence. These assays had potential cross-species amplification, due to sequence similarity between Leishmania species. Previous works demonstrated discrimination between L. (Leishmania) and L. (Viannia) by SYBR green-based qPCR assays designed on kDNA, followed by melting or high-resolution melt (HRM) analysis. Importantly, these approaches cannot fully distinguish L. (L.) infantum from L. (L.) amazonensis, which can coexist in the same geographical area. DNA from 18 strains/isolates of L. (L.) infantum, L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, and 62 clinical samples from L. (L.) infantum-infected dogs were amplified by a previously developed qPCR (qPCR-ML) and subjected to HRM analysis; selected PCR products were sequenced using an ABI PRISM 310 Genetic Analyzer. Based on the obtained sequences, a new SYBR-green qPCR assay (qPCR-ama) intended to amplify a minicircle subclass more abundant in L. (L.) amazonensis was designed. The qPCR-ML followed by HRM analysis did not allow discrimination between L. (L.) amazonensis and L. (L.) infantum in 53.4% of cases. Hence, the novel SYBR green-based qPCR (qPCR-ama) has been tested. This assay achieved a detection limit of 0.1 pg of parasite DNA in samples spiked with host DNA and did not show cross amplification with Trypanosoma cruzi or host DNA. Although the qPCR-ama also amplified L. (L.) infantum strains, the C q values were dramatically increased compared to qPCR-ML. Therefore, the combined analysis of C q values from qPCR-ML and qPCR-ama allowed to distinguish L. (L.) infantum and L. (L.) amazonensis in 100% of tested samples. A new and affordable SYBR-green qPCR-based approach to distinguish between L. (L.) infantum and L. (L.) amazonensis was developed exploiting the major abundance of a minicircle sequence rather than targeting a hypothetical species-specific sequence. The fast and accurate discrimination between these species can be useful to provide adequate prognosis and treatment.

DNA breathing dynamics distinguish binding from nonbinding consensus sites for transcription factor YY1 in cells.

PubMed

Alexandrov, Boian S; Fukuyo, Yayoi; Lange, Martin; Horikoshi, Nobuo; Gelev, Vladimir; Rasmussen, Kim Ø; Bishop, Alan R; Usheva, Anny

2012-11-01

The genome-wide mapping of the major gene expression regulators, the transcription factors (TFs) and their DNA binding sites, is of great importance for describing cellular behavior and phenotypic diversity. Presently, the methods for prediction of genomic TF binding produce a large number of false positives, most likely due to insufficient description of the physiochemical mechanisms of protein-DNA binding. Growing evidence suggests that, in the cell, the double-stranded DNA (dsDNA) is subject to local transient strands separations (breathing) that contribute to genomic functions. By using site-specific chromatin immunopecipitations, gel shifts, BIOBASE data, and our model that accurately describes the melting behavior and breathing dynamics of dsDNA we report a specific DNA breathing profile found at YY1 binding sites in cells. We find that the genomic flanking sequence variations and SNPs, may exert long-range effects on DNA dynamics and predetermine YY1 binding. The ubiquitous TF YY1 has a fundamental role in essential biological processes by activating, initiating or repressing transcription depending upon the sequence context it binds. We anticipate that consensus binding sequences together with the related DNA dynamics profile may significantly improve the accuracy of genomic TF binding sites and TF binding-related functional SNPs.

Temperature gating and competing temperature-dependent effects in DNA molecular wires

NASA Astrophysics Data System (ADS)

Wibowo, Denni; Narenji, Alaleh; Kassegne, Sam

2017-02-01

While recent research in electron-transport mechanism on a double strands DNA seems to converge into a consensus, experiments in direct electrical measurements on a long DNA molecules still lead to a conflicting result This study is the continuation of our previous research in electrical characterization of DNA molecular wires, where we furtherly investigate the effects of temperature on the electrical conductivity of DNA molecular wires by measuring its impedance response. We found that at higher temperatures, the expected increase in charge hopping mechanism may account for the decrease in impedance (and hence increase in conductivity) supporting the 'charge hopping mechanism' theory. UV light exposure, on the other hand, causes damage to GC base pairs reducing the path available for hopping mechanism and hence resulting in increased impedance - this again supporting the 'charge hopping mechanism' theory. We also report that λ-DNA molecular wires have differing impedance responses at two temperature regimes: impedance increases between 4 °C - 40 °C and then decreases between 40 °C - melting point (˜110 °C), after which λ-DNA denatures resulting in no current transduction. We submit that the low impedance of λ-DNA molecular wires observed at moderate to high frequencies may have significant implications to the field of DNA-based bionanoelectronics.

Design, synthesis and DNA interactions of a chimera between a platinum complex and an IHF mimicking peptide.

PubMed

Rao, Harita; Damian, Mariana S; Alshiekh, Alak; Elmroth, Sofi K C; Diederichsen, Ulf

2015-12-28

Conjugation of metal complexes with peptide scaffolds possessing high DNA binding affinity has shown to modulate their biological activities and to enhance their interaction with DNA. In this work, a platinum complex/peptide chimera was synthesized based on a model of the Integration Host Factor (IHF), an architectural protein possessing sequence specific DNA binding and bending abilities through its interaction with a minor groove. The model peptide consists of a cyclic unit resembling the minor grove binding subdomain of IHF, a positively charged lysine dendrimer for electrostatic interactions with the DNA phosphate backbone and a flexible glycine linker tethering the two units. A norvaline derived artificial amino acid was designed to contain a dimethylethylenediamine as a bidentate platinum chelating unit, and introduced into the IHF mimicking peptides. The interaction of the chimeric peptides with various DNA sequences was studied by utilizing the following experiments: thermal melting studies, agarose gel electrophoresis for plasmid DNA unwinding experiments, and native and denaturing gel electrophoresis to visualize non-covalent and covalent peptide-DNA adducts, respectively. By incorporation of the platinum metal center within the model peptide mimicking IHF we have attempted to improve its specificity and DNA targeting ability, particularly towards those sequences containing adjacent guanine residues.

Studies of torsional properties of DNA and nucleosomes using angular optical trapping

NASA Astrophysics Data System (ADS)

Sheinin, Maxim Y.

DNA in vivo is subjected to torsional stress due to the action of molecular motors and other DNA-binding proteins. Several decades of research have uncovered the fascinating diversity of DNA transformations under torsion and the important role they play in the regulation of vital cellular processes such as transcription and replication. Recent studies have also suggested that torsion can influence the structure and stability of nucleosomes---basic building blocks of the eukaryotic genome. However, our understanding of the impact of torsion is far from being complete due to significant experimental challenges. In this work we have used a powerful single-molecule experimental technique, angular optical trapping, to address several long-standing issues in the field of DNA and nucleosome mechanics. First, we utilized the high resolution and direct torque measuring capability of the angular optical trapping to precisely measure DNA twist-stretch coupling. Second, we characterized DNA melting under tension and torsion. We found that torsionally underwound DNA forms a left-handed structure, significantly more flexible compared to the regular B-DNA. Finally, we performed the first comprehensive investigation of the single nucleosome behavior under torque and force. Importantly, we discovered that positive torque causes significant dimer loss, which can have implications for transcription through chromatin.

Species identification in meat products: A new screening method based on high resolution melting analysis of cyt b gene.

PubMed

Lopez-Oceja, A; Nuñez, C; Baeta, M; Gamarra, D; de Pancorbo, M M

2017-12-15

Meat adulteration by substitution with lower value products and/or mislabeling involves economic, health, quality and socio-religious issues. Therefore, identification and traceability of meat species has become an important subject to detect possible fraudulent practices. In the present study the development of a high resolution melt (HRM) screening method for the identification of eight common meat species is reported. Samples from Bos taurus, Ovis aries, Sus scrofa domestica, Equus caballus, Oryctolagus cuniculus, Gallus gallus domesticus, Meleagris gallopavo and Coturnix coturnix were analyzed through the amplification of a 148 bp fragment from the cyt b gene with a universal primer pair in HRM analyses. Melting profiles from each species, as well as from several DNA mixtures of these species and blind samples, allowed a successful species differentiation. The results demonstrated that the HRM method here proposed is a fast, reliable, and low-cost screening technique. Copyright © 2017 Elsevier Ltd. All rights reserved.

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

PubMed Central

Hashemipetroudi, Seyyed Hamidreza; Nematzadeh, Ghorbanali; Ahmadian, Gholamreza; Yamchi, Ahad; Kuhlmann, Markus

2018-01-01

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species. Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family, it could be easily used to other prokaryote and eukaryote species PMID:29443017

Simplicimonas-like DNA in vaginal swabs of cows and heifers cross-reacting in the real-time PCR for T. foetus.

PubMed

Frey, Caroline F; Müller, Norbert; Stäuber, Norbert; Marreros, Nelson; Hofmann, Larissa; Hentrich, Brigitte; Hirsbrunner, Gaby

2017-04-15

Cows on an alpine pasture were presented with severe signs of vaginitis. To rule out infection with Tritrichomonas foetus, vaginal swabs were taken and real-time PCR based on detection via fluorescence resonance energy transfer (FRET) probes and targeting the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA (rDNA) was performed. PCR was positive in 25 of totally 34 assessed cows. However, the melting profiles of the probes targeting the diagnostic PCR products differed from the T. foetus positive control. Subsequent sequencing of the amplicons revealed 91% identity to Simplicimonas sp. sequences deposited in GenBank™. Furthermore, there was no clear association between positive PCR result and presence of vaginitis. To investigate the distribution of this Simplicimonas-like organism in cows, more herds grazing on the same alpine pastures as well as unrelated cows were tested. In total, 133 cows and 16 heifers were sampled, 53 cows and 6 heifers even twice. Vaginitis was evident in 43 cows and 4 heifers. All-over-positivity of PCR was 44%, including nine tests performed on heifers. Melting peak analysis indicated Simplicimonas-like organisms in all positive samples. Culture attempts in bovine InPouch ™ TF failed. No association between a positive PCR result and the presence of vaginitis was found. This is, to the best of our knowledge, the first report on Simplicimonas-like DNA in vaginal swabs of female cattle. Our data suggest that when testing vaginal swabs of cattle by means of T. foetus PCR, false positive reactions due to Simplicimonas-like organisms may occur. Copyright © 2017 Elsevier B.V. All rights reserved.

Deep and persistent melt layer in the Archaean mantle

NASA Astrophysics Data System (ADS)

Andrault, Denis; Pesce, Giacomo; Manthilake, Geeth; Monteux, Julien; Bolfan-Casanova, Nathalie; Chantel, Julien; Novella, Davide; Guignot, Nicolas; King, Andrew; Itié, Jean-Paul; Hennet, Louis

2018-02-01

The transition from the Archaean to the Proterozoic eon ended a period of great instability at the Earth's surface. The origin of this transition could be a change in the dynamic regime of the Earth's interior. Here we use laboratory experiments to investigate the solidus of samples representative of the Archaean upper mantle. Our two complementary in situ measurements of the melting curve reveal a solidus that is 200-250 K lower than previously reported at depths higher than about 100 km. Such a lower solidus temperature makes partial melting today easier than previously thought, particularly in the presence of volatiles (H2O and CO2). A lower solidus could also account for the early high production of melts such as komatiites. For an Archaean mantle that was 200-300 K hotter than today, significant melting is expected at depths from 100-150 km to more than 400 km. Thus, a persistent layer of melt may have existed in the Archaean upper mantle. This shell of molten material may have progressively disappeared because of secular cooling of the mantle. Crystallization would have increased the upper mantle viscosity and could have enhanced mechanical coupling between the lithosphere and the asthenosphere. Such a change might explain the transition from surface dynamics dominated by a stagnant lid on the early Earth to modern-like plate tectonics with deep slab subduction.

Effects of Microsecond Pulse Laser Irradiation on Vis-NIR Reflectance Spectrum of Carbonaceous Chondrite Simulant: Implications for Martian Moons and Primitive Asteroids

NASA Technical Reports Server (NTRS)

Hiroi, T.; Moroz, L. V.; Shingareva, T. V.; Basilevsky, A. T.; Pieters, M.

