polymerase-tm 2.0.0

NEB Tm Calculator + Base Changer: PCR primer Tm/Ta and site-directed mutagenesis primer design for all NEB polymerases

polymerase-tm

Exact Python reproduction of the NEB Tm Calculator and NEB Base Changer for PCR primer melting temperature (Tm), annealing temperature (Ta), and site-directed mutagenesis (SDM) primer design.

Features

• Exact NEB Tm Calculator reproduction -- algorithm reverse-engineered from the NEB Tm Calculator front-end source; verified against the official tool with 0 degC deviation across all tested sequences.
• Zero dependencies for core functions -- tm(), ta(), batch_tm(), check_pair(), and all primer analysis work without any external packages.
• 22 NEB polymerase products with their specific buffer salt concentrations and Ta rules (Q5, Phusion, Taq, OneTaq, LongAmp, Vent, Deep Vent, and more).
• Automatic additive recommendation -- suggests Q5 High GC Enhancer or DMSO based on primer GC, hairpins, and amplicon analysis.
• Batch processing -- process hundreds of primer pairs from CSV files with full Tm/Ta/compatibility analysis.
• PCR protocol generator -- generates complete cycling protocols with polymerase-specific temperatures and extension times. Automatically generates touchdown protocols when primer Tm difference exceeds 3 degC.
• Smart primer design -- find the optimal binding length for a target Tm.
• Primer dimer detection -- checks 3'-end complementarity and self-dimer risk.
• Gibson Assembly overlap design -- generates full primers with overhangs for Gibson/HiFi Assembly.
• Restriction site scanning -- scans primers for ~120 NEB restriction enzyme sites with full IUPAC ambiguity code support (accepts enzyme names or custom dict).
• Primer quality scoring -- comprehensive 0-100 score evaluating GC clamp, runs, repeats, hairpins.
• Hairpin detection -- nearest-neighbor thermodynamic Tm for hairpin stems (SantaLucia 1998), G-T wobble pair tolerance, loop entropy penalty (Jacobson-Stockmayer).
• Site-directed mutagenesis -- reimplementation of the NEB Base Changer v2.7.2 primer design algorithm. Supports AA point mutations, nucleotide substitutions/deletions/insertions, 12 NCBI genetic codes, codon usage/parsimony selection, back-to-back primer design with Owczarzy (2008) bivariate salt correction (Na+ + Mg2+).
• DMSO analysis -- analyses primer hairpins, amplicon GC content, GC-rich hotspots, and template secondary structures (requires [bio] extra for GenBank files).
• Virtual gel visualization -- simulated agarose gel with realistic Ferguson-plot migration physics (requires [viz] extra).
• CLI tool (polymerase-tm) for quick calculations from the terminal.

Optional dependencies: Biopython (for GenBank template analysis), matplotlib + seaborn (for virtual gel).

Installation

# Core package (zero dependencies)
pip install polymerase-tm

# With GenBank template support (adds biopython)
pip install polymerase-tm[bio]

# With virtual gel visualization (adds matplotlib, seaborn)
pip install polymerase-tm[viz]

# Everything
pip install polymerase-tm[all]

# From conda-forge (when available)
mamba install -c conda-forge polymerase-tm

Quick Start

Python API

from polymerase_tm import tm, ta, list_buffers

# Single primer Tm (Q5, 500 nM)
print(tm("ATGTCCCTGCTCTTCTCTCGATGCAA"))          # 72

# Primer pair Ta
result_ta, tm_fwd, tm_rev = ta(
    "ATGTCCCTGCTCTTCTCTCGATGCAA",
    "GTGCCTCCGAGCCAGCACC",
)
print(f"Ta = {result_ta}, Fwd Tm = {tm_fwd}, Rev Tm = {tm_rev}")
# Ta = 72, Fwd Tm = 72, Rev Tm = 75

# Different polymerase
print(tm("ATGTCCCTGCTCTTCTCTCGATGCAA", polymerase="taq"))

# Override buffer (when not using default master mix)
print(tm("ATGTCCCTGCTCTTCTCTCGATGCAA", polymerase="taq", buffer="thermopol"))

