Search Thermo Fisher Scientific
Platinum SuperFi II DNA Polymerase–High-Fidelity PCR Enzyme
For highest confidence in PCR sequence accuracy
Invitrogen Platinum SuperFi II DNA Polymerase is a hot-start, engineered proofreading DNA polymerase, providing superior fidelity and specificity to your PCR. With >300x Taq fidelity and buffer specially formulated for primer annealing at 60°C, Platinum SuperFi II DNA Polymerase offers efficiency and simplicity in PCR applications requiring highest PCR accuracy, such as cloning, sequencing, and mutagenesis.
Highlights
- Exceptional accuracy—Higher than 300x Taq fidelity as determined by next-generation sequencing
- Simplified workflow—Buffer formulated for primer annealing at 60°C (no need for a Tm calculator)
- Increased PCR success—Robust amplification of GC-rich targets, DNA of suboptimal purity, and long sequences
- Enabled automation—High specificity and benchtop stability for 24 hours after reaction setup, enabled by Invitrogen Platinum hot-start technology
- Reduced pipetting—Master mix options available with or without direct gel-loading dyes
Customer stories on Platinum SuperFi II enzyme
Benefits offered by Platinum SuperFi II DNA Polymerase
>300x Taq fidelity
Platinum SuperFi II DNA Polymerase offers the highest level of confidence for preserving DNA sequence accuracy with its extremely low error rate. Using next-generation sequencing, the relative fidelity of Platinum SuperFi II DNA polymerase was calculated to be >300x that of Taq DNA polymerase.
Figure 1. Fidelity comparison across commercially available enzymes relative to Taq enzyme. A 3.9 kb sequence was amplified by PCR using different DNA polymerases, and the resulting PCR amplicons were then fragmented with a MuA transposase. Unique molecular identifiers (UMI), which consist of 12 random nucleotides, were introduced during fragmentation to individually tag each product. After next-generation sequencing, reads were aligned to the correct sequence, grouped by UMI families, and errors were called. Errors were identified only if they were present in all reads in the UMI family; otherwise they were discarded as sequencing errors. The polymerase fidelities were normalized to the Taq DNA polymerase. Read research article here.
Video: What is high-fidelity PCR?
Learn about fidelity of a DNA polymerase, methods for measuring enzyme fidelity, and benefits of using a high-fidelity DNA polymerase in your PCR.
60°C primer annealing
The unique formulation of the Platinum SuperFi II buffer helps reduce tedious optimization step in PCR. Calculation of primer melting temperatures for the annealing step is no longer required with Platinum SuperFi II DNA polymerase. The innovative buffer formulation enables annealing of primers at 60°C regardless of their sequences that follow general primer design rules (Figure 2). The buffer also allows successful amplification when calculated Tms are used in the annealing step (data not shown).
Figure 2.Platinum SuperFi II DNA Polymerase produces PCR products with high specificity and yield following the universal annealing temperature at 60°C. Primer sets of varying annealing temperature were used to amplify 12 targets from 50 ng of human genomic DNA at 60°C annealing temperature. The molecular weight marker is Invitrogen TrackIt 1 Kb Plus DNA Ladder. The annealing temperatures stated were calculated using the Tm calculator for Platinum SuperFi DNA Polymerase.
Learn the importance of the annealing step in PCR, how to circumvent optimization steps using a specially formulated PCR buffer, and the benefits of a universal annealing temperature enabled by the buffer.
Universal protocol
Due to its innovative reaction buffer, Platinum SuperFi II DNA Polymerase allows for a universal annealing temperature and flexible extension time for co-cycling of all assays.
Figure 3. Time saving and assay co-cycling enabled by universal PCR protocol. PCR assays using conventional PCR reagents require specific protocols for amplification of each DNA fragment because of the different primer annealing temperatures and extension steps; therefore, multiple targets often cannot be amplified together in the same PCR run. With Platinum SuperFi II DNA Polymerase, different PCR assays can be cycled together using one protocol with a universal primer annealing temperature and the extension time selected for the longest fragment to be amplified (Figure 4).
