本产品来源于极端耐热菌 Pyrococcus abyssi (P.abyssi)，经重组表达纯化后获得。 热稳定核糖核酸酶HII 是一种核酸内切酶，识别并切割 RNA/DNA 杂合链中的 RNA 链，不切割 sSDNA、dsDNA，对单链 RNA 切割活性极低。 热稳定核糖核酸酶HII 在单核糖核苷酸残基处从 5'端进行切割，切割后产生一个 5'端磷酸基团和一个 3'端羟基。该热稳定核糖核酸酶HII 在 60~80℃具有最佳活性，在37°~95℃间均具有活性，在室温下活性低。本品极度耐热，95℃温育 15 min 后，几乎没有活性损失，可与 PCR 各反应体系兼容。
组分表
| T1522666 | 组件 | 物理外观 | 200U | 储存 | | T1522666A | 热稳定核糖核酸酶HII (2 U/μl) | Liquid | 100μl | -20℃. Avoid freeze/thaw cycle. | | T1522666B | HII 稀释缓冲液 | Liquid | 2×1ml | -20℃. Avoid freeze/thaw cycle. | | T1522666C | 10× RHII 缓冲液 | Liquid | 1ml | -20℃. Avoid freeze/thaw cycle. |
|
活性定义
一个单位（U）是指在 60℃下，30 min 切割 100 pmol 含有单个核糖核苷酸的嵌合 DNA/RNA 杂交双链体底物所需的酶量。
建议反应条件
1× RHII 缓冲液； 60℃温育。
失活条件
终浓度 1%SDS，95℃ 15 min。
产品应用
1. RNase HII 依赖性 PCR (rhPCR)。
2. 减少或去除 PCR 反应中的引物二聚体。
3. 提高多重 PCR 产物的准确性。
4. 区分异种同源基因。
5. SNP 检测和稀有等位基因检测。
6. 降解冈崎片段的 RNA 部分。
7. LAMP 高灵敏度探针法检测。
8. 与 T7 Endo I 一起使用时，在掺入的核糖核苷酸位点产生双链断裂。
质量控制
蛋白纯度
经 SDS-PAGE 凝胶电泳检测，蛋白纯度不低于 95%。
非特异性内切酶活性
37℃下，在 20 μl 反应体系中将 2 U 热稳定核糖核酸酶HII 与 200 ng 超螺旋质粒 DNA 共同温育 4 h，使用琼脂糖凝胶电泳检测， 少于 20% 的质粒 DNA 转变成缺刻或线性状态。
DNase 活性
37℃下，在 20 μl 反应体系中将 2 U 热稳定核糖核酸酶HII 与 15 ng 双链 DNA 片段共同温育 16 h，使用琼脂糖凝胶电泳检测，双链 DNA 片段无变化。
RNase 活性
37℃下，在 10 μl 反应体系中将 2 U 热稳定核糖核酸酶HII 与 500 ng RNA 共同温育 1 h，使用琼脂糖凝胶电泳检测，超过 90% 的 RNA 保持完整。
宿主 DNA 残留
采用中国药典 2020 版四部通则 3407 外源性 DNA 残留量测定 法第三法定量 PCR 法，本品中大肠杆菌宿主细胞 DNA 残留量低于 1 拷贝 /2 U。
使用方法
1. 以 rhPCR 流程为例：
（1）以配合本公司的 Taq-Plus PCR Master Mix (2×)（货号：T295072）使用为例：
| 试剂 | 使用量 |
|---|
| 上游引物（10 μM） | 1 μl | | 下游引物（10 μM） | 1 μl | | 模板 DNA | x μl | | Taq-Plus PCR Master Mix (2×) | 25 μl | 热稳定核糖核酸酶HII
| 20~200 mU | | ddH₂O | 补至 50 μl |
|
（2）轻柔吸打或轻弹管壁以混匀（切勿涡旋），然后瞬时离心以收 集挂壁液滴；
（3）设置 PCR 反应
2. rhPCR 引物设计
注意事项
1. 该酶用于 rhPCR 时，需要使用 3' 端带封闭基团的 PCR 引物。
2. 该酶在 37~80℃均有活性，其用量可根据不同的应用进行调节。
3. 该酶可兼容大多数 PCR 体系，同时对 Mg2+ 浓度无严格要求，在 1~6 mM 范围内均可正常发挥活性。
This product is derived from the extreme thermophilic archaeon Pyrococcus abyssi (P. abyssi) and obtained after recombinant expression and purification.Thermostable RNase HII is an endoribonuclease that recognizes and cleaves the RNA strand in RNA/DNA hybrids, but does not cleave ssDNA or dsDNA, and has very low activity against single-stranded RNA.Thermostable RNase HII cleaves at the 5' side of single ribonucleotide residues, generating a 5'-phosphate group and a 3'-hydroxyl group after cleavage.This Thermostable RNase HII exhibits optimal activity at 60–80 °C, retains activity between 37–95 °C, and shows low activity at room temperature.It is extremely thermostable: after incubation at 95 °C for 15 minutes, there is almost no loss of activity, making it fully compatible with various PCR reaction systems.