# Direct salt concentration (mM)
print(tm("ATGTCCCTGCTCTTCTCTCGATGCAA", salt_mM=50))

# List all available buffers
for b in list_buffers():
    print(f"{b['name']:20s} {b['salt_mM']:>4d} mM")

# Ta with 3% DMSO
ta_dmso, _, _ = ta("ATGTCCCTGCTCTTCTCTCGATGCAA", "GTGCCTCCGAGCCAGCACC", dmso_pct=3)
print(f"Ta with 3% DMSO = {ta_dmso}")  # 70

# List all available polymerases
from polymerase_tm import list_polymerases
for p in list_polymerases():
    print(f"{p['key']:25s} {p['description']}")

Automation & Batch Processing

from polymerase_tm import (
    batch_tm,                 # Bulk Tm for many sequences
    optimal_binding_length,   # Find shortest binding region for target Tm
    check_pair,               # Full primer pair compatibility report
    pcr_protocol,             # Generate complete PCR cycling protocol
    reverse_complement,       # DNA reverse complement
    from_csv, to_csv,         # CSV batch I/O
)

# Batch Tm for multiple primers
results = batch_tm(["ATCGATCGATCG", "GCGCGCGCGCGC", "AATTCCGGAATT"])
for r in results:
    print(f"{r['sequence']}: Tm={r['tm']} degC, GC={r['gc_pct']}%")

# Find optimal binding length for a target Tm
result = optimal_binding_length("ATGTCCCTGCTCTTCTCTCGATGCAA", target_tm=65)
print(f"{result['binding_seq']} ({result['length']} nt, Tm={result['tm']})")
# CCTGCTCTTCTCTCGATGCAA (21 nt, Tm=67)

# Primer pair compatibility check (includes auto additive recommendation)
pair = check_pair("ATGTCCCTGCTCTTCTCTCGATGCAA", "GTGCCTCCGAGCCAGCACC")
print(f"Ta={pair['ta']}, compatible={pair['compatible']}")
if pair["additive"]["recommended"]:
    print(f"Use {pair['additive']['additive']} ({pair['additive']['concentration']})")
    # -> "Use Q5 High GC Enhancer (1x)" for Q5 with high-GC primers
    # -> "Use DMSO (3%)" for Taq with high-GC primers

# Generate full PCR cycling protocol
protocol = pcr_protocol(
    "ATGTCCCTGCTCTTCTCTCGATGCAA",
    "GTGCCTCCGAGCCAGCACC",
    template="...full template sequence...",  # Auto-calculates amplicon_length
)
for step in protocol["cycling"]:
    print(f"{step['step']:25s} {step['temp']} degC  {step['time']}")
# Initial Denaturation       98 degC  30 s
# Denaturation               98 degC  10 s
# Annealing                  72 degC  30 s
# Extension                  72 degC  1 min 15 s
# Final Extension            72 degC  2 min
# Hold                        4 degC  indefinite

# CSV pipeline: read primers, compute everything, write results
results = from_csv("primers.csv")  # expects columns: name, fwd, rev
to_csv(results, "results_with_tm.csv")

Site-Directed Mutagenesis (NEB Base Changer)

from polymerase_tm import BaseChanger

# Design SDM primers for a point mutation
template = "ATGACCATGATTACGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGG..."
bc = BaseChanger(template)
result = bc.point_mutation("T2A")
print(f"FWD: {result.forward.sequence} (Tm={result.forward.tm})")
print(f"REV: {result.reverse.sequence} (Tm={result.reverse.tm})")
print(f"Ta:  {result.ta} degC")

# Multiple mutations
results = bc.batch("T2A A3G K5R")

# Deletion / insertion
result = bc.deletion(start=10, length=3)
result = bc.insertion(position=10, insert_seq="AAAAAA")

# Parsimony codon selection (fewest base changes)
bc = BaseChanger(template, codon_mode="parsimony")

# Alternative genetic code (e.g. vertebrate mitochondrial)
bc = BaseChanger(template, genetic_code=2)

DMSO Analysis

from polymerase_tm import dmso_recommendation, print_dmso_report

report = dmso_recommendation(
    fwd_bind="ATGTCCCTGCTCTTCTCTCGATGCAA",
    rev_bind="GTGCCTCCGAGCCAGCACC",
    template_file="template.gbk",     # optional GenBank template
)
print_dmso_report(report)

