本产品使用酶工程学改造后的热启动Epitech Taq DNA Polymerase，增强了与DNA模板的结合能力，抗干扰能力明显提升。该酶保留了Taq 酶 5’→3’的外切酶活性，可用于探针法荧光定量PCR检测。同时在原酶的基础上加入Taq单克隆抗体进行修饰，增强对Taq酶活性位点的封闭效果，提高特异性。高温加热前，Taq单克隆抗体与Taq聚合酶结合从而抑制聚合酶的活性，达到抑制低温条件下由引物的非特异性退火或引物二聚体引起的非特异性扩增作用。当扩增反应体系被加热到95℃时，聚合酶活性因 Taq 单克隆抗体变性得到恢复，因此无需特殊失活处理，在常规PCR反应条件下即可使用。
配合优化的缓冲体系，Epitech Taq DNA Polymerase适用于亚硫酸氢盐转化处理DNA的PCR反应，对亚硫酸氢盐转化后的含有尿嘧啶的模板DNA有更优的扩增效率、灵敏度。相比于普通DNA，亚硫酸氢盐转化后的DNA损伤严重，会影响PCR反应性能。同样对于扩增受损的DNA样本，胞嘧啶脱氨作用会在一段较长的时间内自行发生，温度升高时脱氨速度加快，并导致DNA和游离核苷酸中尿嘧啶的累积。当其他校正酶无效时， Epitech Taq DNA Polymerase可以对此类含有尿嘧啶的受损DNA模板进行高效扩增。
组分表
FP1508958 | Component | 250U | 1KU | 5KU | Storage |
FP1508958A | Epitech Taq DNA Polymerase (5U/μL) | 50μl | 200μl | 1ml | -20℃. Avoid freeze/thaw cycle. |
FP1508958B | 5×Epitech Taq DNA Polymerase Buffer
| 1ml | 4ml | 20ml | -20℃. Avoid freeze/thaw cycle |
适用范围
适用于亚硫酸氢盐转化处理 DNA 的 PCR 反应。
注意事项
本实验中，亚硫酸氢盐的预处理过程将直接影响模板 DNA 的质量；处理后的模板不能长期储存，-20℃放置不要超过一个月。
其他注意事项
1. 过多的模板 DNA 容易导致非特异性的 PCR 产物。
2. 对于模板的使用给予建议：以哺乳动物和大肠杆菌的基因组 DNA 为例：
| 试剂名称 | 加入量 |
|---|
| 哺乳动物基因组 DNA | 0.1-1 μg |
| 大肠杆菌基因组 DNA | 10-100 ng |
使用方法
反应体系配制前准备：
1. 室温或 4℃融解并混匀反应所需的各种溶液。并放置于冰浴上或冰盒内。建议反应液体分装使用，避免反复冻融；
2. 参考下表配制反应体系，建议反应体系的配制在冰浴或冰盒上进行：
| 组分 | 加入体积 / 人份 | 终浓度 |
|---|
5×Epitech Taq DNA Polymerase Buffer
| 10 μL | 1× |
Epitech Taq DNA Polymerase
| 0.5 μL | 0.05 U/μL |
| dNTPs | 0.5 μL | 0.25 mM |
Primer-probe Mixᵃ
| X μL | —— |
| 模板 | X μL | —— |
ddH₂O
| To 50 μL | —— |
| 终体积 | 50 μL |
注：a.引物、探针以及模板的用量可自行调整。
3. 用移液器轻轻吹打混匀或轻微 Vortex 混匀，室温离心数秒， 使液体积聚于管底；
4. 各设置好的 PCR 反应管置于 PCR 仪上，开始 PCR 反应。
反应程序
荧光定量 PCR
| 步骤名称 | 温度 | 时间 | 循环 |
|---|
| 预变性 | 95 °C | 10 min | 1 |
| 变性ᵃ | 94 °C | 15 s | 40-45 |
| 退火 + 延伸ᵃ | 60 °C | 40 s（收集荧光） | 40-45 |
a. 反应条件根据引物探针和模板情况可自行设置。
实验用例
本实验以结直肠癌相关基因 SDC2 基因和 TFPI2 基因作为检 测的靶基因，ACTB 作为内参基因为例检测结果：(模板浓度为 10ng/μL)
This product uses the engineered hot-start Epitech Taq DNA Polymerase, which has enhanced binding ability to DNA templates and significantly improved anti-interference capability. The enzyme retains the 5’→3’ exonuclease activity of Taq enzyme and can be used for probe-based fluorescent quantitative PCR detection. Meanwhile, Taq monoclonal antibody is added to modify the original enzyme, enhancing the blocking effect on the active site of Taq enzyme and improving specificity. Before high-temperature heating, the Taq monoclonal antibody binds to Taq polymerase to inhibit its activity, thereby suppressing non-specific amplification caused by non-specific annealing of primers or primer dimers under low-temperature conditions. When the amplification reaction system is heated to 95°C, the polymerase activity is restored due to the denaturation of Taq monoclonal antibody, so no special inactivation treatment is required, and it can be used under conventional PCR reaction conditions.
