Snthesis of dna fragmenents linked to a solid support

Two simple procedures for the preparation of DNA fragments covalently and specifically linked to a solid support are presented. The first method consists of the preparation of a nucleoside primer which serves as the initiative site for conventional synthesis of oligomers in either 3' or 5' direction. The second procedure involves the direct attachment of independently synthesized and purified oligomers to a functionalized solid support. The accessibility of such supported oligodeoxynucleotides to enzymes is checked with restriction endonucleases.     

α-(Phenylacetamido)benzylpolystyrene (pab-resin) : A new polymeric support for peptide synthesis with improved acid stability

Solid phase peptide synthesis carried out on conventional chloromethyl polystyrene support has the disadvantage of the acidolysis of the peptide-resin linkage during the deprotection step. In addition, when trifluoroacetic acid is used as deprotecting agent, intrapolymeric trifluoroacetyl transfer may give rise to truncated trifluoroacetylated peptides. A new polymer support devised to minimize these problems, α - (4 - chloromethyl-phenylacetamido) benzylcopoly(styrene-1%-divinylbenzene) (ClCH₂-Pab-resin), has been synthesized. The acetamido bridge between the peptide and the resin increases the acid stability of the new support thus allowing the synthesis of purer peptides in higher yields. Boc-aminoacyl-OCH₂-Pab-resins can be readily prepared in a versatile way by reaction of Boc-amino acid caesium salts with chloromethyl-Pab-resin. This fact contrasts with the rather elaborate procedures required for previously described similar resins.The synthetic viability of the chloromethyl-Pab-resin is shown by the synthesis of Ac-His-Arg-Tyr-Arg-Pro-OH (fragment 39–43 of histone H3). The synthesis and subsequent purification are exhaustively compared with the parallel synthesis carried out on a standard chloromethyl polystyrene support. The presence of the acetamido linkage provokes, as expected, a decrease in the HF cleavage yield. Nevertheless, this disadvantage is balanced by the increase in global purification yields even for the synthesis of a pentapeptide for which only five deprotection steps are required.     

Thermolytic properties of 3-(2-pyridyl)-1-propyl and 2-[N-methyl-N-(2-pyridyl)]aminoethyl phosphate/thiophosphate protecting groups in solid-phase synthesis of oligodeoxyribonucleotides

Thermolytic groups may serve as alternatives to the conventional 2-cyanoethyl group for phosphate/thiophosphate protection in solid-phase oligonucleotide synthesis to prevent DNA alkylation by acrylonitrile generated under the basic conditions used for oligonucleotide deprotection. Additionally, thermolytic groups are attractive in the context of engineering a "heat-driven" process for the synthesis of oligonucleotides on diagnostic microarrays. In these regards, the potential application of pyridine derivatives as thermolytic phosphate/thiophosphate protecting groups has been investigated. Specifically, 2-pyridinepropanol and 2-[N-methyl-N-(2-pyridyl)]aminoethanol were incorporated into deoxyribonucleoside phosphoramidites 7a-d and 9, which were found as efficient as 2-cyanoethyl deoxyribonucleoside phosphoramidites in solid-phase oligonucleotide synthesis. Whereas the removal of 3-(2-pyridyl)-1-propyl phosphate/thiophosphate protecting groups from oligonucleotides is effected within 30 min upon heating at 55 degrees C in concentrated NH4OH or in an aqueous buffer at pH 7.0, cleavage of 2-[N-methyl-N-(2-pyridyl)]aminoethyl groups occurs spontaneously when their phosphate or phosphorothioate esters are formed during oligonucleotide synthesis. The deprotection of these groups follows a cyclodeesterification process generating the bicyclic salts 13 and 14 as side products. These salts do not alkylate or otherwise modify any DNA nucleobases and do not desulfurize a phosphorothioate diester model under conditions mimicking large-scale oligonucleotide deprotection.     

Enhancing the fluorescence intensity of DNA microarrays by using cationic surfactants

DNA microarrays have been used as powerful tools in genomics studies and single nucleotide polymorphisms analysis. However, the fluorescence detection used in most conventional DNA microarrays is still limited by its sensitivity. The aim of this study is to use a cationic surfactant, cetyl trimethylammonium bromide (CTAB), to enhance the fluorescence intensity of 6-carboxy-fluorescene (FAM)-labeled DNA probes immobilized on a DNA microarray. We show that in the presence of CTAB the immobilized FAM-labeled DNA probes is 11-fold brighter than that without exposure to CTAB. Similarly, when we hybridize FAM-labeled DNA targets to a DNA microarray and treat the surface with CTAB solution, the fluorescence intensity shows a 26-fold increase for perfect-match DNA targets. More importantly, the contrast between perfect-match and 1-mismatch DNA is also increased from 1.3-fold to 15-fold. This method offers a simple and efficient technique to enhance the detection limit of DNA microarrays.     

