Degenerate Bases & Spiking Applications

Degenerate Bases & Spiking Applications

The most commonly used degenerate modified base is deoxyinosine, which serves as a more-or-less “universal” base, as it is capable of pairing with all four natural nucleotides, though not with equal affinity (I-C >I-A>I-T~I-G>I-I). Even so, inosine continues to be successfully used in this role in a variety of applications requiring degeneracy at certain base positions of primers and probes, particularly at wobble positions, where degeneracy might be needed to permit annealing to many different, but closely related, sequences (2). For degenerate PCR, incorporating inosine instead of mixed bases into degenerate primers often yields superior amplification results do to inefficient hybridization of the mixed-base degenerate primers (3). When a guanine-rich PCR primer is needed, substitution of inosine for one or more guanines helps reduce undesirable G-quartet formation and primer-dimer artifacts (4). For DNA microarrays, inosine can be used to increase the stability of an oligo library without increasing the library’s diversity, at considerable cost savings (5). Other degenerate bases are useful for certain specialized applications. For example, 5-nitroindole base-pairs indiscriminately with any of the natural nucleotides, a consequence of the fact that it interacts via base-stacking, not hydrogen bonding (6). 5-nitroindole has been incorporated into nested sets of oligo probes to target regions of rRNA in different microorganisms in order to ensure equal probe specificity across them (7). However, its ability to act as a “universal” degenerate base is position-dependent, that is, on where it is located within a primer or probe. The potential uses of other degenerate bases, such as 2-amino purine, iso-dG, and 5-methyliso-dC, can be found in their respective tech sheets.

References

(1) Loakes, D. The applications of universal DNA base analogues.Nucleic Acids Res. (2001), 29: 2437-2447.
(2) Ohtsuka, E., Matsuki, S., Ikehara, M., Takahashi, Y., Matsubara, K. An alternative approach to deoxynucleotides as hybridization probes by insertion of deoxyinosine at ambiguous codon positions. J. Biol. Chem. (1985), 260: 2605-2608.
(3) Liu, H., Nichols, R. PCR amplification using deoxyinosine to replace entire codon and at ambiguous positions.Biotechniques. (1994), 16: 24-26.
(4) Cheng, A., Van Dyke, M.W. Oligodeoxyribonucleotide length and sequence effects on intramolecular and intermolecular G-quartet formation. Gene (1997), 197: 253-260.
(5) Watkins, N.E., SantaLucia, J. Nearest-neighbor thermodynamics of deoxyriboinosine pairs in DNA duplexes. Nucleic Acids Res. (2005), 33: 6258-6267.
(6) Loakes, D.; Brown, D.M. 5-Nitroindole as a universal base analogue. Nucleic Acids Res. (1994), 22: 4039-4043.
(7) Zheng, D.; Raskin, L. Quantification of Methanosaeta species in anaerobic bioreactors using genus- and species-specific hybridization probes. Microb. Ecol. (2000), 39: 246-262.