Glen Report 35-24: Application Note Degenerate Oligonucleotides

A degenerate oligonucleotide is a mixture of sequences that differ in one or more positions. If there is a single degenerate position that contains only purines,  the mixture contains roughly equal amounts of two sequences (21). If there are ten completely random degenerate positions, the mixture contains over a million sequences (410). To describe these degenerate positions, one of eleven possible single-letter, mixed-base codes is used. The standard nomenclature for these degenerate positions is defined by the International Union of Pure and Applied Chemistry (IUPAC, Table 1). PuRine-only positions would be denoted as “R” while completely random (aNy) positions would be denoted as “N”. Degenerate oligonucleotides serve many special applications, and the role these degenerate oligonucleotides play in several applications will be discussed briefly below. 

Symbol Bases Origin of designation
R A, G puRine
Y C, T pYrimidine
M A, C aMino
K G, T Keto
S C, G Strong interaction (3 hydrogen bonds)
W A, T Weak interaction (2 hydrogen bonds)
H A, C, T not G, H follows G in the alphabet
B C, G, T not A, B follows A
V A, C, G not T (not-U), V follows U
D A, G, T not C, D follows C
N A, C, G, T aNy

Table 1. Standard nomenclature for degenerate positions.

1. Degenerate PCR

For certain PCR amplifications, the target template sequence can either be unknown or vary (e.g., SNPs, codon degeneracy, different alleles, etc.). For the former, conserved sequences from related species can be  used for primer design while for the latter, primer  design would try to accommodate all possibilities.  In either scenario, degenerate primers can be used.  The primers are designed to minimize degeneracy,  and the degenerate positions are usually placed away from the 3′-end to promote proper annealing. As an example, an 18 nt primer corresponding to Lys-Ile-Asp-Trp-Phe-Trp would have a degeneracy of 24 to cover all possible codons (Table 2). Degenerate PCR can be very effective. In one multiplex PCR assay investigation, primers of up to 64-fold degeneracy were used to detect a panel of bacteria and viruses, including some that  had significant sequence diversity.1


Application Example Sequences (5′ – 3′)
Lys Ile Asp Trp Phe Trp    
Site-directed Mutagenesis 15nt NNS NNS NNS TDK VVC 15nt  
SELEX (N30) Primer (NNN)10 Primer          

Table 2. Examples of degenerate oligonucleotides.

2. Whole Genome Amplification

Whole genome amplification is a method for indiscriminately amplifying the whole genome rather than small, distinct sections. The method is typically employed in situations where nanograms of material are amplified into micrograms of material for subsequent manipulations. Two popular approaches that use degenerate primers to accomplish this are Primer Extension Preamplification (PEP)2 and Degenerate Oligonucleotide Primed PCR (DOP-PCR).3 The primers used in these two methods are significantly more degenerate than those employed in degenerate PCR. PEP uses completely random 15 nt primers for amplification (Table 2). The one billion primer mixture (415) binds at random locations on the DNA template, and annealing temperatures are initially very low to promote hybridization. For a single human cell, it has been estimated that at least 78% of the genomic material has been replicated at least 30 times over a 50-cycle procedure.2 For DOP-PCR, a partially degenerate primer is used instead (Table 2). Six completely degenerate positions are flanked by ten defined nucleotides on the 5′-end and six defined nucleotides on the 3′-end. Like PEP, the initial annealing temperature is very low, and the 4,096 primers (46) allow species-independent general DNA amplification. As expected, both techniques generate smears of DNA fragments when analyzed by agarose gel electrophoresis.

3. Mutagenesis

Site-directed mutagenesis is a method of introducing specific changes to plasmid DNA, and degenerate oligonucleotides are one way of generating libraries for this process.4 Input oligonucleotides contain a central random codon region with constant 5′- and 3′-ends of 15 nt each that are complementary to regions adjacent to the target plasmid site. While the use of completely degenerate regions (NNN) is possible, this kind of approach has drawbacks due not only to the degeneracy of the genetic code but also to the presence of three  stop codons. Instead, one can use NNS codons  (20 amino acids and only one stop codon) or other more biased degenerate codons such as TDK (hydrophobic amino acids only) or VVC (hydrophilic amino acids only). Fifteen or more codons can be randomized in this manner at a time. For more control over codon composition in libraries, Glen Research also offers  trimer phosphoramidites.5


SELEX (Systematic Evolution of Ligands by EXponential enrichment) is an in vitro selection method for aptamers and nucleic acid enzymes that begin with a very large random pool of sequences.6-8 These sequences typically have a 30-80 nt central random region and primer binding regions to facilitate PCR amplification (Table 2). Libraries can consist of up to 1015 different sequences (~1 nmol), and multiple rounds of selective partitioning and amplification whittles the original library down to a considerably smaller number of sequences that bind to a desired target or catalyze a certain reaction.

