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Universal Bases


Pairing between the standard DNA bases strongly favors A:T and G:C alignment. Mispairing of bases within a DNA duplex is energetically unfavorable and will destabilize the duplex. In most applications that employ synthetic oligonucleotides, the increased stability of a perfectly paired duplex is desired; the ability of even a single mismatch to disrupt or prevent stable hybridization can be exploited in experimental design and can allow oligo based assays to have extremely high specificity. Examples include allelic discrimination assays or single nucleotide polymorphism (SNP) detection.

Universal bases refer to those bases that exhibit the ability to replace any of the four normal bases without significantly destabilizing neighboring base-pair interactions or disrupting the expected functional biochemical utility of the modified oligonucleotide. Oligonucleotides that include a universal base will function as a primer for DNA sequencing or PCR (Loakes, D, et al. 1995). Such oligos will behave as normal substrates for polynucleotide kinase, DNA ligase, and other modifying enzymes (Hill, F. Loakes, D. and Brown D.M., 1998). Two universal bases in common use today are 3-nitropyrrole 2'-deoxynucloside and 5-nitroindole 2'-deoxynucleoside (5-nitroindole); their structures are shown in Figure 1.

However, it is sometimes necessary to use oligonucleotides that allow for imprecise or random base pairing so that imperfect complements are stable. The most common example of this application involves reverse-translation to design oligos (predicting DNA coding sequence) based on a known protein sequence or the insertion of mixed base sites to allow a single oligo probe to hybridize to related but distinct genes (such as HIV sub-strains or allelic variants). Other recent applications include ligase chain reaction, in situ hybridization, in vitro mutagenesis, and motif cloning. Oligo compositions that allow for degenerate hybridization can be achieved via two routes, 1) incorporation of a mixture of standard bases at a given position, or 2) incorporation of an artificial base that permits degenerate base-pairing.

Use of the mixed base incorporation strategy is simple but results in a heterogeneous population of molecules [see FAQ on Mixed Bases]. Even if the number of mixed base sites is limited, a large number of distinct molecules can exist within the population and the functional concentration of one specific desired probe or primer can be quite low. For example, insertion of only 4 "N" bases into a sequence results in 4 x 4 x 4 x 4 = 256 unique species after synthesis.

Alternatively, non-natural bases can be substituted in a sequence that allow for random base-pairing or base-pairing with reduced stringency. Use of such "universal" and "degenerate" bases permits synthesis of oligos that allow degenerate hybridization yet are of low complexity.

Historically, the first "universal" base employed was 2'-deoxyinosine (dI). Deoxyinosine is a naturally occurring base that, while not truly universal, is less destabilizing than mismatches involving the 4 standard bases. Hydrogen bond interactions between dI and dA, dG, dC, and dT are weak and unequal, with the result that some base-pairing bias does exist with dI:dC hybridization>dI:dA> dI:dG> dI:dT (Kawase, Y. et al 1986). Not surprisingly, when present in a DNA template, dI preferentially directs incorporation of dC in the growing nascent strand by DNA polymerase.

More recently, non-natural bases have been engineered that functionally are true universal bases and will not destabilize a Watson-Crick DNA duplex when paired with either dA, dG, dC, or dT. The applications of these universal DNA base analogues have been recently reviewed (Loakes 2001).

Universal and Degenerate Bases

Universal bases refer to those bases that exhibit the ability to replace any of the four normal bases without significantly destabilizing neighboring base-pair interactions or disrupting the expected functional biochemical utility of the modified oligonucleotide. Oligonucleotides that include a universal base will function as a primer for DNA sequencing or PCR (Loakes, D, et al. 1995). Such oligos will behave as normal substrates for polynucleotide kinase, DNA ligase, and other modifying enzymes (Hill, F. Loakes, D. and Brown D.M., 1998). Two universal bases in common use today are 3-nitropyrrole 2'-deoxynucloside and 5-nitroindole 2'-deoxynucleoside (5-nitroindole); their structures are shown in Figure 1.

Figure 1. Structures of universal bases 3-nitropyrrole and 5-nitroindole

The two examples above act as truly random, or "N" bases. Other base modifications have been synthesized that are more specific. Examples include the pyrimidine (C or T) analogue 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one, designated as "p", and the purine (A or G) analogue N6-methoxy-2,6-diaminopurine, designated as "k". The structures of "p" and "k" are illustrated in Figure 2. The "p" base will pair with dA or dG while the "k" base will pair with dT or dC (Bergstrom, D. E., Zhang, P., and Johnson, W.T., 1997).

