Fluorescent detection of single nucleotide polymorphism utilizing a hairpin DNA containing a nucleotide base analog pyrrolo-deoxycytidine as a fluorescent probe

A novel fluorescent method for the detection of single nucleotide polymorphism (SNP) was developed using a hairpin DNA containing nucleotide base analog pyrrolo-deoxycytidine (P-dC) as a fluorescent probe. This fluorescent probe was designed by incorporating a fluorescent P-dC into a stem of the hairpin DNA, whose sequence of the loop moiety complemented the target single strand DNA (ss-DNA). In the absence of the target ss-DNA, the fluorescent probe stays a closed configuration in which the P-dC is located in the double strand stem of the fluorescent probe, such that there is weak fluorescence, attributed to a more efficient stacking and collisional quenching of neighboring bases. In the presence of target ss-DNA, upon hybridizing the ss-DNA to the loop moiety, a stem-loop of the fluorescent probe is opened and the P-dC is located in the ss-DNA, thus resulting in strong fluorescence. The effective discrimination of the SNP, including single base mismatch ss-DNA (A, T, G) and double mismatch DNA (C, C), against perfect complementary ss-DNA was achieved by increased fluorescence intensity, and verified by thermal denaturation and circular dichroism spectroscopy. Relative fluorescence intensity had a linear relationship with the concentration of perfect complementary ss-DNA and ranged from 50 nM to 3.0 μM. The linear regression equation was F/F₀ = 2.73 C (μM) + 1.14 (R = 0.9961) and the detection limit of perfect complementary ss-DNA was 16 nM (S/N = 3). This study demonstrates that a hairpin DNA containing nucleotide base analog P-dC is a promising fluorescent probe for the effective discrimination of SNP and for highly sensitive detection of perfect complementary DNA.     

DNA assay based on monolayer-barcoded nanoparticles for mass spectrometry in combination with magnetic microprobes

Mass spectrometry (MS) based methodology offers simple, fast and sensitive diagnosis. While it has become the predominate approach in biomolecular analysis, it has not been suitable for analyzing nucleic acid due to its low ionization efficiency. We report herein on a DNA assay based on monolayer-barcoded nanoparticles that were encoded with reporter mass molecules, which act as surrogate molecules for the matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF MS) identification of target DNA through mass spectrometry in combination with magnetic microprobes. This assay demonstrated high MS sensitivity, with the ability to detect target DNA at femtomolar (10⁻¹⁵ M) levels. This inaugural effort using combined techniques is significant because it showed an extraordinary analytical capability for differentiating the single nucleotide polymorphism (SNP), which comprises the most abundant source of genetic variation in the human genome. We also report herein the feasibility of MS detection of two target DNAs that have the same mass but different nucleotide base composition, which classic MS methodology is inherently unable to differentiate.     

Control of surface wettability and photomicropatterning with a polymorphic diarylethene crystal upon photoirradiation

A photochromic diarylethene crystal of 1,2‐bis(2‐methyl‐6‐nitro‐1‐benzothiophen‐3‐yl)perfluorocyclopentene ( 1 a ) was found to undergo a thermodynamic phase transition at 180 °C to form a needle‐like crystal, designated as 1 a‐γ . The phase transition involves melting of the initial α‐crystal and growth of the γ‐crystal. The phase transition temperature decreased with the presence of the closed‐ring isomer ( 1 b ) in the crystal because of the decrease in the melting temperature. Upon irradiation with ultraviolet (UV) light, compound 1 a in the α‐crystal was converted into 1 b to an extent of 20 %. Consequently, the α‐crystal containing 20 % of 1 b underwent the phase transition accompanied by melting of the crystal and growth of the γ‐crystal even at 170 °C. Photomicropatterning by the phase transition upon irradiation with UV light using a photomask, followed by heating at 170 °C, was successfully accomplished with a resolution in the microcrystalline pattern of about 20 μm. The contact angle with water on the γ‐microcrystalline phase on a glass substrate was larger than that on the α‐microcrystalline phase by 20°. This can be ascribed to a difference in the roughness of the surface. Furthermore, the γ‐microcrystal was also found to be formed upon heating an amorphous film of 1 a in poly(methyl methacrylate) for 2 min at 130 °C. The crystallized area exhibited a higher water contact angle than the amorphous area. Upon irradiation of the amorphous film with UV light, such crystallization did not take place because of the impurity effect of 1 b in 1 a . Photomicropatterning by the crystallization in the polymer showed a pattern with a higher resolution of about 4 μm, which was much better than that of the neat crystal. This photopatterning process represents a useful tool for controlling the surface wettability in relevant applications.     