5. Next Generation Sequencing

The arrival of next generation sequencing has allowed researchers to obtain massive sets of data, currently up to billions of reads at a time. Looking through these sizable data sets, one may want to quantify individual oligonucleotides based on the number of reads. However,  since most, if not all, sequencing methods require amplification,  and such amplification is not uniform, this does not work well. To address this, Unique Molecular Identifiers (UMIs) can be used.9 A 6-12 random nucleotide region is appended to every original strand of template as a molecular barcode to change every original strand into a unique sequence. The UMI, which is sequenced along with the target, becomes part of the data for processing. Instead of counting reads of a sequence, the count measures how many different UMIs exist  for that same sequence.

Oligonucleotide Synthesis

Degenerate oligonucleotide mixtures are typically synthesized on a single column. When a position requires two bases, then two phosphoramidites are simultaneously injected into the synthesis column. While the synthesizer can draw from two or more phosphoramidite bottles, as needed, the results vary depending on the synthesizer and are rarely homogeneous. A much more effective solution is to prepare a special premixed bottle of phosphoramidites for degenerate positions, and up to eleven of these would be required (Table 1). Over the years, our customers have asked us to prepare DNA phosphoramidite mixes for them, and we have provided them as custom products. Now, as the need for degenerate oligonucleotides continues to grow, we have decided to offer these as standard products to cover all eleven possible DNA combinations in equimolar mixes using the most popular versions of our dA, dC and dG. If one would like customized ratios to compensate for different phosphoramidite coupling rates or different protecting groups on the bases, please reach out to our Customer Service Team. All equimolar and customized ratio phosphoramidite mixes will require at least a two-week lead time.

Figure 1. Degenerate oligonucleotide synthesis with mixes


  1. M.A. Poritz, et al., PLoS One, 2011, 6, e26047.
  2. L. Zhang, et al., Proc Natl Acad Sci U S A, 1992, 89, 5847-51.
  3. H. Telenius, et al., Genomics, 1992, 13, 718-25.
  4. S.S. Sidhu, and G.A. Weiss, Constructing phage display libraries by oligonucleotide-directed mutagenesis. In Phage Display: A practical approach, T. Clackson; H. B. Lowman, Eds. 2004; pp 27-42.
  5. The Glen Report, 2020, 32.1, 8-11.
  6. A.D. Ellington, and J.W. Szostak, Nature, 1990, 346, 818-22.
  7. C. Tuerk, and L. Gold, Science, 1990, 249, 505-10.
  8. D.L. Robertson, and G.F. Joyce, Nature, 1990, 344, 467-8.
  9. T. Kivioja, et al., Nat Methods, 2011, 9, 72-4.


Product Information

Standard Base Mixes                                                                                                                     

Item Catalog No.
Mix R: dA+dmf-dG-CE Phosphoramidite Mix Mix R
Mix Y: Ac-dC+dT-CE Phosphoramidite Mix Mix Y
Mix M: dA+Ac-dC-CE Phosphoramidite Mix Mix M
Mix K: dmf-dG+dT-CE Phosphoramidite Mix Mix K
Mix S: Ac-dC+dmf-dG-CE Phosphoramidite Mix Mix S
Mix W: dA+dT-CE Phosphoramidite Mix Mix W
Mix H: dA+Ac-dC+dT-CE Phosphoramidite Mix Mix H
Mix B: Ac-dC+dmf-dG+dT-CE Phosphoramidite Mix Mix B
Mix V: dA+Ac-dC+dmf-dG-CE Phosphoramidite Mix Mix V
Mix D: dA+dmf-dG+dT-CE Phosphoramidite Mix Mix D
Mix N: dA+Ac-dC+dmf-dG+dT-CE Phosphoramidite Mix Mix N