Application of central composite design for DNA hybridization onto magnetic microparticles

Central composite face-centered (CCF) design and response surface methodologies were used to investigate the effect of probe and target concentration and particle number in immobilization and hybridization on a microparticle-based DNA/DNA hybridization assay. The factors under study were combined according to the CCF design matrix, and the intensity of the hybridization signal was quantified by flow cytometry. A second-order polynomial was fitted to data and validated by analysis of variance. The results showed a complex relationship between variables and response given that all factors as well as some interactions were significant, yet it could explain 95% of the data. Probe and target concentration had the strongest impact on hybridization signal intensity. Increments in initial probe concentration in solution positively affected the hybridization signal until a negative influence of a compact probe layer emerged. This trend was attributed to probe-probe interactions. By manipulating particle number on both immobilization and hybridization, enhancements on the assay sensitivity could be obtained. Under optimized conditions, the limit of detection (LOD) at the 95% confidence level was determined to be 2.3 nM of target solution concentration.     

A new methodology of mesenchymal stem cell expansion using magnetic nanoparticles

Mesenchymal stem cells (MSCs), which can differentiate into multiple mesodermal tissues, may be useful for autologous cell transplantation, if MSCs, which are isolated from bone marrow in small numbers, can be expanded in vitro. We developed a combined methodological approach to enrich and proliferate MSCs in vitro using magnetic nanoparticles. Our magnetite cationic liposomes (MCLs), which have a positive surface charge in order to improve adsorption, accumulated in MSCs at a concentration of 20 pg of magnetite per cell. The MCLs exhibited no toxicity against MSCs in proliferation and differentiation to osteoblasts and adipocytes. The MSCs magnetically labeled by MCLs were enriched using magnets and then cultured, resulting in much higher density (seeding density, 1000 cells/cm²) than in ordinary culture (seeding density, 18 cells/cm²). When MSCs were seeded at high density using MCLs, there was a 5-fold increase in the number of cells, compared to culture prepared without MCLs. Our results suggest that this novel culture method using magnetic nanoparticles can be used to efficiently expand MSCs for clinical application.     

Extraction of DNA from soil using nanoparticles by magnetic bioseparation

Aims: To develop a simple, rapid and inexpensive soil DNA extraction protocol. Methods and Results: The protocol relies on the use of superparamagnetic silica‐magnetite nanoparticles for the isolation and purification of DNA from soil samples. DNA suitable for use in molecular biology applications was obtained from a number of soil samples. Conclusions: The DNA extracted using the tested method successfully permitted the PCR amplification of a fragment of the bacterial 16S rDNA gene. The extracted DNA could also be restriction endonuclease digested. Significance and Impact of the Study: The protocol reported here is simple and permits rapid isolation of PCR‐ready soil DNA. The method requires only small quantities of soil sample, is scalable and suitable for automation.     

Testing the generation time hypothesis using DNA/DNA hybridization between artiodactyls

Heterogeneous rates of molecular change between some mammalian lineages are commonly explained by contrasts in generation time length. Here the generation time hypothesis is tested by comparing the relative rates of molecular change in related artiodactyl taxa differing by their generation time. A demographic model based on allometric relations with the adult body weight is used to estimate the cohort generation time in Bovidae and Cervidae families (Artiodactyla, Mammalia). Two pairs of closely related taxa (two cervids, two bovids) were selected, each showing clear ratios (1.5 to 3.5 times) in their generation time. Rates of genetic change in non-repeated nuclear DNA were estimated by DNA/DNA hybridization experiments performed among these ruminants and a camelid outgroup. Relative rate tests were applied to the two pairs of ingroup taxa differing by their generation time, in order to test if shorter generation time would correspond to higher rate of molecular change. Contradictory statistical results did not show a greater accumulation of nucleotide changes in the lineage leading to the short generation time species. The recorded differences in branch lengths of sister taxa were either conflicting or too small (relative to the contrasted generation times) to reveal a generation time effect. Alternative hypotheses are suggested to explain these preliminary results.     

