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, MDX003
Thermostable high fidelity Hot Start DNA polymerase, generates blunt-ended amplicons up to 5 kb in length making it ideal for high yields in NGS library amplification.
文档与资源
Description
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.
Specifications
| Description | High-fidelity hot start Taq DNA polymerase with separate 10x Pfu Reaction Buffer and MgCl2. |
| Concentration | 2 U/µL |
| Appearance | Clear, colorless solution |
| Hot Start | antibody mediated |
| Application | PCR, NGS library preparation, cloning, protein expression |
| Sample type | cDNA, DNA |
| Presentation | 3 vials |
| Storage | -20 °C |
| 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 |
产品资料
分子诊断的原料试剂解决方案分子诊断的原料试剂解决方案
高特异性 Pfu HS 混合物Pfu高保真热启动聚合酶
下一代测序
Next Generation Sequencing (NGS)
建库分子酶下一代测序
Next Generation Sequencing (NGS)
建库分子酶
常见问答: 高保真Pfu
在自然界中，DNA聚合酶在DNA链延长过程中纠错核苷酸整合的能力对于个体生存至关重要。这被称为“校对活动”，发生在3′到5′方向。聚合酶的这种活性还能去除3'端（A-端）的未配对核苷酸，产生钝端。
如果可能的话，建议对受影响的样品进行清洗以尽可能移除抑制物，或者建议使用高特异性Pfu HS预混液（MDX006），该试剂更适合用于有抑制物存在的情况。
经验证，高保真Pfu HS可对长达5kb的模板进行扩增。
DNA靶标富集是测序前的DNA制备步骤，其中DNA序列被直接扩增（基于多重PCR）或捕获（基于混合捕获），然后可以使用DNA测序仪对这些富集的DNA片段进行测序。
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.
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