Direct immobilization of DNA probes on non-modified plastics by UV irradiation and integration in microfluidic devices for rapid bioassay

DNA microarrays have become one of the most powerful tools in the field of genomics and medical diagnosis. Recently, there has been increased interest in combining microfluidics with microarrays since this approach offers advantages in terms of portability, reduced analysis time, low consumption of reagents, and increased system integration. Polymers are widely used for microfluidic systems, but fabrication of microarrays on such materials often requires complicated chemical surface modifications, which hinders the integration of microarrays into microfluidic systems. In this paper, we demonstrate that simple UV irradiation can be used to directly immobilize poly(T)poly(C)-tagged DNA oligonucleotide probes on many different types of plastics without any surface modification. On average, five- and fourfold improvement in immobilization and hybridization efficiency have been achieved compared to surface-modified slides with aminated DNA probes. Moreover, the TC tag only costs 30% of the commonly used amino group modifications. Using this microarray fabrication technique, a portable cyclic olefin copolymer biochip containing eight individually addressable microfluidic channels was developed and used for rapid and parallel identification of Avian Influenza Virus by DNA hybridization. The one-step, cost-effective DNA-linking method on non-modified polymers significantly simplifies microarray fabrication procedures and permits great flexibility to plastic material selection, thus making it convenient to integrate microarrays into plastic microfluidic systems.     

A new hydrophobic linker effective for the in situ synthesis of DNA-CPG conjugates as tools for SNP analysis

DNA chips consisting of DNA oligonucleotide probes immobilized on the surface of solid supports are very powerful tools for rapid analysis of multiple samples. In this Letter we describe a new method for the efficient synthesis of DNA probes without their serious elimination by use of a new hydrophobic 16-hyroxydecanoic linker and a new non-aqueous reagent of MeNH₂/THF for the deprotection of the base and phosphate protecting groups on CPG resins. The elimination of DNA probes in this new method could be suppressed more than 20-fold compared with the previous method using a hexaethylene glycol linker and concd NH₄OH. Moreover, we carried out SNPs detection by use of our DNA-CPG conjugate to show the utility of our new linker and deprotection conditions.     

Preparation of N3-thymidine–butylene–N3-thymidine interstrand cross-linked DNA via an orthogonal deprotection strategy

A DNA duplex containing an N3-thymidine–butylene–N3-thymidine interstrand cross-link (ICL) was prepared using an on-column orthogonal deprotection strategy to permit different nucleotide sequence composition around the cross-linked site. The conditions used to remove 5′-O-allyloxycarbonyl and 3′-O-tert-butyldimethylsilyl protective groups for various on-column oligonucleotide intermediates did not affect the cross-linked lesion. Efficient removal of these groups enabled successful coupling of 2′-deoxyphosphoramidites to produce the desired duplex with a 31% yield after deprotection and purification.     

DNA微阵列原位合成中的氧化剂体系改进

DNA芯片的制备方法主要有离片合成法和在片合成法．离片合成是用点样法将DNA探针固定在基片上;在片合成是在基片表面直接原位合成寡核苷酸探针阵列．目前国外已有多种DNA芯片原位合成方法的报道,但其中仅Fordor等提出的光脱保护DNA固相原位合成技术已商品化,该方法制备成本高,     

Isostable DNA

The high fidelity detection of multiple DNA sequences in multiplex assays calls for duplexes whose stability is independent of sequence (isostable DNA), forming under universally stringent conditions. Nature did not evolve DNA to form isostable duplexes. Here we report how probe strands can be modified so that an all-A/T target strand is bound with the same or slightly higher affinity than the corresponding all-G/C strand with the same sequence of purines and pyrimidines. We refer to these probes that feature covalently attached ligands as "decorated nucleic acids". Caps, intercalators, and locks were used to stabilize A/T duplexes, and N4-ethylcytosine residues were employed to tune down the stability of G/C duplexes without significantly affecting base pairing selectivity. Near-isostability was demonstrated in solution and on microarrays of high and low density. Further, it is shown that hybridization results involving decorated probes on microarrays can be predicted on the basis of thermodynamic data for duplex formation in solution. Predictable formation of isostable DNA not only benefits microarrays for gene expression analysis and genotyping, but may also improve the sequence-specificity of other applications that rely on the massively parallel formation of Watson-Crick duplexes.     