In situ preparation of magnetic Fe3O4-chitosan nanoparticles for lipase immobilization by cross-linking and oxidation in aqueous solution

A new and simple method has been proposed to prepare magnetic Fe₃O₄-chitosan (CS) nanoparticles by cross-linking with sodium tripolyphosphate (TPP), precipitation with NaOH and oxidation with O₂ in hydrochloric acid aqueous phase containing CS and Fe(OH)₂, and these magnetic CS nanoparticles were used to immobilize lipase. The effects on the sequence of adding NaOH and TPP, the reaction temperature, and the ratio of CS/Fe(OH)₂ were studied. TEM showed that the diameter of composite nanoparticles was about 80 nm, and that the magnetic Fe₃O₄ nanoparticles with a diameter of 20 nm were evenly dispersed in the CS materials. Magnetic measurement revealed that the saturated magnetisation of the Fe₃O₄-CS nanoparticles could reach 35.54 emu/g. The adsorption capacity of lipase onto nanoparticles could reach 129 mg/g; and the maximal enzyme activity was 20.02 μmol min⁻¹ mg⁻¹ (protein), and activity retention was as high as 55.6% at a certain loading amount.     

Hepatic cellular distribution and degradation of iron oxide nanoparticles following single intravenous injection in rats: implications for magnetic resonance imaging

The purpose of this study was to determine the cellular distribution and degradation in rat liver following intravenous injection of superparamagnetic iron oxide nanoparticles used for magnetic resonance imaging (NC100150 Injection). Relaxometric and spectrophotometric methods were used to determine the concentration of the iron oxide nanoparticles and their degradation products in isolated rat liver parenchymal, endothelial and Kupffer cell fractions. An isolated cell phantom was also constructed to quantify the effect of the degradation products on the loss of MR signal in terms of decreased transverse relaxation times, T2*. The results of this study show that iron oxide nanoparticles found in the NC100150 Injection were taken up and distributed equally in both liver endothelial and Kupffer cells following a single 5 mg Fe/kg body wt. bolus injection in rats. Whereas endothelial and Kupffer cells exhibited similar rates of uptake and degradation, liver parenchymal cells did not take up the NC100150 Injection iron oxide particles. Light-microscopy methods did, however, indicate an increased iron load, presumably as ferritin/hemosiderin, within the hepatocytes 24 h post injection. The study also confirmed that compartmentalisation of ferritin/hemosiderin may cause a significant decrease in the MRI signal intensity of the liver. In conclusion, the combined results of this study imply that the prolonged presence of breakdown product in the liver may cause a prolonged imaging effect (in terms of signal loss) for a time period that significantly exceeds the half-life of NC100150 Injection iron oxide nanoparticles in liver.     

Quantitative analysis of DNA hybridization in a flowthrough microarray for molecular testing

Quantitative information about the nucleic acids hybridization reaction on microarrays is fundamental to designing optimized assays for molecular diagnostics. This study presents the kinetic, equilibrium, and thermodynamic analyses of DNA hybridization in a microarray system designed for fast molecular testing of pathogenic bacteria. Our microarray setup uses a porous, nylon membrane for probe immobilization and flowthrough incubation. The Langmuir model was used to determine the reaction rate constants of hybridization with antisense targets specific to Staphylococcus epidermidis and Staphylococcus aureus strains. The kinetic analysis revealed a sequence-dependent reaction rate, with association rate constants on the order of 10⁵ M⁻¹ s⁻¹ and dissociation rate constants of 10⁻⁴ s⁻¹. We found that by increasing the probe surface density from 10¹¹ to 10¹² molecules/cm², the hybridization rate and efficiency are suppressed while the melting temperature of the DNA duplex increases. The maximum fraction of hybridized capture probes at equilibrium did not exceed 50% for hybridization with antisense sequences and was below 6% for hybridization with long targets obtained from PCR. The van’t Hoff analysis of the temperature denaturation data showed that the DNA hybridization in our porous, flowthrough microarray is thermodynamically less favorable than the hybridization of the same sequences in solution.     