Figure 4. Platinum SuperFi II DNA Polymerase enables cycling of shorter and longer amplicons together. 0.7 kb, 2.0 kb, 4.8 kb, and 14 kb fragments were amplified from 100 ng of human genomic DNA using the same protocol for all four targets: 98°C denaturation for 10 sec, 60°C annealing for 10 sec, 72°C extension for 7 min. The extension time was based on length of the longest target.
Direct gel loading
Platinum SuperFi II Green PCR Master Mix offers the convenience of direct gel loading of PCR products, eliminating tedious steps of dye addition to PCR samples and helping reduce pipetting errors. The green buffer is compatible with downstream applications including DNA sequencing, ligation, and restriction digestion.
Figure 5. The green master mix format for loading PCR products directly to a gel for analysis. The master mix of Platinum SuperFi II DNA Polymerase is available in a green buffer format that contains a density reagent and two tracking dyes. DNA migration is easily tracked with two dyes (blue and yellow) that are readily visible during electrophoresis (the lanes for 5 and 15 min in the figure to the right).
>300x Taq fidelity
Platinum SuperFi II DNA Polymerase offers the highest level of confidence for preserving DNA sequence accuracy with its extremely low error rate. Using next-generation sequencing, the relative fidelity of Platinum SuperFi II DNA polymerase was calculated to be >300x that of Taq DNA polymerase.
Figure 1. Fidelity comparison across commercially available enzymes relative to Taq enzyme. A 3.9 kb sequence was amplified by PCR using different DNA polymerases, and the resulting PCR amplicons were then fragmented with a MuA transposase. Unique molecular identifiers (UMI), which consist of 12 random nucleotides, were introduced during fragmentation to individually tag each product. After next-generation sequencing, reads were aligned to the correct sequence, grouped by UMI families, and errors were called. Errors were identified only if they were present in all reads in the UMI family; otherwise they were discarded as sequencing errors. The polymerase fidelities were normalized to the Taq DNA polymerase. Read research article here.
Video: What is high-fidelity PCR?
Learn about fidelity of a DNA polymerase, methods for measuring enzyme fidelity, and benefits of using a high-fidelity DNA polymerase in your PCR.
60°C primer annealing
The unique formulation of the Platinum SuperFi II buffer helps reduce tedious optimization step in PCR. Calculation of primer melting temperatures for the annealing step is no longer required with Platinum SuperFi II DNA polymerase. The innovative buffer formulation enables annealing of primers at 60°C regardless of their sequences that follow general primer design rules (Figure 2). The buffer also allows successful amplification when calculated Tms are used in the annealing step (data not shown).
Figure 2.Platinum SuperFi II DNA Polymerase produces PCR products with high specificity and yield following the universal annealing temperature at 60°C. Primer sets of varying annealing temperature were used to amplify 12 targets from 50 ng of human genomic DNA at 60°C annealing temperature. The molecular weight marker is Invitrogen TrackIt 1 Kb Plus DNA Ladder. The annealing temperatures stated were calculated using the Tm calculator for Platinum SuperFi DNA Polymerase.
Learn the importance of the annealing step in PCR, how to circumvent optimization steps using a specially formulated PCR buffer, and the benefits of a universal annealing temperature enabled by the buffer.
Universal protocol
Due to its innovative reaction buffer, Platinum SuperFi II DNA Polymerase allows for a universal annealing temperature and flexible extension time for co-cycling of all assays.
Figure 3. Time saving and assay co-cycling enabled by universal PCR protocol. PCR assays using conventional PCR reagents require specific protocols for amplification of each DNA fragment because of the different primer annealing temperatures and extension steps; therefore, multiple targets often cannot be amplified together in the same PCR run. With Platinum SuperFi II DNA Polymerase, different PCR assays can be cycled together using one protocol with a universal primer annealing temperature and the extension time selected for the longest fragment to be amplified (Figure 4).