Synthesis of indoles via 6pi-electrocyclic ring closures of trienecarbamates

A new method for the preparation of indoles from readily available alpha-haloenones and alpha-(trialkylstannyl)enecarbamates is described. Following a Stille coupling, trienecarbamate 2 is electronically activated to undergo a facile 6pi-electrocyclic ring closure and subsequent oxidation to afford protected aniline 4. Upon deprotection and reductive amination, acid 5 underwent clean cyclization to N-acetylindole 6 (Ac2O, NEt3, 130 degrees C). This method has been used to construct a variety of substituted indoles that are not easily prepared by conventional indole annelation methods.     

Fabrication of DNA microarrays on nanoengineered polymeric ultrathin film prepared by self-assembly of polyelectrolyte multilayers

Microarray-based technology is in need of flexible and cost-effective chemistry for fabrication of oligonucleotide microarrays. We have developed a novel method for the fabrication of oligonucleotide microarrays with unmodified oligonucleotide probes on nanoengineered three-dimensional thin films that are deposited on glass slides by consecutive layer-to-layer adsorption of polyelectrolytes. Unmodified oligonucleotide probes were spotted and immobilized on these multilayered polyelectrolyte thin films (PET) by electrostatic adsorption and entrapment on the porous structure of the PET film. The PET provides higher probe binding capacity and thus higher hybridization signal than that of the traditional two-dimensional aminosilane and poly-L-lysine coated slides. Immobilized probe densities of 3.4 x 10(12)/cm2 were observed for microarray spots on PET with unmodified 50-mer oligonucleotide probes, which is comparable to the immobilized probe densities of alkyamine-modified 50-mer probes end-tethered on an aldehyde-functionalized slide. The study of hybridization efficiency showed that 90% of immobilized probes on PET film are accessible to target DNA to form duplex format in hybridization. The DNA microarray fabricated on PET film has wider dynamic range (about 3 orders of magnitude) and lower detection limit (0.5 nM) than the conventional amino- and aldehyde-functionalized slides. Oligonucleotide microarrays fabricated on these PET-coated slides also had consistent spot morphology. In addition, discrimination of single nucleotide polymorphism of 16S rRNA genes was achieved with the PET-based oligonucleotide microarrays. The PET microarrays constructed by our self-assembly process is cost-effective, versatile, and well suited for immobilizing many types of biological active molecules so that a wide variety of microarray formats can be developed.     

A convenient synthesis of a 2,7-difunctional tetra(alkoxy)triphenylene involving 4,4′-diacetoxy-3,3′-dialkoxybiphenyl as a key precursor and its conversion to extended hybrid mesogenic compounds

A new rational pathway to 2,7-difunctionalised-β-hexa-substituted triphenylenes is presented, requiring less protection/deprotection and purification steps than more conventional synthetic procedures in the framework of the ‘biphenyl route’. Main improvements are deprotection via alkaline hydrolysis of an ester in ethanol/water medium instead of using toxic and pyrophoric reagents like lithium diphenylphosphide, and the use of easily prepared brominated precursors instead of iodinated reagents for biphenyl synthesis. 4,4′-Diacetoxy-3,3′-bis(hexyloxy)biphenyl has been synthesised under this scheme, and characterised by proton nuclear magnetic resonance (¹H NMR) spectrometry, elemental analysis and single-crystal crystallography. It crystallises in the P-1 space group, and exhibits a layered structure built-up through dipolar, C–H … π and C–H … O=C non-covalent interactions. This compound has been oxidatively coupled with 1,2-bis(hexyloxy)benzene to yield 2,7-dihydroxy-3,6,10,11-tetrakis(hexyloxy)triphenylene, a non-mesogen key precursor for the synthesis of the corresponding liquid-crystalline 2,7-difunctional triphenylenes. Indeed, a reactive 2,7-difunctional mesogen was prepared and used to produce new triphenylene-siloxane hybrid monomeric, trimeric and polymeric mesogens. All of them exhibited columnar hexagonal (Col_h) mesophases.     

