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Advanced method for oligonucleotide deprotection

NUCLEIC ACIDS RESEARCH 2000年第8期28卷 E29页

作者： Surzhikov, Sergey A. Timofeev, Edward N. Chernov, Boris K. Golova, Julia B. Mirzabekov, Andrei D. Argonne Natl Lab Biochip Technol Ctr Argonne IL 60439 USA Russian Acad Sci Engelhardt Inst Mol Biol Joint Human Genome Program Moscow 117984 Russia

A new procedure for rapid deprotection of synthetic oligodeoxynucleotides has been developed. While all known deprotection methods require purification to remove the residual protective groups (e.g. benzamide) and insoluble silicates, the new procedure based on the use of an ammonia-free reagent mixture allows one to avoid the additional purification steps. The method can be applied to deprotect the oligodeoxy-nucleotides synthesized by using the standard protected nucleoside phosphoramidites dG(iBu), dC(Bz) and dA(Bz).

关键词：遗传学技术寡脱氧核糖核苷酸类/化学寡核苷酸序列分析寡核苷酸类/化学合成寡核苷酸类/分离和提纯

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Microscale sample preparation - The demand for microscale analysis has stimulated the development of new solid phase formats in sample preparation.

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NATURE BIOTECHNOLOGY 2000年第1期18卷 104-105页

作者： Pluskal, MG usmgp@***

The demand for microscale analysis has stimulated the development of new solid phase formats in sample *** interest in studying complex patterns of proteins in cells and tissues has led to new developments in analytical technologies. This interest in following changes in the cellular protein phenotype, termed proteomics, has generated a need for rapid and accurate protein identification. Such methods are derived from high-resolution separation approaches, like two-dimensional gel electrophoresis followed by protease digestion. The characterization methodology of choice is no longer Edman-based sequencing, but rather the more rapid and sensitive mass spectrometry (MS) approach. Both methods, however, are compromised by dirty and contaminated samples. Additionally, in MS, much smaller sample volumes and a higher concentration of analyte are required. To meet these challenges, new sample preparation processes are being developed for microscale volumes in miniaturized formats. At a recent meeting^1, microscale methodologies and their increasing application to drug discovery, combinatorial chemistry, and protein/peptide characterization studies were described. This article reviews the development of new formats of microscale sample preparation devices.

关键词：色谱法高压液相/仪器和设备色谱法高压液相/方法色谱法高压液相/发展趋势微量化学/仪器和设备微量化学/方法微量化学/发展趋势寡核苷酸类/分析寡核苷酸类/化学寡核苷酸类/分离和提纯肽类/分析肽类/化学肽类/分离和提纯聚合物蛋白质组标本制备/仪器和设备标本制备/方法标本制备/发展趋势

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Extraction of nuclear proteins with increased DNA binding activity

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BIOTECHNIQUES 2000年第5期28卷 938-+页

作者： Slomiany, BA Kelly, MM Kurtz, DT Med Univ S Carolina Dept Pharmacol Charleston SC 29425 USA

We have developed a method for the rapid extraction of nuclear proteins from cultured cells. The ammonium sulfate method described here extracts larger quantities of proteins that retain DNA binding activity than the modified Dignam method and another popular method used for the extraction of transcription factors. The ammonium sulfate method is rapid and can be used to process a large number of samples for gel shift analysis.

