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PCR Fidelity Estimator.

Predict the fraction of error-free PCR product for any polymerase, amplicon length, and cycle count. Compare Q5, Phusion, KAPA HiFi, Platinum SuperFi II, Pfu, OneTaq, and Taq side-by-side. Runs entirely in your browser.

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Validated2026-04-07
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bp
cycles

Fidelity Estimate

% correct after 30 cycles
98.8%
Error rate
5.30e-7 /bp
Cycles ≥ 95% fidelity
30
Cycles ≥ 99% fidelity
25

Expected molecules after 30 cycles

Total molecules
1.07e+13
Error-free molecules
1.06e+13
Error-containing molecules
1.27e+11

Fidelity across cycles

Comparison across preset polymerases (30 cycles, 1500 bp)

PolymeraseError rateFinal % correctCycles ≥ 95%Cycles ≥ 99%
KAPA HiFi2.8e-799.4%3030
Platinum SuperFi II3.0e-799.3%3030
Phusion High-Fidelity4.4e-799.0%3030
Q5 High-Fidelity5.3e-798.8%3025
Pfu1.3e-697.1%3010
OneTaq Hot Start2.0e-563.5%30
Taq2.9e-552.3%20

When to use

  • Decide how many PCR cycles to run before error accumulation becomes a concern
  • Compare high-fidelity polymerases (Q5, Phusion, KAPA HiFi, Platinum SuperFi II) for a cloning or NGS job
  • Estimate the ceiling for error-free product on long or GC-extreme amplicons
  • Plug in a lab-measured error rate via the Custom option to estimate fidelity for an in-house enzyme
  • Teach the cycle-by-cycle compounding of PCR errors in a methods or reagents class

Do not use for

  • Estimating variant allele frequencies from deep sequencing — the tool does not model sampling noise or strand bias
  • Predicting chimera / recombination rates on mixed-template PCR (e.g. metagenomics)
  • Fidelity of reverse transcription — RT error rates are separate and much higher
  • Error-correcting chemistries like rolling-circle or duplex consensus sequencing, which are modeled differently

Halve e·L per cycle, not multiply

The intuitive "fraction error-free drops by e·L per cycle" is wrong by a factor of 2. Only the half of the population that is newly synthesized each cycle is exposed to error — the other half is inherited. This is why the closed form is (1 − e·L/2)ⁿ.

More cycles is not more data

Past ~25–30 cycles most polymerases are already in plateau and fidelity is dropping. If you need more product, increase starting template copies rather than adding cycles.

Long amplicons amplify the fidelity penalty

Doubling amplicon length doubles the per-cycle error probability. A 4 kb amplicon at 30 cycles with Q5 is still ~98% error-free, but with Taq it drops below 10%. Budget accordingly.

Manufacturer error rates are best-case

Published error rates are measured on optimized buffers and templates. Real-world rates are typically 1.5–3× higher on difficult templates. Treat this calculator as an upper bound on fidelity.

Use starting copies to set sequencing depth

If you plug in starting copies, the tool reports expected error-free molecule counts at the end of the run. This is the ceiling for unique error-free reads, and a useful sanity check for deep sequencing experiments where you need a minimum clean template count.

1

Method

Cycle-by-cycle recursion Xₙ₊₁ = Xₙ·(1 − e·L/2) in closed form: P_correct(n) = (1 − e·L/2)ⁿ. Polymerase presets drawn from Barnes (PNAS 1994), Cline et al. (NAR 1996), and manufacturer product documentation (NEB, Roche/KAPA, Thermo Fisher). Does not model chimera formation, GC bias, or polymerase stalling.

2

Validated

Last validated 2026-04-07. Calculations are designed for planning and documentation support; verify procurement decisions against manufacturer specifications or institutional SOPs.

3

How to cite

How to Cite

ConductScience PCR Fidelity Estimator (v1.7.0). ConductScience, Inc. 2026. Available at: https://conductscience.com/tools/pcr-fidelity-estimator

Barnes WM. PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc Natl Acad Sci USA. 1994;91(6):2216-2220. doi:10.1073/pnas.91.6.2216

Cline J, Braman JC, Hogrefe HH. PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res. 1996;24(18):3546-3551. doi:10.1093/nar/24.18.3546

Sun F. The polymerase chain reaction and branching processes. J Comput Biol. 1995;2(1):63-86. doi:10.1089/cmb.1995.2.63

What is PCR fidelity?

PCR fidelity is the fraction of amplified molecules that are perfect copies of the original template. It is set by two things: the polymerase’s per-base error rate and the number of bases it has to copy each cycle (the amplicon length). Errors compound geometrically across cycles, so even a small per-base error rate can dominate the final population after enough doublings.

This tool estimates that fraction and its behavior across cycles. Use it to decide how many cycles to run, which polymerase to pick for a given cloning/NGS/variant-calling job, and what your realistic ceiling for error-free product is.

The cycle-by-cycle recursion

Let Xₙ be the expected fraction of error-free molecules after n cycles. Each cycle every molecule is kept AND copied once. A newly synthesized molecule is error-free iff its parent was AND the copy itself was clean (probability 1 − e·L). Therefore:

X_{n+1} = (1/2)·Xₙ + (1/2)·Xₙ·(1 − e·L) = Xₙ · (1 − e·L / 2)

Closed form: P_correct(n) = (1 − e·L / 2)ⁿ

This matches the first-order approximation used by NEB’s PCR Fidelity Estimator and by Sun (1995). It assumes independent errors and ideal amplification.

Choosing a polymerase

Cloning / site-directed mutagenesis: use Q5, Phusion, KAPA HiFi, or Platinum SuperFi II. These have error rates ~10⁻⁷ to 10⁻⁶ and produce cloning-grade product at 30–35 cycles.
NGS library prep: KAPA HiFi is the standard choice for amplicon-based libraries due to its very low error rate and GC-tolerant buffer.
Long amplicons (>5 kb): Q5 or Platinum SuperFi II are the most reliable. Fidelity alone does not guarantee yield on long templates — processivity matters just as much.
Colony PCR, genotyping, diagnostic work: OneTaq or Taq is usually fine. You do not need proofreading to confirm the presence or absence of an amplicon.
Avoid Taq for anything downstream of Sanger or NGS: the raw error rate (~3×10⁻⁵) makes every 1 kb amplicon carry at least one substitution in most molecules after 25–30 cycles.

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