Nature of the Ln-Co magnetic interactions in compounds [Ln2Co3(C5H4NPO3)6] · 4H2O with open-framework structures

The reactions of Ln(NO₃)₃ · xH₂O, CoSO₄ · 7H₂O or ZnSO₄ · 6H₂O and 2-pyridylphosphonic acid under hydrothermal conditions result in heterometallic phosphonate compounds with formula [Ln₂M₃(C₅H₄NPO₃)₆] · 4H₂O (Ln₂M₃; M = Co^II or Zn^II; Ln = La^III, Ce^III, Pr^III, Nd^III, Sm^III, Eu^III, Gd^III, Tb^III, Dy^III). These compounds are isostructural and crystallize in a chiral cubic space group I2₁3. Each structure contains the {LnO₉} polyhedra and {MN₂O₄} octahedra which are connected by edge-sharing to form an inorganic open-framework structure with a 3-connected 10-gon (10, 3) topology. The nature of Ln^III-Co^II magnetic interactions in Ln₂Co₃ is investigated by a comparison with their Ln^III-Zn^II analogues. It is found that the Ln^III-Co^II interaction is weak antiferromagnetic for Ln = Ce and ferromagnetic for Ln = Sm, Gd, Tb and Dy. In the cases of Ln = Pr, Nd and Eu, no significant magnetic interaction is observed.     

Comparative study of three magnetic nano-particles (FeSO4, FeSO4/SiO2, FeSO4/SiO2/TiO2) in plasmid DNA extraction

Recent updates on Magnetic Nano-Particles (MNPs) based separation of nucleic acids have received more attention due to their easy manipulation, simplicity, ease of automation and cost-effectiveness. It has been indicated that DNA molecules absorb on solid surfaces via hydrogen-bonding, and hydrophobic and electrostatic interactions. These properties highly depend on the surface condition of the solid support. Therefore, surface modification of MNPs may enhance their functionality and specification. In the present study, we functionalized Fe₃O₄ nano-particle surface utilizing SiO₂ and TiO₂ layer as Fe₃O₄/SiO₂ and Fe₃O₄/SiO₂/TiO₂ and then compare their functionality in the adsorption of plasmid DNA molecules with the naked Fe₃O₄ nano-particles. The result obtained showed that the purity and amount of DNA extracted by Fe₃O₄ coated by SiO₂ or SiO₂/TiO₂ were higher than the naked Fe₃O₄ nano-particles. Furthermore, we obtained pH 8 and 1.5 M NaCl as an optimal condition for desorption of DNA from MNPs. The result further showed that, 0.2 mg nano-particle and 10 min at 55 °C are the optimal conditions for DNA desorption from nano-particles. In conclusion, we recommended Fe₃O₄/SiO₂/TiO₂ as a new MNP for separation of DNA molecules from biological sources.     

DNA hybridization detection in a microfluidic channel using two fluorescently labelled nucleic acid probes

A conceptually new technique for fast DNA detection has been developed. Here, we report a fast and sensitive online fluorescence resonance energy transfer (FRET) detection technique for label-free target DNA. This method is based on changes in the FRET signal resulting from the sequence-specific hybridization between two fluorescently labelled nucleic acid probes and target DNA in a PDMS microfluidic channel. Confocal laser-induced microscopy has been used for the detection of fluorescence signal changes. In the present study, DNA hybridizations could be detected without PCR amplification because the sensitivity of confocal laser-induced fluorescence detection is very high. Two probe DNA oligomers (5'-CTGAT TAGAG AGAGAA-TAMRA-3' and 5'-TET-ATGTC TGAGC TGCAGG-3') and target DNA (3'-GACTA ATCTC TCTCT TACAG GCACT ACAGA CTCGA CGTCC-5') were introduced into the channel by a microsyringe pump, and they were efficiently mixed by passing through the alligator teeth-shaped PDMS microfluidic channel. Here, the nucleic acid probes were terminally labelled with the fluorescent dyes, tetrafluororescein (TET) and tetramethyl-6-carboxyrhodamine (TAMRA), respectively. According to our confocal fluorescence measurements, the limit of detection of the target DNA is estimated to be 1.0 x 10(-6) to 1.0 x 10(-7)M. Our result demonstrates that this analytical technique is a promising diagnostic tool that can be applied to the real-time analysis of DNA targets in the solution phase.     