Optimization of on-chip elongation for fabricating double-stranded DNA microarrays

The sequence-specific recognitions between DNA and proteins are playing important roles in many biological functions. The double-stranded DNA microarrays (dsDNA microarrays) can be used to study the sequence-specific recognitions between DNAs and proteins in highly parallel way. In this paper, two different elongation processes in forming dsDNA from the immobilized oligonucleotides have been compared in order to optimize the fabrication of dsDNA microarrays: (1) elongation from the hairpins formed by the self-hybridized oligonucleatides spotted on a glass; (2) elongation from the complementary primers hybridized on the spotted oligonucleatides. The results suggested that the dsDNA probes density produced by the hybridized-primer extension was about four times lower than those by the self-hybridized hairpins. Meanwhile, in order to reduce the cost of dsDNA microarrays, we have replaced the Klenow DNA polymerase with Taq DNA polymerase, and optimized the reaction conditions of on-chip elongation. Our experiements showed that the elongation temperature of 50 °C and the Mg²⁺ concentration of 2.5 mM are the optimized conditions in elongation with Taq DNA polymerase. A dsDNA microarray has been successfully constructed with the above method to detect NF-kB protein.     

Microarray sampling-platform fabrication using bubble-jet technology for a biochip system

The fabrication of microarrays containing PCR-amplified genomic DNA extracts from mice tumors on a Zetaprobe membrane using a modified thermal ink-jet printer is described. A simple and cost-effective procedure for the fabrication of microarrays containing biological samples using a modified bubble-jet printing system is presented. Because of their mass-produced design, ink-jet printers are a much cheaper alternative to conventional spotting techniques. The usefulness of the biochip microarray platform is illustrated by the detection of human fragile histidine triad (FHIT), a tumor suppressor gene. Subcutaneous carcinomas were induced with MKN/FHIT and MKN/E4 cell lines in immunodeficient mice. Several weeks into their development, the tumors from both groups of mice were removed and subjected to DNA extraction by lysis of tissue samples. The extracted DNA samples were amplified by PCR (30 cycles) using the primers corresponding to nucleotides 2 to 18 of the FHIT sequence. The resulting solution was transferred to the individual reservoirs of a three-color cartridge from a conventional thermal ink-jet printer (HP 694C), and arrays were printed on to a Zetaprobe membrane. After spotting, these membranes were used in a hybridization assay, using fluorescent probes, and detected with a biochip.     

A practical approach for the chemical synthesis of 2′-deoxyguanosine-C8 adducts with mutagenic/carcinogenic amino- or nitro-arenes

Synthetic methods for the preparation of 2′-deoxyguanosine-C8 (dG-C8) adducts with several mutagenic and carcinogenic amino- or nitro-arenes were developed using the palladium-mediated cross-coupling reaction of protected 8-amino-dG with bromoarenes in around 80% yields, followed by conventional deprotection procedures. This approach can be applied to preparation of a variety of authentic dG-C8 adducts with amino or nitro-arenes.     

Thermolytic 4-methylthio-1-butyl group for phosphate/thiophosphate protection in solid-phase synthesis of DNA oligonucleotides

The thermolabile 4-methylthio-1-butyl phosphate/thiophosphate protecting group for DNA oligonucleotides has been investigated for its potential application to a "heat-driven" process for either oligonucleotide synthesis on diagnostic microarrays or, oppositely, to the large-scale preparation of therapeutic oligonucleotides. The preparation of phosphoramidites 10a-d is straightforward, and the incorporation of these amidites into oligonucleotides via solid-phase techniques proceeds as efficiently as that achieved with 2-cyanoethyl deoxyribonucleoside phosphoramidites. The versatility of the 4-methylthio-1-butyl phosphate/thiophosphate protecting group is exemplified by its facile removal from oligonucleotides upon heating for 30 min at 55 degrees C in an aqueous buffer under neutral conditions or within 2 h at 55 degrees C in concentrated NH(4)OH. The deprotection reaction occurs through an intramolecular cyclodeesterification mechanism leading to the formation of sulfonium salt 18. When mixed with deoxyribonucleosides and N-protected 2'-deoxyribonucleosides or with a model phosphorothioate diester under conditions approximating those of large-scale (>50 mmol) oligonucleotide deprotection reactions, the salt 18 did not significantly alter DNA nucleobases or desulfurize the phosphorothioate diester model to an appreciable extent.     