Colorimetric detection of DNA by modulation of thrombin activity on gold nanoparticles

A colorimetric, non-cross-linking aggregation-based gold-nanoparticle (AuNP) probe has been developed for the detection of DNA and the analysis of single-nucleotide polymorphism (SNP). The probe acts by modulating the enzyme activity of thrombin relative to fibrinogen. A thrombin-binding aptamer with a 29-base-long oligonucleotide (TBA(29)) assembled on the nanoparticles (TBA(29)-AuNPs) through sandwich DNA hybridization was found to possess ultra-high anticoagulant potency. The enzyme inhibition of thrombin was determined by thrombin-induced aggregation of fibrinogen-functionalized 56 nm AuNPs (Fib-AuNPs). The potency of the inhibition of TBA(29)-AuNPs relative to thrombin--and thus the degree of aggregation of the Fib-AuNPs--is highly dependent on the concentration of perfectly matched DNA (DNA(pm)). Under optimal conditions [Tris-HCl (20 mM, pH 7.4), KCl (5 mM), MgCl(2) (1 mM), CaCl(2) (1 mM), NaCl (150 mM), thrombin (10 pM), and TBA(29)-AuNPs (20 pM)], the new TBA(29)-AuNP/Fib-AuNP probe shows linear sensitivity to DNA(pm) in the concentration range 20-500 pM with a correlation coefficient of 0.96. The limit of detection for DNA(pm) was experimentally determined to be 12 pM, based on a signal-to-noise ratio (S/N) of 3. The new probe was successfully applied to the analysis of an SNP that is responsible for sickle cell anemia. Relative to conventional molecular-beacon-based probes, the new probe offers the advantages of higher sensitivity and selectivity towards DNA and lower cost, showing its great potential for practical studies of SNPs.     

聚合酶链式反应-限制性片段长度多态性和芯片生物分析技术用于渤海地区鱼种鉴定

以渤海湾具有重要商业价值的9种海鱼(小黄鱼、花鲈、蓝点马鲛、鲐、钝吻黄盖鲽、棘头梅童鱼、许氏平鲉、高眼鲽和长蛇鲻)为研究对象,利用聚合酶链式反应(PCR)-限制性片段长度多态性(RFLP)和芯片生物分析技术对其进行鱼种鉴定。首先提取其基因组脱氧核糖核酸(DNA),对其细胞色素b的特定片段(464 bp)进行扩增,然后选择DdeI、HaeIII和NlaIII3种限制性内切酶进行酶切,并利用芯片生物分析技术得到酶切产物的特异图谱和确切的片段大小,从而有效区分了9种海鱼。研究结果表明,PCR-RFLP和芯片生物分析技术在鱼种鉴定上具有精确、鉴别和快速三大优势,可为鱼类食品检测提供科学依据。     

Single Molecule Genotyping by TIRF Microscopy

As part of a programme to develop a metrological framework for single molecule measurements in biology, we have investigated the applications of single molecule imaging to genomics. Specifically, we have developed a technique for measuring the frequencies of single nucleotide polymorphisms (SNPs) in complex or pooled samples of DNA. We believe that this technique has applications to statistical genotyping—the identification of correlations between SNP frequencies and particular phenotypes—and other areas where it is desirable to track the frequencies of SNPs in complex DNA populations.     

Behavior of LiOH·H2O crystals obtained by evaporation and by drowning out

In the present work, the behavior of crystals derived from two different crystallization methods applied in a concentrated aqueous lithium salt solution was studied. The LiOH·H₂O crystals obtained by a simple evaporation (Crystal I) differed in terms of morphology and solubility from those precipitated from lithium hydroxide solutions by addition of ethanol as a co‐solvent (Crystal II). Solubility of Crystal II at different temperatures (15 to 35°C) and mass ratios of ethanol to water (0 to 0.1) was determined. Polymorphic like behavior of these crystals was evidenced from X‐ray diffraction patterns. Measurement of density, refractive index, absolute viscosity and electrical conductivity of saturated solutions are reported. A thermodynamic analysis in terms of the Chen model for the calculation of activity coefficients, indicate that the polymorphic system in water and in water + ethanol (ethanol/water ratio 0.1) is enantiotropic. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

(E,E)-1,1′-[1,2-Bis(4-chlorophenyl)ethane-1,2-diyl]bis(phenyldiazene) revisited: threefold configurational disorder of (S,S), (R,R) and (S,R) isomers,a detailed critique

Crystal structures described as concomitant triclinic ( I ) and monoclinic ( II ) polymorphs of meso-(E,E)-1,1′-[1,2-bis(4-chlorophenyl)ethane-1,2-diyl]bis(phenyldiazene) [Mohamed et al. (2016). Acta Cryst. C 72 , 57–62] have been re-investigated. The published model for II was distorted due to forcing the symmetry of space group C2/c on an incomplete structure model. It is shown here to be a likely three-component superposition of S,S and R,R enantiomers with a lesser amount of the meso form. A detailed analysis of how the improbable distortion in the published model aroused suspicion and the subsequent construction of undistorted chemically and crystallographically plausible alternatives having the symmetry of Cc and C2/c is presented. For the sake of completeness, an improved model for the triclinic P structure of the meso isomer I , revised to include a minor disorder component, is also given.     

Single Base Pair Mismatch Detection Using Cationic Conjugated Polymers through Fluorescence Resonance Energy Transfer

A simple method using water‐soluble conjugated polymers and a DNA intercalator has been proposed for single base pair mismatch detection with enhanced detection efficiency. Fluorescence resonance energy transfer (FRET) was used as an indicator for unwinding of dsDNA due to base pair mismatch at elevated temperatures. The optical amplification effect of the CPs helps to achieve enhanced detection efficiency.