Primer Analyzer 2025: Professional Primer Analysis Tool

Step 1: Enter Your Sequence

Start by entering your oligonucleotide sequence in the left panel. You can input DNA sequences using A, T, C, G or RNA sequences using A, U, C, G. The sequence can include spaces for readability—they will be automatically removed during calculation. The tool displays the sequence length in real-time as you type.

Step 2: Add Modifications (Optional)

If your oligo includes modifications, use the dropdown buttons above the sequence input:

• 5' MOD: Add 5' modifications like phosphate, biotin, amino groups, or fluorophores (Cy3, Cy5, FAM)
• INTERNAL: Specify internal modifications such as phosphorothioate, 2'-O-Methyl, LNA, or PNA
• 3' MOD: Add 3' modifications including phosphate, amino groups, inverted dT, or spacers
• MIXED BASES: Use degenerate bases (R, Y, M, K, S, W, B, D, H, V, N) for ambiguous positions

Step 3: Adjust Parameters

In the middle panel, configure calculation parameters. Choose a preset or customize settings:

Note: Higher Mg²⁺ in qPCR preset reflects typical reaction conditions for probe-based assays. SpecSheet preset uses manufacturer-recommended conditions for oligo characterization.

Step 4: Choose Analysis Function

Select the appropriate analysis function from the right panel:

• ANALYZE: Comprehensive one-click analysis providing GC content, Tm, molecular weight, extinction coefficient, and more
• HAIRPIN: Predict potential hairpin structures that could interfere with oligo function
• SELF-DIMER: Analyze self-dimerization potential where the oligo binds to itself
• HETERO-DIMER: Evaluate cross-hybridization between two different oligos (requires second sequence input)
• NCBI BLAST: Open NCBI BLAST to search for sequence matches in public databases
• TM MISMATCH: Explore how mismatches at different positions affect melting temperature

Step 5: Interpret Results

Review the calculated results displayed below the analysis buttons. Results include basic properties, OD260 calculations, reverse complement sequence, and analysis-specific outputs based on your selected function.

Basic Properties

• GC Content: Percentage of G and C bases. Optimal range: 40-60% for most applications. Higher GC content increases Tm and stability but may reduce specificity.
• Molecular Weight: Total mass of the oligonucleotide in g/mol. Used for concentration calculations and synthesis yield estimation.
• Length: Number of nucleotides. Typical primers: 18-25 nt; probes: 20-30 nt.

Melting Temperature (Tm)

Tm represents the temperature at which 50% of the oligo is in double-stranded form. Three calculation methods are provided:

• Basic Tm: Simple formula based on length and GC content. Suitable for quick estimates.
• Salt-adjusted Tm: Accounts for Na+ concentration effects. More accurate than basic Tm.
• Nearest-neighbor Tm: Uses 2025 SantaLucia nearest-neighbor thermodynamics—most accurate method. Recommended for critical applications. Incorporates sequence context, salt effects, and oligo concentration.

PCR Annealing Temperature: Typically set 3-5°C below the lowest primer Tm in a pair. For qPCR probes, Tm should be 5-10°C higher than primer Tm.

OD260 Calculations

• Extinction Coefficient (ε): UV absorbance per mole at 260 nm. Higher values indicate stronger absorbance. Used to calculate concentration from OD260 measurements.
• nmol/OD260: Nanomoles per optical density unit. Useful for converting OD measurements to molar concentration.
• µg/OD260: Micrograms per optical density unit. Useful for converting OD measurements to mass concentration.

Secondary Structure Analysis: ΔG Risk Levels

ΔG (Gibbs free energy) values indicate structure stability. Use this guide to interpret hairpin and dimer results:

Tm Calculation: Nearest-Neighbor Method

The nearest-neighbor method calculates Tm by summing thermodynamic contributions from adjacent base pairs. This tool implements parameters from:

• SantaLucia (1998) PNAS: Unified nearest-neighbor thermodynamic parameters for DNA/DNA duplexes
• Owczarzy et al. (2004) Biochemistry: Enhanced salt correction algorithms accounting for monovalent and divalent cations
• Owczarzy et al. (2008) Biochemistry: Mg²⁺ concentration effects and dNTP corrections

This method accounts for: sequence context effects, salt concentration (Na⁺, Mg²⁺, dNTPs), oligo concentration, and terminal base pair stability. The formula: Tm = ΔH° / (ΔS° + R × ln(C/4)) - 273.15 + salt corrections

Secondary Structure Prediction

Hairpin and dimer predictions use established thermodynamic parameters:

• SantaLucia & Hicks (2004): Nearest-neighbor parameters for secondary structure thermodynamics
• Mathews et al. (1999) JMB: Loop entropy and size-dependent penalties
• Algorithm: Scans for complementary regions, calculates ΔG using stacking energies, applies loop penalties, reports most stable structure (lowest ΔG)

ΔG Interpretation: More negative values indicate more stable structures. ΔG < -5 kcal/mol indicates high probability of structure formation at PCR annealing temperatures.

Extinction Coefficient Calculation

The extinction coefficient (ε) is calculated using the nearest-neighbor approximation method:

• Tataurov et al. (2008) Biophys Chem: Nearest-neighbor extinction coefficients for DNA/RNA
• Each dinucleotide contributes a specific ε value (e.g., AA = 13,700 L/(mol·cm))
• Sum of dinucleotide contributions accounts for hypochromicity (base stacking effects)
• More accurate than simple base-counting methods (±2-5% accuracy vs ±10-15%)

Implementation Features

• Owczarzy (2008) salt corrections: Mg²⁺ and dNTP effects on Tm
• Mathews (1999) loop parameters: Size-dependent penalties for hairpin and internal loops
• Modification support: Biotin, fluorophores (Cy3/Cy5/FAM), phosphorothioate, LNA, PNA
• Real-time calculation: Instant results as you type, optimized algorithms
• NCBI BLAST integration: One-click sequence validation and specificity checking

For PCR Primer Design

• Use ANALYZE function to check basic properties (GC content, Tm)
• Ensure primer pair Tm values are within 2-3°C of each other
• Check SELF-DIMER and HETERO-DIMER to avoid primer-dimer formation
• Verify HAIRPIN analysis—avoid primers with stable secondary structures
• Use PCR parameter preset for accurate Tm calculations

For qPCR Probe Design

• Select qPCR parameter preset for optimal conditions
• Probe Tm should be 5-10°C higher than primer Tm
• Check HAIRPIN to ensure probe doesn't form secondary structures
• Verify no cross-hybridization with primers using HETERO-DIMER
• Consider using modified bases (LNA, 2'-O-Methyl) for increased stability

For Multiplex Assays

• Analyze all primer pairs together using HETERO-DIMER
• Ensure all primers have similar Tm values (±2°C)
• Check for cross-hybridization between all primer combinations
• Use TM MISMATCH to understand specificity requirements
• Verify GC content is balanced across all primers

Common Issues & Solutions

• High hairpin ΔG: Redesign sequence to break self-complementary regions
• Low Tm: Increase primer length or GC content (within 40-60% range)
• High self-dimer risk: Avoid palindromic sequences or add mismatches
• Extreme GC content: Redesign to achieve 40-60% GC content
• Cross-hybridization: Modify primer sequences to reduce complementarity