Figure 4. Platinum SuperFi II DNA Polymerase enables cycling of shorter and longer amplicons together. 0.7 kb, 2.0 kb, 4.8 kb, and 14 kb fragments were amplified from 100 ng of human genomic DNA using the same protocol for all four targets: 98°C denaturation for 10 sec, 60°C annealing for 10 sec, 72°C extension for 7 min. The extension time was based on length of the longest target.
Direct gel loading
Platinum SuperFi II Green PCR Master Mix offers the convenience of direct gel loading of PCR products, eliminating tedious steps of dye addition to PCR samples and helping reduce pipetting errors. The green buffer is compatible with downstream applications including DNA sequencing, ligation, and restriction digestion.
Figure 5. The green master mix format for loading PCR products directly to a gel for analysis. The master mix of Platinum SuperFi II DNA Polymerase is available in a green buffer format that contains a density reagent and two tracking dyes. DNA migration is easily tracked with two dyes (blue and yellow) that are readily visible during electrophoresis (the lanes for 5 and 15 min in the figure to the right).
Robustness offered by Platinum SuperFi II DNA Polymerase
Platinum SuperFi II DNA Polymerase amplifies a broad range of target lengths with high specificity and yield due to robustness of the enzyme and superior hot-start technology. The antibody-based Platinum hot-start technology inhibits enzyme activity until the initial PCR denaturation step, preventing nonspecific amplification and primer degradation while allowing greater yield of the target amplicons.
Figure 6. Versatility across broad range of amplicon lengths. Platinum SuperFi II DNA Polymerase (far left panel) provides high specificity and yield across range of DNA fragments from 0.3 kb to 14 kb amplified from 100 ng of human genomic DNA. The same targets were also amplified using competitor DNA polymerases: A—Merck KOD Hot Start, B—KAPA HiFi HotStart PCR Kit, C—PrimeSTAR GXL. The molecular weight marker is TrackIt 1 Kb Plus DNA Ladder.
The high sensitivity of Platinum SuperFi II DNA Polymerase enables detection of low-abundance DNA templates with accurate results. High sensitivity is advantageous in experiments where there is a limited amount of starting material or the target DNA is in low concentration in the sample.
Figure 7. High sensitivity and reliable amplification from low amounts of input DNA. Platinum SuperFi II DNA Polymerase (far left panel) shows reliable amplification of a 2 kb fragment from 0.4 ng, 2 ng, 10 ng, 50 ng, and 250 ng of human genomic DNA (template amounts are indicated above each lane.). The same target was amplified with competitor DNA polymerases: A—PfuUltra II Fusion Hot Start, B—HotStar HiFidelity DNA Polymerase Kit, C—Expand HiFiPLUS Enzyme Blend. The estimated copy number is ~100 copies per 0.4 ng of human genomic DNA. The molecular weight marker is TrackIt 1 Kb Plus DNA Ladder.
Platinum SuperFi II DNA Polymerase is engineered with a DNA-binding domain resulting in high processivity and increased tolerance to common PCR inhibitors such as hemin (red blood cell component), xylan (plant biopolymer), and humic acid (found in soil).
Figure 8. Platinum SuperFi II DNA Polymerase shows high tolerance to common PCR inhibitors. A 2 kb human genomic DNA fragment was amplified from 50 ng of human genomic DNA using Platinum SuperFi II DNA Polymerase or competitor high-fidelity DNA polymerases: A—Q5 Hot Start High-Fidelity, B—PrimeSTAR GXL, C—Merck KOD Hot Start, and D—KAPA HiFi HotStart PCR Kit in reaction mixtures containing 1—no inhibitor, 2—humic acid (4 µg/mL), 3—hemin (20 µM), or 4—bile salt (1 mg/mL). The molecular weight marker is TrackIt 1 Kb Plus DNA Ladder.
Extended stability of the Platinum SuperFi II DNA Polymerase enzyme at room temperature enables high-throughput applications. Its superior Platinum hot-start technology allows benchtop stability and high specificity of the enzyme.
