Incorporation of degenerate base modifications is desirable in cases when either imprecise or random base-pairing is required, and the resulting “mis-matched” complements need to be stable. Application of using degenerate base includes:
- Reverse-translation of known protein sequence
- Development of an in vitro or in vivo probe to hybridize to related but distinct gene
- In viro site-direct mutagenesis and motif cloning.
Mixed Bases (N)
There are three strategies can be use for primer design to dealt with the degeneracy of the genetic code. First, a mixed base addition (N) is used to form the degenerate site. This approach is best if the number of degenerate sites is small.
Universal Bases
Second option is the use of 2’-deoxyInosine, Inosine, or Nebularine or 2’-deoxyNebularine which is capable of pairing with all four natural nucleotides with unequal affinity (I-C >I-A>I-T~I-G>I-I). Regardless, inosine continues to be successfully used in a variety of applications which require 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.
Incorporating inosine instead of mixed bases in the degenerate PCR reaction often yields superior amplification results due toinefficient hybridization of the mixed-base degenerate primers. 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 . 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 .
The third option is the use of a universal nucleoside. In this strategy, the base analog does not hybridize significantly to the other four bases and makes up some of the duplex destabilization by acting as an intercalating agent. 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 . 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 . 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. Other degenerate bases, such as 2-amino purine, iso-dG, and 5-methyliso-dC, can be use as degenerate base.
Rules for the design of primers containig Universal Bases:
- Substitution of universal bases is less destabilizing towards the termini of oligonucleotides than towards the center.
- Grouped substitution s are more easily tolerated than spaced, i.e. contiguous rather than codon third substitutions
- More than 2-3 codon their substitutions in a 20 mer may not realiably yield a sequencing ladder or a PCR product .
- More than a 3 contigous substitutions in a primer may give reduced PCR products or an incorrect sequencing ladder.
- 3'-substitutions may lead to incorrect PCR amplification or failure to give a proper sequencing ladder.
Degenerate Bases: P and K
Some primer/template systems may be unable to tolerate the level of destabilization caused by universal base. Such systems may then be candidates for the use of primers containing P and K degenerate bases. While inosine has functioned relatively well in its role as a universal base but its hybridization properties are not ideal and, when incorporated into PCR primers, it has been reported to code primarily as G. 5-nitroidole and 3-nitropyrrole are categoriezed to the same group of universal bases but their destabilizing effect on duplexes makes them suitable for use in PCR primers with only a few substitution sites. Fully degenerate sites may be formed in an oligonucleotide using an A/C/G/T mix but the complexity of the mixture of oligonucleotides formed in this way obviously rises with each insertion and limits the usefulness of this technique. The use of K-2'-deoxyribose or P-2'-deoxyribose are degenerate bases mimicking a C/T and A/G mix can be used to replace the natural DNA bases with little estabilzation, and it has been shown that they can be substituted independently into oligonucleotide primers for the polymerase chain reaction. They can also be used together in the oligomers to prime DNA syntehsis in PCR and in sequencing reactions more effective than dI.
References:
- R. Nichols, P.C. Andrews, P. Zhang, and D.E. Bergstrom, Nature, 1994, 369, 492-493.
- D.E. Bergstrom, P. Zhang, P.H. Toma, P.C. Andrews, and R. Nichols, J. Am. Chem. Soc., 1995, 117, 1201-1209.
- D. Loakes and D.M. Brown, Nucleic Acids Res., 1994, 22, 4039-4043.
- D. Loakes, D.M. Brown, S. Linde, and F. Hill, Nucleic Acids Res., 1995, In press.
- P. Kong Thoo Lin and D.M. Brown, Nucleic Acids Res., 1992, 20, 5149-5152.
- P. Kong Thoo Lin and D.M. Brown, Nucleic Acids Res., 1989, 17, 10383.
- D.M. Brown and P. Kong Thoo Lin, Carbohydrate Research, 1991, 216, 129-139.
Bio-Synthesis degenerate site modification can be incorporate at any position of an oligonucleotide . Every oligo synthesized is strictly controlled for quality by using either MALDI-TOF mass spectrometry or polyacrylamide gel electrophoresis (PAGE) analysis. Final yields are determined using UV absorbance at OD260 In addition, we perform QC methods tailored to specific modifications, such as OD ratio measurement where appropriate.
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