关键词： CCAAT增强子结合蛋白类细胞培养的 DNA结合蛋白质类/遗传学 DNA结合蛋白质类/分离和提纯电泳聚丙烯酰氨凝胶/方法雌二醇肝/化学肝/细胞学核蛋白质类/遗传学核蛋白质类/分离和提纯寡核苷酸类/遗传学寡核苷酸类/分离和提纯动物小鼠大鼠

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Thermoprecipitation of streptavidin via oligonucleotide-mediated self-assembly with poly (N-isopropylacrylamide)

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BIOCONJUGATE CHEMISTRY 1999年第5期10卷 720-725页

作者： Fong, RB Ding, ZL Long, CJ Hoffman, AS Stayton, PS Univ Washington Dept Bioengn Seattle WA 98195 USA

A versatile strategy has been developed for selectively and sequentially isolating targets in a liquid-phase affinity separation environment. The strategy uses a recently developed approach for joining together molecules in linkages that are defined by the complementary pairing of oligonucleotides conjugated to the different molecules [Niemeyer, C. M., Sane, T., Smith, C. L., and Canter, C. R. (1994) Nucleic Acids Res. 22, 5530-9]. In the work presented here, streptavidin was noncovalently coupled with the temperature-responsive poly(N-isopropylacrylamide) [poly(NIPAAM)] through the sequence-specific hybridization of oligonucleotides conjugated to the protein and polymer. A 20-mer oligonucleotide was covalently linked through a heterobifunctional linker to a genetically engineered streptavidin variant that contained a unique cysteine residue at the solvent-accessible site Glu 116. The complementary DNA sequence was conjugated to the end of a linear eater-activated poly(NIPAAM). The two conjugates were allowed to self-assemble in solution via hybridization of their complementary DNA sequences. The streptavidin-poly(NIPAAM) complex could be used to affinity-precipitate radiolabeled biotin or biotinylated alkaline phosphatase above 32 degrees C through the thermally induced phase separation activity of the poly(NIPAAM). The streptavidin-oligo species could then be reversibly separated from the precipitated polymer-oligo conjugate and recycled by lowering the salt concentration, which results in denaturation of the short double-stranded DNA connection. The use of oligonucleotides to couple polymer to streptavidin allows for selective precipitation of different polymers and streptavidin complexes based on the sequence-specific hybridization of their oligonucleotide appendages.

关键词：丙烯酰胺类/化学合成丙烯酰胺类/化学碱性磷酸酶/化学阴离子生物素/化学生物素化作用化学物理色谱法亲和/方法色谱法离子交换 DNA/化学热指示剂和试剂寡核苷酸类/化学寡核苷酸类/分离和提纯沉淀溶液抗生蛋白链菌素/类似物和衍生物

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Synthesis and characterization of oligonucleotides containing 5′,8-cyclopurine 2′-deoxyribonucleosides:: (5′R)-5′,8-cyclo-2′-deoxyadenosine, (5′S)-5′,8-cyclo-2′-deoxyguanosine, and (5′R)-5′,8-cyclo-2′-deoxyguanosine

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CHEMICAL RESEARCH IN TOXICOLOGY 1999年第5期12卷 412-421页

作者： Romieu, A Gasparutto, D Cadet, J CEA Grenoble Dept Rech Fondamentale Mat Condensee Serv Chim Inorgan & Biol Lab Les Acides Nucl F-38054 Grenoble 9 France

Radiation-induced degradation bf purine and-pyrimidine nucleosides gave rise to carbon-bridged cyclocompounds. Such cyclonucleosides represent a class of tandem lesions in which modification of both the base and 8-deoxyribose has occurred. A solid-phase synthetic method was designed for the incorporation of both 5'R and 5'S diastereoisomers of 5',8-cyclopurine 2'-deoxyribonucleosides into oligodeoxynucleotides to facilitate the assessment of the biochemical and biophysical features of such lesions. We report the preparation of the phosphoramidite synthons of (5'R)-5',8-cyclo-2'-deoxyadenosine (2), (5'S)-5',8-cyclo-2'-deoxyguanosine (3), and (5'R)-5',8-cyclo-2'-deoxyguanosine (4). Fully protected compounds 10, 18, and 25 were then inserted into several oligonucleotides by automated procedures. Analysis of modified DNA oligomers 26-31 by electrospray mass spectrometry and enzymatic digestions with exo- and endonucleases confirmed the base compositions and the integrity of free radical-induced tandem lesions 2-4 that were chemically inserted.