2003-01-01

Goal of this study is to make a progress in understanding the optical effects of space weathering on small bodies believed to be similar in composition to carbonaceous chondrites: C, G, B, F, T, D, and P asteroids and possibly Martian satellites Phobos and Deimos. The companion work focuses on petrological and mineralogical aspects of this process. One of the main factors of space weathering is meteorite and micrometeorite bombardment leading, in particular, to impact melting of components of the regolith. Studies of lunar regolith and laboratory experiments simulating impact melting show that the melting products differ from the unmelted material in mineralogy and distribution of chemical components among different phases that results in spectral changes. We simulate impact melting of CM chondrite by pulse laser irradiation of an artificial analog of such a meteorite. The analog is a mixture of 46 wt.% non-magnetic fraction of L5 ordinary chondrite Tsarev, 47 wt.% serpentine, 5 wt.% kerite, and 2 wt.% calcite. It simulates rather well bulk chemistry, including volatiles such as H2O and CO2, and only approximately the CM chondrite mineralogy. Thus, we do not expect the mixture to be spectrally similar to CM chondrites, but expect the laser melting products to be similar to those formed by impact melting of natural CM chondrites.

MELT RHEOLOGY OF HIGH L-CONTENT POLY(LACTIC ACID). (R826733)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Lunar Cordierite-Spinel Troctolite: Igneous History, and Volatiles

NASA Astrophysics Data System (ADS)

Treiman, A. H.; Gross, J.

2012-03-01

Apollo sample 15295,101 contains a cordierite spinel troctolite (Marvin et al., 1989). The cordierite is volatile-free, at least by EMP — more precise analyses are in progress. The troctolite may be a partial melt of a spinel-rich igneous cumulate.

Should DNA sequence be incorporated with other taxonomical data for routine identifying of plant species?

PubMed

Suesatpanit, Tanakorn; Osathanunkul, Kitisak; Madesis, Panagiotis; Osathanunkul, Maslin

2017-08-31

A variety of plants in Acanthaceae have long been used in traditional Thai ailment and commercialised with significant economic value. Nowadays medicinal plants are sold in processed forms and thus morphological authentication is almost impossible. Full identification requires comparison of the specimen with some authoritative sources, such as a full and accurate description and verification of the species deposited in herbarium. Intake of wrong herbals can cause adverse effects. Identification of both raw materials and end products is therefore needed. Here, the potential of a DNA-based identification method, called Bar-HRM (DNA barcoding coupled with High Resolution Melting analysis), in raw material species identification is investigated. DNA barcode sequences from five regions (matK, rbcL, trnH-psbA spacer region, trnL and ITS2) of Acanthaceae species were retrieved for in silico analysis. Then the specific primer pairs were used in HRM assay to generate unique melting profiles for each plants species. The method allows identification of samples lacking necessary morphological parts. In silico analyses of all five selected regions suggested that ITS2 is the most suitable marker for Bar-HRM in this study. The HRM analysis on dried samples of 16 Acanthaceae medicinal species was then performed using primer pair derived from ITS2 region. 100% discrimination of the tested samples at both genus and species level was observed. However, two samples documented as Clinacanthus nutans and Clinacanthus siamensis were recognised as the same species from the HRM analysis. Further investigation reveals that C. siamensis is now accepted as C. nutans. The results here proved that Bar-HRM is a promising technique in species identification of the studied medicinal plants in Acanthaceae. In addition, molecular biological data is currently used in plant taxonomy and increasingly popular in recent years. Here, DNA barcode sequence data should be incorporated with morphological characters in the species identification.

Carbon nanostructures as immobilization platform for DNA: A review on current progress in electrochemical DNA sensors.

PubMed

Rasheed, P Abdul; Sandhyarani, N

2017-11-15

Development of a sensitive, specific and cost-effective DNA detection method is motivated by increasing demand for the early stage diagnosis of genetic diseases. Recent developments in the design and fabrication of efficient sensor platforms based on nanostructures make the highly sensitive sensors which could indicate very low detection limit to the level of few molecules, a realistic possibility. Electrochemical detection methods are widely used in DNA diagnostics as it provide simple, accurate and inexpensive platform for DNA detection. In addition, the electrochemical DNA sensors provide direct electronic signal without the use of expensive signal transduction equipment and facilitates the immobilization of single stranded DNA (ssDNA) probe sequences on a wide variety of electrode substrates. It has been found that a range of nanomaterials such as metal nanoparticles (MNPs), carbon based nanomaterials, quantum dots (QDs), magnetic nanoparticles and polymeric NPs have been introduced in the sensor design to enhance the sensing performance of electrochemical DNA sensor. In this review, we discuss recent progress in the design and fabrication of efficient electrochemical genosensors based on carbon nanostructures such as carbon nanotubes, graphene, graphene oxide and nanodiamonds. Copyright © 2017 Elsevier B.V. All rights reserved.

The effect of primary versus secondary processes on the volatile content of MORB glasses: An example from the equatorial Mid-Atlantic Ridge (5°N-3°S)

NASA Astrophysics Data System (ADS)

Le Voyer, Marion; Cottrell, Elizabeth; Kelley, Katherine A.; Brounce, Maryjo; Hauri, Erik H.

2015-01-01

We report microanalysis of volatile and trace element compositions, as well as Fe3+/ΣFe ratios, from 45 basaltic glasses from cruise RC2806 along the equatorial Mid-Atlantic Ridge. The along-strike variations in volatiles result from the complex geodynamical setting of the area, including numerous transform faults, variations in ridge depth, melting degree, and source composition. The strongest gradient is centered on 1.7°N and encompasses an increase of H2O, Cl, and F contents as well as high F/Zr ratio spatially coincident with radiogenic isotope anomalies. We interpret these variations as source enrichment due to the influence of the nearby high-μ-type Sierra Leone plume. South of the St. Paul fracture zone, H2O and F contents, as well as H2O/Ce and F/Zr ratios, decrease progressively. This gradient in volatiles is consistent with progressive dilution of an enriched component in a heterogeneous mantle due to the progressive increase in the degree of melting. These two large-scale gradients are interrupted by small-scale anomalies in volatile contents attributed to (1) low-degree melts preferentially sampling enriched heterogeneities near transform faults and (2) local assimilation of hydrothermal fluids in four samples from dredge 16D. Finally, 20 RC2806 samples described as "popping rocks" during collection do not show any difference in volatile content dissolved in the glass or in vesicularity when compared to the RC2806 "nonpopping" samples. Our observations lead us to question the interpretation of the CO2 content in the highly vesicular 2πD43 "popping rock" as being representative of the CO2 content of undegassed mid-ocean ridge basalt.

The System Forsterite-Diopside-Enstatite up to 70 kbar and its Significance to the Genesis of Komatiites

NASA Astrophysics Data System (ADS)

Dasgupta, S.; Gupta, A. K.

2011-12-01

Liquidus phase relations in the system forsterite-diopside-enstatite has been made at 70 kbar under anhydrous conditions using a Walker-type multi-anvil high pressure apparatus. Positions of the pseudoeutectic/ invariant, minimum points and amount of solid solutions of appearing phases are summarized in table 1. Comparison of these phase relations with those conducted by previous investigators at lower pressures and temperatures shows that the fosterite-pyroxene liquidus boundary shifts toward forsterite and away from the diopside apex with increasing pressure. Microprobe analyses indicate that the maximum amount of MgSiO3 that can be incorporated in diopside increases with pressure, and at the solidus (70 kbar, 2010°C), it is about 82%. On the basis of EPMA analyses of coexisting liquid and crystalline phases, three-phase triangles have been constructed. It is observed that at 70 kbar, the early partial melt generated from a model peridotite does not precipitate orthopyroxene. If such a melt instead of crystallizing in-situ, ascend to the surface, then the polybaric-polythermal crystallization path should never intersect the liquidus phase field of orthopyroxene, enstatitess may then appear in the solidus as an exsolution product. Our calculation shows that at 31% partial melting of a model mantle, orthopyroxene should appear as a liquidus phase. With further increase in the degree of partial melting (42-60%), proportion of orthopyroxene crystallizing from the melt progressively increases. With reference to the above discussion we propose that the Gorgona komatiites which are primarily orthopyroxene-deficient komatiites, are an outcome of low degree of partial melting, whereas the orthopyroxene-bearing Commondale komatiites of the southern Kaapvaal Craton, South Africa, are the outcome of a larger degree of partial melting, both generated from melting of an anhydrous mantle.

A simple model for the evolution of melt pond coverage on permeable Arctic sea ice

NASA Astrophysics Data System (ADS)

Popović, Predrag; Abbot, Dorian

2017-05-01

As the melt season progresses, sea ice in the Arctic often becomes permeable enough to allow for nearly complete drainage of meltwater that has collected on the ice surface. Melt ponds that remain after drainage are hydraulically connected to the ocean and correspond to regions of sea ice whose surface is below sea level. We present a simple model for the evolution of melt pond coverage on such permeable sea ice floes in which we allow for spatially varying ice melt rates and assume the whole floe is in hydrostatic balance. The model is represented by two simple ordinary differential equations, where the rate of change of pond coverage depends on the pond coverage. All the physical parameters of the system are summarized by four strengths that control the relative importance of the terms in the equations. The model both fits observations and allows us to understand the behavior of melt ponds in a way that is often not possible with more complex models. Examples of insights we can gain from the model are that (1) the pond growth rate is more sensitive to changes in bare sea ice albedo than changes in pond albedo, (2) ponds grow slower on smoother ice, and (3) ponds respond strongest to freeboard sinking on first-year ice and sidewall melting on multiyear ice. We also show that under a global warming scenario, pond coverage would increase, decreasing the overall ice albedo and leading to ice thinning that is likely comparable to thinning due to direct forcing. Since melt pond coverage is one of the key parameters controlling the albedo of sea ice, understanding the mechanisms that control the distribution of pond coverage will help improve large-scale model parameterizations and sea ice forecasts in a warming climate.

Differential-Integral method in polymer processing: Taking melt electrospinning technique for example

NASA Astrophysics Data System (ADS)

Haoyi, Li; Weimin, Yang; Hongbo, Chen; Jing, Tan; Pengcheng, Xie

2016-03-01

A concept of Differential-Integral (DI) method applied in polymer processing and molding was proposed, which included melt DI injection molding, DI nano-composites extrusion molding and melt differential electrospinning principle and equipment. Taking the melt differential electrospinning for example to introduce the innovation research progress, two methods preparing polymer ultrafine fiber have been developed: solution electro-spinning and melt electro-spinning, between which solution electro-spinning is much simpler to realize in lab. More than 100 institutions have endeavored to conduct research on it and more than 30 thousand papers have been published. However, its industrialization was restricted to some extend because of the existence of toxic solvent during spinning process and poor mechanical strength of resultant fibers caused by small pores on fiber surface. Solvent-free melt electrospinning is environmentally friendly and highly productive. However, problems such as the high melt viscosity, thick fiber diameter and complex equipment makes it relatively under researched compared with solution electrospinning. With the purpose of solving the shortage of traditional electro-spinning equipment with needles or capillaries, a melt differential electro-spinning method without needles or capillaries was firstly proposed. Nearly 50 related patents have been applied since 2005, and systematic method innovations and experimental studies have also been conducted. The prepared fiber by this method had exhibited small diameter and smooth surface. The average fiber diameter can reach 200-800 nm, and the single nozzle can yield two orders of magnitude more than the capillaries. Based on the above principle, complete commercial techniques and equipment have been developed to produce ultra-fine non-woven fabrics for the applications in air filtration, oil spill recovery and water treatment, etc.

KIT mutations in Russian patients with mucosal melanoma.

PubMed

Abysheva, Svetlana N; Iyevleva, Aglaya G; Efimova, Nina V; Mokhina, Yulia B; Sabirova, Feruza A; Ivantsov, Alexandr O; Artemieva, Anna S; Togo, Alexandr V; Moiseyenko, Vladimir M; Matsko, Dmitry E; Imyanitov, Evgeny N

2011-12-01

A single institution series of 48 mucosal melanomas (MMs) has been analyzed for the presence of KIT mutations using high-resolution melting and sequencing of abnormally melted DNA fragments. The analysis of exons 9, 11, 13, and 17 has revealed eight of 48 (17%) nonsynonymous alterations, including zero of seven head and neck, six of 24 anorectal, one of 15 genitourinary, one of one gastric, and zero of one mediastinal MMs. Seven of these mutations were potentially associated with the tumor sensitivity to KIT tyrosine kinase inhibitors. One tumor harbored somatically acquired silent nucleotide substitution c.1383A>G (T461T). This study adds to the evidence that a substantial portion of MMs carry a therapeutically relevant mutation in the KIT oncogene.

Identification of Brucella spp. isolated from human brucellosis in Malaysia using high-resolution melt (HRM) analysis.