Command Line

# Single primer Tm
polymerase-tm ATGTCCCTGCTCTTCTCTCGATGCAA

# Primer pair Ta (includes auto additive recommendation)
polymerase-tm ATGTCCCTGCTCTTCTCTCGATGCAA GTGCCTCCGAGCCAGCACC

# Different polymerase
polymerase-tm -p taq ATGTCCCTGCTCTTCTCTCGATGCAA GTGCCTCCGAGCCAGCACC

# Override buffer (e.g. when not using master mix)
polymerase-tm --buffer thermopol ATGTCCCTGCTCTTCTCTCGATGCAA

# Direct salt concentration override
polymerase-tm --salt 50 ATGTCCCTGCTCTTCTCTCGATGCAA

# With DMSO correction
polymerase-tm --dmso 3 ATGTCCCTGCTCTTCTCTCGATGCAA GTGCCTCCGAGCCAGCACC

# DMSO analysis with template
polymerase-tm --dmso-check --template plasmid.gbk FWD_SEQ REV_SEQ

# Generate PCR Cycler Protocol & Virtual Agarose Gel Plot
polymerase-tm ATGTCCCTGCTCTTCTCTCGATGCAA GTGCCTCCGAGCCAGCACC --template plasmid.gbk --plot-gel out_gel.png

# Generate a standalone virtual gel with multiple custom fragments and a 100bp ladder
polymerase-tm --plot-gel out_multi.png --ladder 100bp --plot-gel-sizes 150 200 400

# Simulate a custom gel run (e.g. 1.5% agarose, 110V, 90 minutes, 15cm gel)
polymerase-tm --plot-gel custom_physics.png --ladder 1kb_plus --plot-gel-sizes 1500 3000 --agarose 1.5 --voltage 110 --time 90 --gel-length 15.0

# Compare topological isomers of the same size plasmid (Linear vs Supercoiled vs Nicked)
polymerase-tm --plot-gel topologies.png --plot-gel-sizes 3000 3000 3000 --topology linear coiled nicked

# List all polymerases / buffers
polymerase-tm --list
polymerase-tm --list-buffers

# Version
polymerase-tm --version

# Site-directed mutagenesis (NEB Base Changer)
polymerase-tm --sdm --mutation M1A TEMPLATE_SEQ
polymerase-tm --sdm --mutation "T2A K5R" --codon-mode parsimony TEMPLATE_SEQ
polymerase-tm --sdm --mode del --mutation 10:3 TEMPLATE_SEQ
polymerase-tm --sdm --mode ins --mutation 10:AAAAAA TEMPLATE_SEQ
polymerase-tm --sdm --mutation T2A --genetic-code 2 TEMPLATE_SEQ

API Reference

Function	Description
tm(seq, polymerase, buffer, salt_mM)	Melting temperature for one primer
ta(seq1, seq2, polymerase, dmso_pct, buffer, salt_mM)	Annealing temperature for a primer pair
list_polymerases()	List all 22 supported polymerases
list_buffers()	List all 17 NEB buffers with salt concentrations
batch_tm(sequences, polymerase)	Batch Tm for multiple sequences
check_pair(fwd, rev, polymerase)	Pair compatibility + additive recommendation
pcr_protocol(fwd, rev, polymerase, template, amplicon_length)	Full PCR cycling protocol (auto-touchdown when Tm diff > 3 degC)
optimal_binding_length(seq, target_tm, polymerase)	Find shortest binding region for target Tm
reverse_complement(seq)	DNA reverse complement
from_csv(path, polymerase)	Read primer pairs from CSV, compute Tm/Ta
to_csv(results, path)	Write results to CSV
primer_dimer(fwd, rev)	Check 3' complementarity / dimer risk
gibson_overlaps(fwd_bind, rev_bind, left_seq, right_seq)	Design Gibson/HiFi Assembly primers
restriction_scan(seq, enzymes)	Scan for restriction sites (~120 NEB enzymes, IUPAC support, accepts name list or dict)
primer_quality(seq)	Quality score 0-100 with issues list
find_hairpins(seq)	Detect hairpin stem-loops with NN Tm
primer_hairpin(seq)	Hairpin scan tuned for primer-length sequences
additive_recommendation(fwd, rev, polymerase)	DMSO / GC Enhancer recommendation
dmso_recommendation(fwd_bind, rev_bind, template_seq, template_file)	Full DMSO/additive analysis
print_dmso_report(report)	Pretty-print a DMSO analysis report
gc_content(seq)	GC content as fraction
plot_virtual_gel(amplicon_lengths, ladder_name, agarose_pct, ...)	Simulated agarose gel image (requires [viz])
BaseChanger(template, orf_start, genetic_code, codon_mode, ...)	SDM primer designer (NEB Base Changer v2.7.2)
select_codon(target_aa, original_codon, mode, genetic_code)	Codon selection (usage/parsimony)
parse_aa_mutation(mutation_str)	Parse AA mutation notation ("M1A", "K2R:CGC")
calc_sdm_tm(seq, mono_mM, divalent_mM, primer_conc_nM)	SDM Tm with bivariate salt correction
owczarzy_bivariate(raw_tm, seq, mono_mM, divalent_mM)	Owczarzy (2008) Na+/Mg2+ correction
GENETIC_CODES	All 12 NCBI genetic codes
get_codon_table(code_id)	Full codon table for any genetic code
get_aa_to_codons(code_id)	Amino acid → codons for any genetic code