关键词：水解作用指示剂和试剂寡核苷酸类/化学合成寡核苷酸类/化学寡核苷酸类/分离和提纯磷酸二酯水解酶类分光光度法紫外线立体异构现象

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Fast and simple purification of chemically modified hammerhead ribozymes using a lipophilic capture tag

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NUCLEIC ACIDS RESEARCH 1999年第8期27卷 1950-1955页

作者： Sproat, BS Rupp, T Menhardt, N Keane, D Beijer, B Innovir GmbH D-37124 Rosdorf Germany

A new type of S-lipophilic capture tag is described, enabling the facile reverse phase HPLC purification of chemically modified hammerhead ribozymes (oligozymes) whilst still carrying the 2'-O-tert.-butyldimethylsilyl protection of the essential riboses, In its most convenient form, the capture tag consists of a simple diol, such as hexan-1,6-diol, which at one end is attached via a silyl residue to a highly lipophilic entity such as tocopherol (vitamin E) or cholesterol, and the other end is functionalized as a phosphoramidite, This lipophilic capture tag is added as the last residue in the solid-phase synthesis of chemically modified hammerhead ribozymes, Cleavage from the support and release of all protecting groups except for the silyl groups is achieved with ethanolamine/ethanol. The crude product is then loaded directly on to a reverse phase HPLC column. Separation of failure peaks from full length product is achieved easily using a short run time. The retarded product peak is collected, lyophilized, desilylated in the normal way and then desalted, This method removes the lipophilic capture tag yet leaves behind the hexanediol entity which helps protect the compound against degradation by 5'-exonucleases. The purity of the product as judged by analytical anion-exchange HPLC and capillary gel electrophoresis is generally better than 95% full-length, and yields of 2-4 mg from a 1 mu mol scale synthesis are routine. In addition, the method can be readily scaled up, an important feature for the development of such chemically modified ribozymes as potential therapeutics.

关键词：胆固醇色谱法高压液相/方法电泳毛细管/方法寡核苷酸类/化学合成寡核苷酸类/分离和提纯 RNA 催化/化学合成 RNA 催化/分离和提纯时间因素维生素E

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Automated purification and quantification of oligonucleotides

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BIOTECHNIQUES 1999年第4期27卷 810-+页

作者： Ivanetich, KM Reid, RC Ellison, R Perry, K Taylor, R Reschenberg, M Mainieri, A Zhu, D Argo, J Cass, D Strickland, C Univ Calif San Francisco Biomol Resource Ctr San Francisco CA 94143 USA Shimadzu Sci Instruments Columbia MD USA

We have developed automated methods for the trityl-on purification and quantification of synthetic oligonucleotides. Oligonucleotide purification is by solid-phase extraction cartridges using Amberchrom(R) CG-50 resin an an XYZ-axis robotic system. Quantification is by OD260nm using an online UV-visible spectrophotometer with sipper. The purification of 20 oligonucleotides requires 5 min of user set-up time, plus 20 min per sample of robot time Fbr a 15-25-mer at the 40 nmol scale of synthesis, the method gives a yield of 2.8 ODs from a load of 10.1 OD, i.e., a 28% average yield. Oligonucleotides purified by this method have proven to be successful for primers for automated DNA sequencing.

关键词：自动分析碱基序列色谱法/经济学色谱法/方法色谱法高压液相成本及成本分析分子序列数据寡核苷酸类/分析寡核苷酸类/化学合成寡核苷酸类/分离和提纯质粒结果可重复性敏感性与特异性序列分析 DNA 分光光度法紫外线模板遗传时间因素

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GRADIENT THIN-LAYER CHROMATOGRAPHY OF OLIGONUCLEOTIDES ON DEAE-CELLULOSE - ALTERNATIVE TO HOMOCHROMATOGRAPHY

