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Nucleic Acid Library Construction Using Synthetic DNA Constructs

This chapter outlines seven synthetic and molecular biology techniques that allow the controlled synthesis of nucleic acid libraries. Specifically: (1) The high-diversity chemical synthesis of point mutations; (2) the high-diversity chemical synthesis of point deletions; (3) ...

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Gene Targeting Using Peptide Nucleic Acid

A brief overview of the properties of the deoxyribonucleic acid mimic peptide nucleic acid (PNA) is given, and the recent progress in cellular delivery of PNA and of using PNA oligomers for antisense and antigene gene targeting is presented.

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DNase I Footprinting of Small Molecule Binding Sites on DNA

Nuclease footprinting techniques were initially developed to investigate protein-deoxyribonucleic acid (DNA) interactions but these tools of molecular biology have also become instrumental for probing sequence-selective binding of small molecules to DNA. Here, the met ...

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Nonenzymatic Template-Directed RNA Synthesis

The aim of this chapter is to provide reliable protocols for the study of nonenzymatic oligomerization reactions of ribonucleic acid and its analogs. Traditional radioactive labels are replaced by fluorescent dyes. Product analysis is either based on reversed-phase high-pressure ...

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High-Throughput Production of Optimized Primers (Fimers) for Whole-Genome Direct Sequencing

Fimers are specifically modified primers selected to inhibit nonspecific interactions occurring in cycle sequencing. They are postsynthetically derived from 2′-methoxyoxalamido or 2′-succinimido precursor oligonucleotides by treatment with appropriate small mo ...

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Design and Optimization of Molecular Beacon Real-Time Polymerase Chain Reaction Assays

During the last few years, several innovative technologies have become available for performing sensitive and accurate genetic analyses. These techniques use fluorescent detection strategies in combination with nucleic acid amplification protocols. Most commonly used is ...

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Fluorescence-Based On-Line Detection as an Analytical Tool in RNA Electrophoresis

In this chapter, we present methods for denaturing and native gel electrophoresis of nucleic acids based on fluorescently labeled probes and automatic signal detection by a deoxyribonucleic acid sequencer. Specific examples are given for the determination of ribonucleic acid (RN ...

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Postsynthetic Functionalization of Triple Helix-Forming Oligonucleotides

The design of molecules that recognize specific sequence on the deoxyribonucleic acid (DNA) double helix would provide interesting tools to interfere with DNA information processing at an early stage of gene expression. This chapter describes in detail the protocol of conjugation be ...

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Universal Labeling Chemistry for Nucleic Acid Detection on DNA Chips

An efficient strategy for nucleic acid labeling and analysis on deoxyribonucleic acid (DNA) chips has been developed. This approach, which combines the fragmentation and the labeling steps, is based on the reactivity of the phosphates of DNA and ribonucleic acid (RNA) fragments and is using ...

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Biotin-Labeled Oligonucleotides With Extraordinarily Long Tethering Arms

We describe synthesis of four novel biotin phosphoramidites with tethering arms ranging from 20 to 74 atoms in length. One of these phosphoramidites is a uridine derivative with a biotin moiety attached through the 2′-position. The biotin phosphoramidites were synthetized based on robu ...

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Chemical Methods for Peptide-Oligonucleotide Conjugate Synthesis

Methods of peptide-oligonucleotide conjugate synthesis are presented that may be useful in the study of cell targeting and delivery of oligonucleotides and their analogs. The first method involves total stepwise solid-phase synthesis on a single support. The second involves prepa ...

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Introduction of Hypermodified Nucleotides in RNA

The anticodon domain of lysine transfer ribonucleic acid (tRNA) is a model system for investigation of the structural and biochemical effects of nucleoside posttranscriptional modification. To enable detailed study of the biophysical and structural effects of hypermodified n ...

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Base-Modified Oligonucleotides With Increased Duplex Stability: Pyrazolo[3,4-d] Pyrimidines Replacing Purines

Oligonucleotides incorporating 8-aza-7-dazapurines (pyrazolopyrimidines) were synthesized. The corresponding nucleosides were prepared and were converted into phosphoramidites. The oligonucleotide duplex stability was studied and was compared to that of the par ...

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Synthesis of DNA Mimics Representing HypNA-pPNA Hetero-Oligomers

The methods for the synthesis and purification of negatively charged peptide nucleic acid (PNA)-relative deoxyribonucleic acid (DNA) mimics containing alternating residues of phosphono peptide nucleic acid (pPNA) monomers and PNA-like monomers on the base of trans-4-hydroxy ...

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Locked Nucleic Acid Synthesis

Methods and protocols for automated synthesis and purification of locked nucleic acid (LNA), a class of oligonucleotides obeying the Watson-Crick base-pairing rules but displaying unprecedented binding affinities toward complementary deoxyribonucleic acid (cDNA) and r ...

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Solid-Phase Synthesis of Circular Oligonucleotides

A protocol for the straightforward preparation of small circular oligodeoxyribonucleotides (2–28 nt) is reported. The assembly of the oligonucleotide chain (standard phosphoramidite chemistry) and cyclization by the phosphotriester method take place on a tailor-made nuc ...

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Di- and Oligonucleotide Synthesis Using H-Phosphonate Chemistry

In this chapter, a concise account of the synthesis of oligonucleotides using the H-phosphonate methodology is given. It includes various methods for the preparation of the starting material, nucleoside 3′-H-phosphonate monoesters, their conversion into dinucleoside H-phosp ...

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Assay for Evaluating Ribonuclease H-Mediated Degradation of RNA-Antisense Oligonucleotide Duplexes

Ribonucleases H are complex enzymes whose functions are not clearly understood, further compounded by the fact that multiple forms of the enzyme are present in various organisms. They are known to recognize and degrade the ribonucleic acid (RNA) strand of numerous deoxyribonucleic acid ...

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Dimethylthiarum Disulfide: New Sulfur Transfer Reagent in Phosphorothioates Oligonucleotide Synthesis

Dimethylthiarum disulfide (DTD) has been developed as a new and efficient sulfur-transfer reagent for automated synthesis of phosphorothioate oligonucleotides using phosphoramidite chemistry. The traditional four-step automated oligonucleotide synthesis has be ...

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Synthesis of DNA Using a New Two-Step Cycle

For the first time a new, two-step method is described for synthesizing deoxyribonucleic acid. The approach uses 5′-carbonate protected 2′-deoxynucleoside-3′-phosphoramidites as synthons and a peroxy anion buffer that removes the carbonate protecting group and oxidizes the int ...

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