Oligonucleotides that are used in various experiments such as PCR, Next Generation Sequencing or quantitative PCR can be synthesized chemically. In contrast to the natural 5’→3’ synthesis, the chemical synthesis generates the strand in a 3’→5’ direction. The first base is coupled covalently to Controlled Pore Glass (CPG). The cyclic elongation reaction consists of five steps: detritylation, tetrazolactivation, coupling, capping and oxidation (figure 1). The synthesis is automated by a synthesizing machine.
Here, we exemplarily synthesized an RNA 20mer with and without a 5’-dimethoxytrityl protection group, respectively, in a 0.2 μmol synthesis scale.
Table of Contents
1 Introduction
2 Material and methods
2.1 Preparations and synthesis
2.2 Cleavage from CPG and deprotection of the base
2.3 RP-HPLC
2.4 DMT cleavage
2.5 Gel analysis
3 Results
3.1 Synthesis
3.2 RP-HPLC
3.3 Gel analysis
4 Discussion
4.1 Synthesis
4.2 RP-HPLC
4.3 DMT cleavage
4.4 Gel analysis
Objectives and Topics
This report documents the chemical synthesis of an RNA 20mer to investigate the impact of the 5'-dimethoxytrityl (DMT) protection group on purification efficiency. The research examines whether the presence of this group is essential for successful isolation via RP-HPLC and provides an analysis of the synthesis and purification results.
- Chemical synthesis of RNA oligonucleotides
- Role of the 5'-dimethoxytrityl (DMT) protection group
- Purification via Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC)
- Comparison of "on" and "off" synthesis versions
- Yield calculation and analysis of synthetic products
Excerpt from the Book
1 Introduction
Oligonucleotides that are used in various experiments such as PCR, Next Generation Sequencing or quantitative PCR can be synthesized chemically. In contrast to the natural 5’→3’ synthesis, the chemical synthesis generates the strand in a 3’→5’ direction. The first base is coupled covalently to Controlled Pore Glass (CPG). The cyclic elongation reaction consists of five steps: detritylation, tetrazolactivation, coupling, capping and oxidation (figure 1). The synthesis is automated by a synthesizing machine.
Here, we exemplarily synthesized an RNA 20mer with and without a 5’-dimethoxytrityl protection group, respectively, in a 0.2 μmol synthesis scale.
Summary of Chapters
1 Introduction: Provides an overview of chemical oligonucleotide synthesis and defines the objective of synthesizing an RNA 20mer to test the 5'-DMT protection group.
2 Material and methods: Describes the technical setup, including the synthesis machine, the specific oligonucleotide sequence, and the protocols for cleavage, deprotection, RP-HPLC purification, and gel analysis.
3 Results: Details the findings regarding repetitive synthesis yields and compares the RP-HPLC chromatography profiles of the "on" and "off" products.
4 Discussion: Evaluates the success of the synthesis and explains why the protection group is critical for purification, while noting limitations in gel analysis.
Keywords
Oligonucleotides, Chemical Synthesis, RNA, 5'-dimethoxytrityl, DMT, RP-HPLC, Controlled Pore Glass, CPG, Detritylation, Tetrazolactivation, Coupling, Capping, Oxidation, Purification, Synthesis yield
Frequently Asked Questions
What is the core subject of this paper?
The paper focuses on the chemical synthesis of an RNA 20mer and explores the necessity of the 5'-dimethoxytrityl (DMT) protection group for the purification of the final product.
What are the primary thematic areas?
The study covers automated oligonucleotide synthesis, purification techniques using RP-HPLC, and analytical methods for verifying synthetic success.
What is the research goal?
The goal is to demonstrate the importance of the 5' protecting group by comparing two synthesis variants: one containing the group ("on") and one lacking it ("off").
Which scientific methods are applied?
The researchers utilized an automated DNA/RNA synthesizer, Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC), optical density measurements, and polyacrylamide gel electrophoresis.
What is covered in the main section?
The main section details the experimental protocol for synthesis, the subsequent chemical cleavage and deprotection steps, the purification results shown via chromatography, and a discussion of the observed outcomes.
Which keywords characterize this work?
Key terms include Oligonucleotides, Chemical Synthesis, RNA, 5'-dimethoxytrityl, RP-HPLC, CPG, and purification.
Why did the "off" version fail during the purification phase?
The "off" version lacked the 5' protection group, causing it to elute faster and overlap with unspecific impurities, which prevented successful purification via RP-HPLC.
What was the outcome of the gel analysis?
The gel analysis was inconclusive as the gel failed to show distinct bands, making further analytical or preparative assessment via electrophoresis impossible.
- Citation du texte
- Anonym (Auteur), 2014, Chemical RNA Synthesis, Munich, GRIN Verlag, https://www.grin.com/document/282479