PubMed

Mohamed Zahidi, Jama'ayah; Bee Yong, Tay; Hashim, Rohaidah; Mohd Noor, Azura; Hamzah, Siti Hawa; Ahmad, Norazah

2015-04-01

Molecular approaches have been investigated to overcome difficulties in identification and differentiation of Brucella spp. using conventional phenotypic methods. In this study, high-resolution melt (HRM) analysis was used for rapid identification and differentiation of members of Brucella genus. A total of 41 Brucella spp. isolates from human brucellosis were subjected to HRM analysis using 4 sets of primers, which identified 40 isolates as Brucella melitensis and 1 as Brucella canis. The technique utilized low DNA concentration and was highly reproducible. The assay is shown to be a useful diagnostic tool, which can rapidly differentiate Brucella up to species level. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Base-Position Error Rate Analysis of Next-Generation Sequencing Applied to Circulating Tumor DNA in Non-Small Cell Lung Cancer: A Prospective Study

PubMed Central

Zonta, Eleonora; Didelot, Audrey; Combe, Pierre; Thibault, Constance; Gibault, Laure; Lours, Camille; Taly, Valérie; Laurent-Puig, Pierre

2016-01-01

Background Circulating tumor DNA (ctDNA) is an approved noninvasive biomarker to test for the presence of EGFR mutations at diagnosis or recurrence of lung cancer. However, studies evaluating ctDNA as a noninvasive “real-time” biomarker to provide prognostic and predictive information in treatment monitoring have given inconsistent results, mainly due to methodological differences. We have recently validated a next-generation sequencing (NGS) approach to detect ctDNA. Using this new approach, we evaluated the clinical usefulness of ctDNA monitoring in a prospective observational series of patients with non-small cell lung cancer (NSCLC). Methods and Findings We recruited 124 patients with newly diagnosed advanced NSCLC for ctDNA monitoring. The primary objective was to analyze the prognostic value of baseline ctDNA on overall survival. ctDNA was assessed by ultra-deep targeted NGS using our dedicated variant caller algorithm. Common mutations were validated by digital PCR. Out of the 109 patients with at least one follow-up marker mutation, plasma samples were contributive at baseline (n = 105), at first evaluation (n = 85), and at tumor progression (n = 66). We found that the presence of ctDNA at baseline was an independent marker of poor prognosis, with a median overall survival of 13.6 versus 21.5 mo (adjusted hazard ratio [HR] 1.82, 95% CI 1.01–3.55, p = 0.045) and a median progression-free survival of 4.9 versus 10.4 mo (adjusted HR 2.14, 95% CI 1.30–3.67, p = 0.002). It was also related to the presence of bone and liver metastasis. At first evaluation (E1) after treatment initiation, residual ctDNA was an early predictor of treatment benefit as judged by best radiological response and progression-free survival. Finally, negative ctDNA at E1 was associated with overall survival independently of Response Evaluation Criteria in Solid Tumors (RECIST) (HR 3.27, 95% CI 1.66–6.40, p < 0.001). Study population heterogeneity, over-representation of EGFR-mutated patients, and heterogeneous treatment types might limit the conclusions of this study, which require future validation in independent populations. Conclusions In this study of patients with newly diagnosed NSCLC, we found that ctDNA detection using targeted NGS was associated with poor prognosis. The heterogeneity of lung cancer molecular alterations, particularly at time of progression, impairs the ability of individual gene testing to accurately detect ctDNA in unselected patients. Further investigations are needed to evaluate the clinical impact of earlier evaluation times at 1 or 2 wk. Supporting clinical decisions, such as early treatment switching based on ctDNA positivity at first evaluation, will require dedicated interventional studies. PMID:28027313

Accelerated age-related cognitive decline and neurodegeneration, caused by deficient DNA repair.

PubMed

Borgesius, Nils Z; de Waard, Monique C; van der Pluijm, Ingrid; Omrani, Azar; Zondag, Gerben C M; van der Horst, Gijsbertus T J; Melton, David W; Hoeijmakers, Jan H J; Jaarsma, Dick; Elgersma, Ype

2011-08-31

Age-related cognitive decline and neurodegenerative diseases are a growing challenge for our societies with their aging populations. Accumulation of DNA damage has been proposed to contribute to these impairments, but direct proof that DNA damage results in impaired neuronal plasticity and memory is lacking. Here we take advantage of Ercc1(Δ/-) mutant mice, which are impaired in DNA nucleotide excision repair, interstrand crosslink repair, and double-strand break repair. We show that these mice exhibit an age-dependent decrease in neuronal plasticity and progressive neuronal pathology, suggestive of neurodegenerative processes. A similar phenotype is observed in mice where the mutation is restricted to excitatory forebrain neurons. Moreover, these neuron-specific mutants develop a learning impairment. Together, these results suggest a causal relationship between unrepaired, accumulating DNA damage, and age-dependent cognitive decline and neurodegeneration. Hence, accumulated DNA damage could therefore be an important factor in the onset and progression of age-related cognitive decline and neurodegenerative diseases.

Chronic inflammation-related DNA damage response: a driving force of gastric cardia carcinogenesis

PubMed Central

Guo, Yi; Tian, Dongping; Yun, Hailong; Chen, Donglin; Su, Min

2015-01-01

Gastric cardia cancer (GCC) is a highly aggressive disease associated with chronic inflammation. To investigate the relationship between DNA damage response (DDR) and chronic inflammation, we collected 100 non-tumor gastric cardia specimens of Chaoshan littoral, a high-risk region for esophageal and gastric cardia cancer. A significantly higher proportion of severe chronic inflammation was found in dysplastic epithelia (80.9%) in comparison with that in non-dysplastic tissues (40.7%) (P<0.001). Immunohistochemical analysis demonstrated that DNA damage response was parallel with the chronic inflammation degrees from normal to severe inflammation (P<0.05). We found that DNA damage response was progressively increased with the progression of precancerous lesions (P<0.05). These findings provide pathological evidence that persistent chronic inflammation-related DNA damage response may be a driving force of gastric cardia carcinogenesis. Based on these findings, DNA damage response in non-malignant tissues may become a promising biomedical marker for predicting malignant transformation in the gastric cardia. PMID:25650663

Chronic inflammation-related DNA damage response: a driving force of gastric cardia carcinogenesis.

PubMed

Lin, Runhua; Xiao, Dejun; Guo, Yi; Tian, Dongping; Yun, Hailong; Chen, Donglin; Su, Min

2015-02-20

Gastric cardia cancer (GCC) is a highly aggressive disease associated with chronic inflammation. To investigate the relationship between DNA damage response (DDR) and chronic inflammation, we collected 100 non-tumor gastric cardia specimens of Chaoshan littoral, a high-risk region for esophageal and gastric cardia cancer. A significantly higher proportion of severe chronic inflammation was found in dysplastic epithelia (80.9%) in comparison with that in non-dysplastic tissues (40.7%) (P<0.001). Immunohistochemical analysis demonstrated that DNA damage response was parallel with the chronic inflammation degrees from normal to severe inflammation (P<0.05). We found that DNA damage response was progressively increased with the progression of precancerous lesions (P<0.05). These findings provide pathological evidence that persistent chronic inflammation-related DNA damage response may be a driving force of gastric cardia carcinogenesis. Based on these findings, DNA damage response in non-malignant tissues may become a promising biomedical marker for predicting malignant transformation in the gastric cardia.

Early In Vitro Differentiation of Mouse Definitive Endoderm Is Not Correlated with Progressive Maturation of Nuclear DNA Methylation Patterns

PubMed Central

Tajbakhsh, Jian; Gertych, Arkadiusz; Fagg, W. Samuel; Hatada, Seigo; Fair, Jeffrey H.

2011-01-01

The genome organization in pluripotent cells undergoing the first steps of differentiation is highly relevant to the reprogramming process in differentiation. Considering this fact, chromatin texture patterns that identify cells at the very early stage of lineage commitment could serve as valuable tools in the selection of optimal cell phenotypes for regenerative medicine applications. Here we report on the first-time use of high-resolution three-dimensional fluorescence imaging and comprehensive topological cell-by-cell analyses with a novel image-cytometrical approach towards the identification of in situ global nuclear DNA methylation patterns in early endodermal differentiation of mouse ES cells (up to day 6), and the correlations of these patterns with a set of putative markers for pluripotency and endodermal commitment, and the epithelial and mesenchymal character of cells. Utilizing this in vitro cell system as a model for assessing the relationship between differentiation and nuclear DNA methylation patterns, we found that differentiating cell populations display an increasing number of cells with a gain in DNA methylation load: first within their euchromatin, then extending into heterochromatic areas of the nucleus, which also results in significant changes of methylcytosine/global DNA codistribution patterns. We were also able to co-visualize and quantify the concomitant stochastic marker expression on a per-cell basis, for which we did not measure any correlation to methylcytosine loads or distribution patterns. We observe that the progression of global DNA methylation is not correlated with the standard transcription factors associated with endodermal development. Further studies are needed to determine whether the progression of global methylation could represent a useful signature of cellular differentiation. This concept of tracking epigenetic progression may prove useful in the selection of cell phenotypes for future regenerative medicine applications. PMID:21779341

Retrospective natural history of thymidine kinase 2 deficiency.

PubMed

Garone, Caterina; Taylor, Robert W; Nascimento, Andrés; Poulton, Joanna; Fratter, Carl; Domínguez-González, Cristina; Evans, Julie C; Loos, Mariana; Isohanni, Pirjo; Suomalainen, Anu; Ram, Dipak; Hughes, M Imelda; McFarland, Robert; Barca, Emanuele; Lopez Gomez, Carlos; Jayawant, Sandeep; Thomas, Neil D; Manzur, Adnan Y; Kleinsteuber, Karin; Martin, Miguel A; Kerr, Timothy; Gorman, Grainne S; Sommerville, Ewen W; Chinnery, Patrick F; Hofer, Monika; Karch, Christoph; Ralph, Jeffrey; Cámara, Yolanda; Madruga-Garrido, Marcos; Domínguez-Carral, Jana; Ortez, Carlos; Emperador, Sonia; Montoya, Julio; Chakrapani, Anupam; Kriger, Joshua F; Schoenaker, Robert; Levin, Bruce; Thompson, John L P; Long, Yuelin; Rahman, Shamima; Donati, Maria Alice; DiMauro, Salvatore; Hirano, Michio

2018-03-30

Thymine kinase 2 (TK2) is a mitochondrial matrix protein encoded in nuclear DNA and phosphorylates the pyrimidine nucleosides: thymidine and deoxycytidine. Autosomal recessive TK2 mutations cause a spectrum of disease from infantile onset to adult onset manifesting primarily as myopathy. To perform a retrospective natural history study of a large cohort of patients with TK2 deficiency. The study was conducted by 42 investigators across 31 academic medical centres. We identified 92 patients with genetically confirmed diagnoses of TK2 deficiency: 67 from literature review and 25 unreported cases. Based on clinical and molecular genetics findings, we recognised three phenotypes with divergent survival: (1) infantile-onset myopathy (42.4%) with severe mitochondrial DNA (mtDNA) depletion, frequent neurological involvement and rapid progression to early mortality (median post-onset survival (POS) 1.00, CI 0.58 to 2.33 years); (2) childhood-onset myopathy (40.2%) with mtDNA depletion, moderate-to-severe progression of generalised weakness and median POS at least 13 years; and (3) late-onset myopathy (17.4%) with mild limb weakness at onset and slow progression to respiratory insufficiency with median POS of 23 years. Ophthalmoparesis and facial weakness are frequent in adults. Muscle biopsies show multiple mtDNA deletions often with mtDNA depletion. In TK2 deficiency, age at onset, rate of weakness progression and POS are important variables that define three clinical subtypes. Nervous system involvement often complicates the clinical course of the infantile-onset form while extraocular muscle and facial involvement are characteristic of the late-onset form. Our observations provide essential information for planning future clinical trials in this disorder. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

3D DNA Crystals and Nanotechnology

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

Paukstelis, Paul; Seeman, Nadrian

DNA's molecular recognition properties have made it one of the most widely used biomacromolecular construction materials. The programmed assembly of DNA oligonucleotides has been used to create complex 2D and 3D self-assembled architectures and to guide the assembly of other molecules. The origins of DNA nanotechnology are rooted in the goal of assembling DNA molecules into designed periodic arrays, i.e., crystals. Here, we highlight several DNA crystal structures, the progress made in designing DNA crystals, and look at the current prospects and future directions of DNA crystals in nanotechnology.

3D DNA Crystals and Nanotechnology

DOE PAGES

Paukstelis, Paul; Seeman, Nadrian

2016-08-18

DNA's molecular recognition properties have made it one of the most widely used biomacromolecular construction materials. The programmed assembly of DNA oligonucleotides has been used to create complex 2D and 3D self-assembled architectures and to guide the assembly of other molecules. The origins of DNA nanotechnology are rooted in the goal of assembling DNA molecules into designed periodic arrays, i.e., crystals. Here, we highlight several DNA crystal structures, the progress made in designing DNA crystals, and look at the current prospects and future directions of DNA crystals in nanotechnology.

