A novel phosphoramidite method for automated synthesis of oligonucleotides on glass supports for biosensor development

Two protocols for functionalization of glass supports with hexaethylene glycol (HEG)-linked oligonucleotides were developed. The first method (standard amidite protocol) made use of the 2-cyanoethyl-phosphoramidite derivative of 4,4′-dimethoxytrityl-protected HEG. This was first coupled to the support by standard solid-phase phosphoramidite chemistry followed by extension with a thymidylic acid icosanucleotide. Stepwise addition of the linker phosphoramidite graduated at 1% (relative to the total sites available) perstep at 50°C resulted in an optimal yield of immobilized oligonucleotides at a density of 2.24 × 10¹⁰ strands/mm². This observed loading maximum lies well below the theoretical maximum loading owing to nonspecific adsorption of HEG on the glass and subsequent blocking of reactive sites. Surface loadings as high as 3.73 × 10¹⁰/mm² and of excellent sequence quality were achieved with a reverse amidite protocol. The support was first modified into a 2-cyanoethyl-N,N-diisopropylphosphoramidite analog followed by coupling with 4,4′-dimethoxytrityl-protected HEG. This protocol is conveniently available when using a conventional DNA synthesizer. The reverse amidite protocol allowed for control of the surface loading at values suitable for subsequent analytical applications that make use of immobilized oligonucleotides as probes for selective hybridization of sample nucleic acids of unknown sequence and concentration.     

直接键连原子间的NMR偶合常数的自然杂化轨道研究V.~(13)C-~(13)C和~(13)C-~(31)P核自旋偶合常数~1J_(CC)和~1J_(CP)

在前文工作的基础上，结合ＭＮＤＯ／ＥＨＭＯ分子轨道方法和自然杂化轨道方法，具体计算了ＣＣ键和ＣＰ键的核自旋偶合常数．计算结果表明，１ＪＣＣ和１ＪＣＰ主要由成键原子的轨道杂化作用和键极性这两种结构因素所决定．为从简单价键理论角度解释和计算１ＪＣＣ和１ＪＣＰ值提供了简便直观的方法．     

Measurement of spin alignment for the 15N32 (6.33 MeV) state from the (16O, 15N) reaction using γ-recoil in flight

High resolution for the ²⁷Al(¹⁶O, ¹⁵N)²⁸Si reaction were measured with a Q3D magnetic spectrometer. The broadened line shape of the ${}^{15}\text{N}{}_{\mathrm{<mtext>3</mtext><mtext>2</mtext>}}$ (6.33 MeV) state after γ-decay in flight was analysed. The m-state population of this state was deduced taking the E2/M1 mixing ratio of δ = +0.1 into account. The connection between spin alignment and the dynamics of the transfer process is discussed.     

Influences of non-selective interactions of nucleic acids on response rates of nucleic acid fiber optic biosensors

The immobilization of oligonucleotides to solid surfaces can provide a platform of chemistry that is suitable for the development of biosensor and microarray technologies. Experiments were performed using a fiber optic nucleic acid biosensor based on total internal reflection fluorescence to examine the effects of the presence of non-complementary DNA on the detection of hybridization of complementary target DNA. The work has focused on the rates and extent of hybridization in the presence and absence of non-selective adsorption using fluorescein-labeled DNA. A stop-flow system of 137 microL volume permitted rapid introduction and mixing of each sample. Response times measured were on the order of seconds to minutes. Non-selective adsorption of non-complementary oligonucleotides (ncDNA) was found to occur at a significantly faster rate than hybridization of complementary oligomers (cDNA) in all cases. The presence of ncDNA oligonucleotides did not inhibit selective interactions between immobilized DNA and cDNA in solution. The presence of high concentrations of non-complementary genomic DNA had little effect on the extent of hybridization of complementary oligonucleotides, but actually reduced the response times of sensors to cDNA oligonucleotides.     

具有切向控制的局部四点插值曲线设计方法

Ｎｉｒａ Ｄｙｎ等提出的四点插值法是一种典型的自由曲线离散造型方法，但该方法不能控制插值点的切向。本文利用薄板样很可能 量的极小化原理给出了具有切向控制的四点分插值条件。用户可以方便地交互控制任一插值点的切向，使得四点插值法更为有效和实用。     

Influences of non-selective interactions of nucleic acids on response rates of nucleic acid fiber optic biosensors

