Oligo - A short piece of synthetic DNA is known as an oligonucleotide, an oligodeoxynucleotide or, simply, an oligo. Oligos are also referred to as primers and probes, alluding to their use in PCR and hybridisation experiments respectively.
Synthesis - VH Bio’s DNA oligos are synthesised using the β-cyanoethyl phosphoroamidite method on high performance controlled pore glass supports in fully automated synthesisers.
Chemistry - VH Bio oligos are synthesised in containers filled with a solid support, usually glass or polystyrene beads, designed to anchor the growing DNA chain. The DNA synthesis consists of a series of chemical reactions:
- Deblocking – the first base at the 3’-end is attached to the solid support via a chemical linker. A trityl protecting group is present on the 5’-OH as a protecting group. This is removed with acid to produce a free 5’-OH group ready for reaction with the next base.
- Coupling – the next base is added and coupled to the first base by means of a phosphate group.
- Capping – 1-2% of the free 5’-OH does not react during the coupling step and is capped with acetic anhydride to ensure that the failed bases take no further part in the synthesis.
- Oxidation – The phosphate group between the bases is oxidised with iodine to produce a stable phosphate linkage.
- Deblocking – the trityl group on the 5’-OH of the second base is removed to produce a free 5’-OH ready for reaction with the next base.
The cycle is repeated until the desired number of bases is added.
Coupling Efficiency
The yield of product after each round of synthesis is called the coupling efficiency. No chemical reaction is 100% efficient, but, by using high quality chemicals and employing good housekeeping procedures on our instruments, we aim to achieve a coupling efficiency of at least 99%.
Trityl Group
Each base added in the DNA synthesis has a dimethoxytrityl (trityl) protecting group attached to protect the 5’-OH during the cycle of chemical reactions. The trityl group is only removed immediately before the next base is added.
Coupling Efficiency Measurement
Removal of the trityl group with acid generates a strong orange colour. The intensity of the colour is proportional to the amount of coupled base and can be measured spectrophotometrically. From the measurement the coupling efficiency can be calculated and related to the quality of the synthesis.
Optical Density (OD) Reading
The optical density reading at 260nm (OD260) is used to quantify an oligo in solution. 1OD260 Unit is approximately 33µg single stranded DNA. The reading includes all DNA in solution, including any failure sequences.
Oligonucleotide Synthesis Impurities
Synthesis of oligonucleotides occurs in the 3’ to 5’ direction by the sequential addition of nucleotide monomers to a solid support derivatised with the first monomer. Each base addition is approximately 99% efficient; this results in the formation of failure sequences in addition to the full-length product. The percentage of failure sequences can be substantial in a non-purified, crude synthesis. This depends on the length of the oligo and the coupling efficiency. The following equation predicts the percentage of full-length product in a crude synthesis:
% Product = E(N-1)
Where E is the base coupling efficiency and N is the length of the oligo.
Based on this equation, a crude 25-mer synthesised with a base coupling efficiency of 99% will be approximately 79% full-length. If the coupling efficiency drops only 1% to 98%, the percentage of full-length product drops to 62%. This variation in purity can have a dramatic effect on experimental outcome, and makes it difficult to compare results between experiments using different lots of oligonucleotides having the same sequence.
It is because of this that VH Bio uses, wherever possible, RPC as its basic method of purification. RPC removes failed sequences from the mixture.
Degenerate Oligonucleotides
Degenerate oligos contain a mixture of bases at one or more sites on the chain. The IUPAC-IUB codes for such mixtures (wobbles) are as follows.
| Symbol |
Mixture |
| R |
A/G |
| Y |
C/T |
| M |
A/C |
| K |
G/T |
| S |
G/C |
| W |
A/T |
| H |
A/C/T |
| B |
G/T/C |
| V |
G/C/A |
| D |
G/T/A |
| N |
G/A/T/C |
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