With an optimized buffer system, Epitech Taq DNA Polymerase is suitable for PCR reactions of DNA samples treated with bisulfite conversion, and has better amplification efficiency and sensitivity for template DNA containing uracil after bisulfite conversion. Compared with ordinary DNA, DNA after bisulfite conversion is severely damaged, which will affect the performance of PCR reactions. Similarly, for amplifying damaged DNA samples, cytosine deamination occurs spontaneously over a long period of time, and the deamination rate accelerates when the temperature rises, leading to the accumulation of uracil in DNA and free nucleotides. When other correction enzymes are ineffective, Epitech Taq DNA Polymerase can efficiently amplify such damaged DNA templates containing uracil.
Component List
FP1508958 | Component | 250U | 1KU | 5KU | Storage |
FP1508958A | Epitech Taq DNA Polymerase (5U/μL)
| 50μl | 200μl | 1ml | -20℃. Avoid freeze/thaw cycle. |
FP1508958B | 5×Epitech Taq DNA Polymerase Buffer
| 1ml | 4ml | 20ml | -20℃. Avoid freeze/thaw cycle |
Scope of Application
Suitable for PCR reactions of DNA samples treated with bisulfite conversion.
Precautions
In this experiment, the pretreatment process of bisulfite will directly affect the quality of template DNA; the treated template cannot be stored for a long time and should not be kept at -20°C for more than one month.
Other Precautions
1.Excessive template DNA is prone to cause non-specific PCR products.
2.Recommendations for the use of templates: Take mammalian and E. coli genomic DNA as examples:
| Reagent Name | Addition Amount |
|---|
| Mammalian Genomic DNA | 0.1-1 μg |
| E. coli Genomic DNA | 10-100 ng |
Usage Method
Preparation before reaction system preparation:
1. Thaw and mix all solutions required for the reaction at room temperature or 4°C. Then place them on an ice bath or in an ice box. It is recommended to aliquot the reaction solutions for use to avoid repeated freeze-thaw cycles.
2. Prepare the reaction system with reference to the following table. It is recommended to prepare the reaction system on an ice bath or in an ice box.
|
Component
| Addition Volume per Reaction
| Final Concentration
|
|---|
5×Epitech Taq DNA Polymerase Buffer
| 10 μL | 1× |
Epitech Taq DNA Polymerase
| 0.5 μL | 0.05 U/μL |
| dNTPs | 0.5 μL | 0.25 mM |
Primer-probe Mixᵃ
| X μL | —— |
Template
| X μL | —— |
ddH₂O
| To 50 μL | —— |
Final Volume
| 50 μL |
|
Note: a. The amounts of primers, probes, and templates can be adjusted according to actual needs.
3. Gently pipette to mix or vortex slightly, then centrifuge at room temperature for a few seconds to collect the liquid at the bottom of the tube.
4. Place each prepared PCR reaction tube in a PCR instrument and start the PCR reaction.
Reaction Procedure
Fluorescent Quantitative PCR
Step Name
| Temperature
| Time
| Cycles
|
|---|
Pre-denaturation
| 95 °C | 10 min | 1 |
Denaturationᵃ
| 94 °C | 15 s | 40-45 |
Annealing + Extensionᵃ
| 60 °C | 40 s (Collect Fluorescence)
| 40-45 |
a. Reaction conditions can be adjusted according to the primers, probes, and templates.
Experimental Example
In this experiment, the colorectal cancer-related genes SDC2 and TFPI2 were used as target genes for detection, and ACTB was used as an internal reference gene. The detection results are as follows (template concentration: 10 ng/μL):
ZJ26F0535446
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