Figure 9. Assembled reactions with Platinum SuperFi II DNA Polymerase are stable room temperature. A 0.5 kb fragment was amplified form 50 ng of human genomic DNA. PCR reactions were set up and left at room temperature for 0 hr and 24 hr before loading to the Applied Biosystems ProFlex thermal cycler. Even after 24 hr of room-temperature setup, Platinum SuperFi II DNA Polymerase (lane P) produces results with high specificity and yields. The same experiment was also performed with competitor DNA polymerases: A—Q5 Hot Start High-Fidelity, B—KAPA HiFi HotStart PCR Kit, C—PrimeSTAR GXL. The molecular weight marker is TrackIt 1 Kb Plus DNA Ladder.
PCR of different templates with Platinum SuperFi II DNA Polymerase
Platinum SuperFi II DNA Polymerase amplifies a broad range of sequence content with high specificity due to robustness of the enzyme and its specially formulated buffer. The Platinum SuperFi II buffer enables amplification of GC-rich targets without a need for supplementary DNA-melting additives.
Figure 10. Robust amplification of AT-rich and GC-rich targets by Platinum SuperFi II DNA polymerase. Fifteen targets of varying GC content were amplified from 50 ng of human genomic DNA without any supplementary buffer additives that help with DNA denaturation. The molecular weight marker is TrackIt 1 Kb Plus DNA Ladder.
Figure 11. Enhanced amplification of GC-rich targets. Platinum SuperFi II DNA Polymerase provides high specificity and yield of difficult GC-rich targets (far left panel) without any supplementary DNA-melting additives. Four GC-rich fragments (0.74 kb, 0.58 kb, 0.71 kb and 0.72 kb in length; GC content indicated above) were amplified from 50 ng of human genomic DNA. The same targets were also amplified using competitor DNA polymerases according to manufacturers‘ recommended protocols for GC-rich PCR: A—PrimeSTAR GXL DNA Polymerase, B—KAPA HiFi HotStart PCR Kit in specialized reaction buffer for GC rich fragments, C—Merck KOD Hot Start DNA Polymerase with 10% DMSO added. The molecular weight marker is TrackIt 1 Kb Plus DNA Ladder.
Due its high processivity and extremely low error rate, Platinum SuperFi II DNA Polymerase is ideal for accurately amplifying long fragments (up to 20 kb) with high yields and specificity. Amplification of longer targets (up to 40 kb) is possible but may require additional optimization such as using high-quality templates (pure, fresh, and intact) and fresh primer solutions. Reducing primer concentration to 0.2 μM may also improve the results.
Figure 12. Amplification of long fragments. Platinum SuperFi II DNA Polymerase (lane P) successfully amplifies 20 kb targets from 200 ng of human genomic DNA. Using the same primer sets, competitor DNA polymerases were also tested: A—Q5 Hot Start High-Fidelity, B—KAPA HiFi HotStart PCR Kit, C—Merck KOD Hot Start, E - D—PrimeSTAR GXL and E—PfuUltra II Fusion HotStart. The molecular weight marker is TrackIt 1 Kb Plus DNA Ladder.
Figure 13. Amplification of targets >20 kb. Platinum SuperFi II DNA Polymerase (lane P) successfully amplifies 30 kb targets and 40 kb targets from 50 ng of E. coli genomic DNA. Using the same primer sets, competitor DNA polymerases were also tested: A—Q5 Hot Start High-Fidelity, B—KAPA HiFi HotStart PCR Kit, C—Merck KOD Hot Start, E - D—PrimeSTAR GXL and E—PfuUltra II Fusion HotStart. The molecular weight marker is TrackIt 1 Kb Plus DNA Ladder.
With high specificity and processivity, Platinum SuperFi II DNA Polymerase can multiplex over a broad range of template concentrations in the buffer provided, without the need for significant optimization.
