Synthesis and Antibody Mediated Detection of 2,4-Dinitrophenyl (DNP) Labelled Oligonucleotides

Abstract

Different DNP phosphoramidites based on non-nucleoside and nucleoside backbone molecules are developed and used in the multiple labelling of oligonucleotides during the solid phase synthesis. It is demonstrated that the antibody mediated detection of DNP labelled oligonucleotides is comparable to that of digoxigenin, biotin and fluorescein.     

Synthesis of Spirannic Nucleosides and Their Incorporation into Oligonucleotides

Abstract

The spirannic nucleosides dH _α, dH _β, sB and cB were synthesized and incorporated into oligonucleotides using the cpg-oxalyl solid support strategy.     

Synthesis and Properties of New Fluorescein-Labeled Oligonucleotides

Abstract

2,4-Dinitroaniline is an efficient intramolecular fluorescence-quencher for fluorescein - labeled oligonucleotides and interacts with the heterocyclic bases on duplex formation. Consequently, intramolecular fluorescence quenching is disturbed in double labeled oligonucleotides of this type, and fluorescein shows strong fluorescence in a duplex form. There is a substential increase of the fluorescence-quantum yield when the marker and quencher is attached to a single guanosine residue. Two kinds of doubly labeled oligonucleotides have been synthesized, using the NPE/NPEOC strategy.     

Substrate Properties of New Fluorescently Labeled Deoxycytidine Triphosphates in Enzymatic Synthesis of DNA with Polymerases of Families A and B

The efficiency of the incorporation of fluorescently labeled derivatives of 2'-deoxycytidine in DNA synthesized de novo has been studied using PCR with Taq and Tth polymerases of family A and Vent (exo–) and Deep Vent (exo–) polymerases of family B. Four derivatives of 5'-triphosphate-2'-deoxycytidine (dCTP) have different chemical structures of the indodicarbocyanine dye and Cy5 analogue attached to position 5 of cytosine. The kinetics of the accumulation of the PCR products and the intensity of the fluorescent signals in the hybridization analysis with immobilized DNA probes depend on the modification of the fluorescently labeled dCTP counterpart, its concentration, and the type of DNA polymerase. All labeled triphosphates showed some inhibitory effects on PCR. The best balance between the efficiency of incorporating labeled cytidine derivatives and the negative effect on the PCR kinetics has been shown in the case of Hot Taq polymerase in combination with the Cy5-dCTP analogue, which contains containing electrically neutral chromophore, the axis of which is a continuation of the linker between the chromophore and the pyrimidine base.     

Design and Synthesis of New Acid Cleavable Linkers for DNA Sequencing by Synthesis

A new kind of acid sensitive tetrahydrofuranyl (THF) linker was synthesized and then reacted with 5-(6)-carboxytetramethylrhodaminesuccinimidyl ester (5(6)-TAMRA, SE), followed by di(N-succinimidyl) carbonate (DSC) and modified 2′-deoxyuridine triphosphate (dUTP); the final product, as a reversible terminator for DNA sequencing by synthesis (DNA SBS), was given obtained and confirmed by ¹H-NMR, ³¹P-NMR, and HRMS with purity of up to 99%. The synthesized dye-labeled terminator incorporated into DNA strand successfully, and the fluorophore was cleaved completely under acidic conditions. The preliminary results encourage us to explore more acid-sensitive linkers for DNA SBS to increase the cleavage efficiency under weakly acidic conditions.     

