Many different applications demand different purity or scale to work well when it comes to custom DNA oligo synthesis. Here are some guidelines for purity and synthesis scale selection for different applications.


Understanding Why Oligos Sometimes Require Purification

Following DNA synthesis, the completed DNA chain is released from the solid support by incubation in basic solutions such as ammonium hydroxide. This solution contains the required full-length oligo but also contains all of the DNA chains that were aborted during synthesis (failure sequences). If a 20-mer was synthesized, the solution would also contain 19 mer failures, 18 mer failures, 17 mer failures etc. The amount of failure sequences present is influenced by the coupling efficiency. These failure sequences can compete with the full-length product in some applications such as PCR, and may therefore need removing before the oligo can be used successfully. 

Purification Options Offered

Purification Method Description Benefit
(40 nm, 7-30 bp; 50 nm-1 µm, 7-100 bp)
Oligos are processed through normal phase chromatography column which removes salts but not failure sequences. A salt-free DNA solution, ready-to-use; suitable for many PCR and sequencing applications without further purification.
(50 nm-1 µm, 7-100 bp)
Based on reverse phase chromatography; removes failure sequences from the completed synthesis. Provides full-length sequences needed in some applications
(50 nm-1 µm, 7-65 bp)
Reverse Phase High Performance Liquid Chromatography (HPLC) removes failure sequences or unincorporated label the same way as cartridge purification. Guarantees highly purified primer required in some applications (>=85% full length).
(50 nm, 20-100 bp; 200nm-1 µm, 7-100bp)
Method used to differentiate full-length product from failure sequences based on size and conformation. Provides the highest percentage of full-length oligos (>=85%) required for certain demanding applications such as mutagenesis or adapter production.


Application-Purity Guide

Application Suggested Purity
AFLP™ Analysis Desalted oligos have been used successfully for Amplification Restriction Fragment Polymorphisms.
Antisense HPLC-purified oligos are cited most frequently in references for antisense studies. See Minimum Yields chart for HPLC purification yields.
First-Strand cDNA Synthesis for Generation of Libraries Generally oligos for first strand cDNA synthesis for library construction has some sequence at the end which codes for 5´ restriction endonuclease cloning sites. Therefore, it is best to use full-length, Cartridge, HPLC, or PAGE-purified oligos.
Fluorescent Sequencing All four purity grades have worked successfully for Invitrogen scientists.
Gel Shift Assays Cartridge, HPLC, and PAGE-Purified oligos are recommended for gel shift assays, so as to have a homogeneous population of DNA fragments.
GENETRAPPER® Screening PAGE-purified oligos are recommended. Primers should be phenol extracted and ethanol precipitated prior to use in the tailing reaction in GeneTrapper® System. If Desalted Purity oligos are purchased they can be PAGE-Purified using the PAGE purification protocol.
Isothermal Sequencing Desalted oligos are sufficient for this application, along with Cartridge, HPLC, and PAGE-purified.
Microarrays Standard desalted oligos are sufficient for printing onto arrays.
PCR Desalted oligos work fine for standard PCR. Higher purity options will also work.
PCR using oligos with critical 5´ sequences (e.g., restriction endonuclease sites, RNA polymerase promoters) Cartridge, HPLC, and PAGE-purified oligos are best for the greatest efficiency. Since oligos are synthesized 3´ to 5´, incomplete oligos (n-x oligos) will be missing the 5´ sequence. It is important to use full-length oligos that have the 5´ sequence present, otherwise there will be a population of PCR products missing the sequence intended to be installed before PCR.
Production of Cloning Adapters Full-length oligos work best for efficient cloning. Utilize cartridge, HPLC, or PAGE-Purified oligos for full length.
Site-Directed Mutagenesis Full-length (e.g., Cartridge, HPLC, and PAGE-purified) oligos as a rule tend to give the highest percentage of mutagenized clones (especially if the intended mutation is close to the 5´ end of the oligo). Desired mutations have been obtained using Desalted oligos. However, some wild-type parental vector clones tend to carry over.