Mechanisms for radiation damage in DNA. Progress report, January 1, 1980-December 31, 1980

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

Sevilla, M D

1980-09-01

In this project several mechanisms are proposed for radiation damage to DNA constituents and DNA, and a series of experiments detailed utilizing electron spin resonance spectrometry to test the proposed mechanisms. Under current investigation are irradiated systems of DNA constituents which may shed light on indirect effects. In addition, studies of radiation effects on lipids have been undertaken which will shed light on the only other proposed site for cell kill, the membrane. Studies completed during the past year are: (1) ..pi.. cations produced in DNA bases by attack of oxidizing radicals; (2) INDO studies of radicals produced in peptidesmore » and carboxylic acid model compounds; (3) electron reactions with carboxylic acids, ketones and aldehydes; and (4) ..gamma..-irradiation of esters and triglycerides. Progress has been made this year in a study of radicals generated in model compounds for the sugar-phosphate backbone.« less

Atomic force microscopy on chromosomes, chromatin and DNA: a review.

PubMed

Kalle, Wouter; Strappe, Padraig

2012-12-01

The purpose of this review is to discuss the achievements and progress that has been made in the use of atomic force microscopy in DNA related research in the last 25 years. For this review DNA related research is split up in chromosomal-, chromatin- and DNA focused research to achieve a logical flow from large- to smaller structures. The focus of this review is not only on the AFM as imaging tool but also on the AFM as measuring tool using force spectroscopy, as therein lays its greatest advantage and future. The amazing technological and experimental progress that has been made during the last 25 years is too extensive to fully cover in this review but some key developments and experiments have been described to give an overview of the evolution of AFM use from 'imaging tool' to 'measurement tool' on chromosomes, chromatin and DNA. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

Drug delivery systems based on nucleic acid nanostructures.

PubMed

de Vries, Jan Willem; Zhang, Feng; Herrmann, Andreas

2013-12-10

The field of DNA nanotechnology has progressed rapidly in recent years and hence a large variety of 1D-, 2D- and 3D DNA nanostructures with various sizes, geometries and shapes is readily accessible. DNA-based nanoobjects are fabricated by straight forward design and self-assembly processes allowing the exact positioning of functional moieties and the integration of other materials. At the same time some of these nanosystems are characterized by a low toxicity profile. As a consequence, the use of these architectures in a biomedical context has been explored. In this review the progress and possibilities of pristine nucleic acid nanostructures and DNA hybrid materials for drug delivery will be discussed. For the latter class of structures, a distinction is made between carriers with an inorganic core composed of gold or silica and amphiphilic DNA block copolymers that exhibit a soft hydrophobic interior. Copyright © 2013 Elsevier B.V. All rights reserved.

Prediction of the efficacy of immunotherapy by measuring the integrity of cell-free DNA in plasma in colorectal cancer.

PubMed

Kitahara, Masahiro; Hazama, Shoichi; Tsunedomi, Ryouichi; Takenouchi, Hiroko; Kanekiyo, Shinsuke; Inoue, Yuka; Nakajima, Masao; Tomochika, Shinobu; Tokuhisa, Yoshihiro; Iida, Michihisa; Sakamoto, Kazuhiko; Suzuki, Nobuaki; Takeda, Shigeru; Ueno, Tomio; Yamamoto, Shigeru; Yoshino, Shigefumi; Nagano, Hiroaki

2016-12-01

We previously reported a phase II study of a cancer vaccine using five novel peptides recognized by HLA-A*2402-restricted CTL in combination with oxaliplatin-containing chemotherapy (FXV study) as first-line therapy for patients with metastatic colorectal cancer and demonstrated the safety and promising potential of our five-peptide cocktail. The objective of this analysis was to identify predictive biomarkers for identifying patients who are likely to receive a clinical benefit from immunochemotherapy. Circulating cell-free DNA (cfDNA) in plasma has been reported to be a candidate molecular biomarker for the efficacy of anticancer therapy. Unlike uniformly truncated small-sized DNA released from apoptotic normal cells, DNA released from necrotic cancer cells varies in size. The integrity of plasma cfDNA (i.e. the ratio of longer fragments [400 bp] to shorter fragments [100 bp] of cfDNA), may be clinically useful for detecting colorectal cancer progression. We assessed plasma samples collected from 93 patients prior to receiving immunochemotherapy. The cfDNA levels and integrity were analyzed by semi-quantitative real-time PCR. Progression-free survival was significantly better in patients with a low plasma cfDNA integrity value than in those with a high value (P = 0.0027). Surprisingly, in the HLA-A*2402-matched group, patients with a low plasma cfDNA integrity value had significantly better progression-free survival than those with a high value (P = 0.0015). This difference was not observed in the HLA-A*2402-unmatched group. In conclusion, the integrity of plasma cfDNA may provide important clinical information and may be a useful predictive biomarker of the outcome of immunotherapy in metastatic colorectal cancer. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Mechanical anisotropy control on strain localization in upper mantle shear zones

NASA Astrophysics Data System (ADS)

Herwegh, Marco; Mercolli, Ivan; Linckens, Jolien; Müntener, Othmar

2016-05-01

Mantle rocks at oceanic spreading centers reveal dramatic rheological changes from partially molten to solid-state ductile to brittle deformation with progressive cooling. Using the crustal-scale Wadi al Wasit mantle shear zone (SZ, Semail ophiolite, Oman), we monitor such changes based on quantitative field and microstructural investigations combined with petrological and geochemical analyses. The spatial distribution of magmatic dikes and high strain zones gives important information on the location of magmatic and tectonic activity. In the SZ, dikes derived from primitive melts (websterites) are distributed over the entire SZ but are more abundant in the center; dikes from more evolved, plagioclase saturated melts (gabbronorites) are restricted to the SZ center. Accordingly, harzburgite deformation fabrics show a transition from protomylonite (1100°C), mylonite (900-800°C) to ultramylonite (<700°C) and a serpentine foliation (<500°C) from the SZ rim to the center. The spatial correlation between solid-state deformation fabrics and magmatic features indicates progressive strain localization in the SZ on the cooling path. Three stages can be discriminated: (i) Cycles of melt injection (dunite channels and websterite dikes) and solid-state deformation (protomylonites-mylonites; 1100-900°C), (ii) dominant solid-state deformation in harzburgite mylonites (900-800°C) with some last melt injections (gabbronorites) and ultramylonites (<700°C), and (iii) infiltration of seawater inducing a serpentine foliation (<500°C) followed by cataclasis during obduction. The change of these processes in space and time indicates that early dike-related ridge-parallel deformation controls the onset of the entire strain localization history promoting nucleation sites for different strain weakening processes as a consequence of changing physicochemical conditions.

Reactive Transport of Slab-Derived Carbonatitic Melts in the Deep Upper Mantle and Generation of Kimberlites

NASA Astrophysics Data System (ADS)

Sun, C.; Dasgupta, R.

2017-12-01

Kimberlite is a diamond-bearing CO2-rich ultramafic magma from the mantle at depths of >200 km, featured by enrichment of incompatible elements [1]. It has been considered significant for understanding mantle geochemistry and particularly for providing information of deep carbon cycle. Recent experimental studies suggested that partial melts of carbonated peridotites at high pressures and temperatures could resemble the MgO (>20 wt%) and enriched incompatible elements in kimberlites only when the source experienced refertilization with perhaps prior depletion (e.g., [2]). Although addition of CO2 and incompatible elements in the deep mantle is often linked to subducted components, partial melts directly from carbonated oceanic crusts do not have high enough MgO (e.g., ≤8.2 wt%; [3]). A crucial question is how slab-derived CO2-rich melt evolves in reaction with ambient mantle, which may provide a feasible mechanism for kimberlite generation. To investigate the fate of slab-derived carbonatitic melt in the deep ambient mantle, we have performed multi-anvil experiments at 7-10 GPa and 1400-1450 °C. The starting compositions were synthesized by mixing a fertile peridotite composition, KLB-1, with variable proportions (0-45 wt.%) of Ca-rich carbonatitic melt similar to those derived from a carbonated ocean crust at 13-21 GPa [3]. Experiments were performed in Pt, Pt/Gr, Au-Pd and Au-Pd/Gr capsules, and the experimental phases include olivine ± opx + cpx + majoritic garnet ± carbonated silicate melt. With the increase of melt-rock ratios, experimental melts become progressively enriched in CaO (13.0-23.1 wt%) and CO2 (14.2-38.7 wt%) but depleted in MgO (28.9-19.9 wt%), SiO2 (33.1-7.9 wt%), and Al2O3 (2.7-0.2 wt%). The net flux of melt increases with the increase of infiltrating carbonatitic melt proportion and with the decrease of pressure. Kimberlite melts were produced from experiments with 5-25 wt% infiltrating carbonatitic melts by dissolution of olivine and orthopyroxene and precipitation of clinopyroxene. Thus, a localized influx of slab-derived CO2-rich melts can enlarge the mantle porosity, enhance melt focusing, and initiate a channelized flow of kimberlite melts. [1] Becker & Le Roex (2006) J. Pet. 47: 673-703; [2] Brey et al. (2008) J. Pet. 49: 797-821; [3] Thomson et al. (2016) Nature 529: 76-79.

SUPRAMOLECULAR MORPHOLOGY OF TWO-STEP MELT-SPUN POLY(LACTIC ACID) FIBERS. (R826733)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

MELT RHEOLOGY OF POLY(LACTIC ACID): CONSEQUENCES OF BLENDING CHAIN ARCHITECTURES. (R826733)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

MELT RHEOLOGY OF POLY(LACTIC ACID): ENTANGLEMENT AND CHAIN ARCHITECTURE EFFECTS. (R826733)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Complementary rare earth element patterns in unique achondrites, such as ALHA 77005 and shergottites, and in the earth

NASA Technical Reports Server (NTRS)

Ma, M.-S.; Schmitt, R. A.; Laul, J. C.

1982-01-01

Abundances of major, minor, and trace elements are determined in the Antarctic achondrite Allan Hills (ALHA) 77005 via sequential instrumental and radiochemical neutron activation analysis. The rare earth element (REE) abundances of ALHA 77005 reveal a unique chondritic normalized pattern; that is, the REEs are nearly unfractionated from La to Pr at approximately 1.0X chondrites, monotonically increased from Pr to Gd at approximately 3.4X with no Eu anomaly, nearly unfractionated from Gd and Ho and monotonically decreased from Ho to Lu at approximately 2.2X. It is noted that this unique REE pattern of ALHA 77005 can be modeled by a melting process involving a continuous melting and progressive partial removal of melt from a light REE enriched source material. In a model of this type, ALHA 77005 could represent either a crystallized cumulate from such a melt or the residual source material. Calculations show that the parent liquids for the shergottites could also be derived from a light REE enriched source material similar to that for ALHA 77005.

Conjunctival flora of healthy and diseased eyes of grey seals (Halichoerus grypus): implications for treatment.

PubMed

Fleming, M; Bexton, S

2016-07-23

Ocular pathology is relatively common in stranded seals admitted to wildlife rehabilitation hospitals. Some have pre-existing problems, while others develop eye problems in captivity, and in particular ulcerative keratitis, due to factors such as large prominent eyes, suboptimal water quality, trauma and infighting. Despite treatment, corneal ulcerations can rapidly progress to 'melting' ulcers with subsequent rupture of the globe. In this case series, 32 grey seals (Halichoerus grypus) had conjunctival swabs taken on admission to a UK wildlife hospital to identify ocular bacterial flora and nine had subsequent swabs taken after four weeks to see if this changed in captivity. Additionally, nine seals with ocular pathology were also swabbed. Although a wide range of bacteria were cultured on admission, the most common isolates were Gemella haemolysans, Escherichia coli and Clostridium perfringens All 'melting' ulcers were associated with Pseudomonas aeruginosa, which suggests this bacterial species may be significant in the pathogenesis of progressive stromal ulceration in grey seals. British Veterinary Association.

[RESEARCH PROGRESS OF THREE-DIMENSIONAL PRINTING TECHNIQUE FOR SPINAL IMPLANTS].

PubMed

Lu, Qi; Yu, Binsheng

2016-09-08

To summarize the current research progress of three-dimensional (3D) printing technique for spinal implants manufacture. The recent original literature concerning technology, materials, process, clinical applications, and development direction of 3D printing technique in spinal implants was reviewed and analyzed. At present, 3D printing technologies used to manufacture spinal implants include selective laser sintering, selective laser melting, and electron beam melting. Titanium and its alloys are mainly used. 3D printing spinal implants manufactured by the above materials and technology have been successfully used in clinical. But the problems regarding safety, related complications, cost-benefit analysis, efficacy compared with traditional spinal implants, and the lack of relevant policies and regulations remain to be solved. 3D printing technique is able to provide individual and customized spinal implants for patients, which is helpful for the clinicians to perform operations much more accurately and safely. With the rapid development of 3D printing technology and new materials, more and more 3D printing spinal implants will be developed and used clinically.