A New Solid-Phase Chemical DNA Sequencing Method Which Uses Streptavidin-Coated Magnetic Beads

To simplify the chemical DNA sequencing protocol, we developeda new solid-phase method which uses streptavidin-coated magneticbeads. This method is based on the finding that the biotinylatedDNA-streptavidin complex was stable under the conditions forsome chemical sequencing reactions. The 5'-biotinylated DNAgenerated by the polymerase chain reaction was first capturedby streptavidin-coated magnetic beads and then subjected toa set of simplified chemical sequencing reactions on the beadsat room temperature. Followed by the piperidine cleavage reaction,the products were resolved by gel electrophoresis, transferredonto a nylon membrane and visualized by chemiluminescent detection.As a consequence, highquality sequencing ladders were obtained,due to complete removal of contaminating chemicals, withoutthe time-consuming precipitation/centrifugation steps used inthe conventional chemical sequencing protocol     

A Rapid Method for Simultaneous Chemical Synthesis of Oligonucleotides in Large Nufiber

Abstract

A rapid manual method i s described for simultaneous synthem o v e r one hundred oligonucleotides on a microscale by the phosphotriester approach on cellulose paper.     

Synthesis of Novel Tyrosinyl FRET Cassettes,Terminators, and Their Potential Use in DNA Sequencing

Abstract

Fluorescence resonance energy transfer (FRET) dye labeled cassettes and terminators with one or more donor dyes (fluorescein) and acceptor dye (rhodamine dyes) with benzofuran or tyrosine linker moieties were synthesized. These terminators were evaluated for their energy transfer and DNA sequencing potential using thermostable DNA polymerase.     

Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a population always have accumulated mutations that are either detrimental or beneficial for the cell. If the fitness effect of a mutation provides the subpopulation with a strong selective growth advantage, the individuals of this subpopulation may rapidly outcompete and even completely eliminate their immediate fellows. Thus, small genetic changes and selection-driven accumulation of cells that have acquired beneficial mutations may lead to a complete shift of the genotype of a cell population. Here we present a procedure to monitor the rapid clonal expansion and elimination of beneficial and detrimental mutations, respectively, in a bacterial cell population over time by cocultivation of fluorescently labeled individuals of the Gram-positive model bacterium Bacillus subtilis. The method is easy to perform and very illustrative to display intraspecies competition among the individuals in a bacterial cell population.     

A Solid-Phase Method for the Synthesis of Small to Medium-Sized Cyclic Oligonucleotides

Abstract

Cyclic oligonucleotides (2- to 30-mer) are synthesized by a solid-phase method, for both chain elongation and cyclization, employing a new linker and standard phosphoramidite chemistry. Fairly pure crude products (>90% by HPLC) are obtained.     

A Rapid Method for Manual or Automated Purification of Fluorescently Labeled Nucleic Acids for Sequencing,Genotyping, and Microarrays

Fluorescent dyes provide specific, sensitive, and multiplexed detection of nucleic acids. To maximize sensitivity, fluorescently labeled reaction products (e.g., cycle sequencing or primer extension products) must be purified away from residual dye-labeled precursors. Successful high-throughput analyses require that this purification be reliable, rapid, and amenable to automation. Common methods for purifying reaction products involve several steps and require processes that are not easily automated. Prolinx^®, Inc. has developed RapXtract^™ superparamagnetic separation technology, affording rapid and easy-to-perform methods that yield high-quality product and are easily automated. The technology uses superparamagnetic particles that specifically remove unincorporated dye-labeled precursors. These particles are efficiently pelleted in the presence of a magnetic field, making them ideal for purification because of the rapid separations that they allow. RapXtract-purified sequencing reactions yield data with good signal and high Phred quality scores, and they work with various sequencing dye chemistries, including BigDye and near-infrared fluorescence IRDyes. RapXtract technology can also be used to purify dye primer sequencing reactions, primer extension reactions for genotyping analysis, and nucleic acid labeling reactions for microarray hybridization. The ease of use and versatility of RapXtract technology makes it a good choice for manual or automated purification of fluorescently labeled nucleic acids.     

Synthesis of Oligonucleotides Bearing Polyamine Groups for Recognition of DNA Sequences

Abstract

A 2-sperminoguanosine nucleotide has been synthesized and incorporated into oligonucleotides which showed increased duplex melting termperature.     

