PowerResist Taq Polymerase 是通过基因工程技术对野生型 Taq 酶进行多位点突变获得的突变体酶。
与野生型相比，该酶对模板亲和性更高、抗抑制性更强，对临床样品中的内源和外源干扰物具有很强的耐受性，可实现全血直扩、粪便样本粗提物直扩、植物样本粗提物直扩，能有效减少样本前处理和 DNA 提取等步骤。
该酶扩增速率快，适用于快速 PCR；检测灵敏度高，适用于低模板检测；单碱基识别能力强，可用于检测 SNP（单核苷酸多态性）。
可用于探针法荧光定量 PCR 检测项目。
采用两个 Taq 单克隆抗体进行修饰，可同时封闭 Taq 酶的 5’-3’聚合活性和 5’-3’外切活性。在高温加热前，抗体对聚合酶活性的封闭作用，能有效抑制低温条件下因引物非特异性退火或引物二聚体引发的非特异性扩增，提升扩增的特异性和扩增效率；抗体对外切活性的封闭作用，可减少低温条件下对荧光探针的切割，保证 qPCR（实时荧光定量 PCR）的基线平稳，获得较好的 S 型扩增曲线。
当扩增反应体系被加热至 95℃并持续 2.5 分钟时，修饰抗体变性脱落，释放出 Taq 酶的聚合活性和外切活性，因此无需特殊失活处理，在常规 PCR 反应条件下即可使用。
与改良的扩增缓冲液配合使用时，该酶能快速激活，有效增加反应产物量，并提高 PCR 反应的灵敏度、特异性和抗干扰性。
组分表
| FP1509109 | Component | 250U | 1KU | 2.5KU | 4×2.5KU | Storage | | FP1509109A | PowerResist Taq Polymerase (5U/μL) | 50μl | 200μl | 500μl | 4×500μl | -20℃. Avoid freeze/thaw cycle. | | FP1509109B | 5×GN Buffer（Without Mg²⁺） | 1ml | 4×1ml | 10×1ml | 40×1ml | -20℃. Avoid freeze/thaw cycle. | | FP1509109C | 100 mM Mg²⁺ | 100μl | 400μl | 1ml | 4×1ml | -20℃. Avoid freeze/thaw cycle. |
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适用范围适用于热启动法 qPCR 扩增、全血直扩、抗干扰扩增、快速 PCR、ARMS（扩增阻碍突变系统）。
注意事项
1. 对于大多数扩增反应，起始变性时间设为 95℃、2.5 分钟即可；若模板复杂，可适当调整变性时间。
2. 延伸时间需根据 PCR 产物长度确定，例如扩增人基因组 DNA 的 2kb 片段时，延伸时间最快可达 15 秒；若为复杂模板（如选用全血模板扩增），可适当延长延伸时间。
其他注意事项
1. 以全血为模板进行 PCR 扩增时，全血加入体积建议控制在反应体系体积的 20%（v/v）以内。
2. 用于荧光定量 PCR 时，全血加入体积建议≤反应体系体积的 5%（v/v），过多血样加入会影响荧光信号的收集。
使用方法
1. 反应体系配制前准备
1.1 将反应所需的各种溶液在室温或 4℃条件下溶解并混匀，之后置于冰浴或冰盒内；建议将反应液体分装使用，避免反复冻融。
1.2 可根据实验项目需求按比例调整反应体系体积，确保各组分终浓度一致即可。
1.3 参考下表设置荧光定量 PCR 反应体系，建议在冰浴或冰盒上完成体系配制：
| 组分 | 加入体积 | 终浓度 |
|---|
| 5×GN Buffer（without Mg²⁺） | 10 μL | 1× | | dNTPs（25 mM each） | 0.4 μL | 0.2 mM | | Template DNA（模板 DNA） | 5~10 μL | — | | Forward Primer（上游引物，10 µM） | 1 μL | 0.2 µM | | Reverse Primer（下游引物，10 µM） | 1 μL | 0.2 µM | | Probe（探针，10 µM） | 0.5 μL | 0.1 µM | | PowerResist Taq Polymerase（5U/μL） | 0.5 μL | 2.5U/50μL | | MgCl₂（100 mM） | 1 μL | 2 mM | | ddH₂O（超纯水） | 补足至 50μL | — | | 总体积 | 50μL | — |
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* 不同类型模板在 50µL 反应体积中的推荐用量如下：
哺乳动物基因组 DNA：0.1~1 µg
大肠杆菌基因组 DNA：10~100 ng
质粒 DNA：0.1~10 ng
1.4 Mg²⁺推荐用量为 2mM~6mM。
1.5 配制过程中避免剧烈震荡，可用移液器轻轻吹打混匀或轻微涡旋振荡（Vortex）混匀，之后在室温下离心数秒。
反应程序
1. 常规定性 PCR（以扩增 1 kb 目的片段为例）
| 步骤名称 | 温度 | 时间 | 循环数 |
|---|
| 热激 | 95℃ | 2min30s | 1 | | 变性 | 95℃ | 20s | 30~35 | | 退火 | 55℃ | 20s | 30~35 | | 延伸 | 72℃ | 40s | 30~35 | | 延伸 | 72℃ | 7min | 1 | | 保存 | 12℃ | ∞（无限期） | 1 |
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2. 荧光定量 PCR
| 步骤名称 | 温度 | 时间 | 循环数 |
|---|
| 热激 | 95℃ | 2min30s | 1 | | 变性 | 95℃ | 15s | 35~40 | | 退火 + 延伸 | 55℃ | 40s | 35~40 |
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a. PCR 反应条件（包括温度、时间、循环数等）需根据模板、引物、PCR 产物长度及 GC 含量等因素灵活调整。