(Boiling water reactor (BWR) CORA experiments)

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

Ott, L.J.

To participate in the 1990 CORA Workshop at Kernforschungszentrum Karlsruhe (KfK) GmbH, Karlsruhe, FRG, on October 1--4, and to participate in detailed discussions on October 5 with the KfK CORA Boiling Water Reactor (BWR) experiments. The traveler attended the 1990 CORA Workshop at KfK, FRG. Participation included the presentation of a paper on work performed by the Boiling Water Reactor Core Melt Progression Phenomena Program at Oak Ridge National Laboratory (ORNL) on posttest analyses of CORA BWR experiments. The Statement of Work (November 1989) for the BWR Core Melt Progression Phenomena Program provides for pretest and posttest analyses of themore » BWR CORA experiments performed at KfK. Additionally, it is intended that ORNL personnel participate in the planning process for future CORA BWR experiments. For these purposes, meetings were held with KfK staff to discuss such topics as (1) experimental test schedule, (2) BWR test conduct, (3) perceived BWR experimental needs, and (4) KfK operational staff needs with respect to ORNL support. 19 refs.« less

Prospects and progress of high Tc superconductivity for space applications

NASA Technical Reports Server (NTRS)

Romanofsky, Robert R.; Sokoloski, Marty M.

1991-01-01

Current research in the area of high temperature superconductivity is organized around four key areas: communications and data, sensors and cryogenics, propulsion and power, and space materials technology. Recently, laser ablated YBa2Cu3O(7-x) films on LaAlO3 produced far superior RF characteristics when compared to metallic films on the same substrate. The achievement has enabled a number of unique microwave device applications, such as low insertion loss phase shifters and high-Q filters. Melt texturing and melt-quenched techniques are being used to produce bulk material with optimized magnetic properties. These yttrium-enriched materials possess enhanced flux pinning characteristics and could lead to prototype cryocooler bearings. Significant progress has also occurred in bolometer and current lead technology. Studies were conducted to evaluate the effect of high temperature superconducting materials on the performance and life of high power magnetoplasma-dynamic thrusters. Extended studies were also performed to evaluate the benefit of superconducting magnetic energy storage for LEO space station, lunar, and Mars mission applications.

Spectroscopic studies of the interaction between pirimicarb and calf thymus DNA.

PubMed

Zhang, Guowen; Hu, Xing; Pan, Junhui

2011-02-01

The interaction between pirimicarb and calf thymus DNA in physiological buffer (pH 7.4) was investigated with the use of Neutral Red (NR) dye as a spectral probe by UV-vis absorption, fluorescence and circular dichroism (CD) spectroscopy, as well as viscosity measurements and DNA melting techniques. The results revealed that an intercalation binding should be the interaction mode of pirimicarb to DNA. CD spectra indicated that pirimicarb induced conformational changes of DNA. The binding constants of pirimicarb with DNA were obtained by the fluorescence quenching method. The thermodynamic parameters, enthalpy change (ΔHθ) and entropy change (ΔSθ) were calculated to be -52.13±2.04 kJ mol(-1) and -108.8±6.72 J mol(-1) K(-1) according to the van't Hoff equation, which suggested that hydrogen bonds and van der Waals forces might play a major role in the binding of pirimicarb to DNA. Further, the alternative least squares (ALS) method was applied to resolve a complex two-way array of the absorption spectra data, which provided simultaneously the concentration information for the three reaction components, pirimicarb, NR and DNA-NR. This ALS analysis indicated that the intercalation of pirimicarb into the DNA by substituting for NR in the DNA-NR complex. Copyright © 2010 Elsevier B.V. All rights reserved.

Promoter Melting Plays Critical Role in Lymphocyte Activation | Center for Cancer Research

Cancer.gov

Transcription in eukaryotic cells is a precisely timed ballet that consists of RNA polymerase II (pol II) recruitment to gene promoters, assembly of the multiprotein preinitiation complex, opening of the DNA, escape of pol II from the promoter, pol II pausing downstream, mRNA elongation, and, eventually, termination. The two main points of regulation are thought to be

Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini using real-time PCR and high resolution melting analysis.

PubMed

Cai, Xian-Quan; Yu, Hai-Qiong; Li, Rong; Yue, Qiao-Yun; Liu, Guo-Hua; Bai, Jian-Shan; Deng, Yan; Qiu, De-Yi; Zhu, Xing-Quan

2014-01-01

Clonorchis sinensis and Opisthorchis viverrini are both important fish-borne pathogens, causing serious public health problem in Asia. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis for the specific detection and rapid identification of C. sinensis and O. viverrini. Primers targeting COX1 gene were highly specific for these liver flukes, as evidenced by the negative amplification of closely related trematodes. Assays using genomic DNA extracted from the two flukes yielded specific amplification and their identity was confirmed by sequencing, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit below 1 pg of purified genomic DNA, 5 EPG, or 1 metacercaria of C. sinensis. Moreover, C. sinensis and O. viverrini were able to be differentiated by their HRM profiles. The method can reduce labor of microscopic examination and the contamination of agarose electrophoresis. Moreover, it can differentiate these two flukes which are difficult to be distinguished using other methods. The established method provides an alternative tool for rapid, simple, and duplex detection of C. sinensis and O. viverrini.

High-resolution melt and morphological analyses of mealybugs (Hemiptera: Pseudococcidae) from cacao: tools for the control of Cacao swollen shoot virus spread.

PubMed

Wetten, Andy; Campbell, Colin; Allainguillaume, Joël

2016-03-01

Mealybugs (Hemiptera: Coccoidea: Pseudococcidae) are key vectors of badnaviruses, including Cacao swollen shoot virus (CSSV), the most damaging virus affecting cacao (Theobroma cacao L.). The effectiveness of mealybugs as virus vectors is species dependent, and it is therefore vital that CSSV resistance breeding programmes in cacao incorporate accurate mealybug identification. In this work, the efficacy of a CO1-based DNA barcoding approach to species identification was evaluated by screening a range of mealybugs collected from cacao in seven countries. Morphologically similar adult females were characterised by scanning electron microscopy, and then, following DNA extraction, were screened with CO1 barcoding markers. A high degree of CO1 sequence homology was observed for all 11 individual haplotypes, including those accessions from distinct geographical regions. This has allowed the design of a high-resolution melt (HRM) assay capable of rapid identification of the commonly encountered mealybug pests of cacao. HRM analysis readily differentiated between mealybug pests of cacao that cannot necessarily be identified by conventional morphological analysis. This new approach, therefore, has potential to facilitate breeding for resistance to CSSV and other mealybug-transmitted diseases. © 2015 Society of Chemical Industry.

pH-independent triple-helix formation with 6-oxocytidine as cytidine analogue.

PubMed

Parsch, U; Engels, J W

2000-07-03

The syntheses of six different phosphoramidite building blocks of 6-oxocytosine and 5-allyl-6-oxocytosine as analogues of N(3)-protonated cytosine are described. These compounds have been incorporated into oligonucleotides by standard solid-phase synthesis. Hybridization of 15-mer Hoogsteen strands with target 21-mer duplexes was investigated. Comparison of the triplex-forming abilities of the different building blocks revealed that: i) 5-allyl substitution has a negative influence on triplex stability, ii) a uniform backbone of the Hoogsteen strand stabilizes triplexes relative to mixed backbones; iii) RNA strands with 6-oxocytidine or 5-allyl-6-oxocytidine do not form a triple helix with the DNA target duplex, probably due to backbone torsional constraints; and (iv) a 15-mer DNA sequence with three isolated 2'-deoxy-6-oxocytidines has the highest T(m) of all cytidine analogues investigated in this study. CD experiments provided further evidence for the presence or absence of triplex structures. In the course of these temperature-dependent CD measurements we were able to detect duplex and triplex melting independent from each other at selected wavelengths. This methodology is especially interesting in cases where UV melting curves show only one transition owing to spectral overlap.

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) for the characterisation of pathogenic leptospires: intra-serovar divergence, inter-serovar convergence, and evidence of attenuation in Leptospira reference collections.

PubMed

Tulsiani, S M; Craig, S B; Graham, G C; Cobbold, R C; Dohnt, M F; Burns, M-A; Jansen, C C; Leung, L K-P; Field, H E; Smythe, L D

2010-07-01

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) is a novel technology that has emerged as a possible method to characterise leptospires to serovar level. RAPD-HRM has recently been used to measure intra-serovar convergence between strains of the same serovar as well as inter-serovar divergence between strains of different serovars. The results indicate that intra-serovar heterogeneity and inter-serovar homogeneity may limit the application of RAPD-HRM in routine diagnostics. They also indicate that genetic attenuation of aged, high-passage-number isolates could undermine the use of RAPD-HRM or any other molecular technology. Such genetic attenuation may account for a general decrease seen in titres of rabbit hyperimmune antibodies over time. Before RAPD-HRM can be further advanced as a routine diagnostic tool, strains more representative of the wild-type serovars of a given region need to be identified. Further, RAPD-HRM analysis of reference strains indicates that the routine renewal of reference collections, with new isolates, may be needed to maintain the genetic integrity of the collections.

High resolution melting analytical platform for rapid prenatal and postnatal diagnosis of β-thalassemia common among Southeast Asian population.

PubMed

Prajantasen, Thanet; Fucharoen, Supan; Fucharoen, Goonnapa

2015-02-20

High resolution melting (HRM) analysis is a powerful technology for scanning sequence alteration. We have applied this HRM assay to screen common β-thalassemia mutations found among Southeast Asian population. Known DNA samples with 8 common mutations were used in initial development of the methods including -28 A-G, codon 17 A-T, IVSI-1G-T, IVSI-5G-C, codon 26G-A (Hb E), codons 41/42 -TTCT, codons 71/72+A and IVSII-654 C-T. Further validation was done on 60 postnatal and 6 prenatal diagnoses of β-thalassemia. Each mutation has specific HRM profile which could be used in rapid screening. Apart from those with DNA deletions, the results of HRM assay matched 100% with those of routine diagnosis made by routine allele specific PCR. In addition, the HRM assay could initially recognize three unknown mutations including a hitherto un-described one in Thai population. The established HRM assay should prove useful for rapid and high throughput platform for screening and prenatal diagnosis of β-thalassemia common in the region. Copyright © 2014 Elsevier B.V. All rights reserved.

Interaction of the Retinoblastoma Protein with Orc1 and Its Recruitment to Human Origins of DNA Replication

PubMed Central

Mendoza-Maldonado, Ramiro; Paolinelli, Roberta; Galbiati, Laura; Giadrossi, Sara; Giacca, Mauro

2010-01-01

Background The retinoblastoma protein (Rb) is a crucial regulator of cell cycle progression by binding with E2F transcription factor and repressing the expression of a variety of genes required for the G1-S phase transition. Methodology/Principal Findings Here we show that Rb and E2F1 directly participate in the control of initiation of DNA replication in human HeLa, U2OS and T98G cells by specifically binding to origins of DNA replication in a cell cycle regulated manner. We show that, both in vitro and inside the cells, the largest subunit of the origin recognition complex (Orc1) specifically binds hypo-phosphorylated Rb and that this interaction is competitive with the binding of Rb to E2F1. The displacement of Rb-bound Orc1 by E2F1 at origins of DNA replication marks the progression of the G1 phase of the cell cycle toward the G1-S border. Conclusions/Significance The participation of Rb and E2F1 in the formation of the multiprotein complex that binds origins of DNA replication in mammalian cells appears to represent an effective mechanism to couple the expression of genes required for cell cycle progression to the activation of DNA replication. PMID:21085491

Imaging chromatin nanostructure with binding-activated localization microscopy based on DNA structure fluctuations

PubMed Central

Szczurek, Aleksander; Klewes, Ludger; Xing, Jun; Gourram, Amine; Birk, Udo; Knecht, Hans; Dobrucki, Jurek W.; Mai, Sabine

2017-01-01

Abstract Advanced light microscopy is an important tool for nanostructure analysis of chromatin. In this report we present a general concept for Single Molecule localization Microscopy (SMLM) super-resolved imaging of DNA-binding dyes based on modifying the properties of DNA and the dye. By careful adjustment of the chemical environment leading to local, reversible DNA melting and hybridization control over the fluorescence signal of the DNA-binding dye molecules can be introduced. We postulate a transient binding as the basis for our variation of binding-activated localization microscopy (BALM). We demonstrate that several intercalating and minor-groove binding DNA dyes can be used to register (optically isolate) only a few DNA-binding dye signals at a time. To highlight this DNA structure fluctuation-assisted BALM (fBALM), we applied it to measure, for the first time, nanoscale differences in nuclear architecture in model ischemia with an anticipated structural resolution of approximately 50 nm. Our data suggest that this approach may open an avenue for the enhanced microscopic analysis of chromatin nano-architecture and hence the microscopic analysis of nuclear structure aberrations occurring in various pathological conditions. It may also become possible to analyse nuclear nanostructure differences in different cell types, stages of development or environmental stress conditions. PMID:28082388

Influence of Ionic Liquids on Thermodynamics of Small Molecule-DNA Interaction: The Binding of Ethidium Bromide to Calf Thymus DNA.

