HiFi Seq Hotstart DNA polymerase 是通过基因工程改造的工程酶，在无需辅助蛋白及额外的 DNA 结合功能域的条件下提高了对 DNA 模板的亲和力。相比于其他野生型的 B 族 DNA 聚合酶，HiFi Seq Hotstart DNA 聚合酶强大的持续合成能力能够显著的提高扩增产物量、扩增速率以及灵敏度，同时长片段扩增和复杂模板的扩增能力（高 AT 或者高 GC 模板）也有明显的提高。
HiFi Seq Hotstart DNA polymerase 有 5’→3’聚合酶活性和 3’→5’外切酶活性（校对活性），无 5’→3’外切酶活性。强大的 3’→5’ 外切酶活性保证了 DNA 扩增过程中的准确率，HiFi Seq Hotstart DNA polymerase 保真性是野生型 Taq DNA 聚合酶的 100 倍，是 其他 B 族 DNA 聚合酶的 10 倍。
组分表
FP1509106 | 组分 | 100U | 500U | 存储温度 |
FP1509106A | HiFi Seq Hotstart DNA polymerase (1U/μL) | 100 μL | 500 μL | -20℃ |
FP1509106B | 5×HiFi seq buffer | 1.0 mL | 5×1.0 mL | -20℃ |
FP1509106C | 10×Enhancer | 500 μL | 2×1.25 mL | -20℃ |
适用范围
NGS DNA 文库的富集、基因表达克隆、基因定点突变、细胞内基因点突变的分析。
使用方法
1. 提前取出 5×HiFi seq buffer、10×Enhancer 置于室温融化，如果有沉淀析出，请彻底混匀溶解后使用，HiFi Seq Hotstart DNA polymerase 置于冰上备用。
2. 按照下表配制 PCR 体系，注意此步骤需于冰浴中操作。
| 组分 | 体积（μL） |
|---|
| 5×HiFi seq buffer | 10 |
| 10×Enhancer | 5 |
| 10mM dNTP | 1 |
| HiFi Seq Hotstart DNA polymerase | 1 |
| Primer(each 5μM) | 2-5 |
| templates | — |
| ddH2O | — |
| Total | 50 |
1) 反应体系可以设置在 10-50μl 之间，试剂使用量可以按照比例调整，不推荐使用 > 50μl 体系；
2) 1×HiFi seq buffer 包含 2.5mM Mg²⁺(1×)，如果体系需要更高浓度的 Mg²⁺可以额外的添加；
3) 在初次测试时，50μl 反应体系建议基因组模板使用量为 10-100ng，稍微复杂模板使用量为 0.1-1ng。
反应程序
3. 按下表设置 PCR 仪反应程序，开启热盖，温度设置于 105℃。
1) 预变性时间 95℃ 3min 对于常规模板是足够的，对于 GC≧70% 模板建议将预变性时间延长至 5min；
2) 体系中高盐离子浓度会影响 DNA 溶解，为保证复杂 GC 模板 能够完全变性，建议将变性温度调整到 98℃。高盐 buffer 同时也 会影响引物的退火，优化后的退火温度要略高于常规的 PCR 体 系，初次反应建议使用 56℃为退火温度,若反应不理想,则可以每 调整 3℃测试一次的方法来确定最适的退火温度；
3) 两步法扩增程序，建议退火/延伸范围 65-68℃，延伸时间按 30-60 sec/kb 计算；
4) 常规条件下目的片段≦1kb，延伸时间按 15sec/kb 计算，当目 的片段>1kb，或者要提高产物量，延伸时间按 30 sec/kb 计算；
5) 为获得较高的保真性，建议扩增循环数≦25cycles，扩增循环 数越高，错配率越高。
HiFi Seq Hotstart DNA polymerase is an engineered enzyme modified via genetic engineering. It enhances the affinity for DNA templates without the need for auxiliary proteins or additional DNA-binding domains. Compared to other wild-type B-family DNA polymerases, HiFi Seq Hotstart DNA polymerase features robust processivity, which can significantly increase the yield, amplification rate, and sensitivity of amplified products. Meanwhile, its ability to amplify long fragments and complex templates (such as those with high AT or high GC content) is also notably improved.
HiFi Seq Hotstart DNA polymerase possesses 5’→3’ polymerase activity and 3’→5’ exonuclease activity (proofreading activity), but lacks 5’→3’ exonuclease activity. The powerful 3’→5’ exonuclease activity ensures the accuracy during DNA amplification. The fidelity of HiFi Seq Hotstart DNA polymerase is 100 times that of wild-type Taq DNA polymerase and 10 times that of other B-family DNA polymerases.
Component List
FP1509106 | Components | 100U | 500U | Storage |
FP1509106A | HiFi Seq Hotstart DNA polymerase (1U/μL) | 100 μL | 500 μL | -20℃ |
FP1509106B | 5×HiFi seq buffer | 1.0 mL | 5×1.0 mL | -20℃ |
FP1509106C | 10×Enhancer | 500 μL | 2×1.25 mL | -20℃ |
Application Scope
Enrichment of NGS DNA libraries, gene expression cloning, site-directed mutagenesis, and analysis of intracellular gene point mutations.
Usage Method
1. Take out 5×HiFi seq buffer and 10×Enhancer in advance and thaw them at room temperature. If precipitation occurs, mix thoroughly until dissolved before use. Keep HiFi Seq Hotstart DNA polymerase on ice for later use.
2. Prepare the PCR system according to the table below. Note that this step should be performed on an ice bath.
Component
| Volume (μL) |
|---|
| 5×HiFi seq buffer | 10 |
| 10×Enhancer | 5 |
| 10mM dNTP | 1 |
| HiFi Seq Hotstart DNA polymerase | 1 |
| Primer (each 5μM) | 2-5 |
| templates | — |
| ddH2O | — |
| Total | 50 |
① The reaction volume can be set between 10–50 μl, and the reagent dosage can be adjusted proportionally. Using a volume > 50 μl is not recommended.
② 1×HiFi seq buffer contains 2.5 mM Mg²⁺ (1×). Additional Mg²⁺ can be added if a higher concentration is required for the system.
③ For initial testing, the recommended amount of genomic template in a 50 μl reaction system is 10–100 ng, and 0.1–1 ng for slightly complex templates.
Reaction Procedure
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3. Set the PCR instrument program according to the table below, turn on the heated lid, and set the temperature to 105℃.
① A pre-denaturation step of 3 min at 95℃ is sufficient for regular templates. For templates with GC content ≥ 70%, extend the pre-denaturation time to 5 min.
② High salt ion concentration in the system can affect DNA dissolution. To ensure complete denaturation of complex GC-rich templates, adjust the denaturation temperature to 98℃. High-salt buffers also affect primer annealing, so the optimized annealing temperature should be slightly higher than that of conventional PCR systems. For the first reaction, use 56℃ as the annealing temperature; if the reaction result is unsatisfactory, adjust the temperature in 3℃ increments to determine the optimal annealing temperature.
③ For the two-step amplification program, the recommended annealing/extension temperature range is 65–68℃, and the extension time is calculated as 30–60 sec/kb.
④ Under conventional conditions, if the target fragment ≤ 1 kb, calculate the extension time as 15 sec/kb; if the target fragment > 1 kb or higher product yield is required, calculate the extension time as 30 sec/kb.
⑤ To achieve high fidelity, the number of amplification cycles is recommended to be ≤ 25. The higher the number of amplification cycles, the higher the mismatch rate.
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