Kod Polymerase Error Rate
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Q5 Error Rate
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between polymerases can be expressed in absolute terms, often by the number of errors per 1,000 or 10,000 nucleotides, or expressed as the number of theoretical errors dna polymerase error rate per base. The level of fidelity can also be expressed in relative terms,
Pcr Fidelity Calculator
often using Taq DNA polymerase as the relative standard (Table 1).Note that while single base substitution errors are the
Dna Polymerase Fidelity Comparison
easiest to assess, polymerases are also capable of insertion/ deletion errors that can lead to frameshift mutations, a highly deleterious type of error. Comparison of High-Fidelity Polymerases PRODUCT NAME (Supplier) POLYMERASE FIDELITY https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/pcr-fidelity-calculator.html (Reported by supplier) MAXIMUM AMPLICON LENGTH6 EXTENSION TIME6 (For simple templates5) EXTENSION TIME6 (For complex templates5) Q5 High-Fidelity DNA Polymerase (NEB) >100X Taq1,2 20 kb simple; 10 kb complex 10 s/kb 10 s/kb (<1 kb) 20–30 s/kb (>1 kb) Phusion High-Fidelity DNA Polymerase (NEB) >50X Taq1,2 20 kb simple; 10 kb complex 15 s/kb 30 s/kb AccuPrime™ Pfx (Life) 26X Taq1 12 kb4 60 s/kb4 https://www.neb.com/products/pcr-polymerases-and-amplification-technologies/q5-high-fidelity-dna-polymerases/q5-high-fidelity-dna-polymerases/how-is-fidelity-measured PfuUltra™ II Fusion HS (Agilent) 20X Taq1 19 kb4 15 s/kb (<10 kb4) 30 s/kb (>10 kb4) PfuUltra High-Fidelity DNA Polymerase (Agilent) 19X Taq1 17 kb simple; 6 kb complex 60 s/kb (<10 kb) 120 s/kb (>10 kb) 60 s/kb (<6 kb) 120 s/kb (>6 kb) Platinum Taq HiFi (Life) 6X Taq1 20 kb4 60 s/kb4 KOD DNA Polymerase (EMD) 4X Taq3 6 kb simple; 2 kb complex 10–20 s/kb 30–60 s/kb 1 PCR-based mutation screening in lacZ (NEB), lacI (Agilent) or rpsL (Life). 2 Due to the very low frequency of misincorporation events being measured, the error rate of high-fidelity enzymes like Q5 is difficult to measure in a statistically significant manner. Although measurements from assays done side-by-side with Taq yield Q5 fidelity values from 100-200 X Taq, we report “>100X Taq" as a conservative value. 3 Takagi et al (1997) Appl. Env. Microbiol. 63, 4504-4510. 4 Template not specified. 5 Simple templates include plasmid, viral and E. coli genomic DNA. Complex templates include plant, human and other mammalian genomic DNA. 6 Values provided by individual manufacturers. Polymerase fidelity assays take many forms and have been used extensively for comparing high-fidelity polymerase
$0.97 link Phusion HF The following table lists a variety of polymerases, some immediately available in lab and some not, and useful information about each of them. http://barricklab.org/twiki/bin/view/Lab/ProtocolsTaq Taq Platinum Taq KOD PFU Vent Phusion Species Thermophilis Aquaticus Mix of Taq, Pyrococcus species GB-D polymerase and Platinum® Taq Antibody Thermococcus kodakaraensis Pyrococcus furiosis Thermococcus litoralis Pyrococcus + Processivity domain Fidelity (err/bp) 10–4 to 10–5 10–5 to 10–6 2.6x10–6 1-3 x 10–6 10–6 10–6 to 10–7 Elongation Rate 1 min/kb 1 min/kb 20-30 sec/kb 1 min/kb 1 min/kb 20-30 sec/kb Processivity ~ 50 error rate bases > 50 bases >300 bases 20 ? 35 Nicking Activity None None None ? < 10% ? Amplicon Size < 5kbp Up to 20 kbp Plasmid/Linear = 6kbp Genome = 2kbp Vector: Up to 15 kbp Genome = Up to 19 kbp ? Genome = 10 kbp Plasmid/Linear = 20 kbp 3' → 5' exonuclease? (proofreading) No Yes (Pyrococcus) Yes Yes Yes Yes 5' polymerase error rate → 3' exonuclease? Yes Yes No No No No Strand displacing? Yes Yes No No Yes No Overhang? 3' - A 3' - A No No No No GC-Rich Performance Low Low High Medium High Medium Hot Start? No Yes Available Available No Available Applications Standard Taq Diagnostic PCR Since Taq is the cheapest polymerase available, it is the most appropriate for diagnostic applications requiring multiple reactions, such as checking if your cloning or genomic insertion worked via PCR of the intended insert region. Colony PCR Taq is especially well-suited to PCR on the unpurified genome, as in colony PCR. Taq will more successfully (and less expensively) amplify a single product off a small number of cells than Phusion. Phusion High Fidelity High-fidelity PCR The error rate of Phusion DNA Polymerase is 4.4 x 10^-7; therefore, Phusion DNA Polymerase is suitable for all PCR applications requiring great accuracy. Cloning Phusion DNA Polymerase amplifies templates with an accuracy and speed previously unattainable with a single enzyme. This makes it a superior choice for cloning. Phusion DNA Polymerase possesses 5' - 3' polymerase activity and 3'-5' exonuclease activity and will generate blunt ended p