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DNA-Free DNA Polymerases
Thermo Fisher Scientific has developed an enzyme manufacturing process using Single-Use System (SUS)-based technology with extensive quality control testing to help reduce the risk of DNA contaminates in the PCR reagents for commercial supply. These reagents are an exceptional choice for PCR assays where higher sensitivity and specificity are needed to help minimize ambiguous or false-positive results.
DNA-free enzyme manufacturing with single-use technology
To avoid the potential risks and implications associated with contaminated PCR reagents, we have adopted the use of SUS technology to manufacture PCR reagents. Watch below how SUS technology helped minimize the risks of DNA contamination in production of PCR reagents.
Video: DNA-free PCR reagents
Watch how SUS technology helps minimize the risks of DNA contamination in production of PCR reagents.
How SUS manufacturing is helping solve the problem of DNA contamination in qPCR/PCR-based assays
Click image to enlarge
Read about the distinctions between traditional and single-use system of manufacturing PCR enzymes.
Advantages of SUS manufacturing
- Entirely closed system throughout the manufacturing process—No exposure to environment and human operators
- Sterile single-use bags, tubing, and connectors—No potential cross-contamination from common-use equipment
- Manufacturing in facilities that meet Class D and Class C cleanroom standards—Dedicated purpose-built space helps prevent exposure to DNA contaminants
- Stringent quality control testing
- Adherence to ISO 13485 quality standards
Differences between SUS manufacturing and conventional manufacturing of DNA-free enzymes
Conventional manufacturing
The manufacturing process for commercially available DNA polymerases typically consists of multiple steps handled by a scientist or operator in an open environment (Figure 1). In addition, this equipment may be shared for manufacturing other proteins and enzymes; the purity of a preparation relies on efficient decontamination steps between processes.
Contaminating DNA commonly found in enzyme preparations can occur during one or all these many steps throughout the manufacturing process. During testing, these DNA polymerases have shown detectable levels of DNA contaminants [1]. This poses a risk for commercial-scale production of DNA polymerase that Thermo Fisher Scientific has addressed with SUS-based manufacturing.
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Figure 1. A conventional manufacturing workflow for recombinant enzymes illustrates the risk of DNA contamination. The typical process for enzyme preparation includes repeated opportunities for potential DNA contamination from an open environment to handling by an operator between steps. There is also a potential risk for the carryover of DNA contaminants when utilizing equipment that is shared with other manufacturing processes for the preparation of other enzymes and proteins.
SUS manufacturing
At Thermo Fisher Scientific, we have adapted our SUS technology for the commercial-scale production of recombinant enzymes (Figure 2). All stages of SUS-based manufacturing are performed within a closed environment and rely on sterile single-use bags, tubing, and connectors throughout production. All buffers and washing solutions are prepared in single-use bags and filtered for sterilization. With closed SUS-based manufacturing, the probability of DNA contamination has been reduced to a negligible risk level.
Click image to enlarge
Figure 2. Closed SUS-based process for the manufacture of recombinant enzymes. This process represents a completely closed system that relies on single-use bags, tubes, and connectors, which help reduce the potential for DNA contamination to a negligible risk level whether from the environment or the operator. Moreover, because it is single-use, and equipment or materials are not shared, it helps eliminate the manufacturing workflow as a source of cross-contamination.
Benefits of single-use technology in the production of DNA-free PCR enzymes
Invitrogen Platinum Taq DNA polymerase, manufactured utilizing the SUS technology, is subject to rigorous quality control testing, such as application-specific functional assays. This DNA-free enzyme delivers exceptional performance.
Platinum Taq DNA polymerase, DNA-free, helps in minimizing false positives from PCR based assays. False positives are signals generated in no template control (NTC) reaction in absence of template DNA. A highly specific test should not produce false positives or misclassify the identity of DNA target.
Figure 3. Uncompromised specificity of Platinum Taq DNA polymerase, DNA-free, as compared to competitor. Performance of Platinum Taq DNA polymerase, DNA-free (blue curves), and Promega GoTaq MDx Hot start Polymerase (green curves) in qPCR assays were evaluated using primers targeting E. coli 16S rRNA gene and varying amount of E. coli input DNA. In 80 wells of negative control (no DNA template added), there was no E. coli DNA detected.
Real-time PCR assays performed with Platinum Taq DNA polymerase manufactured using SUS technology demonstrates the same performance as compared to Platinum Taq DNA polymerase manufactured using conventional manufacturing scheme.
Figure 4. Sensitive, specific, and reproducible amplification with DNA-free Taq DNA polymerase. Performance of Platinum Taq DNA polymerase, DNA-free (blue curves), and Platinum Taq DNA polymerase (red curves) in qPCR assays were evaluated using primers targeting E. coli 16S rRNA gene and varying amount of E. coli input DNA.
NA-free enzymes are analyzed following high standards to help ensure the absence of nucleases and DNA contamination (Table 1). Additional testing demonstrated that Platinum Taq DNA polymerase, DNA-free, is the only enzyme with less than 1 copy of contaminating DNA in 100 units of enzyme (Table 2).