Figure 14. Multiplex PCR over a broad range of template concentrations using Platinum SuperFi II DNA Polymerase. 15 targets (99 bp; 131 bp; 160 bp; 199 bp; 251 bp; 300 bp; 345 bp; 400 bp; 516 bp; 613 bp; 735 bp; 908 bp; 1,005 bp; 1,190 bp; and 1,606 bp) were amplified from 2.5 ng, 25 ng, and 250 ng of human genomic DNA (template amounts are indicated above each lane). The molecular weight marker is TrackIt 100 bp DNA Ladder.
Due to high specificity and high inhibitor tolerance, Platinum SuperFi II DNA Polymerase enables efficient amplification of DNA of suboptimal quality such as formalin fixed paraffin-embedded (FFPE) samples.
Figure 15. Amplification of FFPE-extracted DNA. Platinum SuperFi II DNA Polymerase successfully amplifies targets up to 0.4 kb from 10 ng of mouse FFPE DNA extracted using Invitrogen RecoverAll Total Nucleic Acid Isolation Kit for FFPE. The molecular weight marker is TrackIt 100 bp DNA Ladder.
Differences between Platinum SuperFi II DNA Polymerase and Platinum SuperFi DNA Polymerase
Platinum SuperFi II DNA Polymerase comes with a reaction buffer that is specially formulated with iso-stabilizing components. This unique buffer composition offers several advantages: No requirement of Tm calculation for primers, more robust amplification of GC-rich target, enhanced amplification of long sequences, and a universal PCR protocol for high-throughput PCR (Table 1).
Table 1. Comparison of Platinum SuperFi II and Platinum SuperFi DNA Polymerases.
| Platinum SuperFi II DNA Polymerase | Platinum SuperFi DNA Polymerase | |
|---|---|---|
| Fidelity (vs. Taq) | >300x | >300x |
| Hot-start modification | Yes | Yes |
| Tm calculator needed | No (primers anneal at 60°C) | Yes |
| Universal PCR protocol | Yes | No |
| GC-rich amplification | Yes (GC enhancer not required) | Yes (GC enhancer recommended) |
| Long-range amplification | Up to 20 kb (enhanced performance) | Up to 20 kb |
| Benchtop stability | Up to 24 hr | Up to 24 hr |
| Inhibitor tolerance | High | High |
| 3′ end of amplicons | Blunt | Blunt |
| Certified low level of residual DNA (per 50 μL rxn) | ≤1 copy of bacterial DNA ≤0.3 copy of human DNA | ≤1 copy of bacterial DNA ≤0.2 copy of human DNA |
Benchmarking data of Platinum SuperFi II and Platinum SuperFi DNA polymerases
References
Viral research
Other microbes
| Usage | Reference |
|---|---|
| Multiplex PCR for detection of S. aureus genes | Allam A, Fakhr A, Mahmoud M et al. (2021) Staphylococcus aureus nasal colonization among health care workers at an Egyptian tertiary care hospital. Microbes and Infectious Diseases 2(1):108–118. |
| PCR with gDNA and cDNA of fungal samples, followed by Sanger sequencing
| Ferrara M, Gallo A, Perrone G et al. (2020) Comparative genomic analysis of ochratoxin A biosynthetic cluster in producing fungi: new evidence of a cyclase gene involvement. Front Microbiol 11:581309. |
Metagenomics
| Usage | Reference |
|---|---|
| Amplification of bacterial full-length genes, followed by Sanger sequencing | Ferrara M, Haidukowski M, D'Imperio M et al. (2021) New insight into microbial degradation of mycotoxins during anaerobic digestion. Waste Manag 119:215–225. |
| Metabarcoding of fish DNA from pumped deep-sea water | Kawato M, Yoshida T, Miya M et al. (2021) Optimization of environmental DNA extraction and amplification methods for metabarcoding of deep-sea fish. MethodsX 8: 101238. |
| Rare mutation detection by molecular barcoding; fidelity measurement of different DNA polymerases | Mielinis P, Sukackaite R, Serapinaite A et al. (2021) MuA-based molecular indexing for rare mutation detection by next-generation sequencing. J Mol Bio 443 (19):167209. |
Immunology
| Usage | Reference |
|---|---|
