Degenerate Bases

Degenerate Bases & Mixed‑Nucleotide Oligonucleotides

Build diversity with IUPAC degenerate bases and per‑cycle mixture control. From randomers/UMIs and barcodes to NNK/NNS codon mutagenesis and targeted biased mixes, we manufacture high‑complexity pools with validated composition and full QC.

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Overview

Bio‑Synthesis provides end‑to‑end degenerate base (IUPAC) & mixed‑nucleotide services for UMIs/barcodes, randomers, and NNK/NNS codon mutagenesis. We handle design consultation (IUPAC schemes, biased ratios, NNK/NNS vs. trimer strategies), per‑site dosing with compensation for chemistry bias, custom synthesis from µmol → multi‑gram, and a full QC package.

Deliverables can include composition reports (trityl monitoring), optional NGS validation (sentinel or full‑length), CoA & method summaries, and RUO → GMP‑like documentation. We also offer kitting/aliquoting, barcoding, and logistics support. Inosine and universal base options are available where pairing tolerance is required.

IUPAC: R Y S W K M B D H V N
Custom doping (%, mol)
NNK/NNS schemes
Inosine • universal base
QC: LC‑MS • NGS option
Design & Dosing
Equimolar or biased
IUPAC per site
Use Cases
UMIs • barcodes •
NNK/NNS • trimer mixes
Validation
LC‑MS •
sentinel/full‑length NGS
Supply
Desalt → HPLC/UPLC •
RUO→GMP‑like

IUPAC Degenerate Codes

Code Meaning Base set Default mix Notes
R purine A or G 50:50 Biased on request
Y pyrimidine C or T 50:50
S strong G or C 50:50
W weak A or T 50:50
K keto G or T 50:50
M amino A or C 50:50
B not A C/G/T ≈33:33:33 Bias to avoid G if needed
D not C A/G/T ≈33:33:33
H not G A/C/T ≈33:33:33
V not T(U) A/C/G ≈33:33:33
N any A/C/G/T ≈25:25:25:25 Custom e.g., A:10/C:40/G:40/T:10
Custom schemes: NNK (N,N,K=G/T) → 32 codons, 1 stop (TAG). NNS (S=G/C) similar; both reduce stops vs NNN. Trimer codon mixes available.

Technology & Methods

Per‑Cycle Dosing

During each coupling, we meter A/C/G/T according to your ratio. Ratios can vary by position in one sequence.

Bias Management

Compensate for reactivity differences; trityl monitoring keeps composition near spec. Optional NGS confirms base distributions.

Pools & Purification

For maximum diversity, desalt is typical. If HPLC is required, expect some diversity loss; we can tune gradients to mitigate.

Design tip: For UMIs/barcodes, sample ≥10× the unique space (e.g., 12N ≈ 16.7M combos ⇒ ≥167M reads). For codons, NNK/NNS minimize stops; trimer mixes provide strict control.

Applications

UMIs & Barcodes

Error‑correction, deduplication, and molecule tracking with NL randomers or structured barcodes.

Protein Mutagenesis

NNK/NNS codon randomization for libraries; trimer codon mixes reduce bias and stop codons.

Probe/Assay Robustness

Degenerate positions improve inclusivity; inosine/universal bases can be used where tolerance is needed.

Products & Ordering

Product / Modification Description Function Application Code
Equimolar Degenerate Mix (R,Y,S,W,K,M,B,D,H,V,N) Standard IUPAC mixes at specified positions. Controlled variability UMIs/barcodes, inclusivity primers [IUPAC‑Std]
Biased Degenerate Mix (Custom %) Non‑equimolar ratios per position (e.g., A:10/C:40/G:40/T:10). Tuned distributions Directed evolution, motif tiling [IUPAC‑Bias]
NNK / NNS Codon Mix Per‑codon dosing to minimize stops (NNK/NNS); position‑specific. Codon randomization Protein libraries [NNK]/[NNS]
Trimer Codon Mix (Defined) Pre‑mixed codon trimers for uniform amino‑acid coverage. Reduce codon bias High‑quality libraries [TriCodon]
Inosine (dI/rI) at Variable Sites Wobble base to tolerate multiple pairings. Pairing tolerance Inclusive primers/probes [Ino]
Universal Base (e.g., 5‑Nitroindole) Stacking “universal” analog; note potential Tm reduction. Position flexibility Uncertain positions; scanning [UB]
Combinatorial Pools (Windowed) Degenerate windows tiled across regions with per‑site mix control. Coverage scanning Promoter/aptamer discovery [Pool‑Tile]
Customizations: per‑site ratios, barred bases (e.g., “not G”), length/scale options, desalting vs HPLC/UPLC, aliquots & kitting, RUO→GMP‑like docs.

Technical Notes

  • Complexity & sampling. Plan ≥10× sampling of unique space (e.g., 12N ≈ 16.7M → ≥167M reads) to avoid collisions.
  • Chemistry bias. We compensate for base reactivity differences during dosing; verify composition via sentinel or full‑length NGS.
  • PCR representation. Minimize cycles; use high‑fidelity enzymes; UMIs assist with deduplication.
  • Purification impact. Desalt maximizes diversity; HPLC/UPLC improves purity but can reduce representation—choose per assay.
  • Tm & hybridization. Degenerate positions broaden Tm; inosine/universal bases may lower Tm; adjust length/ionic strength.

FAQ

What are IUPAC degenerate bases?

IUPAC codes define base mixtures (e.g., R=A/G, Y=C/T). We meter A/C/G/T per cycle to create controlled variability.

What’s the difference between NNK and NNS schemes?

NNK (K=G/T) and NNS (S=G/C) both reduce stop codons vs NNN; we can also supply trimer codon mixes for uniform AA coverage.

Can you produce biased (non‑equimolar) mixes at specific sites?

Yes. We dose custom percentages per position (e.g., A:10/C:40/G:40/T:10) and report composition on request.

Will HPLC purification reduce diversity in randomers?

Somewhat. Desalt maximizes representation; tuned gradients can mitigate loss if HPLC/UPLC is required.

Do you offer NGS composition validation?

Yes. Sentinel‑site or full‑length NGS can quantify base distributions and confirm dosing accuracy.

What’s the role of inosine and universal bases?

Inosine provides wobble pairing tolerance; universal bases (e.g., 5‑nitroindole) help at uncertain positions but may lower Tm.

Ready to specify your degenerate design?

Send positions, IUPAC codes or % ratios, desired complexity, purification, and any NNK/NNS/trimer scheme.

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References

  1. IUPAC nucleotide ambiguity codes and usage in synthesis.
  2. NNK/NNS randomization and trimer‑codon strategies.
  3. UMIs/barcodes for sequencing error correction and deduplication.

On request, we’ll align mixture specs and validation plans to your assay and instrument.

Why Choose Bio-Synthesis

Trusted by biotech leaders worldwide for over 40+ years of delivering high quality, fast and scalable synthetic biology solutions.