Triazinylamidophosphate Oligonucleotides: Synthesis and Study of Their Interaction with Cells and DNA-Binding Proteins

Russian Journal of Bioorganic Chemistry - In this work, representatives of the class of triazinylamidophosphate oligonucleotide derivatives were obtained for the first time. A scheme for the...     

A Novel and Convenient Method for the Synthesis of Free 5′-Thiol Modified Oligonucleotides

Abstract

The synthesis of free 5′-thiol-modified oligonucleotides using a 4,4′,4″-trimethoxytrityl (TMTr)-protected linker and standard Poly-Pak^TM purification has been described.     

伞菌DNA简易序列分析引出的思考

目的:伞菌物种以子实体形态特征为分类依据,研究以菌丝体代替子实体进行种质资源鉴定的遗传证据。方法:以常见的食用伞菌香菇、平菇子实体的不同部位组织及其分离菌丝体为供试材料,制备了12个随机引物介导的RAPD-PCR指纹,把DNA指纹图谱转化为简易的序列数据,经生物信息处理软件比较分析。结果:香菇不同菌株子实体DNA相似性系数在0.886～0.986之间,平菇不同菌株子实体DNA相似系数在0.779～0.976之间。对于供试的单个子实体而言,香菇和平菇子实体的菌盖、菌褶、菌柄及其组织分离菌丝体,与以此为菌种栽培得到的子实体相比较,所获得的有限DNA指纹图谱全部相同。结论:揭示了香菇和平菇不同菌株的遗传多样性,初步反映了伞菌不同发育阶段在分子水平上的遗传变异和亲缘关系,争论了以菌丝体代替子实体使用RAPD手段进行种质资源鉴定与系统发育分析引出的真菌遗传问题。     

A Rapid Method for Purification of Oligonucleotides

Abstract

A simple, rapid and novel method for purification of the oligonucleotide using silica gel matrix is described.     

The Application of H-Phosphonate Chemistry in the HELP Synthesis of Oligonucleotides

Abstract

The use of the H-phosphonate chemistry for its possible utilization in the liquid phase synthesis of oligonucleotides has been investigated.     

Development of a New DNA Sequencing Method: 3′-Ester Cleavage Catalyzed by Taq DNA Polymerase

Abstract

A new 3′-esterified dTTP is incorporated into DNA by Taq DNA polymerase but does not act as a chain terminator. The esterase activity of the polymerase seems to be template dependent and occurs only if the next correct nucleotide is present.     

Turnover of Labelled DNA in Differentiated Collenchyma

The incorporation of ³H-thymidine into the nuclear DNA of differentiated, non-dividing collenchyma tissue in cut shoots of Lycopersicon esculentum was studied autoradiographically. When the fed shoots were transferred for up to 168 h to a non-radioactive solution, a significant loss of label was observed following a chase for 48 h. Thereafter, the only further change in the labelling pattern was demonstrated by a 168 h chase. When fed shoots were alternatively re-rooted and sampled over a longer chase period, there was a consistent decline in the mean grain count per nucleus until 14 weeks after re-rooting. A final sampling at 32 weeks revealed that almost all of the labelled DNA had been lost or turned over from this tissue.     

Oligonucleotides containing substituted 4-nitroindoles: Synthesis and study of their DNA duplexes

The synthesis of oligonucleotides containing 1-(2-deoxy-β-D-ribofuranosyl)-2-methyl-4-nitroindole and 1-(2-deoxy-β-D-ribofuranosyl)-2-phenyl-4-nitroindole is described. The synthesized modified oligonucleotides were used for studying the stability of intermolecular DNA duplexes with one unnatural strand and for evaluation of discriminating potential of 2-methyl-and 2-phenyl-4-nitroindoles toward nucleic bases. For comparison, an unmodified oligonucleotide and oligonucleotides bearing 5-nitroindole were used. It was shown that 2-methyl-4-nitroindole was only insignificantly inferior in stability to 5-nitroindole and characterized by a similar discriminating potential. 2-Phenyl-4-nitroindole provided a more pronounced duplex destabilization, the discrimination toward natural bases being decreased.