Dissecting the DNA damage response using functional genomics approaches in S. cerevisiae

The faithful transmission of genetic information from a mother to daughter cells can only occur if the integrity of the genome is preserved. DNA transactions within cells are tightly regulated to prevent DNA damage. When events that challenge genome integrity do occur, a complex web of DNA damage response pathways comes into play. Studies in model organisms have contributed significantly to the understanding of these pathways. In the last decade, the development of functional genomics techniques in S.cerevisiae has ushered in systematic approaches for the study of complex cellular processes. These methods have enabled the systematic interrogation of the DNA damage response.     

Analysis of GAA/TTC DNA triplexes using nuclear magnetic resonance and electrospray ionization mass spectrometry

The formation of a GAA/TTC DNA triplex has been implicated in Friedreich's ataxia. The destabilization of GAA/TTC DNA triplexes either by pH or by binding to appropriate ligands was analyzed by nuclear magnetic resonance (NMR) and positive-ion electrospray mass spectrometry. The triplexes and duplexes were identified by changes in the NMR chemical shifts of H8, H1, H4, 15N7, and 15N4. The lowest pH at which the duplex is detectable depends upon the overall stability and the relative number of Hoogsteen C composite function G to T composite function A basepairs. A melting pH (pHm) of 7.6 was observed for the destabilization of the (GAA)2T4(TTC)2T4(CTT)2 triplex to the corresponding Watson-Crick duplex and the T4(CTT)2 overhang. The mass spectrometric analyses of (TTC)6.(GAA)6 composite function(TTC)6 triplex detected ions due to both triplex and single-stranded oligonucleotides under acidic conditions. The triplex ions disappeared completely at alkaline pH. Duplex and single strands were detectable only at neutral and alkaline pH values. Mass spectrometric analyses also showed that minor groove-binding ligands berenil, netropsin, and distamycin and the intercalating ligand acridine orange destabilize the (TTC)6.(GAA)6 composite function (TTC)6 triplex. These NMR and mass spectrometric methods may function as screening assays for the discovery of agents that destabilize GAA/TTC triplexes and as general methods for the characterization of structure, dynamics, and stability of DNA and DNA-ligand complexes.     

Utilizing RNA/DNA hybridization to directly quantify mRNA levels in microbial fermentation samples

mRNA quantification has become a research hotspot. Quantitative real-time RT-PCR is a popular method but is known to lack precision. To rapidly monitor the kinetics of mRNA levels for the control of microbial fermentation processes, we developed an SYBR Green I-based universal method to directly quantify mRNA from fermentation samples. After total RNA was extracted, the mRNA was hybridized and protected by a longer DNA oligonucleotide. The probe length determined the strength of signal amplification. S1 nuclease and RNase A were used to remove excess probe, single-stranded RNA, and mis-matched RNA/DNA hybrids. Finally, the perfect-matched RNA/DNA hybrid was quantified by SYBR Green I dye. The conditions of liquid hybridization and enzyme digestion were systemically optimized. The kinetic tendency of phzC mRNA levels during phenazine-1-carboxylic acid fermentation was consistent with the results from MB hybridization in our previous report. The detection of mRNA levels of ten genes in Pseudomonas sp. M18G proved that this method is universal and feasible for mRNA quantification, and has great potential for application in mRNA quantification in various organisms.     

Two new genomes in the Oryza complex identified on the basis of molecular divergence analysis using total genomic DNA hybridization