PubMed

Mishra, Arpit; Ekka, Mary Krishna; Maiti, Souvik

2016-03-17

Ionic liquids (ILs) are salts with poor ionic coordination, resultantly remaining in liquid state below 100 °C and some may retain liquid state even at room temperature. ILs are known to provide a conducive environment for many biological enzymatic reactions, but their interaction with biomacromolecules are poorly understood. In the present study, we investigate the effect of various ionic liquids on DNA-small molecule interaction using calf thymus DNA (ctDNA)-ethidium bromide (EB) as a model system. The effect of various ionic liquids on these interactions is studied by an array of techniques such as circular dichroism (CD), UV melting, fluorescence exclusion and isothermal titration calorimetry. Interestingly, we observed that presence of IL increased the stability of ctDNA without altering its structure. The binding affinities Kbs for EB binding to ctDNA in the presence of 300 mM ILs are about half order of magnitude smaller than the Kbs in absence of ILs and correspond to a less favorable free energy. We noted that, when adjusted to corresponding buffer condition, the unfavorable shift in ΔG of ctDNA-EB interaction is attributed to decreased entropy in the case of ILs, whereas the same effect by NaCl was due to increased enthalpy.

Synthesis and characterization of 6,6’-bis(2-hydroxyphenyl)-2,2’-bipyridine ligand and its interaction with ct-DNA

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

Selamat, Norhidayah; Heng, Lee Yook; Hassan, Nurul Izzaty

2015-09-25

The tetradentate ligand with four donor atoms OONN was synthesized. Bis(phenoxy)bipyridine ligand was prepared by Suzuki coupling reaction between 6,6’-dibromo-2,2’-bipyridyl and 2-hydroxyphenylboronic acid with presence of palladium (II) acetate. Bis(phenoxy)bipyridine ligand was also synthesized by demethylating of 6,6’-bis(2-methoxyphenyl)-2,2’-bipyridyl ligand through solvent free reaction using pyridine hydrocloride. The formation of both phenoxy and methoxy ligands was confirmed by {sup 1}H, 2D cosy and {sup 13}C NMR spectroscopy, ESI-MS spectrometry, FTIR spectroscopy. The purity of the ligand was confirmed by melting point. Binding studies of small molecules with DNA are useful to understand the reaction mechanism and to provide guidance for themore » application and design of new and more efficient drugs targeted to DNA. In this study, the binding interaction between the synthesized ligand with calf thymus-DNA (ct-DNA) has been investigated by UV/Vis DNA titration study. From the UV/Vis DNA study, it shows that bis(phenoxy)bipyridine ligand bind with ct-DNA via outside binding with binding contant K{sub b} = 1.19 × 10{sup 3} ± 0.08 M{sup −1}.« less

Challenges and progress in making DNA-based AIS early ...

EPA Pesticide Factsheets

The ability of DNA barcoding to find additional species in hard-to-sample locations or hard-to-identify samples is well established. Nevertheless, adoption of DNA barcoding into regular monitoring programs has been slow, in part due to issues of standardization and interpretation that need resolving. In this presentation, we describe our progress towards incorporating DNA-based identification into broad-spectrum aquatic invasive species early-detection monitoring in the Laurentian Great Lakes. Our work uses community biodiversity information as the basis for evaluating survey performance for various taxonomic groups. Issues we are tackling in bringing DNA-based data to bear on AIS monitoring design include: 1) Standardizing methodology and work flow from field collection and sample handling through bioinformatics post-processing; 2) Determining detection sensitivity and accounting for inter-species differences in DNA amplification and primer affinity; 3) Differentiating sequencing and barcoding errors from legitimate new finds when range and natural history information is limited; and 4) Accounting for the different nature of morphology- vs. DNA-based biodiversity information in subsequent analysis (e.g., via species accumulation curves, multi-metric indices). not applicable

A DNA methylation map of human cancer at single base-pair resolution

PubMed Central

Vidal, E; Sayols, S; Moran, S; Guillaumet-Adkins, A; Schroeder, M P; Royo, R; Orozco, M; Gut, M; Gut, I; Lopez-Bigas, N; Heyn, H; Esteller, M

2017-01-01

Although single base-pair resolution DNA methylation landscapes for embryonic and different somatic cell types provided important insights into epigenetic dynamics and cell-type specificity, such comprehensive profiling is incomplete across human cancer types. This prompted us to perform genome-wide DNA methylation profiling of 22 samples derived from normal tissues and associated neoplasms, including primary tumors and cancer cell lines. Unlike their invariant normal counterparts, cancer samples exhibited highly variable CpG methylation levels in a large proportion of the genome, involving progressive changes during tumor evolution. The whole-genome sequencing results from selected samples were replicated in a large cohort of 1112 primary tumors of various cancer types using genome-scale DNA methylation analysis. Specifically, we determined DNA hypermethylation of promoters and enhancers regulating tumor-suppressor genes, with potential cancer-driving effects. DNA hypermethylation events showed evidence of positive selection, mutual exclusivity and tissue specificity, suggesting their active participation in neoplastic transformation. Our data highlight the extensive changes in DNA methylation that occur in cancer onset, progression and dissemination. PMID:28581523

Genetic and epigenetic profiling of CLL disease progression reveals limited somatic evolution and suggests a relationship to memory-cell development.

PubMed

Smith, E N; Ghia, E M; DeBoever, C M; Rassenti, L Z; Jepsen, K; Yoon, K-A; Matsui, H; Rozenzhak, S; Alakus, H; Shepard, P J; Dai, Y; Khosroheidari, M; Bina, M; Gunderson, K L; Messer, K; Muthuswamy, L; Hudson, T J; Harismendy, O; Barrett, C L; Jamieson, C H M; Carson, D A; Kipps, T J; Frazer, K A

2015-04-10

We examined genetic and epigenetic changes that occur during disease progression from indolent to aggressive forms of chronic lymphocytic leukemia (CLL) using serial samples from 27 patients. Analysis of DNA mutations grouped the leukemia cases into three categories: evolving (26%), expanding (26%) and static (47%). Thus, approximately three-quarters of the CLL cases had little to no genetic subclonal evolution. However, we identified significant recurrent DNA methylation changes during progression at 4752 CpGs enriched for regions near Polycomb 2 repressive complex (PRC2) targets. Progression-associated CpGs near the PRC2 targets undergo methylation changes in the same direction during disease progression as during normal development from naive to memory B cells. Our study shows that CLL progression does not typically occur via subclonal evolution, but that certain CpG sites undergo recurrent methylation changes. Our results suggest CLL progression may involve developmental processes shared in common with the generation of normal memory B cells.

Strategies for Detecting Biological Molecules on Titan.

PubMed

Neish, Catherine D; Lorenz, Ralph D; Turtle, Elizabeth P; Barnes, Jason W; Trainer, Melissa G; Stiles, Bryan; Kirk, Randolph; Hibbitts, Charles A; Malaska, Michael J

2018-05-02

Saturn's moon Titan has all the ingredients needed to produce "life as we know it." When exposed to liquid water, organic molecules analogous to those found on Titan produce a range of biomolecules such as amino acids. Titan thus provides a natural laboratory for studying the products of prebiotic chemistry. In this work, we examine the ideal locales to search for evidence of, or progression toward, life on Titan. We determine that the best sites to identify biological molecules are deposits of impact melt on the floors of large, fresh impact craters, specifically Sinlap, Selk, and Menrva craters. We find that it is not possible to identify biomolecules on Titan through remote sensing, but rather through in situ measurements capable of identifying a wide range of biological molecules. Given the nonuniformity of impact melt exposures on the floor of a weathered impact crater, the ideal lander would be capable of precision targeting. This would allow it to identify the locations of fresh impact melt deposits, and/or sites where the melt deposits have been exposed through erosion or mass wasting. Determining the extent of prebiotic chemistry within these melt deposits would help us to understand how life could originate on a world very different from Earth. Key Words: Titan-Prebiotic chemistry-Solar system exploration-Impact processes-Volcanism. Astrobiology xx, xxx-xxx.

Challenges and progress in making DNA-based AIS early detection monitoring operational

EPA Science Inventory

The ability of DNA barcoding to find additional species in hard-to-sample locations or hard-to-identify samples is well established. Nevertheless, adoption of DNA barcoding into regular monitoring programs has been slow, in part due to issues of standardization and interpretation...

DNA and histone methylation in gastric carcinogenesis

PubMed Central

Calcagno, Danielle Queiroz; Gigek, Carolina Oliveira; Chen, Elizabeth Suchi; Burbano, Rommel Rodriguez; Smith, Marília de Arruda Cardoso

2013-01-01

Epigenetic alterations contribute significantly to the development and progression of gastric cancer, one of the leading causes of cancer death worldwide. Epigenetics refers to the number of modifications of the chromatin structure that affect gene expression without altering the primary sequence of DNA, and these changes lead to transcriptional activation or silencing of the gene. Over the years, the study of epigenetic processes has increased, and novel therapeutic approaches that target DNA methylation and histone modifications have emerged. A greater understanding of epigenetics and the therapeutic potential of manipulating these processes is necessary for gastric cancer treatment. Here, we review recent research on the effects of aberrant DNA and histone methylation on the onset and progression of gastric tumors and the development of compounds that target enzymes that regulate the epigenome. PMID:23482412

Melt production in large-scale impact events: Implications and observations at terrestrial craters

NASA Technical Reports Server (NTRS)

Grieve, Richard A. F.; Cintala, Mark J.

1992-01-01

The volume of impact melt relative to the volume of the transient cavity increases with the size of the impact event. Here, we use the impact of chondrite into granite at 15, 25, and 50 km s(sup -1) to model impact-melt volumes at terrestrial craters in crystalline targets and explore the implications for terrestrial craters. Figures are presented that illustrate the relationships between melt volume and final crater diameter D(sub R) for observed terrestrial craters in crystalline targets; also included are model curves for the three different impact velocities. One implication of the increase in melt volumes with increasing crater size is that the depth of melting will also increase. This requires that shock effects occurring at the base of the cavity in simple craters and in the uplifted peaks of central structures at complex craters record progressively higher pressures with increasing crater size, up to a maximum of partial melting (approx. 45 GPa). Higher pressures cannot be recorded in the parautochthonous rocks of the cavity floor as they will be represented by impact melt, which will not remain in place. We have estimated maximum recorded pressures from a review of the literature, using such observations as planar features in quartz and feldspar, diaplectic glasses of feldspar and quartz, and partial fusion and vesiculation, as calibrated with estimates of the pressures required for their formation. Erosion complicates the picture by removing the surficial (most highly shocked) rocks in uplifted structures, thereby reducing the maximum shock pressures observed. In addition, the range of pressures that can be recorded is limited. Nevertheless, the data define a trend to higher recorded pressures with crater diameter, which is consistent with the implications of the model. A second implication is that, as the limit of melting intersects the base of the cavity, central topographic peaks will be modified in appearance and ultimately will not occur. That is, the peak will first develop a central depression, due to the flow of low-strength melted materials, when the melt volume begins to intersect the transient-cavity base.

Engineering artificial machines from designable DNA materials for biomedical applications.

PubMed

Qi, Hao; Huang, Guoyou; Han, Yulong; Zhang, Xiaohui; Li, Yuhui; Pingguan-Murphy, Belinda; Lu, Tian Jian; Xu, Feng; Wang, Lin

2015-06-01

Deoxyribonucleic acid (DNA) emerges as building bricks for the fabrication of nanostructure with complete artificial architecture and geometry. The amazing ability of DNA in building two- and three-dimensional structures raises the possibility of developing smart nanomachines with versatile controllability for various applications. Here, we overviewed the recent progresses in engineering DNA machines for specific bioengineering and biomedical applications.

