Understanding Why Oligos Sometimes Require Purification

There is often some confusion surrounding the issues of synthesis scale and synthesis yield in custom oligonucleotide manufacturing. Simply put, scale refers to the amount of starting material which is composed the attachment of last 3' base to a solid support and housed within the column used to make the oligonucleotide. Yield, on the other hand, refers to the amount of final product recovered after all of the synthesis and purification steps associated with the oligonucleotide have been completed.

Custom oligonucleotide synthesis begins with specification of the desired sequence in an oligonucleotide synthesis platform. Specification is composed of three crucial elements. First is the actual sequence that is to be made. Second is the identification of modifications, if any, that are desired. Third is verification of the scale at which the synthesis is to be carried out. This third element bears upon the choice of a column in which the synthesis will be performed. Synthesis columns permit a one-way flow of reagents from the synthesis platform through a precisely defined physical space containing and confining the growing oligonucleotide. Columns are prepared with a fixed amount of the last 3' base attached to a solid support, the controlled pore glass, or CPG, bead. It is the amount of the last 3' base nucleotide present in the column, in nanomoles, that constitutes synthesis scale.

Subsequent to placing the column, properly prepared with the correct 3’ nucleotide at the desired scale, in the synthesis platform, the rest of the oligonucleotide is synthesized by adding each base of the specified sequence one at a time in the 3’15’ direction. In a perfect world; i.e., one in which chemical and physical reality is suspended, each base added will couple with 100% efficiency. In such a scenario, scale and yield will be equal. In reality, coupling efficiency is less than 100% at each step in the synthesis. Moreover, coupling efficiency varies for each base both by type and position in the growing oligonucleotide (cf., Temsamani et al., 1995; Hecker and Rill, 1998).

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. At Bio-Synthesis we do our best to optimize this efficiency using modified programming and paying special attention to reagent preparation. The results of a drop in coupling efficiency of just a few percentage points on the yield can be devastating. This is easily demonstrated by calculating the theoretical yield with the following formula:

where (eff) is average coupling efficiency and n is the number of bases in the oligonucleotide. For example, a synthesis of a 30mer (which requires 29 couplings) with an average coupling efficiency of 99% theoretically yields 75% of product (0.9929). That same synthesis at 98% efficiency will have a maximum yield of only 55%. That one percent costs nearly half the material. Consider the difficulty of making a 70mer. Even at 99%, the best one could hope for is 50% yield. At 98% it becomes an abysmal 25% yield. Monitoring efforts at Bio-Synthesis confirm that our average coupling efficiency exceeds 99% for all oligonucleotides. Thus, theoretical yield for a 24-mer will be 89.1% full-length product (FLP) at 99.5% average coupling efficiency and 79.4% FLP at 99.0% average coupling efficiency.

As a general rule, Bio-Synthesis recommends that any oligonucleotide longer than 40 bases should receive further purification. In addition, for demanding applications such as site directed mutagenesis, cloning, and gel-shift protein-binding assays, additional purification is recommended even for oligonucleotides shorter than 40 bases. Our experience over the past ten years indicates that taking the time to purify an oligonucleotide used in the more demanding applications saves far more in terms of the precious commodities of time and research funds on the other end than it costs on the front end. Thus, additional purification should be considered for any oligonucleotide that is to be used for any application other than routine PCR or DNA sequencing.

Purification Options Offered

Application-Purity Guide