Source: https://guthlabs.ai/docs/instruments/primer-melting-temperature-tm-calculator/mechanism/

instrument mechanism / verified-clean

# Primer Melting Temperature (Tm) Calculator mechanism

The inspectable method record for Primer Melting Temperature (Tm) Calculator, kept separate from implementation and qualification.

Status: **VERIFIED_CLEAN**Lifecycle: **verified-clean**

## Model scope

The verdict is NEEDS_REVIEW because two alternative algorithms are named but the nearest-neighbor implementation is not fully defined. Vectors 1-3 exercise exact standard Wallace-rule coefficient entries, including zero-GC and high-GC boundary behavior. The nearest-neighbor vector uses the stated SantaLucia 1998 convention and explicitly supplies every table value needed for reproduction.

- The Wallace rule Tm=4(G+C)+2(A+T) is a standard rough estimate for short, relatively simple oligonucleotides, but its applicability limits should be stated explicitly; it is not generally reliable for long primers, unusual salt conditions, mismatches, secondary structure, or modified bases.

- The salt-adjusted nearest-neighbor method is underspecified: it must define the exact SantaLucia 1998 parameter convention, salt model, oligo concentration convention, self-complementarity treatment, terminal AT corrections, initiation terms, and whether divalent ions are supported.

- The nearest-neighbor formula must specify whether concentration is total strand concentration and whether the non-self-complementary correction ln(Ct/4) or a self-complementary correction is used.

- GC% is not mathematically defined in the specification; the usual definition is 100*(G+C)/sequence length, with ambiguity for empty or ambiguous-base sequences.

- The specification calls the instrument a lookup-table engine but does not state how missing or ambiguous nucleotide symbols are handled.

## Units and conventions

- Sequence contains only uppercase or case-normalized A, C, G, and T bases; ambiguous symbols, gaps, and modified bases are rejected unless separately modeled.

- Wallace-rule coefficients are 4 degrees C per G/C base and 2 degrees C per A/T base; the result is in degrees C.

- GC% is reported as a percentage, not a fraction.

- For the nearest-neighbor vector, SantaLucia-style enthalpies are in kcal/mol, entropies are in cal/(mol*K), R=1.9872 cal/(mol*K), oligo concentration is total strand concentration in mol/L, and monovalent sodium concentration is in mol/L.

- The nearest-neighbor salt correction used here is +16.6*log10([Na+]) degrees C, with [Na+] in mol/L; this is a monovalent-salt approximation and does not independently model Mg2+ or dNTP binding.

- The nearest-neighbor example assumes a non-self-complementary duplex and uses Tm(K)=1000*DeltaH/(DeltaS+R*ln(Ct/4)), followed by conversion to degrees C and the sodium correction.

## Inspectable vector

One of 4 source-bound review vectors:

````
{
  "inputs": {
    "sequence": "ATGCGTACGATCGATCGTAC",
    "method": "Wallace rule"
  },
  "expected_outputs": {
    "Tm_C": 60,
    "GC_percent": 50
  },
  "reference_data": {
    "wallace_GC_coefficient_C_per_base": 4,
    "wallace_AT_coefficient_C_per_base": 2,
    "source_convention": "widely tabulated standard Wallace-rule values"
  },
  "working": "Length = 20. Count G+C = 10 and A+T = 10. Tm = 4*10 + 2*10 = 40 + 20 = 60 deg C. GC% = 100*(10/20) = 50%."
}
````

## Failure boundary

**BREAK_CLAIMED** · SELF_FLAGGED

The specification permits hazardous ambiguity: an empty sequence can produce a misleading raw Wallace value of 0 °C while GC% is undefined, and the nearest-neighbor branch cannot produce a numeric result without the required parameter table and explicit method, unit, and convention validation.

## Other records

[01**Overview**](<https://guthlabs.ai/docs/instruments/primer-melting-temperature-tm-calculator/>)[03**Implementation**](<https://guthlabs.ai/docs/instruments/primer-melting-temperature-tm-calculator/implementation/>)[04**Qualification**](<https://guthlabs.ai/docs/instruments/primer-melting-temperature-tm-calculator/qualification/>)

## Sources and evidence

These pointers bind the page to evidence. They do not grant authority or collapse verification into deployment.

- `VERIFIED_LEDGER.json`

- `evidence-tranche7/tranche7-results.json`

- `BOUNDARY_HAZARD_CATALOG.json`