Engineering Artificial Machines from Designable DNA Materials for Biomedical Applications

PubMed Central

Huang, Guoyou; Han, Yulong; Zhang, Xiaohui; Li, Yuhui; Pingguan-Murphy, Belinda; Lu, Tian Jian; Xu, Feng

2015-01-01

Deoxyribonucleic acid (DNA) emerges as building bricks for the fabrication of nanostructure with complete artificial architecture and geometry. The amazing ability of DNA in building two- and three-dimensional structures raises the possibility of developing smart nanomachines with versatile controllability for various applications. Here, we overviewed the recent progresses in engineering DNA machines for specific bioengineering and biomedical applications. PMID:25547514

Two familial ALS proteins function in prevention/repair of transcription-associated DNA damage.

PubMed

Hill, Sarah J; Mordes, Daniel A; Cameron, Lisa A; Neuberg, Donna S; Landini, Serena; Eggan, Kevin; Livingston, David M

2016-11-29

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron dysfunction disease that leads to paralysis and death. There is currently no established molecular pathogenesis pathway. Multiple proteins involved in RNA processing are linked to ALS, including FUS and TDP43, and we propose a disease mechanism in which loss of function of at least one of these proteins leads to an accumulation of transcription-associated DNA damage contributing to motor neuron cell death and progressive neurological symptoms. In support of this hypothesis, we find that FUS or TDP43 depletion leads to increased sensitivity to a transcription-arresting agent due to increased DNA damage. Thus, these proteins normally contribute to the prevention or repair of transcription-associated DNA damage. In addition, both FUS and TDP43 colocalize with active RNA polymerase II at sites of DNA damage along with the DNA damage repair protein, BRCA1, and FUS and TDP43 participate in the prevention or repair of R loop-associated DNA damage, a manifestation of aberrant transcription and/or RNA processing. Gaining a better understanding of the role(s) that FUS and TDP43 play in transcription-associated DNA damage could shed light on the mechanisms underlying ALS pathogenesis.

Two familial ALS proteins function in prevention/repair of transcription-associated DNA damage

PubMed Central

Hill, Sarah J.; Mordes, Daniel A.; Cameron, Lisa A.; Neuberg, Donna S.; Landini, Serena; Eggan, Kevin; Livingston, David M.

2016-01-01

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron dysfunction disease that leads to paralysis and death. There is currently no established molecular pathogenesis pathway. Multiple proteins involved in RNA processing are linked to ALS, including FUS and TDP43, and we propose a disease mechanism in which loss of function of at least one of these proteins leads to an accumulation of transcription-associated DNA damage contributing to motor neuron cell death and progressive neurological symptoms. In support of this hypothesis, we find that FUS or TDP43 depletion leads to increased sensitivity to a transcription-arresting agent due to increased DNA damage. Thus, these proteins normally contribute to the prevention or repair of transcription-associated DNA damage. In addition, both FUS and TDP43 colocalize with active RNA polymerase II at sites of DNA damage along with the DNA damage repair protein, BRCA1, and FUS and TDP43 participate in the prevention or repair of R loop-associated DNA damage, a manifestation of aberrant transcription and/or RNA processing. Gaining a better understanding of the role(s) that FUS and TDP43 play in transcription-associated DNA damage could shed light on the mechanisms underlying ALS pathogenesis. PMID:27849576

Silicate melts density, buoyancy relations and the dynamics of magmatic processes in the upper mantle

NASA Astrophysics Data System (ADS)

Sanchez-Valle, Carmen; Malfait, Wim J.

2016-04-01

Although silicate melts comprise only a minor volume fraction of the present day Earth, they play a critical role on the Earth's geochemical and geodynamical evolution. Their physical properties, namely the density, are a key control on many magmatic processes, including magma chamber dynamics and volcanic eruptions, melt extraction from residual rocks during partial melting, as well as crystal settling and melt migration. However, the quantitative modeling of these processes has been long limited by the scarcity of data on the density and compressibility of volatile-bearing silicate melts at relevant pressure and temperature conditions. In the last decade, new experimental designs namely combining large volume presses and synchrotron-based techniques have opened the possibility for determining in situ the density of a wide range of dry and volatile-bearing (H2O and CO2) silicate melt compositions at high pressure-high temperature conditions. In this contribution we will illustrate some of these progresses with focus on recent results on the density of dry and hydrous felsic and intermediate melt compositions (rhyolite, phonolite and andesite melts) at crustal and upper mantle conditions (up to 4 GPa and 2000 K). The new data on felsic-intermediate melts has been combined with in situ data on (ultra)mafic systems and ambient pressure dilatometry and sound velocity data to calibrate a continuous, predictive density model for hydrous and CO2-bearing silicate melts with applications to magmatic processes down to the conditions of the mantle transition zone (up to 2773 K and 22 GPa). The calibration dataset consist of more than 370 density measurements on high-pressure and/or water-and CO2-bearing melts and it is formulated in terms of the partial molar properties of the oxide components. The model predicts the density of volatile-bearing liquids to within 42 kg/m3 in the calibration interval and the model extrapolations up to 3000 K and 100 GPa are in good agreement with results from ab initio calculations. The density model has been applied to examine the mineral-melt buoyancy relations at depth and the implications of these results for the dynamics of magma chambers, crystal settling and the stability and mobility of magmas in the upper mantle will be discussed.

Multiple episodes of partial melting, depletion, metasomatism and enrichment processes recorded in the heterogeneous upper mantle sequence of the Neotethyan Eldivan ophiolite, Turkey

NASA Astrophysics Data System (ADS)

Uysal, Ibrahim; Ersoy, E. Yalçın; Dilek, Yildirim; Kapsiotis, Argyrios; Sarıfakıoğlu, Ender

2016-03-01

The Eldivan ophiolite along the Izmir-Ankara-Erzincan suture zone in north-central Anatolia represents a remnant of the Neotethyan oceanic lithosphere. Its upper mantle peridotites include three lithologically and compositionally distinct units: clinopyroxene (cpx)-harzburgite and lherzolite (Group-1), depleted harzburgite (Group-2), and dunite (Group-3). Relics of primary olivine and pyroxene occur in the less refractory harzburgites, and fresh chromian spinel (Cr-spinel) is ubiquitous in all peridotites. The Eldivan peridotites reflect a petrogenetic history evolving from relatively fertile (lherzolite and cpx-harzburgite) toward more depleted (dunite) compositions through time, as indicated by (i) a progressive decrease in the modal cpx distribution, (ii) a progressive increase in the Cr#s [Cr / (Cr + Al)] of Cr-spinel (0.15-0.78), and (iii) an increased depletion in the whole-rock abundances of some magmaphile major oxides (Al2O3, CaO, SiO2 and TiO2) and incompatible trace elements (Zn, Sc, V and Y). The primitive mantle-normalized REE patterns of the Group-1 and some of the Group-2 peridotites display LREE depletions. Higher YbN and lower SmN/YbN ratios of these rocks are compatible with their formation after relatively low degrees (9-25%) of open-system dynamic melting (OSDM) of a Depleted Mid-ocean ridge Mantle (DMM) source, which was then fluxed with small volumes of oceanic mantle-derived melt [fluxing ratio (β): 0.7-1.2%]. Accessory Cr-spinel compositions (Cr# = 015-0.53) of these rocks are consistent with their origin as residual peridotites beneath a mid-ocean ridge axis. Part of the Group-2 harzburgites exhibit lower YbN and higher SmN/YbN ratios, LREE-enriched REE patterns, and higher Cr-spinel Cr#s ranging between 0.54 and 0.61. Trace element compositions of these peridotites can be modeled by approximately 15% OSDM of a previously 17% depleted DMM, which was then fluxed (β: 0.4%) with subduction-influenced melt. The Group-3 dunite samples contain Cr-spinel with elevated Cr#s (0.73-0.78) and low-TiO2 contents (< 0.13 wt.%), implying higher degrees of melting (21-24%) of an already depleted DMM that was triggered by infiltration of low-Ti boninite melt with fluxing rates of 0.4-4.0%. The existence of interstitial, idiomorphic Cr-spinel (high Cr# and low Ti) in the Group-3 dunites is consistent with this interpretation. The occurrence of both MOR- and SSZ-type peridotites in the Eldivan ophiolite suggests that its heterogeneous upper mantle was produced as a result of different partial melting and melt-rock reaction processes in different tectonic settings within the Neotethyan realm.

EFFECTS OF MOLECULAR ARCHITECTURE ON TWO-STEP MELT-SPUN POLY(LACTIC ACID) FIBERS. (R826733)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Chemostratigraphy of Subduction Initiation: Boninite and Forearc Basalt from IODP Expedition 352

NASA Astrophysics Data System (ADS)

Shervais, John; Haugen, Emily; Godard, Marguerite; Ryan, Jeffrey G.; Prytulak, Julie; Li, Hongyan; Chapman, Timothy; Nelson, Wendy R.; Heaton, Daniel E.; Kirchenbaur, Maria; Shimizu, Kenji; Li, Yibing; Whattam, Scott A.; Almeev, Renat; Sakuyama, Tetsuya; Reagan, Mark K.; Pearce, Julian A.

2017-04-01

The Izu-Bonin forearc has been the focus of several recent IODP (International Ocean Discovery Program) expeditions studying the geophysical, petrologic, and chemical response to subduction initiation and its potential relationship to ophiolite genesis. IODP Expedition 352 cored four holes in the Izu-Bonin forearc near Chichi Jima in order to document the petrologic and chemical evolution of nascent subduction zones. Holes U1440 and U1441, drilled closest to the trench, sampled forearc basalt (FAB). U1439 and U1442, drilled stratigraphically up-section and farther from the trench, sampled boninite, high-Mg andesite, and basalt. FAB are characterized by MORB-like compositions, with relatively constant Ti, Zr, and Ti/Zr. In general, more primitive FAB are found in the lower part of the section. In detail, FAB have lower Na, Ti, P, and Zr, lower Ti/V ratios, and are LREE-depleted relative to MORB. Best fit models for the least evolved FAB and a depleted MORB mantle (DMM) source require extraction of 1% melt in the garnet lherzolite field and 19% melt extraction in the spinel lherzolite field (relative to 8-10% melt of DMM to produce MORB). Three types of boninite were found: high silica boninite (HSB), low silica boninite (LSB), and basaltic boninite (BB), as well as high Mg andesites (HMA). HSB, the youngest unit in both U1439 and U1442, is underlain by LSB-BB-HMA lavas, which often occur in mixed magma zones with evolved boninite and basalt. Boninites are distinguished by co-variations in SiO2-MgO and TiO2-MgO, and by Ti/Zr ratios, which increase from HSB through LSB to BB. HSB, LSB and BB define parallel trends in TiO2-MgO space: a low Ti trend represented by LSB and BB, and a lower Ti trend represented by HSB. All of the boninite suite rocks are slightly LREE-rich relative to MORB. LSB and BB have flat REE patterns relative to primitive mantle, whereas HSB are slightly LREE-rich. These trends require distinct source compositions in HSB relative to LSB/BB. The decrease in Ti/Zr from BB to HSB suggests a slab melt component. Melting models (non-modal, fractional) for boninites require additional partial melting of a residual source more depleted than DMM, and mixing with less depleted melts. The data require a heterogeneous source during subduction initiation, tapping progressively more refractory mantle through time, and showing progressive enrichment in slab components.

Consequences of magma eruption dynamics: Intraflow variations in petrography and mineral chemistry within a single eruptive unit from Whitewater Canyon, Oregon

NASA Astrophysics Data System (ADS)

Ustunisik, G. K.; Nielsen, R. L.

2012-12-01

Individual lava flows are sometimes characterized by progressive changes in petrography and mineral chemistry which have been attributed to progressive magma chamber evacuation. In the case of Whitewater Canyon flow, a glacially quenched andesite unit on the NW flank of Mt. Jefferson, significant changes have been observed in phenocryst content and mineral chemistry within a transect from the early erupted components (inferred by flow morphology to be quenched against glacial ice ~10000 ybp), to the top of the 30 m thick flow unit. With the increasing distance from the quenched interface, the matrix changes from glassy to microcrystalline. The matrix material is generally similar in composition to the glassy melt inclusions rhyolitic in composition yet relatively degassed (lower Cl, S). Based on their morphology, we have identified at least 4 populations of plagioclase phenocrysts within the single flow: (1) Relatively unzoned high An cores (>An80) with oscillatory overgrowth, (2) Lower An cores (An50-60), associated with dacitic melt inclusions, (3) Cellular low An cores (An50-60) with higher An overgrowths (~An65-75), and (4) Lath shaped, sometimes oscillatory zoned moderately high An phenocrysts (An65-75) -often associated with olivine:cpx:plagioclase glomerocrysts. Melt inclusions are present in orthopyroxene and plagioclase, but only in the earliest erupted samples (within 5-10 meters of the quenched interface). This mafic component, characterized by olivine, intermediate plagioclase (An60-75), clinopyroxene, orthopyroxene, and oxides, was present at a range of scales from glomerocrysts to 10 cm+ enclaves. Amphibole and quartz are present only in samples from the interior of the flow unit. The width of reaction rims on amphibole increase as one progress upwards towards the flow interior. Our initial conclusions are this eruptive unit represents the progressive evacuation of a shallow magma chamber where the upper parts of the chamber had already been partially degassed. This is supported by the absence of amphibole phenocrysts in the first erupted, quenched samples. However, the presence of volatiles in glassy melt inclusions and phenocrysts suggests that the system had not completely degassed prior to the eruption. Also, the petrographic heterogeneity of the unit demonstrates that complete overturn and mixing did not happen at Mt. Jefferson. Therefore, eruption may have been triggered by the injection of mafic material; however, the petrographic and field evidence suggests that overturn is not required as part of that triggering event.

