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 can be used for creating pools of oligos that include matches to a variable or unknown template sequence. They can also be used to create diversity in clone libraries and in site directed mutagenesis.

Two types of randomization are available—standard and custom mixed bases, both of which can be ordered through IDT’s online Oligo Entry tool by selecting the Bases tab.

Standard mixing is a popular and economical way of incorporating mixed bases into oligo sequences. Standard mixed bases can be inserted at any/all positions 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, standard mixed bases will potentially have variation from the designated ratio due to the difference in coupling rates between base sites.

By requesting custom mixed bases, defined ratios can be added during synthesis, which includes unequal ratios. Pricing for custom mixed bases is calculated by the number of unique ratios per order with a maximum of 4 distinct formulations per oligonucleotide. Custom mixed base services include additional fees in addition to the standard charges for synthesis and/or purification.

Standard mixed bases are designated using a capital IUB (International Union of Biochemistry) code. To include standard 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). Some examples include:

- 5’ GTA CTG CAA TAG
**rNrNrN**TGA TCG AGA 3’ - 5’
**NNN**CTG CAA TAG TGA TCG AGA 3’ - 5’ CTG CAA TAA TAG T
**K**C TT**R**TT**N**GC**N**3’

Mixed bases required | Mixed-base code for DNA | Mixed-base code for RNA |
---|---|---|

A, G | R | rR |

C, T/U | Y | rY |

A, C | M | rM |

G, T/U | K | rK |

G, C | S | rS |

A, T | W | rW |

A, C, T/U | H | rH |

G, C, T (or U) | B | rB |

A, C, G | V | rV |

A, G, T/U | D | rD |

A, C, G, T/U | N | rN |

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 custom mixed base must name and define the ratio, all subsequent identical insertions only need to include the name.

For equimolar ratios, our site will use only the IUB symbol to represent the ratio in the sequence. Non-equimolar ratios will default to N1, N2, N3, or N4, based on the number of unique ratios in the sequence.

For example, an N mixed base with 25% of each base would be written as: (N:25252525). Each mixed base in the sequence with this ratio can be notated with (N). See below for what this looks like when incorporated in a sequence.

- 5′ ACT GTA CCG TAT TCC (N:25252525)(N)(N) TTA (N)(N)(N) ATA 3′

Up to 4 unique custom-mixed ratios can be included in an oligo sequence. Each of these ratios must also include a unique name. See the two examples below.

**Example 1:**5′ CAT (N:25252525)(N)T (N1:20202040)(N1)(N) G(N1)A 3′This oligo has three insertions of a 25% for each base mix (labeled N) and three insertions of a 20% A, 20% C, 20% G, 40% T mix (labeled N1).

**Example 2:**5′ AGG (K:00005050)(K)(K) (N1:10002070)(N1)A GTA 3′This oligo has three insertions of a 50% G, 50% T mix (labeled K) and two insertions of a 10% A, 20% G, 70% T mix (labeled N1).

In addition to IDT standard and custom mixed options for randomizing bases, we offer multiple base types (e.g., DNA, RNA, Affinity Plus™/locked nucleic acid, or 2'-O-methyl). This allows the incorporation of multiple base types or modifications from the IDT list of modified base offerings into a single position on an oligo. For example, a custom mixed base order for a single position can be 50% T Affinity Plus (locked nucleic acid) and 50% G Affinity Plus modification. Custom oligos can also contain some nucleotide positions that are mixed bases with standard DNA and other positions with Affinity Plus modified bases.

As with standard and nonmodified bases, for equimolar ratios, our site uses the IUB symbol to represent the ratio in the sequence. Non-equimolar ratios will default to N1, N2, N3, or N4, based on the number of unique ratios in the sequence.

**Example 1:**5’ CAG +(N:25252525)+(N)T CTA CAT GTA TAA GTA 3’This oligo has two insertions of a mix of 25% for each base (labeled N) with Affinity Plus (locked nucleic acid) modifications (designated with a + before the left parentheses).

**Example 2:**5’ AGG (N1:00004060)(N1)(N1) +(N2:10002070)+(N2)A GTA 3’This oligo has three insertions of a 40% G, 60% T mix (labeled N1) as standard DNA and two insertions of a 10% A, 20% G, 70% T mix (labeled N2) with Affinity Plus modifications.

Please note that modified mixed bases are considered custom and are subjected to the maximum of 4 distinct custom formulations per oligonucleotide. Custom services include additional fees in addition to the standard costs for the oligo synthesis and/or purifications.

RUO23-2006_001