Table 1. Stringent quality testing to validate DNA-free Platinum Taq DNA polymerase.
| Purity test | Requirement |
|---|---|
| Exonucleases and endonucleases | undetected |
| RNases | undetected |
| Bacterial gDNA (16S rRNA) | ≤0.01 copy/enzyme unit |
| Human gDNA (Alu) | ≤0.001 copy/enzyme unit |
| Plasmid DNA (ori1) | ≤0.01 copy/enzyme unit |
Table 2. Quality control standards for low-DNA contamination and DNA-free Taq DNA polymerases.
| Product | Copy # of bacterial gDNA/100 units | Copy # of plasmid DNA/100 units | Copy 3 of human gDNA/100 units |
|---|---|---|---|
| Platinum Taq DNA polymerase, DNA-free | 0.4 | 0.4 | 0.00 |
| Eurogentec HGS Diamond Taq polymerase | 11.7 | 300 | 0.04 |
| Roche Taq DNA polymerase, GMP grade | 18 | 80 | 0.12 |
| Roche AptaTaq DNA polymerase, LDx, glycerol-free | 4.1 | n.d. in 50 Units | 0.17 |
| Sigma MTP Taq DNA polymerase | 13.2 | 11,600 | 0.12 |
| Promega GoTaq MDx Hot Start Polymerase | 18.5 | 400 | 0.06 |
Platinum Taq DNA polymerase, DNA-free aids in accurate detection of low-copy (i.e., one copy) of target DNA. Uncompromised sensitivity is the advantage in PCR-based assays where the target DNA is in low concentration in the sample or when there is a limited amount of starting material (Figure 5).
Figure 5. Uncompromised sensitivity of Platinum Taq DNA polymerase, DNA-free, as compared to competitor. Performance of Platinum Taq DNA polymerase, DNA-free (blue curves), and Promega GoTaq MDx Hot Start Polymerase (green curves) in qPCR assays were evaluated using primers targeting E. coli 16S rRNA gene and varying amount of E. coli input DNA.
Platinum Taq DNA polymerase, DNA-free, helps in minimizing false positives from PCR based assays. False positives are signals generated in no template control (NTC) reaction in absence of template DNA. A highly specific test should not produce false positives or misclassify the identity of DNA target.
Figure 3. Uncompromised specificity of Platinum Taq DNA polymerase, DNA-free, as compared to competitor. Performance of Platinum Taq DNA polymerase, DNA-free (blue curves), and Promega GoTaq MDx Hot start Polymerase (green curves) in qPCR assays were evaluated using primers targeting E. coli 16S rRNA gene and varying amount of E. coli input DNA. In 80 wells of negative control (no DNA template added), there was no E. coli DNA detected.
Real-time PCR assays performed with Platinum Taq DNA polymerase manufactured using SUS technology demonstrates the same performance as compared to Platinum Taq DNA polymerase manufactured using conventional manufacturing scheme.
Figure 4. Sensitive, specific, and reproducible amplification with DNA-free Taq DNA polymerase. Performance of Platinum Taq DNA polymerase, DNA-free (blue curves), and Platinum Taq DNA polymerase (red curves) in qPCR assays were evaluated using primers targeting E. coli 16S rRNA gene and varying amount of E. coli input DNA.
NA-free enzymes are analyzed following high standards to help ensure the absence of nucleases and DNA contamination (Table 1). Additional testing demonstrated that Platinum Taq DNA polymerase, DNA-free, is the only enzyme with less than 1 copy of contaminating DNA in 100 units of enzyme (Table 2).
Table 1. Stringent quality testing to validate DNA-free Platinum Taq DNA polymerase.
| Purity test | Requirement |
|---|---|
| Exonucleases and endonucleases | undetected |
| RNases | undetected |
| Bacterial gDNA (16S rRNA) | ≤0.01 copy/enzyme unit |
| Human gDNA (Alu) | ≤0.001 copy/enzyme unit |
| Plasmid DNA (ori1) | ≤0.01 copy/enzyme unit |
Table 2. Quality control standards for low-DNA contamination and DNA-free Taq DNA polymerases.
| Product | Copy # of bacterial gDNA/100 units | Copy # of plasmid DNA/100 units | Copy 3 of human gDNA/100 units |
|---|---|---|---|
| Platinum Taq DNA polymerase, DNA-free | 0.4 | 0.4 | 0.00 |
| Eurogentec HGS Diamond Taq polymerase | 11.7 | 300 | 0.04 |
| Roche Taq DNA polymerase, GMP grade | 18 | 80 | 0.12 |
| Roche AptaTaq DNA polymerase, LDx, glycerol-free | 4.1 | n.d. in 50 Units | 0.17 |
| Sigma MTP Taq DNA polymerase | 13.2 | 11,600 | 0.12 |
| Promega GoTaq MDx Hot Start Polymerase | 18.5 | 400 | 0.06 |
Platinum Taq DNA polymerase, DNA-free aids in accurate detection of low-copy (i.e., one copy) of target DNA. Uncompromised sensitivity is the advantage in PCR-based assays where the target DNA is in low concentration in the sample or when there is a limited amount of starting material (Figure 5).
Figure 5. Uncompromised sensitivity of Platinum Taq DNA polymerase, DNA-free, as compared to competitor. Performance of Platinum Taq DNA polymerase, DNA-free (blue curves), and Promega GoTaq MDx Hot Start Polymerase (green curves) in qPCR assays were evaluated using primers targeting E. coli 16S rRNA gene and varying amount of E. coli input DNA.
SUS manufacturing services
We offer a wide range of DNA polymerases and reverse transcriptases with high quality and exceptional performance for your PCR- or qPCR-based assays. For more information about Platinum Taq DNA polymerase, DNA-free, or other DNA-free enzymes, please contact us and we will work with you to help meet your requirements.
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