Degenerate Bases & Spiking Design and Protocols

Degenerate Bases & Spiking Design / Protocol

The ability of inosine to act as a degenerate base makes it particularly useful as a way to reduce the overall degeneracy of degenerate PCR primer sets. Such sets are commonly used for DNA amplification of regions where only a gene's protein sequence is known, or when the goal is to amplify similar genes from different species. Since inosine is capable of base-pairing with any natural nucleotide, it can be used to substitute for any "N" (A,C,G,T) degenerate position (see Designing Degenerate Primers and Degenerate primers). When using inosine in this manner, be aware that because this base does not base-pair with natural nucleotides with equal affinity (I-C>I-A>I-T~I-G), there will be some difference in priming efficiency between the members of the degenerate primer set. However, in most cases, the overall increase in priming efficiency afforded by the 4-fold reduction in degeneracy per inosine substitution outweighs this, as such substitution both increases the effective concentration of these primers in the pool and also reduces the amount required optimization of the reaction conditions.

5-nitroindole functions as a non-hydrogen bonding universal base, and pairs indiscriminately with any natural nucleotide by base-stacking interactions. 5-nitroindole is particularly useful as a universal base in degenerate hybridization probes. Thus, it can sometimes serve as a useful alternative to inosine in cases where avoidance of bias in base-pairing is critical.

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.