A series of dialkylformamidine protected deoxyadenosine phosphoramidites were prepared for automated, solid-support DNA synthesis. The set of Abz, Gdmf, Cbz, T phosphoramidites gave high purity and high yield oligodeoxynucleotides, with complete deprotection at 65 °C in one hour. Different times and temperatures of exposure to concentrated ammonium hydroxide were examined to establish the optimum conditions for deprotection of oligodeoxynucleotides.

N-6-Dialkylformamidine-2′-deoxyadenosine Phosphoramidites in Oligodeoxynucleotide Synthesis Raped Deprotection of Oligodeoxynucleotides

Oxidative conversion of N-dimethylformamidine nucleosides to N-cyano nucleosides

Synthetic nucleic acids are produced routinely for a wide variety of applications, including biological and chemical research, and diagnostic or therapeutic applications. To ensure an adequate level of quality and purity, rapid and convenient analytical methods are necessary. This unit discusses basic principles to guide in the selection of appropriate purification and analysis protocols.

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Overview of purification and analysis of synthetic nucleic acids.

1 Chemical Synthesis of Oligonucleotides: From Dream to Automation

Synthesis of Phosphonoacetate RNA and a Two-Step RNA Synthesis

AutoAnalysis is a new method for detecting and quantitating the trityl cation released each cycle on automated DNA synthesizers. The trityl (dimethoxytrityl) cation is removed from the growing oligonucleotide after each base addition and is a useful measure of synthesis efficiency. The traditional absorbance method of collecting each trityl effluent with a fraction collector, followed by dilution with an acid solution and careful quantitation by UV/VIS spectroscopy is costly, tedious and prone to error. The absorbance method for trityl cation analysis must usually wait until the synthesis is complete. Interruption of a failed operation, for a variety of reasons, such as an empty reagent reservoir, is thus not possible. Taking advantage of the conductive properties of the trityl cation, immediate and real-time quantitation is now possible by integrating the total conductance of the flowing stream during the detritylation step after each nucleoside addition in DNA synthesis. A conductivity cell is mounted downstream, past the synthesis column. The conductivity signal is processed and displayed as the current average stepwise yield and overall yield. If the yield drops below a pre-set threshold value because of a failure situation, the synthesizer will interrupt, preserving reagents. AutoAnalysis allows trityl monitoring with complete automation on the Applied Biosystems Models 392 and 394 DNA/RNA Synthesizers.

Ross G

Papers

Trityl monitoring of automated DNA synthesizer operation by conductivity : a new method of real-time analysis