b. 延伸时间需依据 PCR 产物长度设置，通常每 kb 产物的最快延伸时间为 15 秒。例如：PCR 产物长度为 1kb 时，延伸时间可设为 15 秒；产物长度为 2kb 时，延伸时间可设为 30 秒；若扩增效果不佳，可适当延长至 45 秒，其他长度片段以此类推。
实验用例
1. 在高、低浓度模板下，比较竞品和 PowerResist Taq Polymerase对腐殖酸的耐受性，结果显示 PowerResist Taq Polymerase 拥有更强的抑制剂耐受性和更出色的扩增能力。
2. 以全血为模板，扩增人基因组 2kb 目的片段，PowerResist TaqPolymerase 耐全血抑制能力与竞品相比，具有明显优势。
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3. 以人基因组 DNA 为模板，扩增 2 kb 目的片段，PowerResist TaqPolymerase 相较于竞品，可以在更短的延伸时间内扩增出目的片段。
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PowerResist Taq Polymerase is a mutant enzyme obtained by site-directed mutagenesis of wild-type Taq enzyme using genetic engineering techniques.
Compared with the wild-type, this enzyme has higher affinity for templates and stronger resistance to inhibitors. It exhibits high tolerance to endogenous and exogenous interferents in clinical samples, enabling direct amplification of whole blood, crude extracts from fecal samples, and crude extracts from plant samples. This effectively reduces steps such as sample pretreatment and DNA extraction.
This enzyme has a fast amplification rate, making it suitable for rapid PCR; it also has high detection sensitivity, which is applicable for low-template detection. Additionally, it possesses strong single-base recognition ability and can be used for SNP (Single Nucleotide Polymorphism) detection.
It can be used in probe-based quantitative real-time PCR (qPCR) detection assays.
The enzyme is modified with two Taq monoclonal antibodies, which can simultaneously block the 5’-3’ polymerase activity and 5’-3’ exonuclease activity of the Taq enzyme. Before high-temperature heating, the blocking effect of the antibodies on the polymerase activity can effectively inhibit non-specific amplification caused by non-specific annealing of primers or primer dimers under low-temperature conditions, thereby improving the specificity and efficiency of amplification. Meanwhile, the blocking effect on the exonuclease activity can reduce the cleavage of fluorescent probes under low-temperature conditions, ensuring a stable baseline in qPCR and obtaining a good S-shaped amplification curve.
When the amplification reaction system is heated to 95°C for 2.5 minutes, the modified antibodies denature and dissociate, releasing the polymerase activity and exonuclease activity of the Taq enzyme. Therefore, no special inactivation treatment is required, and the enzyme can be used under conventional PCR reaction conditions.
When used in conjunction with the improved amplification buffer, the enzyme can be activated quickly, effectively increasing the amount of reaction products and enhancing the sensitivity, specificity, and anti-interference ability of the PCR reaction.
