热敏感性双链脱氧核糖核酸酶，即 Thermolabile dsDNase， 是一种核酸内切酶，能够裂解 DNA 中的磷酸二酯键， 生成带有 5'- 磷酸和 3'- 羟基末端的寡核苷酸。dsDNase 能够特异性 的消化双链 DNA（double-stranded DNA, dsDNA）而不会消化单链 DNA、引物、探针和 RNA。dsDNase 具有热敏感性，可在 55℃条件 下快速失活。dsDNase 主要用于反转录实验前快速去除 RNA 样本中 的基因组 DNA 污染，与传统的使用 DNase I 去除基因组 DNA 污染 的方法相比，无需额外加入 EDTA 失活，可减少对 RNA 的损伤，同 时节省实验时间，保证 RNA 水平定量的准确性。
组分和说明
| T751064 | 组分
| 物理外观 | 50T | 5*50T | 储存 | | T751064A | Thermolabile dsDNase | Liquid | 50 μl | 5×50 μl | -20 ℃. Avoid freeze/thaw cycles.
| | T751064B | 10× dsDNase Buffer | Liquid | 200 μl | 5×200 μl | -20 ℃. Avoid freeze/thaw cycles. |
|
活性定义
根据 Kunitz 实验方法，在 25℃ pH 5.0 的条件下，以过量的大分 子 DNA 为底物，在 260 nm 波长处每分钟能引起吸光度增加 0.001 的 酶量定义为 1 个活性单位（U）。
抑制与失活
抑制条件：金属离子、EDTA、SDS、DTT、β- 巯基乙醇、高盐离子浓度等会抑制 dsDNase 的活性；
失活条件：55℃温育 5 min。
RNA 酶活性检测
将酶液与 RNA 底物 在 37℃温育 1 h，通过凝胶电泳检测没有发现 RNA 底物降解。
功能检测
该产品被用于测试去除来自 RNA 样本中的基因组 DNA 并进行了 RT-qPCR 扩增，发现去除率 ≥99.9%。RNA 数量不受 dsDNase 处理的影响。
使用方法
1. 于冰上配制如下反应体系：
| 试剂 | 使用量 |
|---|
| dsDNase | 1 μl | | 10× dsDNase Buffer | 1 μl | | 模板 RNA | x μl | | 总 RNA | 1 pg~5 μg/10 μl | | mRNA | 0.1 pg~500 ng/10 μl | | 特异性 RNA | 0.01 ng~500 ng/10 μl | | Nuclease-Free Water | To 10 μl |
|
2. 轻轻吹打混匀反应体系后，将以上混合液在 37℃温育 2~5 min；
3. 65℃热失活 2 min，迅速将获得的 RNA 置于冰上，用于后续实验。 长期保存请置于 -80℃，避免反复冻融。
注意事项
1. 若 RNA 样本下游用于 RT-PCR，且目的基因长度 ≥3 kb，失活步骤前需添加终浓度为 10 mM 的 DTT；
2. 为避免 RNA 降解，可在反应体系中加入适量的 RNase Inhibitor。
常见问题
问题描述： RT-qPCR 实验 NTC 对照有扩增
原因：
1. gDNA 污染较为严重，反应时间不足；
2. RNA 模板中存在较高浓度抑制剂影响 dsDNase 酶活。
解决方案：
1. 延长孵育时间至 5 min；
2. 使用 75% 无水乙醇洗涤 RNA 模板，并溶解于无核酸酶水中。
Thermolabile dsDNase is a nuclease endonuclease that cleaves phosphodiester bonds in DNA, generating oligonucleotides with 5'-phosphate and 3'-hydroxyl termini. dsDNase specifically digests double-stranded DNA (dsDNA) without digesting single-stranded DNA, primers, probes, or RNA. dsDNase is thermolabile and can be rapidly inactivated at 55°C. dsDNase is mainly used for rapid removal of genomic DNA contamination from RNA samples prior to reverse transcription experiments. Compared with the traditional method of removing genomic DNA contamination using DNase I, it eliminates the need for additional EDTA inactivation, reduces RNA damage, saves experimental time, and ensures the accuracy of RNA quantification.
Components and Specifications
| T751064 | Component | Appearance | 50T | 5*50T
| Storage | | T751064A | Thermolabile dsDNase | Liquid | 50 μl | 5×50 μl | -20 ℃. Avoid freeze/thaw cycles.
| | T751064B | 10× dsDNase Buffer | Liquid | 200 μl | 5×200 μl | -20 ℃. Avoid freeze/thaw cycles. |
|
Activity Definition
One unit (U) of activity is defined as the amount of enzyme that causes an increase in absorbance of 0.001 per minute at 260 nm, using excess high-molecular-weight DNA as substrate, at 25 °C and pH 5.0, according to the Kunitz method.
Inhibition and Inactivation
Inhibition conditions: Metal ions, EDTA, SDS, DTT, β-mercaptoethanol, high salt concentration, etc. will inhibit the activity of dsDNase.
Inactivation conditions: Incubate at 55°C for 5 min.
RNase Activity Assay
The enzyme was incubated with RNA substrate at 37°C for 1 hour, and no degradation of the RNA substrate was detected by gel electrophoresis.
Functional Assay
The product was tested for removal of genomic DNA contamination from RNA samples followed by RT-qPCR amplification. The efficiency of genomic DNA removal is ≥99.9%, and RNA quantity is not affected by dsDNase treatment.
Usage
1. Prepare the following reaction mixture on ice:
| Reagent | Volume |
|---|
| dsDNase | 1 μl | | 10× dsDNase Buffer | 1 μl | | Template RNA | x μl | | Total RNA | 1 pg~5 μg/10 μl | | mRNA | 0.1 pg~500 ng/10 μl | | Specific RNA | 0.01 ng~500 ng/10 μl | | Nuclease-Free Water | To 10 μl |
|
2. Mix the reaction mixture gently by pipetting, then incubate the mixture at 37°C for 2–5 min.
3. Heat-inactivate at 65°C for 2 min, then immediately place the obtained RNA on ice for subsequent experiments. For long-term storage, keep at -80°C and avoid repeated freeze-thaw cycles.
Notes
1. If the RNA sample is used for downstream RT-PCR with a target gene length ≥3 kb, add DTT to a final concentration of 10 mM before the inactivation step.
2. To avoid RNA degradation, an appropriate amount of RNase Inhibitor can be added to the reaction system.
Troubleshooting
Problem Description: Amplification observed in NTC control of RT-qPCR experiment
Cause:
1. Severe gDNA contamination with insufficient reaction time;
2. High concentration of inhibitors in RNA template affecting dsDNase activity.
Solution:
1. Extend incubation time to 5 min;
2. Wash the RNA template with 75% anhydrous ethanol and dissolve in nuclease-free water.
ZJ26F0535134
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