Components List| T1522666 | Components
| Appearance
| 200U | Storage
|
| T1522666A | Thermostable RNase HII (2 U/μl)
| Liquid | 100μl | -20℃. Avoid freeze/thaw cycle. |
| T1522666B | HII Dilution Buffer
| Liquid | 2×1ml | -20℃. Avoid freeze/thaw cycle. |
| T1522666C | 10× RHII Buffer
| Liquid | 1ml | -20℃. Avoid freeze/thaw cycle. |
Unit Definition
One unit (U) is defined as the amount of enzyme required to cleave 100 pmol of chimeric DNA/RNA hybrid duplex containing a single ribonucleotide in 30 minutes at 60 °C.
Recommended Reaction Conditions
1× RHII Buffer; incubation at 60 °C.
Inactivation Conditions
1% (final concentration) SDS, incubation at 95 °C for 15 minutes.
Applications
RNase HII-dependent PCR (rhPCR).
Reducing or eliminating primer-dimer formation in PCR reactions.
Improving the accuracy of multiplex PCR products.
Distinguishing paralogous homologous genes.
SNP detection and rare allele detection.
Degrading the RNA portion of Okazaki fragments.
LAMP high-sensitivity probe-based detection.
Generating double-strand breaks at incorporated ribonucleotide sites when used with T7 Endonuclease I.
Quality Control
Protein Purity
SDS-PAGE analysis confirms protein purity of no less than 95%.
Non-specific Endonuclease Activity
2 U of Thermostable RNase HII was incubated with 200 ng supercoiled plasmid DNA in a 20 μL reaction system at 37 °C for 4 hours. Agarose gel electrophoresis showed less than 20% of the plasmid DNA was converted to nicked or linear form.
DNase Activity
2 U of Thermostable RNase HII was incubated with 15 ng double-stranded DNA fragments in a 20 μL reaction system at 37 °C for 16 hours. Agarose gel electrophoresis showed no degradation of the dsDNA fragments.
RNase Activity
2 U of Thermostable RNase HII was incubated with 500 ng RNA in a 10 μL reaction system at 37 °C for 1 hour. Agarose gel electrophoresis showed more than 90% of the RNA remained intact.
Host DNA Residue
The residual host DNA level in this product is less than 1 copy per 2 U, as determined by quantitative PCR (Method III) according to the Chinese Pharmacopoeia 2020 Edition Part IV General Rule 3407.
Usage Protocol
- Example protocol for rhPCR:(1) Reaction system using the company's Taq-Plus PCR Master Mix (2×) (Cat. No.: T295072):
Reagent
| Volume
|
|---|
Forward Primer (10 μM)
| 1 μl | Reverse Primer (10 μM)
| 1 μl | Template DNA
| x μl | | Taq-Plus PCR Master Mix (2×) | 25 μl | Thermostable RNase HII
| 20~200 mU | | ddH₂O | Up to 50 μl
|
|
（2）Mix gently by pipetting up and down or flicking the tube (do not vortex), then briefly centrifuge to collect all liquid droplets on the tube wall.
（3）Set up the PCR reaction.
2. rhPCR Primer Design
Precautions
- When using this enzyme for rhPCR, PCR primers with a 3'-blocking group are required.
- This enzyme is active at 37–80 °C, and its dosage can be adjusted according to different applications.
- This enzyme is compatible with most PCR systems and has no strict requirements on Mg²⁺ concentration; it can function normally within the range of 1–6 mM.
ZJ26F0636732
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