https://zenodo.org/record/1259717/files/article.pdf

Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach

Deoxyoligonucleotides can now be synthesized rapidly and in high yield because of recent advances in nucleic acid chemistry. Key innovations include solid-phase synthesis on silica-based supports and the development of stable deoxynucleoside phosphoramidites as synthons. When incorporated into manual, semiautomatic, or automatic instruments, these new procedures can be used to prepare probes, mixed probes, deoxyoligonucleotides for priming DNA synthesis, analogues of deoxyoligonucleotides, and DNA segments containing more than 100 deoxynucleotides.

https://science.sciencemag.org/content/sci/230/4723/281.full.pdf

Gene synthesis machines: DNA chemistry and its uses

For over 60 years, the synthetic production of new DNA sequences has helped researchers understand and engineer biology. Here we summarize methods and caveats for the de novo synthesis of DNA, with particular emphasis on recent technologies that allow for large-scale and low-cost production. In addition, we discuss emerging applications enabled by large-scale de novo DNA constructs, as well as the challenges and opportunities that lie ahead.

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Large-scale de novo DNA synthesis: technologies and applications

DNA sequencing by parallel oligonucleotide extensions

We have achieved the ability to synthesize thousands of unique, long oligonucleotides (150mers) in fmol amounts using parallel synthesis of DNA on microarrays. The sequence accuracy of the oligonucleotides in such large-scale syntheses has been limited by the yields and side reactions of the DNA synthesis process used. While there has been significant demand for libraries of long oligos (150mer and more), the yields in conventional DNA synthesis and the associated side reactions have previously limited the availability of oligonucleotide pools to lengths <100 nt. Using novel array based depurination assays, we show that the depurination side reaction is the limiting factor for the synthesis of libraries of long oligonucleotides on Agilent Technologies' SurePrint DNA microarray platform. We also demonstrate how depurination can be controlled and reduced by a novel detritylation process to enable the synthesis of high quality, long (150mer) oligonucleotide libraries and we report the characterization of synthesis efficiency for such libraries. Oligonucleotide libraries prepared with this method have changed the economics and availability of several existing applications (e.g. targeted resequencing, preparation of shRNA libraries, site-directed mutagenesis), and have the potential to enable even more novel applications (e.g. high-complexity synthetic biology).

Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process

A method is provided for capping failure sequences in oligonucleotide synthesis by phosphitylation. A phosphite monoester is reacted with the 5' or 3' hydroxyl of the failure sequence between successive condensation steps in a synthesis procedure to form a 5' or 3' phosphite diester with the failure sequence. The phosphite diester substituent is inert with respect to subsequent reaction steps in the synthesis of the desired oligonucleotide product.

Chemical capping by phosphitylation during oligonucleotide synthesis

Use of a C4 column for reversed-phase high-performance liquid chromatographic purification of synthetic oligonucleotides

The synthesis in our laboratory of very long (80–106 bases) oligodeoxynucleotides using the phosphite method of Matteucci and Caruthers in an automated DNA synthesizer indicates the true efficiency of this multiple step process. The coupling efficiency as measured by trityl cation release i s 99.5–99.8%. However the yield obtained is still below the theoretical yield calculated from the above efficiency. We have attempted t o elucidate yield decreasing mechanisms in long ol igodeoxynucleotide synthesis. Crude reaction mixtures of phosphate-deblocked oligonucleotides which had no 5′-trityl group and which were examined immediately after cleavage from the support show one major species by high resolution PAGE. Harsher hydrolysis generates a new pattern of products superimposed upon the initial ladder representing cleavage at purine residues internal to the full length oligonucleotide. In many cases the cleavage ladder is significantly more intense than the coupling ladder. Hydrolysis a t apurinic s...

J. William Efcavitch

Papers

Chemical capping by phosphitylation during oligonucleotide synthesis

Use of a C4 column for reversed-phase high-performance liquid chromatographic purification of synthetic oligonucleotides

Depurination As a Yield Decreasing Mechanism in Oligodeoxynucleotide Synthesis