| Cloning of VH and VL regions of an immunoglobulin | Granel J, Lemoine R, Morello E (2020) 4C3 human monoclonal antibody: a proof of concept for non-pathogenic proteinase 3 anti-neutrophil cytoplasmic antibodies in granulomatosis with polyangiitis. Front Immunol 11:573040. |
| RT-PCR with human liver and testis samples for microRNA expression analysis, followed by Sanger sequencing | Rubino E, Cruciani M, Tchitchek N (2021) Human ubiquitin-specific peptidase 18 is regulated by microRNAs via the 3'untranslated region, a sequence duplicated in long intergenic non-coding RNA genes residing in chr22q11.21. Front Genet 11:627007. |
Additional areas
Viral research
Other microbes
| Usage | Reference |
|---|---|
| Multiplex PCR for detection of S. aureus genes | Allam A, Fakhr A, Mahmoud M et al. (2021) Staphylococcus aureus nasal colonization among health care workers at an Egyptian tertiary care hospital. Microbes and Infectious Diseases 2(1):108–118. |
| PCR with gDNA and cDNA of fungal samples, followed by Sanger sequencing
| Ferrara M, Gallo A, Perrone G et al. (2020) Comparative genomic analysis of ochratoxin A biosynthetic cluster in producing fungi: new evidence of a cyclase gene involvement. Front Microbiol 11:581309. |
Metagenomics
| Usage | Reference |
|---|---|
| Amplification of bacterial full-length genes, followed by Sanger sequencing | Ferrara M, Haidukowski M, D'Imperio M et al. (2021) New insight into microbial degradation of mycotoxins during anaerobic digestion. Waste Manag 119:215–225. |
| Metabarcoding of fish DNA from pumped deep-sea water | Kawato M, Yoshida T, Miya M et al. (2021) Optimization of environmental DNA extraction and amplification methods for metabarcoding of deep-sea fish. MethodsX 8: 101238. |
| Rare mutation detection by molecular barcoding; fidelity measurement of different DNA polymerases | Mielinis P, Sukackaite R, Serapinaite A et al. (2021) MuA-based molecular indexing for rare mutation detection by next-generation sequencing. J Mol Bio 443 (19):167209. |
Immunology
| Usage | Reference |
|---|---|
| Cloning of VH and VL regions of an immunoglobulin | Granel J, Lemoine R, Morello E (2020) 4C3 human monoclonal antibody: a proof of concept for non-pathogenic proteinase 3 anti-neutrophil cytoplasmic antibodies in granulomatosis with polyangiitis. Front Immunol 11:573040. |
| RT-PCR with human liver and testis samples for microRNA expression analysis, followed by Sanger sequencing | Rubino E, Cruciani M, Tchitchek N (2021) Human ubiquitin-specific peptidase 18 is regulated by microRNAs via the 3'untranslated region, a sequence duplicated in long intergenic non-coding RNA genes residing in chr22q11.21. Front Genet 11:627007. |
Additional areas
Ordering information
Resources
Flyers
Manuals/protocols
Application notes
White papers
Spotlight article
Support
PCR education
Learn about the history of PCR, setup considerations, importance of DNA polymerase choice, common methods and applications, and troubleshooting.
PCR and cDNA Synthesis support center
Find tips, troubleshooting help, and resources for your end-point PCR and cDNA applications.
OEM & customized PCR solutions
Discover customizable manufacturing solutions, product labeling, and packaging capabilities for your specific requirements.
Contact us
Email or call our Technical Application Scientists for additional questions regarding PCR enzymes and master mixes.
All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Q5 is a trademark of New England BioLabs Inc. PrimeSTAR is a trademark of Takara-Clontech Laboratories, Inc. Merck is a trademark of Merck KGaA. KAPA HiFi HotStart is a trademark of Roche Inc. HotStar is a trademark of Qiagen GmbH. Roche Expand is a trademark of Roche Molecular Systems Inc. PfuUltra is a trademark of Agilent, Inc.
For Research Use Only. Not for use in diagnostic procedures.