High-resolution melting analysis (HRM) for differentiation of four major Taeniidae species in dogs Taenia hydatigena, Taenia multiceps, Taenia ovis, and Echinococcus granulosus sensu stricto.

PubMed

Dehghani, Mansoureh; Mohammadi, Mohammad Ali; Rostami, Sima; Shamsaddini, Saeedeh; Mirbadie, Seyed Reza; Harandi, Majid Fasihi

2016-07-01

Tapeworms of the genus Taenia include several species of important parasites with considerable medical and veterinary significance. Accurate identification of these species in dogs is the prerequisite of any prevention and control program. Here, we have applied an efficient method for differentiating four major Taeniid species in dogs, i.e., Taenia hydatigena, T. multiceps, T. ovis, and Echinococcus granulosus sensu stricto. High-resolution melting (HRM) analysis is simpler, less expensive, and faster technique than conventional DNA-based assays and enables us to detect PCR amplicons in a closed system. Metacestode samples were collected from local abattoirs from sheep. All the isolates had already been identified by PCR-sequencing, and their sequence data were deposited in the GenBank. Real-time PCR coupled with HRM analysis targeting mitochondrial cox1 and ITS1 genes was used to differentiate taeniid species. Distinct melting curves were obtained from ITS1 region enabling accurate differentiation of three Taenia species and E. granulosus in dogs. The HRM curves of Taenia species and E .granulosus were clearly separated at Tm of 85 to 87 °C. In addition, double-pick melting curves were produced in mixed infections. Cox1 melting curves were not decisive enough to distinguish four taeniids. In this work, the efficiency of HRM analysis to differentiate four major taeniid species in dogs has been demonstrated using ITS1 gene.

Aza-macrocyclic Triphenylamine Ligands for G-Quadruplex Recognition.

PubMed

García-España, Enrique Victor; Pont, Isabel; González-García, Jorge; Inclán, Mario; Reynolds, Matthew; Delgado-Pinar, Estefanía; Albelda, M Teresa; Vilar, Ramon

2018-05-16

A new series of triphenylamine-based ligands with one (TPA1PY), two (TPA2PY) or three pending aza-macrocycle(s) (TPA3PY) have been synthesised and studied by means of pH-metric titrations, UV/Vis spectroscopy and fluorescence experiments. The affinity of these ligands for G-quadruplex (G4) DNA and its selectivity over duplex DNA were investigated by FRET melting assays, fluorimetric titrations and circular dichroism (CD) spectroscopy. Interestingly, the interaction of the bi- and specially the tri-branched ligand with G4 leads to a very intense red-shifted fluorescence emission band which may be associated with intermolecular aggregation between the molecule and the DNA. This light-up effect allows the application of the ligands as fluorescence probes to selectivity detect G4. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A self-assembled nanoscale robotic arm controlled by electric fields

NASA Astrophysics Data System (ADS)

Kopperger, Enzo; List, Jonathan; Madhira, Sushi; Rothfischer, Florian; Lamb, Don C.; Simmel, Friedrich C.

2018-01-01

The use of dynamic, self-assembled DNA nanostructures in the context of nanorobotics requires fast and reliable actuation mechanisms. We therefore created a 55-nanometer–by–55-nanometer DNA-based molecular platform with an integrated robotic arm of length 25 nanometers, which can be extended to more than 400 nanometers and actuated with externally applied electrical fields. Precise, computer-controlled switching of the arm between arbitrary positions on the platform can be achieved within milliseconds, as demonstrated with single-pair Förster resonance energy transfer experiments and fluorescence microscopy. The arm can be used for electrically driven transport of molecules or nanoparticles over tens of nanometers, which is useful for the control of photonic and plasmonic processes. Application of piconewton forces by the robot arm is demonstrated in force-induced DNA duplex melting experiments.

Human Mitochondrial Transcription Factor B2 Is Required for Promoter Melting during Initiation of Transcription.

PubMed

Posse, Viktor; Gustafsson, Claes M

2017-02-17

The mitochondrial transcription initiation machinery in humans consists of three proteins: the RNA polymerase (POLRMT) and two accessory factors, transcription factors A and B2 (TFAM and TFB2M, respectively). This machinery is required for the expression of mitochondrial DNA and the biogenesis of the oxidative phosphorylation system. Previous experiments suggested that TFB2M is required for promoter melting, but conclusive experimental proof for this effect has not been presented. Moreover, the role of TFB2M in promoter unwinding has not been discriminated from that of TFAM. Here we used potassium permanganate footprinting, DNase I footprinting, and in vitro transcription from the mitochondrial light-strand promoter to study the role of TFB2M in transcription initiation. We demonstrate that a complex composed of TFAM and POLRMT was readily formed at the promoter but alone was insufficient for promoter melting, which only occurred when TFB2M joined the complex. We also show that mismatch bubble templates could circumvent the requirement of TFB2M, but TFAM was still required for efficient initiation. Our findings support a model in which TFAM first recruits POLRMT to the promoter, followed by TFB2M binding and induction of promoter melting. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular differentiation of three canine and feline hookworms in South China through HRM analysis.

PubMed

Wang, M W; Yan, X X; Hang, J X; Shi, X L; Fu, Y Q; Zhang, P; Yang, F; Pan, W D; Li, G Q

2018-02-05

To investigate the prevalence of canine and feline hookworms in South China, and to assess the risk of zoonotic hookworms to humans, one pair of primers (HRM-F/HRM-R) was designed to establish a high-resolution melting (HRM) method based on internal transcribed spacer 1 (ITS-1) rDNA for the detection of Ancylostoma ceylanicum, A. caninum and A. tubaeforme infection. The results showed that the HRM for the three hookworms produced different melting-curve profiles, where melting temperature (T m) values were 84.50°C for A. ceylanicum, 82.25°C for A. caninum and 81.73°C for A. tubaeforme, respectively. The reproducibility of intra- and inter-assay melting curves was almost perfect. The lowest concentration detected was about 5.69 ×10-4 g/μl. The HRM detection results from 18 canine and feline hookworm samples were in complete accordance with their sequencing results. The HRM method was more sensitive than the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique in the detection of 98 clinical samples. It is concluded that the HRM method can differentiate between A. ceylanicum, A. caninum, A. tubaeforme and their mixed infections, which may provide important technical support for the zoonotic risk assessment and molecular epidemiological survey of canine and feline hookworms.

High resolution melting analysis to genotype the most common variants in the HFE gene.

PubMed

Marotta, Roberta V; Turri, Olivia; Morandi, Antonella; Murano, Manuela; d'Eril, Gianlodovico Melzi; Biondi, Maria Luisa

2011-09-01

High resolution melting (HRM) analysis of PCR amplicons was recently introduced as a closed-tube, rapid, and inexpensive method of genotyping. This study evaluated this system as an option for detecting the three most common mutations in the HFE gene (C282Y, H63D, S65C), accounting for the main form of hereditary haemochromatosis. Ninety samples, previously screened by direct sequencing, and 27 controls were used. The analysis were performed on the Rotor Gene Q, using the commercial HRM mix containing the Eva Green dye (Qiagen). Specific primers allowed the amplification of the regions of interest in the HFE gene. Following amplification, a HRM analysis was conducted to detect DNA variants. The thermal denaturation profiles of the samples were compared with those of the controls. One hundred percent of heterozygous and homozygous samples were readily identified. Heterozygotes were easily identified because heteroduplexes altered the shape of the melting curves, but significant differences were also present in the melting curves of the homozygous carries compared with those of the wild-type subjects. HRM analysis is an appealing technology for HFE gene screening. It is a robust technique that can be widely adopted in diagnostic laboratories to facilitate gene mutation screening.

Challenges and progress in making DNA-based monitoring operational AIS early detection as testbed

EPA Science Inventory

The ability of DNA barcoding to find additional species in hard-to-sample locations or hard-to-identify samples is well established. Nevertheless, adoption of DNA barcoding into regular monitoring programs has been slow, in part due to issues of standardization and interpretation...

Mechanistic Differences in DNA Nanoparticle Formation in the Presence of Oligolysines and Poly-L-lysine†

PubMed Central

Nayvelt, Irina; Thomas, Thresia; Thomas, T. J.

2008-01-01

We studied the effectiveness of trilysine (Lys3)-, tetralysine (Lys4)-, pentalysine (Lys5)-, and poly-L-lysine (PLL) (MW: 50,000) on λ-DNA nanoparticle formation, and characterized the size, shape and stability of nanoparticles. Light scattering experiments showed EC50 (lysine concentration at 50% DNA compaction) values of ~0.0036, 2, and 20 μmoles/liter, respectively, for PLL, Lys5, and Lys4 at 10 mM [Na+]. Plots of log[EC50] versus log[Na+] showed positive slopes of 1.09 and 1.7, respectively, for Lys4 and Lys5 and a negative slope of −0.1 for PLL. Hydrodynamic radii of oligolysine-condensed particles increased (48–173 nm) with increasing [Na+], whereas no significant change occurred to nanoparticles formed with PLL. There was an increase in the size of nanoparticles formed with Lys5 at >40 °C, whereas no such change occurred with PLL. DNA melting temperature increased with oligolysine concentration. These results indicate distinct differences in the mechanism(s) by which oligolysines and PLL provoke DNA condensation to nanoparticles. PMID:17291071

Ultralocalized thermal reactions in subnanoliter droplets-in-air.

PubMed

Salm, Eric; Guevara, Carlos Duarte; Dak, Piyush; Dorvel, Brian Ross; Reddy, Bobby; Alam, Muhammad Ashraf; Bashir, Rashid

2013-02-26

Miniaturized laboratory-on-chip systems promise rapid, sensitive, and multiplexed detection of biological samples for medical diagnostics, drug discovery, and high-throughput screening. Within miniaturized laboratory-on-chips, static and dynamic droplets of fluids in different immiscible media have been used as individual vessels to perform biochemical reactions and confine the products. Approaches to perform localized heating of these individual subnanoliter droplets can allow for new applications that require parallel, time-, and space-multiplex reactions on a single integrated circuit. Our method positions droplets on an array of individual silicon microwave heaters on chip to precisely control the temperature of droplets-in-air, allowing us to perform biochemical reactions, including DNA melting and detection of single base mismatches. We also demonstrate that ssDNA probe molecules can be placed on heaters in solution, dried, and then rehydrated by ssDNA target molecules in droplets for hybridization and detection. This platform enables many applications in droplets including hybridization of low copy number DNA molecules, lysing of single cells, interrogation of ligand-receptor interactions, and rapid temperature cycling for amplification of DNA molecules.

A multiple-alignment based primer design algorithm for genetically highly variable DNA targets

PubMed Central

2013-01-01

Background Primer design for highly variable DNA sequences is difficult, and experimental success requires attention to many interacting constraints. The advent of next-generation sequencing methods allows the investigation of rare variants otherwise hidden deep in large populations, but requires attention to population diversity and primer localization in relatively conserved regions, in addition to recognized constraints typically considered in primer design. Results Design constraints include degenerate sites to maximize population coverage, matching of melting temperatures, optimizing de novo sequence length, finding optimal bio-barcodes to allow efficient downstream analyses, and minimizing risk of dimerization. To facilitate primer design addressing these and other constraints, we created a novel computer program (PrimerDesign) that automates this complex procedure. We show its powers and limitations and give examples of successful designs for the analysis of HIV-1 populations. Conclusions PrimerDesign is useful for researchers who want to design DNA primers and probes for analyzing highly variable DNA populations. It can be used to design primers for PCR, RT-PCR, Sanger sequencing, next-generation sequencing, and other experimental protocols targeting highly variable DNA samples. PMID:23965160