Degenerate Bases & Spiking - Introduction

Introduction to Degenerate Bases & Spiking

Degenerate base means more than one base possibility at a particular position, this is usually the case when a DNA sequence is derived from amino acid sequence with codon based sequence. An oligo sequence can be synthesized with multiple bases at the same position, this is termed as degenerate base also sometime referred as "wobble" position or "mixed base".
IUB (International Union of Biochemistry) has established single letter codes for all possible degenerate possibilities. An example is "R" that is A+G at the same position with 50% of the oligo sequence will have an A at that position, and the other 50% have G. A degenerate base position may have any combination of two, three, or four bases.

Chemical synthesis of oligos using IUB degenerate bases is programmed and automated to deliver the percentage of each base for reaction at that specific base position; example for the letter "N", 25% of each base will be delivered for coupling. The delivery and coupling may not be 100% accurate and efficient for each base and thus approximately 10% deviation should be expected and considered in the final oligo sequence.

For degenerate (mixed bases) positions use the following IUB codes.
R=A+G
Y=C+T
M=A+C
K=G+T
S=G+C
W=A+T
H=A+T+C
B=G+T+C
D=G+A+T
V=G+A+C
N=A+C+G+T

Custom spiking is the addition of differing molar concentration of bases at a single position, this is different from degeneracy at a position based on codons. Codon based degeneracy is usually equimolar concentration of each base at the same position (done at no extra charge for all internal and 5' position, see order form for single letter IUB codes). Custom spiking (example, 10% A, 75% G, 5% C & 10% T) has to be specified as required on the order form.

Custom column has to be prepared when the degeneracy and custom spiking is at the 3' position.Customers who wish custom spiking at certain positions of their oligo must include the relevant specifics position and spiking composition) in the comments section of the on-line order form for that particular oligo. The prices listed below is for one 3' site or up to 8 internal sites in the same oligo.

Degenerate bases, in the context of modified bases, refers to their ability to form a reasonably stable base pair with more than one base, for example, with all pyrimidines (C and T) or purines (A and G). Examples of degenerate bases include deoxyinosine (dI) and 5-nitroindole, both of which can pair with all four naturally-occurring bases. Incorporation of degenerate base modifications is desirable in cases when either imprecise or random base-pairing is required, and the resulting "mismatched" complements need to be stable. Examples include reverse-translation of known protein sequence for oligo design (oligos to be used as primers or probes), development of an in vitro or in vivo oligo probe able to hybridize to related but distinct genes (such as viral sub-strains or allelic variants (SNPs), indels, etc), in vitro site-directed mutagenesis and motif cloning (1).