Component
| FP1509109 | Component | 250U | 1KU | 2.5KU | 4×2.5KU | Storage | | FP1509109A | PowerResist Taq Polymerase (5U/μL) | 50μl | 200μl | 500μl | 4×500μl | -20℃. Avoid freeze/thaw cycle. | | FP1509109B | 5×GN Buffer（Without Mg²⁺） | 1ml | 4×1ml | 10×1ml | 40×1ml | -20℃. Avoid freeze/thaw cycle. | | FP1509109C | 100 mM Mg²⁺ | 100μl | 400μl | 1ml | 4×1ml | -20℃. Avoid freeze/thaw cycle. |
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Scope of ApplicationSuitable for hot-start qPCR amplification, direct whole blood amplification, anti-interference amplification, rapid PCR, and ARMS (Amplification Refractory Mutation System).
Precautions
- For most amplification reactions, the initial denaturation time can be set at 95°C for 2.5 minutes. If the template is complex, the denaturation time can be adjusted appropriately.
- The extension time should be determined according to the length of the PCR product. For example, when amplifying a 2kb fragment of human genomic DNA, the extension time can be as fast as 15 seconds. If it is a complex template (such as using whole blood as a template for amplification), the extension time can be appropriately prolonged.
Other Precautions
- When performing PCR amplification using whole blood as a template, the volume of whole blood added is recommended to be controlled within 20% (v/v) of the reaction system volume.
- When used for fluorescent quantitative PCR, the volume of whole blood added is recommended to be ≤ 5% (v/v) of the reaction system volume. Excessive addition of blood will affect the collection of fluorescent signals.
Usage Method
Preparation before Reaction System Configuration
1.1 Dissolve and mix various solutions required for the reaction at room temperature or 4°C, and then place them in an ice bath or ice box. It is recommended to aliquot the reaction liquids for use to avoid repeated freezing and thawing.
1.2 The reaction system volume can be adjusted in proportion according to the experimental project requirements to ensure that the final concentration of each component is consistent.
1.3 Refer to the following table to set up the fluorescent quantitative PCR reaction system. It is recommended to complete the system configuration on an ice bath or ice box.
| Component | Volume Added | Final Concentration |
|---|
| 5×GN Buffer (without Mg²⁺) | 10 μL | 1× | | dNTPs (25 mM each) | 0.4 μL | 0.2 mM | | Template DNA | 5 - 10 μL | — | | Forward Primer (10 μM) | 1 μL | 0.2 μM | | Reverse Primer (10 μM) | 1 μL | 0.2 μM | | Probe (10 μM) | 0.5 μL | 0.1 μM | | PowerResist Taq Polymerase (5 U/μL) | 0.5 μL | 2.5 U/50 μL | | MgCl₂ (100 mM) | 1 μL | 2 mM | | ddH₂O | Make up to 50 μL | — | | Total Volume | 50 μL | — |
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*Recommended amounts of different types of templates in a 50 μL reaction volume are as follows:
Mammalian genomic DNA: 0.1 - 1 μg
E. coli genomic DNA: 10 - 100 ng
Plasmid DNA: 0.1 - 10 ng
1.4 The recommended amount of Mg²⁺ is 2 mM - 6 mM.
1.5 Avoid vigorous shaking during preparation. It can be mixed by gently pipetting or slightly vortexing, and then centrifuged at room temperature for a few seconds.
Reaction Procedure
1. Conventional Qualitative PCR (taking the amplification of a 1 kb fragment as an example)
| Step Name | Temperature | Time | Number of Cycles |
|---|
| Heat Shock | 95°C | 2min30s | 1 | | Denaturation | 95°C | 20s | 30 - 35 | | Annealing | 55°C | 20s | 30 - 35 | | Extension | 72°C | 40s | 30 - 35 | | Extension | 72°C | 7min | 1 | | Storage | 12°C | ∞ (Indefinite) | 1 |
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2. Fluorescent Quantitative PCR
| Step Name | Temperature | Time | Number of Cycles |
|---|
| Heat Shock | 95°C | 2min30s | 1 | | Denaturation | 95°C | 15s | 35 - 40 | | Annealing + Extension | 55°C | 40s | 35 - 40 |
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a. PCR reaction conditions (including temperature, time, number of cycles, etc.) need to be flexibly adjusted according to factors such as templates, primers, PCR product length, and GC content.
b. The extension time should be set according to the length of the PCR product. Usually, the fastest extension time for each kb product is 15 seconds. For example: when the length of the PCR product is 1kb, the extension time can be set to 15 seconds; when the product length is 2kb, the extension time can be set to 30 seconds; if the amplification effect is not good, it can be appropriately extended to 45 seconds, and so on for fragments of other lengths.
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