Oligonucleotide Synthesis

A collection of powerful new techniques for oligonucleotide synthesis and for the use of modified oligonucleotides in biotechnology. Among the protocol highlights are a novel two-step process that yields a high purity, less costly, DNA, the synthesis of phosphorothioates using new sulfur transfer agents, the synthesis of LNA, peptide conjugation methods to improve cellular delivery and cell-specific targeting, and triple helix formation. The applications include using molecular beacons to monitor the PCR amplification process, nuclease footprinting to study the sequence-selective binding of small molecules of DNA, nucleic acid libraries, and the use of small interference RNA (siRNA) as an inhibitor of gene expression.

Includes supplementary material: sn.pub/extras

Autorentext
Piet Herdewijn is Professor at the Katholieke Universiteit Leuven, Belgium, and head of the laboratory of medicinal chemistry at the Rega Institute. He obtained his Ph.D. and habilitation from the same university. He was a postdoctoral fellow of the Humboldt Foundation at the University of Konstanz. He has conducted research in bioorganic and medicinal chemistry, mainly in the field of nucleosides, nucleotides, and nucleic acids, at the University of Leuven, the University of Ghent, and the Université d'Evry-Val-d'Essonne.

Klappentext
Nucleic acids chemistry has progressed rapidly in the last 10 years with the introduction of many new techniques and the development of many novel therapeutic entities. In Oligonucleotide Synthesis: Methods and Applications, laboratory experts describe in step-by-step detail the powerful new techniques they have developed for oligonucleotide synthesis and for the use of modified oligonucleotides in biotechnology. Among the protocol highlights are the synthesis of phosphorothioates via new sulfur transfer agents, the synthesis of LNA, peptide conjugation methods to improve cellular delivery and cell-specific targeting, triple helix formation, and a novel two-step process that yields a high purity DNA that is less costly. Novel applications include the use of molecular beacons to monitor the PCR amplification process, nuclease footprinting to study the sequence-selective binding of small molecules to DNA, nucleic acid libraries, optimization of RNA synthesis for siRNA applications, and the use of small interference RNA (siRNA) as an inhibitor of gene expression. The protocols follow the successful Methods in Molecular Biology™ series format, each one offering step-by-step laboratory instructions, an introduction outlining the principle behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
Cutting-edge and highly practical, Oligonucleotide Synthesis: Methods and Applications offers today's investigators not only insight into important new biotechnologies, but also a full range of the detailed protocols needed to work successfully in DNA synthesis and its applications today.

Inhalt
Synthesis of DNA Using a New Two-Step Cycle.- RNA Synthesis Using 2?-O-(Tert-Butyldimethylsilyl) Protection.- RNA Oligonucleotide Synthesis Via 5?-Silyl-2?-Orthoester Chemistry.- Dimethylthiarum Disulfide.- Assay for Evaluating Ribonuclease H-Mediated Degradation of RNA-Antisense Oligonucleotide Duplexes.- Di- and Oligonucleotide Synthesis Using H-Phosphonate Chemistry.- Solid-Phase Synthesis of Circular Oligonucleotides.- Locked Nucleic Acid Synthesis.- Synthesis of DNA Mimics Representing HypNA-pPNA Hetero-Oligomers.- Base-Modified Oligonucleotides With Increased Duplex Stability.- of Hypermodified Nucleotides in RNA.- Chemical Methods for Peptide-Oligonucleotide Conjugate Synthesis.- Biotin-Labeled Oligonucleotides With Extraordinarily Long Tethering Arms.- Universal Labeling Chemistry for Nucleic Acid Detection on DNA Chips.- Postsynthetic Functionalization of Triple Helix-Forming Oligonucleotides.- Fluorescence-Based On-Line Detection as an Analytical Tool in RNA Electrophoresis.- Design and Optimization of Molecular Beacon Real-Time Polymerase Chain Reaction Assays.- High-Throughput Production of Optimized Primers (Fimers) for Whole-Genome Direct Sequencing.- Nonenzymatic Template-Directed RNA Synthesis.- DNase I Footprinting of Small Molecule Binding Sites on DNA.- Gene Targeting Using Peptide Nucleic Acid.- Nucleic Acid Library Construction Using Synthetic DNA Constructs.- In Vitro Selection From Combinatorial Nucleic Acid Libraries.- In Vitro Selection Procedures for Identifying DNA and RNA Aptamers Targeted to Nucleic Acids and Proteins.- Short Double-Stranded Ribonucleic Acid as Inhibitor of Gene Expression by the Interference Mechanism.