Bsu DNA Polymerase (Large Fragment) 来源于嗜热脂肪芽孢杆 菌 (Bacillus subtilis )，系将 Bacillus subtilis DNA 聚合酶 I 的 N 端 296 个氨基酸截去而得。该酶保留了 Bacillus subtilis DNA 聚合酶 I 的 5'→3' 聚合酶活性，但不具有 5'→3' 核酸外切酶和 3'→5' 核酸外切酶活性。该酶最适反应温度为 37℃，具有很强的链置换活性，适用于 DNA 等温扩增或 cDNA 二链合成。
质量控制
1. 蛋白纯度检测
使用 SDS-PAGE 凝胶电泳检测，蛋白纯度不低于 95%。
2. 核酸内切酶活性检测
将 5 μg Bsu DNA Polymerase 与 200 ng 超螺旋质粒 DNA 在37℃下，共同温育 4 h 后，使用琼脂糖凝胶电泳检测，少于 10% 的质粒 DNA 转变成缺刻或线性状态。
3. 非特异性核酸酶活性检测
将 5 μg Bsu DNA Polymerase 与 15 ng 双链 DNA 片段在 37 ℃温育 16 h，使用琼脂糖凝胶电泳检测双链 DNA 底物无变化。
4. RNase 活性检测
将 5 μg Bsu DNA Polymerase 与 500 ng 总 RNA 在 37 ℃温育1 h，使用琼脂糖凝胶电泳检测超过 90% 的 RNA 仍保持完整。
5. 宿主 DNA 残留检测
使用大肠杆菌 16S rDNA 特异性引物探针组， 采用荧光定量 PCR法检测 5 μg Bsu DNA Polymerase，大肠杆菌宿主基因组 DNA 残留低于 10 copies。
失活条件
75℃温浴 20 min 即可失活。
主意事项
1. 由于缺乏 3'→5' 核酸外切酶活性，Bsu DNA Polymerase (Large fragment) 不能切除 3' 未配对的突出末端，因而不适用 于生成平齐末 端；
2. Bsu DNA Polymerase (Large fragment)，在 25℃条件下具有 50% 活性，是 Klenow Fragment (3'→5' exo- ) 的两倍。
Bsu DNA Polymerase (Large Fragment) is derived from Bacillus subtilis. It is obtained by truncating the N-terminal 296 amino acids of Bacillus subtilis DNA Polymerase I. This enzyme retains the 5'→3' polymerase activity of Bacillus subtilis DNA Polymerase I, while lacking both 5'→3' and 3'→5' exonuclease activities. It exhibits an optimal reaction temperature of 37 °C and possesses strong strand displacement activity, making it suitable for DNA isothermal amplification and second-strand cDNA synthesis.
Quality Control
1. Protein Purity
Determined by SDS-PAGE; purity ≥ 95%.
2. Endonuclease Activity Assay
5 μg Bsu DNA Polymerase was incubated with 200 ng supercoiled plasmid DNA at 37 °C for 4 h. Agarose gel electrophoresis showed less than 10% of plasmid DNA was converted into nicked or linear form.
3. Non-specific Nuclease Activity Assay
5 μg Bsu DNA Polymerase was incubated with 15 ng double-stranded DNA fragment at 37 °C for 16 h. No alteration of dsDNA substrate was observed by agarose gel electrophoresis.
4. RNase Activity Assay
5 μg Bsu DNA Polymerase was incubated with 500 ng total RNA at 37 °C for 1 h. Agarose gel electrophoresis confirmed over 90% of RNA remained intact.
5. Host Genomic DNA Residue
Quantitative real-time PCR with E. coli 16S rDNA-specific primers and probes was used to detect 5 μg Bsu DNA Polymerase; residual E. coli genomic DNA was less than 10 copies.
Inactivation Condition
Incubation at 75 °C for 20 min completely inactivates the protein.
Precautions：
- Due to the absence of 3'→5' exonuclease activity, Bsu DNA Polymerase (Large Fragment) cannot remove unpaired 3' overhangs, and thus is not suitable for blunt-end generation.
- Bsu DNA Polymerase (Large Fragment) retains 50% of its activity at 25 °C, which is twice that of Klenow Fragment (3'→5' exo⁻).
ZJ26F0736962
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