热敏UDG酶

产品说明： 热敏 UDG 酶可通过催化水解含有 U-DNA 位点 DNA 链的尿嘧啶糖苷键（碱基切除），释放尿嘧啶并产生碱敏感的无嘧啶基位点（AP-DNA），该酶可作用于含尿嘧啶的单链和双链 DNA。热敏 UDG 酶对双链 DNA 的活性低于单链 DNA。该酶对小的 U-DNA 寡核苷酸和 dUMP 有活性，但对 RNA 或正常的无尿嘧啶 DNA 无活性。由于热敏 UDG 酶无金属离子要求，所以该酶在 Mg2 +或 EDTA 的存在下是有活性的。然而， 甘油，Mg2 +和高离子强度可以降低该酶的活性。 来自 BMTU 3346 的热敏 UDG 酶比来自大肠杆菌的相应的酶（95℃10min）可以更快灭活（在 95℃ 2min）。 产品内容： Heat-labile Uracil-DNA Glycosylase （1U/μl） 来源：克隆有热敏UDG酶基因的BMTU 3346菌株。 活性定义： 一个单位定义为在 37℃，60min 内完全降解 1μg 纯化的单链尿嘧啶 DNA（噬菌体M13，大肠杆菌 CJ236dut-ung-中生长）所需的热敏 UDG 酶的量。 酶储存液： 20mM Tris-HCl，pH 8.0（4℃），0.1mM EDTA，100mM KCl，1mM DTT，50%甘油，0.5%Nonidet， 0.5% Tween 20。 在缺乏稳定剂的缓冲液（例如 PCR 缓冲液：10mM Tris-HCl，pH8.3, 50mM KCl，1.5mM MgCl2）中，酶在升高温度时可以被快速灭活。 应用： 1) 在已经掺入脱氧尿苷酸残基位点切割 DNA。通过碱处理，高温或内切核酸酶（如 T4 核酸内切酶） 特异性切割无嘧啶位点，可催化水解产生的 AP-DNA。 2) 含尿嘧啶的 DNA（U-DNA）可以用体外方法制备。可以使用热敏 UDG 酶实现位点特异性，链特异性或普通切割，这取决于如何制备 U-DNA。 3) 该酶可用于提高定向诱变方法的效率和用来获得高度标记的寡核苷酸探针。 4) 热敏 UDG 酶可与 dUTP 一起使用，以消除 DNA 合成反应的 PCR 污染。为了使 PCR 产物易于降解，在 PCR 反应混合物中，必须用 dUTP 替换 dTTP。在 PCR 之前，PCR 反应混合物必须用热敏 UD 酶进行预处理，以降解含尿嘧啶的 DNA。天然 DNA 不含尿嘧啶，因此样品不会因此而降解。 使用方法： 反应体系(50μl) 1) 在 15℃-25℃下孵育产物 10min。 2) 95℃加热 2min 使热敏 UDG 酶灭活。 3) 进行 PCR。 4) 短期保存 PCR 产物可于 2℃-8℃储存。长期储存，请将 PCR 产物置于-15℃至-25℃冷冻储存。 热失活：95℃加热 2min。（若用于 RT-PCR，55℃加热 10min 进行热失活。） Product description: Thermosensitive UDG enzyme can release uracil and produce alkali sensitive pyrimidine free sites (AP-DNA) by catalytic hydrolysis of uracil glycoside bonds (base excision) of DNA strands containing U-DNA sites. This enzyme can act on single and double stranded DNA containing uracil. The activity of heat sensitive UDG enzyme to double stranded DNA is lower than that to single stranded DNA. The enzyme is active to small U-DNA oligonucleotides and dUMP, but inactive to RNA or normal uracil free DNA. As the thermal UDG enzyme has no requirement for metal ions, it is active in the presence of Mg2+or EDTA. However, glycerol, Mg2+and high ionic strength can reduce the activity of this enzyme. The thermostable UDG enzyme from BMTU 3346 can be inactivated faster (at 95 ℃ for 2 min) than the corresponding enzyme from E. coli (at 95 ℃ for 10 min). Product content: Heat-labile Uracil-DNA Glycosylase （1U/ μ l） Source: BMTU 3346 strain with heat sensitive UDG enzyme gene was cloned. Activity definition: one unit is defined as complete degradation within 60 min at 37 ℃ μ G The amount of heat sensitive UDG enzyme required for purified single stranded uracil DNA (phage M13, E. coli CJ236dut ung). Enzyme storage solution: 20mM Tris HCl, pH 8.0 (4 ℃), 0.1mM EDTA, 100mM KCl, 1mM DTT, 50% glycerin, 0.5% Nonidet, 0.5% Tween 20. In the buffer without stabilizer (such as PCR buffer: 10mM Tris HCl, pH8.3, 50mM KCl, 1.5mM MgCl2), the enzyme can be rapidly inactivated at elevated temperature. Application: 1) DNA was cleaved at sites where deoxyuridine residues had been incorporated. Through alkali treatment, high temperature or endonuclease (such as T4 endonuclease) specifically cleaves pyrimidine free sites, which can catalyze the hydrolysis of AP-DNA. 2) Uracil containing DNA (U-DNA) can be prepared in vitro. Thermosensitive UDG enzymes can be used to achieve site specific, strand specific or general cleavage, depending on how U-DNA is prepared. 3) The enzyme can be used to improve the efficiency of directed mutagenesis and obtain highly labeled oligonucleotide probes. 4) Thermosensitive UDG enzyme can be used together with dUTP to eliminate PCR contamination of DNA synthesis reaction. In order to make the PCR product easy to degrade, dTTP must be replaced by dUTP in the PCR reaction mixture. Before PCR, the PCR reaction mixture must be pretreated with heat sensitive UD enzyme to degrade the DNA containing uracil. Natural DNA does not contain uracil, so the sample will not be degraded. usage method: Reaction system (50 μ l) 1) Incubate the product at 15 ℃ - 25 ℃ for 10min. 2) Heat at 95 ℃ for 2min to inactivate heat sensitive UDG enzyme. 3) Perform PCR. 4) Short term preservation of PCR products can be stored at 2 ℃ - 8 ℃. For long-term storage, please place the PCR products in - 15 ℃ to - 25 ℃ for frozen storage. Thermal deactivation: heating at 95 ℃ for 2min. (If used for RT-PCR, heat at 55 ℃ for 10min for thermal deactivation.)

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