Electrochemical Synthesis of Carbon Nanodots Directly from Alcohols

Carbon nanodots (C‐dots) show great potential as an important material for biochemical sensing, energy conversion, photocatalysis, and optoelectronics because of their water solubility, chemical inertness, low toxicity, and photo‐ and electronic properties. Numerous methods have been proposed for the preparation of C‐dots. However, complex procedures and strong acid treatments are often required, and the as‐prepared C‐dots tend to be of low quality, and in particular, have a low efficiency for photoluminescence. Herein, a facile and general strategy involving the electrochemical carbonization of low‐molecular‐weight alcohols is proposed. As precursors, the alcohols transited into carbon‐containing particles after electrochemical carbonization under basic conditions. The resultant C‐dots exhibit excellent excitation‐ and size‐dependent fluorescence without the need for complicated purification and passivation procedures. The sizes of the as‐prepared C‐dots can be adjusted by varying the applied potential. High‐quality C‐dots are prepared successfully from different small molecular alcohols, suggesting that this research provides a new, highly universal method for the preparation of fluorescent C‐dots. In addition, luminescence microscopy of the C‐dots is demonstrated in human cancer cells. The results indicate that the as‐prepared C‐dots have low toxicity and can be used in imaging applications.     

Iterative Synthesis of Nucleoside Oligophosphates with Phosphoramidites

P‐Amidites can be used in iterative couplings to selectively give mixed P^III–P^V anhydrides. These intermediates can be oxidized followed by a rapid removal of the two terminal fluorenylmethyl groups. An iterative synthesis (coupling, oxidation, deprotection) of nucleoside oligophosphates can be carried out in solution and on a solid support. The coupling rates and yields are high, the procedures convenient (non‐dry reagents and solvents, ambient conditions, unprotected nucleotides), and the purification is very simple. The method works with all canonical nucleosides and holds promise for significant simplification of the usually cumbersome process of P‐anhydride bond construction.     

Effective preparation of montmorillonite/polyurethane nanocomposites by introducing cationic groups into the polyurethane main chain

In this study, the effect of introducing a small amount of cationic groups into the polymer main chain on the exfoliation of montmorillonite (MMT) and the physical properties of the subsequent MMT/polymer nanocomposites were investigated. As a matrix polymer, a polyurethane cationomer (PUC) containing 3 mol% of quaternary ammonium groups was synthesized and MMT/PUC nanocomposites containing various amounts of MMT were prepared by the solution intercalation method. From the WAXS and TEM analyses, it was found that the MMT layers were completely exfoliated and dispersed in the PUC matrix. The Young’s modulus of the MMT/PUC nanocomposites significantly increased with increasing MMT content, but their elongation at break and maximum stress were maintained at a level close to that of the PU only at an MMT content of 1 wt% and decreased as the content of MMT increased above this level. The phase separation of the MMT/PUC nanocomposites was retarded with increasing content of MMT, due to the strong interactions between the PUC chains and the exfoliated MMT layers. It was found that the presence of small amounts of cationic groups in the main chain of the matrix polymer was very effective in facilitating the preparation of the MMT/polymer nanocomposites.     

Microwave-assisted in situ synthesis of fluorescent gold nanoclusters with BSA/montmorillonite and application on latent fingermark imaging

A method for in situ preparation of fluorescent gold nanoclusters(Au NCs) with bovine serum albumin/montmorillonite composite powder(Au NC-BSA/MMT) was developed, and the products were used to detect latent fingermarks. In this work, Au NCs were "grown" both inside and on the surface of BSA/MMT clay using one-step reduction of HAu Cl4 by BSA. The as-prepared Au NC-BSA/MMT nanocomposites emit intensive red fluorescence under the excitation of UV-visible light and show stable chemical features and low toxicity. The obtained fluorescent powders were characterized by UV-visible absorption spectroscopy,fluorescence spectroscopy, infrared spectroscopy, transmission electron microscopy/high-resolution transmission electron microscopy, scanning electron microscopy, X-ray photoelectron spectroscopy and X-ray diffraction to depict their sizes, structural information and optical features. Given their environmentally friendly preparation, simple operation, low cost, efficient UVvisible radiation-dependent photoluminescence and good affinity with finger residues, the in situ synthesized Au NC-BSA/MMT nanocomposite powders were used as an alternative fluorescent developing reagent for developing latent fingermarks deposited on various object surfaces(such as glass, aluminum foil, painted metal, plastic products and weighing papers) for individual identification. As results, the developed fingermarks with clear patterns and satisfactory level-2(minutiae points) and level-3(sweat pores) ridge details were obtained. Notably, treated prints could be excited by red light and emitted near infrared fluorescence, which was beneficial to avoid background interference and reduce the damage caused by UV light. With the advantages of the simple preparation process and good enhancement performance for latent fingermarks, the proposed method might be used in the preparation of various fluorescent probes for detecting trace evidence in forensic sciences.