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ANALYTICAL BIOCHEMISTRY 1979年第2期98卷 346-352页

作者： DOMDEY, H GROSS, HJ Max-Planck-Inst. Biochem. D-8033 Martinsried bei Munchen Germany

Homochromatography fingerprints are widely used for sequence studies of labeled RNA;the more general use of this method is restricted in several aspects by the large excess of RNA introduced by the homomix. Gradient TLC on DEAE-cellulose plates using ammonium formate gradients in 9 M urea provides an efficient procedure for preparing fingerprints of labeled and unlabeled RNA, and allows the isolation of oligonucleotides free of salt, urea and carrier RNA. This method produces fingerprints similar to those obtained by homochromatography under appropriate conditions. Consequently, gradient TLC is a convenient alternative to homochromatography without some of its limitations.

关键词：碱基序列色谱法 DEAE纤维素/仪器和设备色谱法 DEAE纤维素/方法色谱法薄层/仪器和设备色谱法薄层/方法肝/分析分子量寡核苷酸类/分离和提纯寡核糖核苷酸类/分离和提纯 RNA 核糖体 RNA 转移核糖核酸酶T1 动物兔

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SEPARATION OF OLIGONUCLEOTIDES, NUCLEOTIDES, AND NUCLEOSIDES ON COLUMNS OF POLYSTYRENE ANION-EXCHANGERS WITH SOLVENT SYSTEMS CONTAINING ETHANOL

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ANALYTICAL BIOCHEMISTRY 1976年第1期70卷 64-74页

作者： ASTERIADIS, GT ARMBRUSTER, MA GILHAM, PT PURDUE UNIV DEPT BIOL SCI BIOCHEM DIV LAFAYETTE IN 47907 USA

A study of the absorption of oligonucleotides by polystyrene anion-exchangers showed that a large component of the total binding forces consists of nonionic interactions, and that these interactions can be substantially reduced in the ion-exchange chromatography of these molecules by adding ethanol to the eluting solvents. This technique permits the separation of oligonucleotide species at pH values near neutrality and with solvent systems containing gradients of relatively low concentrations of Cl-. Chromatography of oligonucleotides with solvents containing 40% ethanol yields reproducible retention volumes that are dependent on their chain lengths and, to some extent, on their base compositions and the presence of 3''-terminal or 5''-terminal PO42- groups in the molecules. The flexibility in the use of solvent systems with 3 variable factors: pH, ethanol concentrations, and gradient of salt concentration, can also be exploited in the separation of mixtures of nucleosides this observation permitted the development of simple procedures for the characterization of ribo- and deoxyribooligonucleotides with respect to their base compositions, chain lengths and the identity of their 3''- and 5''-terminal nucleosides. Oligonucleotides were obtained from: a pig liver nuclei RNase digest of poly(A) and poly(U);a pancreatic RNase digest of wheat germ ribosomal RNA;and RNase T, digest of bacteriophage GA RNA (phage grown in Escherichia coli).

关键词：色谱法离子交换/方法乙醇寡核苷酸类/分离和提纯寡核糖核苷酸类/分离和提纯 RNA 核糖核酸酶类核糖核苷类/分离和提纯核糖核苷酸类/分离和提纯

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SEPARATION OF OLIGONUCLEOTIDES BY THIN-LAYER ELECTROPHORESIS

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ANALYTICAL BIOCHEMISTRY 1975年第1期64卷 214-220页

作者： GROHMANN, K SINSHEIMER, RL CALTECH DIV BIOL PASADENA CA 91109 USA

A method for fingerprinting of 32P labeled oligonucleotides is described. It involves separation by electrophoresis on cellulose thin layers in the first dimension, followed by chromatography on DEAE cellulose in the ... 详细信息

关键词：放射自显影术色谱法 DEAE纤维素大肠杆菌噬菌体 DNA病毒 DNA 病毒/分析电泳寡核苷酸类/分离和提纯

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