Figure 2. Structures of degenerate bases "p" and "k"

Properties of oligonucleotides containing universal bases

Melting Behavior

The thermodynamic effect of substituting sites in primers with universal bases has been studied using thermal melt experiments. Not surprisingly, the presence of a universal base in a duplex was destabilizing compared to the perfect match duplex, however this duplex was more stable than those having mismatches between standard bases; substitution with 3-nitropyrrole and 5-nitroindole near the ends of oligonucleotides was less destabilizing than substitution in the center (Nichols et al., 1994; Loakes and Brown, 1994). Modified oligonucleotides are more stable if universal bases are grouped together rather than dispersed throughout the sequence. 5-nitroindole is the most stable of the available universal bases. In one study, six insertions of 5-nitroindole into an oligonucleotide were more stable than three insertions of 3-nitropyrrole based on stacking enthalpy measurements (Loakes and Brown, 1994).

A recent thermal melting study reported Tm values and thermodynamic parameters on oligonucleotides substituted with universal bases (Bergstrom et al., 1997). Each nucleoside analog displayed a unique pattern of base pairing preferences. The least discriminating analog was 3-nitropyrrole, for which Tm values differed by 5C (19.4 -24.5C) and ΔG25C ranged from -6.1 to -6.5 kcal/mol. 5-Nitroindole gave duplexes significantly higher thermal stability, with Tm differing by 11.4C (35.0 to 46.5C) and ΔG25C ranging from -7 to -8.5 kcal/mol. Incorporation of deoxyinosine (dI) although often used as a universal nucleoside, showed a stronger base pair bias than any of the nitroazole derivatives; Tm values ranged from 35.4C when paired with G to 62.3C when paired with C demonstrating a bias for base pairing with C.

Another study (Vallone and Benight, 1999) reported on the effects of 5-nitroindole on the thermodynamic stability of DNA hairpins. Incorporation of 5-nitroindole bases in the duplex stem or loop regions of short DNA hairpins significantly affects both their enthalpic and entropic melting components in a compensating manner, while the transition free energy varies linearly with the transition temperature. Results of circular dichroism measurements also revealed slight differences between the modified hairpins and the control in both the duplex and melted states, suggesting subtle structural differences between the control and DNA hairpins containing a 5-nitroindole base.

Universal bases in primers: DNA sequencing and PCR

Nichols et al. (1994) compared perfect-match primers with primers containing mixed bases and universal bases in DNA sequencing. Primers were modified at the third position in 4 codons with either an "N" mix (resulting in a 256-fold degenerate population), with dI, and with 3-nitropyrrole. The sequencing ladder obtained using "N"-mix was unreadable and that obtained using dI was only partially readable. Results obtained using the 3-nitropyrrole primer were not only clear, it was indistinguishable from that obtained using the control primer.

In a separate study, Loakes et al. (1995) reported that the introduction of more than one 3-nitropyrrole or 5-nitroindole universal base at dispersed positions into primers significantly reduced their efficiency in sequencing reactions. In both cases, only a small number of substitutions were tolerated. However, primers containing up to four consecutive 5-nitroindole bases performed well in sequencing reactions. Consecutive 3-nitropyrrole substitutions were tolerated, but less well in comparable reactions. Similarly these authors reported that the incorporation of universal bases 3-nitropyrrole and 5-nitroindole in primers reduced their efficiency in PCR similar to their effects on sequencing reactions. In addition, in PCR experiments neither base, when incorporated into primers in codon third positions, was as effective as deoxyinosine, which was incorporated in six codon third positions in a 20-mer oligomer. P and K appear to replace natural DNA bases with little destabilization and can be used in primers for PCR (Lin and Brown, 1992).

Degenerate bases in primers: DNA sequencing and PCR

Template properties

To examine the behavior of the degenerate bases when copied by Thermus aquaticus (Taq) DNA polymerase, two oligonucleotide templates were synthesized. One template contained six dP residues, and the other six-dK residues. Each template was amplified using two flanking primers by Taq polymerase. When the resulting PCR products were sequenced and analyzed, results indicated that P directed incorporation of dA 60% and dG in 40%, whereas K directed incorporation of dC 13% and dT 87% (Hill et al., 1998).

Sequencing and PCR primers

To test the use of P and K in primers for DNA synthesize (Hill et al., 1998), the dUTPase gene of Caenorhabditis elegans, which contains three nonidentical homologous repeats, was used as a model system. The C. elegans dUTPase gene was sequenced as well as amplified using a pair of oligodeoxyribonucleotides, each 20 residues long and containing an equamolar mixture of P and K at six positions. Results shown that degenerate bases P and K can be used together in oligomers to prime DNA synthesis in PCR using Taq polymerase and in sequencing reactions using T7 DNA polymerase. No nonspecific amplification was obtained on genomic DNA of C. elegans. To compare primers containing P/K mixtures (which is equivalent to a universal base) with the equivalent primers containing dI in PCR, a pair of primers containing six modified bases was synthesized. In a direct comparison, P/K pair was more effective in primers than dI.