The immobilization of oligonucleotides to solid surfaces can provide a platform of chemistry that is suitable for the development of biosensor and microarray technologies. Experiments were performed using a fiber optic nucleic acid biosensor based on total internal reflection fluorescence to examine the effects of the presence of non-complementary DNA on the detection of hybridization of complementary target DNA. The work has focused on the rates and extent of hybridization in the presence and absence of non-selective adsorption using fluorescein-labeled DNA. A stop-flow system of 137 μL volume permitted rapid introduction and mixing of each sample. Response times measured were on the order of seconds to minutes. Non-selective adsorption of non-complementary oligonucleotides (ncDNA) was found to occur at a significantly faster rate than hybridization of complementary oligomers (cDNA) in all cases. The presence of ncDNA oligonucleotides did not inhibit selective interactions between immobilized DNA and cDNA in solution. The presence of high concentrations of non-complementary genomic DNA had little effect on the extent of hybridization of complementary oligonucleotides, but actually reduced the response times of sensors to cDNA oligonucleotides. Received: 26 September 2000 / Revised: 24 November 2000 / Accepted: 30 November 2000     

Spin alignment and polarisation in the (16O,15N) transfer reactions

The spin alignment of the¹⁵N ₃ ^2/* (6.33 MeV) state was measured in the⁸⁸Sr(¹⁶O,¹⁵N)⁸⁹Y reaction atE _L =96 MeV using theγ-recoil method. Angular distributions of excited states in¹⁵N and⁸⁹Y were measured with high accuracy. The analysis in terms of DWBA shows that the spin alignment is correctly described by the usual reaction models. The polarisation of the outgoing¹⁵N_1/2(GS) and¹⁵N ₃ ^2/* (6.33 MeV) is discussed. It is shown that cross section differences for transitions to final states with different configurations are sensitive to a spin-orbit potential of¹⁵N. The strength and sign of the spin-orbit potential for¹⁵N is determined.     

Them-state population of12C2+ in inelastic scattering

The spin alignment of the¹²C₂ ⁺ state in inelastic scattering of¹²c on⁶⁴zn at E_L=65 MeV has been measured using the γ-recoil-in-flight method. Good agreement with coupled channel calculations are obtained. The sensitivity of the spin alignment to reaction models is discussed.     

Considerations for the quantitative transduction of hybridization of immobilized DNA

Immobilized single-stranded DNA (ssDNA) can be used as a selective ‘reagent’ to bind complementary DNA or RNA for applications such as the detection of pathogenic organisms, gene therapy agents and genetic mutations. The density of ssDNA on a surface will determine the charge density due to ionizable phosphate groups. Such a negatively charged interface will attract positive counter-ions from solution, which may result in a local ionic strength, pH and dielectric constant on the surface that is substantially different from that in bulk electrolyte solution. It is the local conditions which influence the thermodynamics of hybridization, and this can studied by the melt temperature (T_m) of double-stranded DNA (dsDNA). Experimental work and theoretical models have been used to examine whether hybridization reactions on a surface can cause dynamic changes in local charge density, and therefore, changes in selectivity and drift in calibration for quantitative analysis. Organosilane chemistry has been used to covalently immobilize hexaethylene glycol linkers and to control the subsequent density of dT₂₀ that was prepared by automated synthesis. Fiber-optic biosensors based on fused silica that was coated with DNA were used in a total internal reflection fluorescence instrument to determine T_m from the dissociation of duplexes of fluorescein-labeled dA₂₀ : dT₂₀. The experimental results suggest that the thermodynamic stability of duplexes that are immobilized on a surface is dependent on the density of immobilized DNA and on the extent of hybridization of DNA. The experimental results show that the thermodynamic stability of immobilized dsDNA is significantly different than that of dsDNA in bulk solution, and include observations of the variation of enthalpy at different ionic strengths, asymmetry in the melt curves, and the possibility of a reduced dielectric constant within a DNA layer relative to that in bulk solution.     

Measurement of spin-alignment of excited12C2+ nuclei in interactions of16O with12C usingγ-decay in flight

Measuring energy spectra of nuclei afterγ-decay of excited states in flight the spin alignment of¹²C₂₊ states has been measured. Inelastic scattering,¹⁶O(¹⁶C,¹²C₂₊)¹⁶O and the reaction¹²C(¹⁶O,¹²C₂₊)¹⁶O leading to¹²C₂₊ (4.43 MeV) state have been studied. Characteristic line shapes of the¹²C₂₊ peak were observed using a Q3D magnetic spectrometer. The magnetic substate (m-states) population has been deduced from the spectra as function of reaction angle. A comparison of the measuredm-state population with reaction models shows that the first reaction is consistent with inelastic scattering although discrepancies remain. Discrepancies are also obtained if the reaction¹²C(¹⁶O,¹²C₂₊)¹⁶O is interpreted using a FRDWBA transfer calculation. At least 1/3 of the cross section can be attributed toα-transfer. A calculation which couples transfer and inelastic scattering channels seems to be necessary.