Synthesis and Purification of Oligonucleotides

  title={Synthesis and Purification of Oligonucleotides},
  author={Andrew Ellington and Jack D. Pollard},
  journal={Current Protocols in Molecular Biology},
This unit provides strategies on the maximization of synthetic yield, the generation of sequences containing site‐specific modifications, and the isolation of synthetic oligonucleotides. Protocols describe monitoring the progress of synthesis via the trityl assay and methods for deprotection of DNA and RNA oligonucleotides. 

Overview of Purification and Analysis of Synthetic Nucleic Acids

Basic principles are discussed to guide in the selection of appropriate purification and analysis protocols for synthetic nucleic acids.

Highly parallel oligonucleotide purification and functionalization using reversible chemistry

A cost-effective, highly parallel method for purification and functionalization of 5-labeled oligonucleotides based on 5′-hexa-His phase tag purification, followed by exchange of the hexa-His tag for a functional group using reversible reaction chemistry is developed.

Synthesis of mixed-sequence oligonucleotides on mesoporous silicon: chemical strategies and material stability

The stability of functionalized mesoporous silicon supports in the solid-phase synthesis of oligonucleotides is studied in aminosilane-modified porous silicon photonic structures.

Simultaneous and stoichiometric purification of hundreds of oligonucleotides

A high-throughput method for oligo purification that also normalises the concentrations of the oligos in the final samples is developed and uses a rationally designed randomer capture probe to enrich for oligos with perfect 5′ sequences.

Aminosilane-modified mesoporous oxidized silicon for in situ oligonucleotides synthesis and detection

Direct solid phase of oligonucleotides (ONs) requires high chemical stability of the support material. In this work, we investigate the passivation ability of porous oxidized silicon multilayered

A High‐Throughput Process for the Solid‐Phase Purification of Synthetic DNA Sequences

The simulated high‐throughput and scalability features of the solid‐phase purification process are demonstrated without sacrificing purity of the DNA sequences.

Solid-phase synthesis of multivalent glycoconjugates on a DNA synthesizer.

The synthesis of multivalent glycoconjugates on solid-phase allows custom tailoring of their structure to the requirements of biological assays within hours, as opposed to traditional approaches that require weeks or months of work in the laboratory.

A systematic comparison of error correction enzymes by next-generation sequencing

A method to quantify errors in synthetic DNA by next-generation sequencing and is able to quantify differential specificities such as ErrASE preferentially corrects C/G → G/C transversions whereas T7 Endonuclease I preferently corrects A/T → T/A transversions.

Synthesis of 4-Thiouracil KPGEPGPK Analogues as Potential TIIICBP Identification Tools

Three octapeptides analogues obtained by the incorporation of photochemical 4-thiouracil (s4U) probes selectively activables could be used to identify a new receptor for type III collagen named Type III Collagen Binding Protein (TIIICBP).

An Improved PEG‐Linked Solid Support for Minimizing Process‐Related Impurities During Solid‐Phase Synthesis of DNA and RNA Sequences

Functionalization of a CPG support with five hexaethylene glycol spacers led to a 42% reduction in process‐related impurities contaminating oligonucleotide sequences, compared to that obtained using the commercial long‐chain alkylamine (LCAA) CPGSupport.



Syringe method for stepwise chemical synthesis of oligonucleotides.

Pertinent supporting data on the effect of variations in the detritylation, condensation, oxidation, capping and cleavage steps in the synthetic approach and in isolation procedures are presented.

Synthesis, deprotection, analysis and purification of RNA and ribozymes.

Improvements in the synthesis, deprotection and purification of oligoribonucleotides are described. These advances allow for reduced synthesis and deprotection times, while improving product yield.


An efficient anion-exchange HPLC method has been developed for the purification of chemically synthesized RNA and the resulting product precipitated directly by the addition of 1-propanol and a new activator, 5-ethylthio-1H-tetrazole significantly enhances the synthesis quality and yield of oligoribonucleotides.

Oligonucleotide synthesis : a practical approach

An introduction to modern methods of DNA synthesis and chromatographic purification of synthetic oligonucleotides is presented.

Solid supported hydrolysis of apurinic sites in synthetic oligonucleotides for rapid and efficient purification on reverse-phase cartridges.

It is found that the problem is due to the presence of truncated 5'-DMT fragments generated from apurinic sites within the target product during NH4OH deprotection, which can be purified by RPC to near homogeneity.

Improved chemistry for oligodeoxyribonucleotide synthesis substantially improves restriction enzyme cleavage of a synthetic 35mer.

Improvements in chemistry increased the restriction efficiency of synthetic DNA up to 10-fold and contained no detectable modified bases while DNA synthesized by the original chemistry had a large number of modifications.

The final deprotection step in oligonucleotide synthesis is reduced to a mild and rapid ammonia treatment by using labile base-protecting groups.

The stability of N 6-phenoxyacetyl-deoxyadenosine versus depurination in acidic conditions used in the detritylation step was favorably compared with that of the classic N6-benzoyl protected adenine.

Fast cleavage and deprotection of oligonucleotides

Covalent attachment of DNA oligonucleotides to glass.

Standard molecular biology procedures, such as ligation and restriction digest, were efficiently performed on the glass synthesized oligonucleotide, with the added benefit of extreme ease of handling.