Buffer / salt override: tm() and ta() accept optional buffer (NEB buffer name) or salt_mM (direct mM value) to override the polymerase default. Priority: salt_mM > buffer > polymerase default.

Algorithm

Component	Method	Reference
Nearest-neighbor Tm	SantaLucia (1998)	PNAS 95:1460-5
Salt correction (mono)	Owczarzy et al. (2004)	Biochemistry 43:3537-54
Salt correction (bivariate)	Owczarzy et al. (2008)	Biochemistry 47:5336-53
Ta rules	Polymerase-specific	NEB Tm Calculator v1.16
SDM primer design	Back-to-back	NEB Base Changer v2.7.2
DMSO correction	-0.6 degC per 1%	NEB Tm Calculator v1.16

Buffer Salt Concentrations

Buffer	[Monovalent] (mM)	Used by
Q5	150	Q5, Q5 Hot Start, Q5 Blood Direct
Q5U	170	Q5U Hot Start
Q5 Master Mix	150	Q5 2X Master Mix
Phusion HF / GC	222	Phusion, Phusion Hot Start Flex
Standard Taq	55	Taq, Hot Start Taq, EpiMark
ThermoPol	40	Vent, Deep Vent
OneTaq Std	54	OneTaq (Standard Buffer)
OneTaq GC	80	OneTaq (GC Buffer)
LongAmp	100	LongAmp, LongAmp Hot Start
Crimson Taq	55	Crimson Taq
Hemo KlenTaq	70	Hemo KlenTaq
Multiplex	90	Multiplex PCR Master Mix

Ta Calculation Rules

Polymerase family	Rule	Cap
Q5	min(Tm1, Tm2) + 1	72 degC
Q5U	min(Tm1, Tm2) + 2	72 degC
Phusion	0.93 * min(Tm1, Tm2) + 7.5	72 degC
Taq / OneTaq	min(Tm1, Tm2) - 5	68 degC
LongAmp	min(Tm1, Tm2) - 5	65 degC
Vent / Deep Vent	min(Tm1, Tm2) - 2	72 degC

Disclaimer

This package is not affiliated with New England Biolabs (NEB). The algorithms were reverse-engineered from the publicly available JavaScript sources of the NEB Tm Calculator and NEB Base Changer for research and educational purposes. Always verify critical calculations against the official tools.

Module Structure

polymerase_tm/
├── __init__.py      # Re-exports (backward compatible)
├── constants.py     # NN_PARAMS, BUFFERS, POLYMERASES, GENETIC_CODES
├── core.py          # tm(), ta(), owczarzy_bivariate(), calc_sdm_tm()
├── mutagenesis.py   # BaseChanger, SDMPrimer, MutagenesisResult
├── dmso.py          # DMSO analysis, hairpins, GC analysis
├── batch.py         # batch_tm(), pcr_protocol(), CSV I/O
├── analysis.py      # restriction_scan(), primer_dimer(), primer_quality()
├── gel.py           # Virtual agarose gel visualization
└── cli.py           # Command-line interface (Tm/Ta + SDM)

All functions are re-exported from __init__.py — from polymerase_tm import tm and from polymerase_tm import BaseChanger work directly.

License

MIT