The genus Oryza to which cultivated rice belongs has 24 species (2n = 24 or 48), representing seven genomes (AA, BB, CC, EE, FF, BBCC and CCDD). The genomic constitution of five of these species is unknown. These five species have been grouped into two species complexes, the tetraploid ridleyi complex (O. ridleyi, O.␣longiglumis) and the diploid meyeriana complex (O.␣granulata, O. meyeriana, O. indandamanica). To evaluate the genomic structure of these species in terms of divergence at the molecular level vis-à-vis other known genomes of Oryza, we used the total genomic DNA hybridization approach. Total genomic DNA (after restriction digestion) of 79 accessions of 23 Oryza species, 6 related genera, 5 outgroup taxa (2 monocots, 3 dicots) and 6 F₁s and BC₁s derived from crosses of O.␣sativa with wild species were hybridized individually with ³²P-labeled total genomic DNA from 12 Oryza species: O. ridleyi, O. longiglumis, O. granulata, O.␣meyeriana, O. brachyantha, O. punctata, O. officinalis, O. eichingeri, O. alta, O. latifolia, O. australiensis, and O.␣sativa. The labeled genomic DNAs representing the ridleyi and meyeriana complexes cross-hybridized best to all the accessions of their respective species, less to those representing other genomes of Oryza and related genera, and least to outgroup taxa. In general, the hybridization differential measured in terms of signal intensities was >50-fold under conditions that permit detection of 70–75% homologous sequences, both in the presence and in the absence of O. sativa DNA as competitor. In contrast, when total DNAs representing other Oryza genomes were used as probes, species of the O.␣ridleyi and O.␣meyeriana complexes did not show any significant cross-hybridization (<5%). These results demonstrate that the genome(s) of both of these complexes are highly diverged and distinct from all other known genomes of Oryza. We, therefore, propose new genomic designations for these two species complexes: GG for the diploid O. meyeriana complex and HHJJ for the allotetraploid O. ridleyi complex. The results also suggest that the uniqueness of these genomes is not restricted to species-specific highly repetitive DNA sequences, but also applies to dispersed sequences present in single or low to moderate copy numbers. Furthermore these appear to share relatively more genome-specific repeat sequences between themselves than with other genomes of rice. The study also demonstrates the potential of total genomic DNA hybridization as a simple but powerful tool, complementary to existing approaches, for ascertaining the genomic makeup of an organism. Received: 26 July 1996 / Accepted: 17 September 1996     

Effect of local sugar and base geometry on <Superscript>13</Superscript>C and <Superscript>15</Superscript>N magnetic shielding anisotropy in DNA nucleosides

Density functional theory was employed to study the dependence of 13C and 15N magnetic shielding tensors on the glycosidic torsion angle (chi) and conformation of the sugar ring in 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, and 2'-deoxythymidine. In general, the magnetic shielding of the glycosidic nitrogens and the sugar carbons was found to depend on both the conformation of the sugar ring and chi. Our calculations indicate that the magnetic shielding anisotropy of the C6 atom in pyrimidine and the C8 atom in purine bases depends strongly on chi. The remaining base carbons were found to be insensitive to both sugar pucker and chi re-orientation. These results call into question the underlying assumptions of currently established methods for interpreting residual chemical shift anisotropies and 13C and 15N auto- and cross-correlated relaxation rates and highlight possible limitations of DNA applications of these methods.     

Immobilization of Serratia marcescens lipase onto amino-functionalized magnetic nanoparticles for repeated use in enzymatic synthesis of Diltiazem intermediate

Magnetic Fe₃O₄ nanoparticles were prepared by chemical coprecipitation method and subsequently coated with 3-aminopropyltriethoxysilane (APTES) via silanization reaction. The synthesized materials were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). With glutaraldehyde as the coupling agent, the lipase from Serratia marcescens ECU1010 (SmL) was successfully immobilized onto the amino-functionalized magnetic nanoparticles. The results showed that the immobilized protein load could reach as high as 35.2 mg protein g⁻¹ support and the activity recovery was up to 62.0%. The immobilized lipase demonstrated a high enantioselectivity toward (+)-MPGM (with an E-value of 122) and it also displayed the improved thermal stability as compared to the free lipase. When the immobilized lipase was employed to enantioselectively hydrolyze (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM] in water/toluene biphasic reaction system for 11 consecutive cycles (totally 105 h), still 59.6% of its initial activity was retained, indicating a high stability in practical operation.     

In situ hybridization to metaphase chromosomes in six species ofPhaseolus andVigna using ribosomal DNA as the probe

In situ hybridization with a biotin-labeled rice ribosomal DNA (rDNA) probe to the somatic metaphase chromosomes of six species ofPhaseolus andVigna (P. angularis, P. calcaratus, P. coccineus, P. vulgaris, V. sesquipedalis andV. sinensis) was done to determine the sites of rDNA. Hybridization signals were present in the terminal and subterminal chromosome regions of each of the six species. The number of rDNA sites was two inP. angularis andP. calcaratus, four inP. coccineus andP. vulgaris, and six inV. sesquipedalis andV. sinensis.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30 kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.