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Mixed bases

IDT offers oligonucleotides containing randomized or “mixed” bases. The final order contains a population of oligos with a different base at the randomized position. Mixed bases are ideal for creating pools of binding primers that include matches to a variable or unknown template sequence(s). They can also be used to create diversity in clone libraries and in site-directed mutagenesis.

Two types of randomization are available—machine mixing and hand mixing, both of which can be ordered through online oligo entry tools by selecting the Bases tab.

Standard machine mixing is the most popular and economical way of incorporating mixed bases into oligo sequences. Machine-mixed bases can be inserted at any/all base sites within a sequence at no additional charge. At the mixed base position(s), the synthesizer dispenses an equal ratio of the desired bases (e.g., a mix of A:C:G:T would be dispensed at 25:25:25:25, a mix of A:C:G would be 33:33:33:00, and a mix of A:C would be 50:50:00:00); however, each base could be added at slightly different efficiency, so there is no guarantee that all bases will equally represented in final oligo population.

By requesting hand mixing of bases, custom ratios can be added during synthesis, which includes non-equimolar ratios. Additionally, hand-mixed base ratios are adjusted to compensate for the slight coupling variations each base has during synthesis. Pricing for hand-mixed bases is calculated by the number of unique ratios per order.

Formatting mixed bases

Machine-mixed bases

Machine-mixed bases are designated using a capital IUB (International Union of Biochemistry) code. To include machine-mixed bases into your oligo sequence, use the DNA or RNA Oligo Entry tool and enter the appropriate mixed base code at the desired position(s).

Mixed bases requiredMixed-base code for DNAMixed-base code for RNA
A, C, T/UHrH
G, C, T (or U)BrB
A, C, GVrV
A, G, T/UDrD
A, C, G, T/UNrN

Hand-mixed bases

If custom ratios are required, then for each mixed base position, the IUB code followed by a colon with a listing of the desired ratio are both included in parentheses. The ratio must be written in order of A, C, G, T (or A, C, G, U for RNA). The first instance of the hand-mixed base must name and define the ratio, all subsequent identical insertions only need to include the name. For example, an N mixed base with 25% of each base would be written as: (N1:25252525). Each mixed base in the sequence with this ratio can be notated with (N1). See below for what this looks like when incorporated in a sequence.

  • 5′ ACT GTA CCG TAT TCC (N1:25252525)(N1)(N1) TTA (N1)(N1)(N1) ATA 3′

Up to four unique hand-mixed ratios can be included in an oligo sequence. Each of these ratios must also include a unique name.  See below for two examples.

  • Example 1: 5′ CAT (N1:25252525)(N1)T (N2:20202040)(N2)(N1) G(N2)A 3′

This oligo has three insertions of a 25% for each base mix (labeled N1) and three insertions of a 20% A, 20% C, 20% G, 40% T mix (labeled N2).

  • Example 2: 5′ AGG (K1:00004060)(K1)(K1) (D1:10002070)(D1)A GTA 3′

This oligo has three insertions of a 40% G, 60% T mix (labeled K1) and two insertions of a 10% A, 20% G, 70% T mix (labeled D1).