Oligonucleotide Synthesis: Methods and Applications

✍ Scribed by Piet Herdewijn

• Publisher: Humana Press
• Year: 2004
• Tongue: English
• Leaves: 452
• Series: Methods in Molecular Biology
• Edition: 1
• Category: Library

✦ Synopsis

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.

✦ Table of Contents

Preface
......Page 6
Contents
......Page 10
1. Synthesis of DNA Using a New Two-Step Cycle
......Page 18
2. RNA Synthesis Using 2'-O-(Tert-Butyldimethylsilyl) Protection......Page 34
3. RNA Oligonucleotide Synthesis Via
5'-Silyl-2'-Orthoester Chemistry......Page 50
4. Dimethylthiarum Disulfide:
New Sulfur Transfer Reagent in Phosphorothioates Oligonucleotide Synthesis......Page 68
5. Assay for Evaluating Ribonuclease H-Mediated Degradation of RNA–Antisense Oligonucleotide Duplexes......Page 82
6. Di- and Oligonucleotide Synthesis Using H-Phosphonate
Chemistry......Page 98
7. Solid-Phase Synthesis of Circular Oligonucleotides......Page 118
8. Locked Nucleic Acid Synthesis......Page 144
9. Synthesis of DNA Mimics Representing HypNA–pPNA
Hetero-Oligomers......Page 164
10. Base-Modified Oligonucleotides With Increased Duplex Stability:
Pyrazolo[3,4-d] Pyrimidines Replacing Purines......Page 182
11. Introduction of Hypermodified Nucleotides in RNA......Page 204
12. Chemical Methods for Peptide–Oligonucleotide Conjugate Synthesis......Page 222
13. Biotin-Labeled Oligonucleotides With Extraordinarily Long Tethering Arms......Page 242
14. Universal Labeling Chemistry for Nucleic Acid
Detection on DNA Chips......Page 258
15. Postsynthetic Functionalization of Triple Helix-Forming Oligonucleotides......Page 268
16. Fluorescence-Based On-Line Detection as an Analytical Tool in RNA Electrophoresis......Page 278
17. Design and Optimization of Molecular Beacon Real-Time Polymerase Chain Reaction Assays......Page 290
18. High-Throughput Production of Optimized Primers (Fimers) for Whole-Genome Direct Sequencing......Page 308
19. Nonenzymatic Template-Directed RNA Synthesis......Page 322
20. DNase I Footprinting of Small Molecule Binding Sites on DNA......Page 336
21. Gene Targeting Using Peptide Nucleic Acid......Page 360
22. Nucleic Acid Library Construction Using Synthetic DNA Constructs......Page 376
23. In Vitro Selection From Combinatorial Nucleic Acid
Libraries......Page 396
24. In Vitro Selection Procedures for Identifying DNA and RNA Aptamers Targeted to Nucleic Acids and Proteins......Page 408
25. Short Double-Stranded Ribonucleic Acid as Inhibitor of Gene Expression by the Interference Mechanism......Page 428
Index......Page 448