Improved cloning efficiency of polymerase chain reaction (PCR) products after proteinase K digestion
Improved cloning efficiency of polymerase chain reaction (PCR) products after proteinase K digestion
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提高蛋白酶 K 消化后聚合酶链式反应 (PCR) 产物的克隆效率
DOI：
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发表时间：
1991
期刊：
影响因子：
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通讯作者：
D. Gewert
中科院分区：
文献类型：
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作者：
J. Crowe;H. Cooper;M. A. Smith;M. Sims;D. Parker;D. Gewert
我们和其他人在克隆PCR衍生的DNA片段时偶尔会遇到困难(1)。即使在用苯酚和苯酚：氯仿提取DNA，然后用乙醇沉淀和/或柱纯化条带之后，也会发生这种情况。为了验证Taq聚合酶仍然与DNA结合从而抑制限制性内切酶活性的假设，我们在内切酶消化之前加入了蛋白酶K消化步骤，并比较了PCR产物的克隆频率。我们在这里报告了一个例子，在加入蛋白酶K消化步骤后，我们获得了更高的克隆效率。用人免疫球蛋白K链cDNA模板进行聚合酶链式反应。反应在可编程加热块(Hybaid)中进行，使用25轮温度循环(94℃1分钟，50℃2分钟和72℃3分钟)，然后在72℃下进行最后10分钟的步骤。按照制造商的建议，在100年的最终体积中使用800毫微克的每个引物、100 ng的模板和2.5个单位的Taq聚合酶(Perkin Elmer CETUS)。人工合成的寡核苷酸在7500DNA合成仪(Milligen)上被设计用于扩增K链c DNA的355bp恒定区。该引物寡核苷酸是非磷酸化的，并且在3‘端与模板具有20个核苷酸的互补性，在此具有HindU1位点5’和HindU1位点的4碱基延伸5‘。反应完成后，将扩增产物等分成2份。非蛋白酶K对照用苯酚：氯仿和氯仿提取，然后用乙醇沉淀。蛋白水解酶K组分还经过苯酚：氯仿和氯仿提取，然后调整为5 mM EDTA，10 mM Tris(pH 8.0)，0.5%十二烷基硫酸钠。加入终浓度为50个/g/ml的蛋白酶K，37℃孵育30分钟，68℃孵育10分钟。反应混合物用苯酚：氯仿提取一次，然后用氯仿提取一次，然后乙醇沉淀。非蛋白酶K和蛋白酶K反应随后被同等处理，用HindUl消化，并在3%NuSieve琼脂糖凝胶中进行电泳。从凝胶中取出PCR产物条带，在68℃下融化10分钟，然后连接到去磷酸化的Mndm Cut PUC 18(Pharmacia)。此外，还建立了仅与载体连接的对照。用等量的每一次连接转化感受态DH5大肠杆菌。
We and others have occasionally experienced difficulty in cloning PCR derived DNA fragments (1). This has occurred even after extraction of the DNA with phenol and phenol:chloroform, followed by precipitation with ethanol and/or column purification of the bands. To test the hypothesis that the Taq polymerase remains bound to the DNA and therefore inhibits restriction endonuclease activity, we incorporated a proteinase K digestion step prior to endonuclease digestion and compared the frequency of cloning of the PCR derived products. We report here an example of the increased cloning efficiency we have achieved after the incorporation of the proteinase K digestion step. PCR reactions were carried out using a human immunoglobulin K chain cDNA template. The reactions were performed in a programmable heating block (Hybaid) using 25 rounds of temperature cycling (94°C for 1 min, 50°C for 2 min and 72°C for 3 min) followed by a final 10 min step at 72°C. Eight hundred nanograms of each primer, 100 ng of template and 2.5 Units of Taq polymerase (Perkin Elmer Cetus) were used in a final volume of 100 y\ with the reaction buffer as recommended by the manufacturer. Synthetic oligonucleotides were made on a 7500 DNA Synthesiser (Milligen), and were designed to amplify the 355 bp constant region of the K chain cDNA. The primer oligonucleotides were unphosphorylated and had a complementarity of 20 nucleotides with the template at the 3' end, a HindUl site 5' to this and a 4 base extension 5' of the HindUl site. On completion of the reaction the PCR product was divided into 2 equal parts. The non-proteinase K control was subjected to phenol:chloroform and chloroform extraction follwed by ethanol precipitation. The proteinase K fraction also received phenol:chloroform and chloroform extractions and was subsequently adjusted to 5 mM EDTA, 10 mM Tris (pH 8.0), 0.5% SDS. Proteinase K was added to a final concentration of 50 /ig/ml and incubated for 30 minutes at 37°C and then 68°C for 10 minutes. The reaction mix was then extracted once with phenol:chloroform and once with chloroform before ethanol precipitation. The non-proteinase K and proteinase K reactions were subsequently treated identically and were digested with HindUl and subjected to electrophoresis in 3 % NuSieve agarose. The PCR product bands were excised from the gel and melted at 68°C for 10 minutes befure ligation to dephosphorylated Mndm cut pUC 18 (Pharmacia). A control ligation with vector only was also set up. An equal volume of each ligation was used to transform competent DH5 E. coli.