Beginner’s Walkthrough: Designing Your First Probe with OligoLocator

How OligoLocator Streamlines Oligonucleotide Design for PCR and ASODesigning effective oligonucleotides—short DNA or RNA sequences used as primers, probes, or therapeutic antisense oligonucleotides (ASOs)—is a critical step in molecular biology workflows. Poorly chosen oligos can cause failed PCR reactions, off-target binding, low assay sensitivity, or ineffective therapeutics. OligoLocator is a specialized design tool that simplifies and accelerates this process by combining sequence analysis, thermodynamic prediction, specificity screening, and practical design rules into a single, user-friendly interface. This article explains the core challenges of oligonucleotide design, how OligoLocator addresses them, and practical workflows for PCR and ASO projects.

Key challenges in oligonucleotide design

Designers face several recurring problems:

• Sequence specificity: avoiding off-target binding to similar genomic regions.
• Melting temperature ™ control: matching primer or probe Tm for robust annealing.
• Secondary structures: hairpins and self-dimers reduce effective concentration.
• GC content and sequence composition: extreme GC or AT stretches reduce performance.
• Location constraints: for ASOs, targeting functional regions (splice sites, UTRs) matters; for PCR, primer spacing and amplicon size are critical.
• Workflow speed and reproducibility: manual checking across tools is time-consuming and error-prone.

How OligoLocator solves these problems

OligoLocator streamlines design by integrating four core capabilities:

1. Sequence-aware candidate generation

   • Automatically scans input target sequences to propose candidate oligos across specified windows and lengths.
   • Applies customizable positional filters (e.g., avoid SNPs, target exon–exon junctions, exclude repetitive regions).

2. Thermodynamic and secondary-structure prediction

   • Computes accurate melting temperatures using nearest-neighbor thermodynamics (with salt and oligo concentration adjustments).
   • Predicts hairpins and self-/cross-dimers, flagging problematic sequences.

3. Specificity and off-target screening

   • Rapidly screens candidate oligos against reference genomes or transcriptomes to detect potential off-target hybridization.
   • Reports alignment locations, mismatch tolerance, and predicted off-target Tm to prioritize safe candidates.

4. Practical rule enforcement and scoring

   • Enforces common best-practice rules (GC content ranges, terminal base preferences, avoiding homopolymers).
   • Assigns composite scores combining Tm match, secondary-structure penalties, and specificity metrics to rank candidates.

These features reduce manual steps and centralize decision-making so users can move quickly from sequence to validated oligo lists.

Specific workflows

PCR primer design

• Input target sequence or genomic coordinates; specify desired amplicon length (e.g., 100–400 bp).
• OligoLocator scans for primer pairs whose forward and reverse primers:
  • Have matched Tm within a user-defined tolerance (commonly ±1–2 °C).
  • Lack strong hairpins or dimers (ΔG thresholds).
  • Produce minimal off-target alignments and uniquely map to the intended locus.
• The tool can suggest primer pairs with product-specific checks such as avoiding primer–primer complementarity across the pair, and predicting expected amplicon size and sequence.
• Output includes ranked primer pairs with detailed metrics, allowing quick selection and exporting of ready-to-order sequences.

Practical tip: for qPCR assays, select primers that avoid long amplicons and target exon–exon junctions when amplifying cDNA to reduce genomic DNA amplification.

Antisense oligonucleotide (ASO) design

• Input the target mRNA or pre-mRNA sequence and annotate regions to prioritize (e.g., translation start site, splice sites, regulatory motifs).
• OligoLocator generates candidate ASOs considering:
  • Optimal length for the chosen chemistry (e.g., 15–20 nt for many gapmers).
  • Local RNA secondary structure propensity and accessibility predictions, so candidates target accessible loop regions or single-stranded stretches.
  • Off-target transcriptome screening to avoid unintended knockdown.
• The tool can integrate chemical-modification rules (e.g., LNA, 2’-O-Me placements) so melting temperature and potency predictions reflect modified bases.
• A scoring system balances predicted binding affinity, specificity, and structural accessibility to prioritize candidates for in vitro testing.

Practical tip: combine OligoLocator accessibility predictions with experimental validation (e.g., RNase H mapping or tiled ASO screens) to confirm effective binding sites.

Usability features that save time

• Batch processing: design many oligos across multiple targets in one run.
• Export formats: support for CSV, FASTA, and order-ready sheets for oligo vendors.
• Customizable parameter presets: save templates for common assay types (PCR, qPCR, Sanger sequencing, ASO gapmers).
• Visual reports: coverage maps, predicted secondary-structure diagrams, and specificity hit lists help users interpret results quickly.
• API access: integrate OligoLocator into automated pipelines for high-throughput projects.

Example case study (PCR)

A lab needed primers for 150 human gene targets for expression validation by qPCR. Using OligoLocator with a preset qPCR template:

• Generated primer pairs for all 150 targets in under an hour.
• Automatic off-target screening eliminated pairs mapping to pseudogenes.
• Final selection required only minimal manual curation; ordering and validation PCRs succeeded for >90% of assays on first attempt.

Limitations and complementary strategies

• In silico predictions are not perfect—empirical validation remains necessary.
• RNA accessibility predictions are approximate; experimental methods (e.g., SHAPE, RNase H assays) improve confidence for ASO targets.
• Off-target predictions depend on the completeness and accuracy of the reference genome/transcriptome used.

Use OligoLocator to narrow down robust candidate lists, then validate experimentally with small-scale screens and controls.

Conclusion

OligoLocator accelerates oligonucleotide design for PCR and ASO workflows by integrating candidate generation, thermodynamic modeling, off-target screening, and practical design rules into a single streamlined platform. It reduces manual checks, improves reproducibility, and shortens time-to-experiment while supporting high-throughput and customizable workflows. When paired with targeted experimental validation, OligoLocator helps teams move from sequence to reliable oligos faster and with fewer costly failures.