LAMP Primer Tm + GC Checker

How LAMP amplification works

LAMP (Loop-mediated isothermal AMPlification) uses a strand-displacing DNA polymerase (typically Bst) and 4–6 primers to amplify DNA at a constant temperature of 60–65 °C. Unlike PCR, LAMP does not require thermal cycling.

The reaction proceeds through a series of self-priming steps: 1. F3/B3 (outer primers) initiate strand displacement 2. FIP/BIP (inner primers) create dumbbell-shaped intermediates 3. LF/LB (loop primers) accelerate amplification by priming within loops

The result is a concatemer of alternating inverted repeats, producing 10⁹–10¹⁰ copies in 30–60 minutes.

Notomi primer design guidelines

LAMP primers must satisfy several constraints for reliable amplification:

• F3/B3 (outer): 18–22 nt, Tm 55–67 °C, GC 40–65%
• FIP/BIP (inner): 40–60 nt total (F1c+F2 or B1c+B2), Tm 60–70 °C
• LF/LB (loop): 15–25 nt, Tm 55–67 °C
• Tm difference between any primer pair should be ≤5 °C
• GC content 40–65% for all primers
• Strong 3′-end stability (ΔG < −8 kcal/mol) for efficient extension

These guidelines are from Notomi et al. (2000) and NEB LAMP technical resources.

Nearest-neighbour Tm calculation

This tool uses the SantaLucia (1998) unified nearest-neighbour model:

Where ΔH and ΔS are summed from all dinucleotide steps plus initiation parameters. Salt correction adjusts ΔS:

Default conditions: [Na⁺] = 50 mM, [oligo] = 0.25 µM, matching typical LAMP buffer conditions.