PCR is used for the amplification of a next-generation sequencing (NGS) library. In addition to increasing the amount of DNA, the PCR amplification step is also used for DNA target enrichment for fragments that have an adaptor-ligated to each end. These will be the fragments that attach to the flow cell and will be sequenced, so it is important that this PCR amplification step does not introduce mutations to the NGS library, as a lower error rate means that less sequence coverage is required, saving expense and time.
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NGS library amplification
Four NGS libraries were created using E. coli DNA. These libraries were amplified using High-Fidelity Pfu prior to being placed on flow cells and being sequenced. For each sample, the fraction of uniquely mapped (mapped to only one place on a reference sequence), ambiguously mapped and unmapped reads relative to the total number of reads per sample is shown. The results illustrate that with E. coli DNA, High-Fidelity Pfu allowed for over 90% unique reads that could be aligned to the reference sequence and as such these libraries are considered very good libraries.
High-Fidelity Pfu is a thermostable high fidelity DNA polymerase enzyme, isolated from Pyrococcus furiosus. The Pfu polymerase enzyme replicates DNA in the 5´→3´ direction in the presence of magnesium, but also possesses 3´→5´ exonuclease (proofreading) activity. Base misincorporations that may occur during polymerization are rapidly excised by this proofreading activity. High-Fidelity has an error rate of 3.0 x 10-6 generating blunt-ended amplicons up to 5 kb in length. Glycerol-Free High-Fidelity Pfu (HC) is supplied in a glycerol-free storage buffer and is accompanied by a 5x Reaction Buffer that contains magnesium, dNTPs and excipients required for lyophilization.
|Description||MDX003 High-fidelity hot start Taq DNA polymerase with separate 10x Pfu Reaction Buffer and MgCl2. MDX203 high concentration, high-fidelity Taq DNA polymerase with separate Pfu Polymerase Dilution Buffer and10x Pfu Reaction Buffer.|
|Concentration||2 U/µL and 20 U/µL|
|Appearance||Clear, colorless solution|
|Hot Start||MDX003 antibody mediated|
|Application||PCR, NGS library preparation, cloning, protein expression|
|Sample type||cDNA, DNA|
|Mix stability||See outer label|
|Consistency||± 0.5 Ct variance between test and reference sample|
|DNA Contamination||None detected in PCR amplification with traces overlay with the negative control on E. coli and mouse genomic DNA specific targets.|
|DNase Contamination||No detectable degradation|
FAQs: High-Fidelity Pfu
In nature, the ability of a DNA polymerase to correct misincorporation of nucleotides in the DNA strand being elongated is often crucial to the survival of the host organism. This ability is termed “proofreading activity” and occurs in the 3′ to 5′ direction. This activity also leads to the polymerase removing unpaired nucleotides overhanging at the 3’end (A-overhangs), creating blunt ends.
If possible, we would recommend an additional clean-up of affected samples, alternatively High-Specificity Pfu HS Mix (MDX006) would be recommended, as it is designed to work in the presence of inhibitors.
High-Fidelity Pfu has been validated for templates up to 5 kb.
DNA target enrichment is a pre-sequencing DNA preparation step where DNA sequences are either directly amplified (multiplex PCR-based) or captured (hybrid capture-based). These enriched DNA fragments can then be sequenced using DNA sequencers.
During PCR amplification setup at room temperature, standard polymerases will have some activity. When using a hot-start polymerase, this enzyme will have no activity at room temperature, thus reducing the risk of unspecific products in the reaction due to these mis-primed oligonucleotides. This feature makes this type of polymerase ideally suited to high-throughput applications, where the reactions may be left sitting at room temperature for prolonged periods of time.
High-Fidelity Pfu should be stored at -20°C for optimal retention of activity. We do not recommend the storage of High-Fidelity Pfu at -80 °C, although MDX003 contains glycerol, at such a low temperature ice crystals could form on the active site, which may affect or destroy the activity of the enzyme.
A lyophilized PCR molecular test will remain stable for up to 2 years at ambient temperature if correctly stored in sealed pouches.
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