Roche PCR Reagents Selection Guide for High Fidelity, Difficult Templates, and Long PCR Amplification
Cutting-edge science shouldn’t have to compromise for the sake of economy. Since the introduction of PCR technology, Roche has provided the gold standard in PCR reagents. Our advanced isolation, purification, and manufacturing techniques ensure that you receive not only the clearest, most reliable results, but also convenience and affordability to fit any laboratory budget.
PCR-Grade Nucleotides ensure optimal performance.
PCR success is highly dependent on your selection of both enzymes and nucleotides. Roche PCR enzymes are available in convenient dNTPacks, which include premixed solutions of additive-free sodium salt nucleotides. These function tested PCR-Grade Nucleotides are manufactured by enzymatic synthesis and purified by a unique process and are an ideal contributor to the quality of your PCR results.
Affordable PCR characterized by robust amplification with minimal template requirements.
Roche Taq DNA Polymerase is produced under GMP conditions. This highly purified enzyme passes several stringent tests for functionality and purity, ensuring reliable, consistent results with every lot. For additional information, please view our standard PCR protocol.
Figure 1. Lot-to-lot consistency ensures reproducible results. Five different lots of Taq DNA Polymerase were tested for the ability to amplify a 0.5 kb fragment of lambda DNA. Reliable, consistent results are obtained with every lot of Roche Taq DNA Polymerase that was tested.
The Taq DNA Polymerase, GMP Grade belongs to the family of high-performance, validated amplification enzymes and is manufactured using evaluated production, quality control, and filling procedures.
Choose the polymerase researchers trust, as with qPCR and the LightCycler® Systems.
FastStart™ Taq DNA Polymerase is a thermostable, chemically modified form of recombinant Taq DNA polymerase. Due to its modification, FastStart™ Taq DNA polymerase is only activated at high temperatures, making it the ideal enzyme when your assay’s amplification is delayed.
Figure 4. Amplification of the human erythropoietin gene from 3-300 ng cDNA after immediate (E0) and 24 hours after reaction setup at room temperature (E24).
Multiplexing and sequencing stringent hot start and improved fidelity is key in complex endpoint PCR applications, such as multiplexing or sequencing. Choose FastStart™ High Fidelity PCR System for complex hot start PCR up to 5 kb with higher fidelity, as required for multiplexing and sequencing. It is up to six times more accurate compared to both standard Taq DNA polymerase and FastStart™ Taq DNA Polymerase.
Figure 5. Sensitivity test in 18-plex (74 bp – 470 bp) PCR. Set of 18 multiplexed primers was applied to various concentrations of human genomic DNA.
Expand™ High Fidelity PCR reagents are designed for robust amplification across a broad range of assays and amplicon types, consisting of an enzyme blend of Taq DNA Polymerase and a polymerase with proofreading activity for robust PCR. Choose the Expand™ High FidelityPLUS PCR System for robust PCR up to 5 kb with PCR carryover prevention.
The Expand™ High FidelityPLUS PCR System is ideal for robust high-fidelity applications, such as cloning and labeling of DNA fragments with radioactively or nonradioactively modified nucleotides. In addition, combine dUTP incorporation with uracil-DNA glycosylase to prevent PCR cross-contamination.
Figure 6. Comparison of Expand™ High FidelityPlus PCR System with four commercially available polymerase mixes. Various amounts (ng) of human genomic DNA were used to amplify a 4.8 kb fragment from the tissue plasminogen activator (tPA) gene, in accordance with each manufacturer's recommended conditions. Expand High FidelityPLUS PCR System produces the best specificity, sensitivity, and yield, even from as little as 1 ng human genomic DNA.
Supplier A: Mixture of Taq DNA polymerase (deleted at N-terminus), a proofreading polymerase, and a hot start antibody.
Supplier B and D: Mixture of Taq DNA polymerase and a proofreading polymerase.
Supplier C: Mixture of Taq DNA polymerase, a proofreading polymerase, and an enhancing factor.
High fidelity PCR is required in applications where sequence accuracy is crucial without sacrificing yield. Use Pwo SuperYield DNA Polymerase to obtain high yields of PCR product with consistent high fidelity. Choose maximum fidelity and avoid sequence errors, base exchanges, and frame shifts when combined with a proofreading reverse transcriptase, such as in the Transcriptor High Fidelity cDNA Synthesis Kit. The combination is ideal for applications such as cloning, site-directed mutagenesis, and gene expression analysis.
Figure 7. Improved accuracy in RT-PCR. Combination of the proofreading reverse transcriptase, Transcriptor High Fidelity Reverse Transcriptase (Roche), and a proofreading polymerase, Pwo SuperYield DNA Polymerase (Roche) for amplification. Data compared to a commonly used M-MuLV reverse transcriptase and Taq DNA polymerase. Error rate was determined after reverse transcription and 25 PCR cycles using the 454 Sequencing System. The error rates for the Roche enzymes was the mean value of four independent experiments in which at least 3.1 × 106 bases were sequenced. For M-MuLV reverse transcriptase and Taq DNA Polymerase, 4.5 × 106 bases were sequenced. The accuracy is represented as error rate -1.
Choose the GC-RICH PCR System, a blend of a proofreading polymerase and Taq DNA Polymerase to power through templates that are difficult or impossible to amplify with other polymerases or other blends of polymerases and additives. The enhanced processivity of the blend and the unique GC-RICH Resolution Solution are combined to deliver superior performance.
Figure 8. Successfully amplify GC-rich templates with the GC-RICH PCR System. Amplification of a 264 bp template (74% GC content) within the human ApoE gene using the GC-RICH PCR System or the Expand High Fidelity PCR System.
Result: The GC-RICH PCR System amplifies the GC-rich fragment with high specificity and yield using GC-RICH Resolution Solution.
Rely on this next-generation system from Roche, the company that pioneered long-template PCR. Choose Expand™ Long Range dNTPack for consistent amplification of PCR products of 5 to 25 kb from genomic DNA. When amplifying fragments longer than 20 kb, use Expand™ 20 kbPLUS PCR System. This unique system features an optimized buffer and enzyme blend mixture.
Figure 9. Amplification of various genomic templates using Expand™ Long Range, dNTPack
Products for PCR Optimization
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