{"success":true,"version":"v1","request":{"tool":"get_calculator_schema","calculator_id":"pcr-primer-tm"},"result":{"entity_type":"calculator","id":"pcr-primer-tm","calculator_id":"pcr-primer-tm","canonical_url":"https://tttkmbb.com/science/pcr-primer-tm","name":"PCR Primer Tm Calculator","title":"PCR Primer Tm Calculator – Melting Temperature (Wallace and Basic Formulas), GC Content, Molecular Weight and Reverse Complement","category":"science","category_name":"Biology, Earth & Space","tool_name":"calculate_pcr_primer_tm","featured_mcp_tool":false,"description":"Analyses a DNA primer sequence: length, GC content, melting temperature by the Wallace rule 2(A+T) + 4(G+C) and by the basic GC formula 64.9 + 41 × (G+C − 16.4) / N, single-stranded molecular weight, complement, reverse complement, self-complementarity and simple design checks.","use_when":"You are designing or checking PCR or sequencing primers and need a quick Tm estimate, GC %, the reverse complement or the oligo's molecular weight.","do_not_use_when":"You need salt- and concentration-corrected nearest-neighbour Tm values (as used by commercial primer tools), degenerate primers beyond N, or modified bases.","inputs":[{"name":"sequence","label":"Primer sequence (5'→3')","type":"string","required":true,"description":"8–60 nucleotides using A, C, G, T (U is treated as T) and N; case-insensitive; spaces, digits and 5'/3' markers are ignored.","example":"ATGCATGCATGCATGCATGC"}],"outputs":[{"name":"length","label":"Length","type":"integer","unit":"nt","decimals":4,"description":"Number of nucleotides including N."},{"name":"gc_content_percent","label":"GC content","type":"number","unit":"%","decimals":2,"description":"100 × (G + C) / length."},{"name":"tm_wallace","label":"Tm (Wallace rule)","type":"number","unit":"°C","decimals":1,"description":"2 × (A + T) + 4 × (G + C); intended for primers up to about 13–14 nt but always reported."},{"name":"tm_basic","label":"Tm (basic GC formula)","type":"number","unit":"°C","decimals":2,"description":"64.9 + 41 × (G + C − 16.4) / N; intended for primers of 14 nt or more."},{"name":"tm_recommended","label":"Tm (recommended formula)","type":"number","unit":"°C","decimals":2,"description":"Wallace value for sequences up to 13 nt, basic value for 14 nt or more."},{"name":"molecular_weight_g_mol","label":"Molecular weight (ssDNA)","type":"number","unit":"g/mol","decimals":2,"description":"A × 313.21 + C × 289.18 + G × 329.21 + T × 304.2 − 61.96 (linear, 5'-OH, anhydrous); omitted when the sequence contains N."},{"name":"complement","label":"Complement (3'→5')","type":"string","decimals":4,"description":"Base-by-base complement in the same orientation."},{"name":"reverse_complement","label":"Reverse complement (5'→3')","type":"string","decimals":4,"description":"Complement read in the opposite direction: the sequence of the opposite strand."},{"name":"self_complementary","label":"Self-complementary","type":"boolean","decimals":4,"description":"true when the sequence equals its own reverse complement (palindromic), which favours primer-dimers."},{"name":"gc_clamp","label":"3' GC clamp","type":"boolean","decimals":4,"description":"true when the 3'-terminal base is G or C."},{"name":"max_homopolymer_run","label":"Longest run of one base","type":"integer","decimals":4,"description":"Length of the longest run of identical consecutive bases."},{"name":"notes","label":"Design notes","type":"string_list","decimals":4,"description":"Deviations from common primer guidelines (18–30 nt, GC 40–60 %, Tm 50–65 °C, no runs of 4+, not self-complementary)."}],"input_schema":{"type":"object","properties":{"sequence":{"description":"8–60 nucleotides using A, C, G, T (U is treated as T) and N; case-insensitive; spaces, digits and 5'/3' markers are ignored.","type":"string","examples":["ATGCATGCATGCATGCATGC"]}},"additionalProperties":false,"required":["sequence"]},"output_schema":{"type":"object","properties":{"length":{"description":"Number of nucleotides including N. Unit: nt.","type":"integer","x-unit":"nt"},"gc_content_percent":{"description":"100 × (G + C) / length. Unit: %.","type":"number","x-unit":"%"},"tm_wallace":{"description":"2 × (A + T) + 4 × (G + C); intended for primers up to about 13–14 nt but always reported. Unit: °C.","type":"number","x-unit":"°C"},"tm_basic":{"description":"64.9 + 41 × (G + C − 16.4) / N; intended for primers of 14 nt or more. Unit: °C.","type":"number","x-unit":"°C"},"tm_recommended":{"description":"Wallace value for sequences up to 13 nt, basic value for 14 nt or more. Unit: °C.","type":"number","x-unit":"°C"},"molecular_weight_g_mol":{"description":"A × 313.21 + C × 289.18 + G × 329.21 + T × 304.2 − 61.96 (linear, 5'-OH, anhydrous); omitted when the sequence contains N. Unit: g/mol.","type":"number","x-unit":"g/mol"},"complement":{"description":"Base-by-base complement in the same orientation.","type":"string"},"reverse_complement":{"description":"Complement read in the opposite direction: the sequence of the opposite strand.","type":"string"},"self_complementary":{"description":"true when the sequence equals its own reverse complement (palindromic), which favours primer-dimers.","type":"boolean"},"gc_clamp":{"description":"true when the 3'-terminal base is G or C.","type":"boolean"},"max_homopolymer_run":{"description":"Length of the longest run of identical consecutive bases.","type":"integer"},"notes":{"description":"Deviations from common primer guidelines (18–30 nt, GC 40–60 %, Tm 50–65 °C, no runs of 4+, not self-complementary).","type":"array","items":{"type":"string"}}}},"formula":"Tm_Wallace = 2 × (A + T) + 4 × (G + C); Tm_basic = 64.9 + 41 × (G + C − 16.4) / N; GC % = 100 × (G + C) / N; MW = 313.21·A + 289.18·C + 329.21·G + 304.2·T − 61.96","method":"Wallace rule (Wallace et al. 1979) and the GC-content formula (Marmur–Doty type) assume about 50 mM Na⁺ and ignore primer concentration and mismatches; they typically differ by several °C from nearest-neighbour values. N counts toward the length only. The molecular weight uses anhydrous nucleotide residue masses with −61.96 for the missing terminal phosphate and added water.","sources":[{"name":"Wallace RB et al. (1979) Hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 DNA: the effect of single base pair mismatch, Nucleic Acids Res 6(11):3543-3557","url":"https://pubmed.ncbi.nlm.nih.gov/158748/","type":"peer_reviewed","retrieved_at":"2026-09-24"},{"name":"Wikipedia – Nucleic acid thermodynamics","url":"https://en.wikipedia.org/wiki/Nucleic_acid_thermodynamics","type":"reference","retrieved_at":"2026-09-24"},{"name":"Wikipedia – Primer (molecular biology)","url":"https://en.wikipedia.org/wiki/Primer_(molecular_biology)","type":"reference","retrieved_at":"2026-09-24"}],"freshness":{"type":"static","max_age_seconds":null,"note":"Deterministic formula with fixed constants; results never go stale. Inputs supplied by the caller determine the output."},"examples":[{"name":"20-mer ATGCATGCATGCATGCATGC","inputs":{"sequence":"ATGCATGCATGCATGCATGC"},"expected":{"length":20,"gc_content_percent":50,"tm_wallace":60,"tm_basic":51.78,"tm_recommended":51.78,"molecular_weight_g_mol":6117.04,"reverse_complement":"GCATGCATGCATGCATGCAT","complement":"TACGTACGTACGTACGTACG","self_complementary":false,"gc_clamp":true},"url":"https://tttkmbb.com/api/v1/calculate/pcr-primer-tm?sequence=ATGCATGCATGCATGCATGC"},{"name":"12-mer palindrome ACGTACGTACGT","inputs":{"sequence":"acgt acgt acgt"},"expected":{"length":12,"gc_content_percent":50,"tm_wallace":36,"tm_basic":29.37,"tm_recommended":36,"molecular_weight_g_mol":3645.44,"reverse_complement":"ACGTACGTACGT","self_complementary":true,"max_homopolymer_run":1},"url":"https://tttkmbb.com/api/v1/calculate/pcr-primer-tm?sequence=acgt+acgt+acgt"}],"faq":[{"q":"Which Tm should I use?","a":"For a first estimate use tm_recommended (Wallace up to 13 nt, basic formula from 14 nt); for annealing temperatures rely on a nearest-neighbour calculation with your actual salt and primer concentrations and start about 3–5 °C below the lower primer Tm."},{"q":"Why is the Wallace Tm so high for a 20-mer?","a":"The rule adds 2–4 °C per base without bound, so it overestimates Tm for long primers; that is why the GC formula is used from 14 nt."},{"q":"What does the reverse complement give me?","a":"The sequence of the opposite strand written 5'→3', i.e. what a reverse primer targeting this site must look like."}],"tags":["primer tm","melting temperature","gc content","reverse complement","oligo molecular weight","pcr primer design"],"related":[{"calculator_id":"dna-concentration","reason":"Quantify the primer or template from an A260 reading."},{"calculator_id":"molar-mass","reason":"Molar mass of other molecules from a chemical formula."},{"calculator_id":"molarity","reason":"Prepare a primer stock of known molarity from its mass and molecular weight."}],"links":{"html":"https://tttkmbb.com/science/pcr-primer-tm","markdown":"https://tttkmbb.com/science/pcr-primer-tm.md","json":"https://tttkmbb.com/science/pcr-primer-tm.json","api":"https://tttkmbb.com/api/v1/calculate/pcr-primer-tm","schema":"https://tttkmbb.com/api/v1/calculators/pcr-primer-tm","openapi":"https://tttkmbb.com/openapi.json","mcp":"https://tttkmbb.com/mcp"},"version":"v1","updated_at":"2026-09-24"},"timestamp":"2026-09-24T03:47:22Z"}