Practical Applications and Recommended Uses

Clearly, universal and degenerate bases have their place in the molecular biologist's toolbox. A number of potential uses and recommendations can be summarizes as follows:

Primer Design using Universal and Degenerate Bases

  1. 5-nitroindole 2'-deoxynucleoside (5-nitroindole) is preferable to 2'-deoxyinosine as a universal base although dI has the advantage of being less expensive and historically has been the most common universal base used in primer and probe synthesis.
  2. Increased specificity can be obtained by using the degenerate bases 6H, 8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one, designated as "p" as a pyrimidine (C or T) analogue, and N6-methoxy-2,6-diaminopurine, designated as "k" as a purine (A or G) analogue
  3. Substitution of universal bases is less destabilizing towards the termini of oligonucleotides than towards the center.
  4. Grouped substitutions are more easily tolerated than spaced substitutions. Specifically, designing a probe spanning several codons, each with a substitution in the third position to account for the "wobble" base can be particularly unstable.
  5. More than 2-3 contiguous substitutions in a primer may give reduced PCR products or an incorrect sequencing ladder.
  6. 3' Substitutions may lead to incorrect PCR amplification or failure to give a proper sequencing ladder.

Probe Design using Universal and Degenerate Bases

Amino Acid Abbreviations Codons(s) Sense
Alanine Ala A 5'-GCA-3'
5'-GCN-3' 5'-IGC-3'
Arginine Arg R CGA
Asparagine Asn N AAU
Aspartic Acid Asp D GAU
Cysteine Cys C UGU
Glutamic Acid Glu E GAA
Glutamine Gln Q CAA
Glycine Gly G GGA
Histidine His H CAU
Isoleucine Ile I AUU
Leucine Leu L UUA
Lysine Lys K AAA
Methionine Met M AUG ATG CAG
Phenylalanine Phe F UUU
Proline Pro P CCA
Serine Ser S UCA
Threonine Thr T ACA
Valine Val V GUA
Termination     UAA
I=inosine or 5-nitroindole, p=the pyrimidine (C or T) analogue 6H,8H-3,4-dihydropyrimido[4,5-C][1,2]oxazin-7-one, k=the purine analogue N6-methoxy-2,6-diaminopurine, otherwise standard IUPAC abbreviations for multiple bases apply.

*We recommend that one try to avoid back translating from codons for Arg, Leu and Ser, the degeneracy inherent in codons specifying these amino acids will greatly increase the number of resulting oligonucleotide sequence permutations making the functional concentration of one specific desired probe or primer very low.


Primer heterogeneity can be reduced or eliminated by using universal or degenerate bases in synthetic oligonucleotides. However, some primer/template systems may be destabilized by the incorporation of a universal base, these systems may be candidates for the use of the degenerate bases "p" or "k" where "p" is equivalent to a pyrimidine mixture (C& T) while "k" is equivalent to a purine mixture (A & G).


Bergstrom D.E., Zhang, P. and Johnson W.T. (1997) "Comparison of the base pairing properties of a series of nitroazole nucleobase analogs in the oligodeoxyribonucleotide sequence 5'-d(CGCXAATTYGCG)-3'." Nucleic Acids Res. 25:1935-1942.

Hill F., Loakes D. and Brown D.M. (1998) "Polymerase recognition of synthetic oligodeoxyribonucleotides incorporating degenerate pyrimidine and purine bases." Proc Natl Acad Sci U S A., 95:4258-4263.

Kawase, Y., Iwai, S., Inoue, H., Miura, K. and Ohtsuka E. (1986) " Studies on nucleic acid interactions. I. Stabilities of mini-duplexes (dG2A4XA4G2-dC2T4YT4C2) and self-complementary d(GGGAAXYTTCCC) containing deoxyinosine and other mismatched bases." Nucleic Acids Res., 1919:7727-7736.

Lin, P. K. and Brown, D.M. (1989) "Synthesis and duplex stability of oligonucleotides containing cytosine-thymine analogues." Nucleic Acids Res., 17:10373-10383.

Lin, P. K. and Brown D.M. (1992) "Synthesis of oligodeoxyribonucleotides containing degenerate bases and their use as primers in the polymerase chain reaction." Nucleic Acids Res., 20:5149-5152.

Loakes D. and Brown D.M. (1994) "5-Nitroindole as an universal base analogue.", Nucleic Acids Res., 22:4039-4043.

Loakes D., Brown, D.M., Linde, S. and Hill F. (1995) "3-Nitropyrrole and 5-nitroindole as universal bases in primers for DNA sequencing and PCR." Nucleic Acids Res., 23:2361-2366.

Loakes, D. (2001) "The applications of universal DNA base analogues" Nucleic Acids Res., 29:2437-2447.

Nichols, R., Andrews, P.C. Zhang, P. and Bergstrom D.E. (1994) "A universal nucleoside for use at ambiguous sites in DNA primers.", Nature, 369:492-493.

Vallone P.M. and Benight A.S. (1999) "Melting studies of short DNA hairpins containing the universal base 5-nitroindole." Nucleic Acids Res., 27:3589-3596.

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