Generation of cohesive ends on PCR products by UDG-mediated excision of dU, and application for cloning into restriction digest-linearized vectors.
Generation of cohesive ends on PCR products by UDG-mediated excision of dU, and application for cloning into restriction digest-linearized vectors.
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DOI：
10.1101/gr.2.4.328
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发表时间：
1993-05-01
期刊：
影响因子：
--
通讯作者：
Walker, M R
中科院分区：
文献类型：
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作者：
Smith, C;Day, P J;Walker, M R
我们已经研究了使用尿嘧啶N-糖基化酶（UDG）的dU切除来在PCR片段上产生粘性末端，以“模仿”限制性内切酶产生的那些。通过将牛碱性成纤维细胞生长因子的383 - 388-bp片段亚克隆到限制性酶线性化的pT 7 T318 U中，证明了使用粘性末端模仿SacI或PstI末端进行定向和非定向克隆的这种方法的可行性。UDG介导的粘性末端与PstI产生的载体末端不完全匹配，得到合理的克隆效率和准确性，表明该方法可以扩展到模拟产生3'突出端的其他限制性酶。通过UDG快速和特异性切除dU（在37 ° C下30分钟内）与使用限制性位点修饰的引物相比具有几个潜在的优点，包括避免在PCR产物内的内部位点处的限制性切割。此外，在连接之后，所述方法可用于防止连接的DNA区段随后被限制性酶切割，即，通过不在连接处重新产生限制性酶识别序列，这可在基因工程中找到应用。通过调整该方法以使用含dU的接头或“载体”，该方法可用于克隆未知序列（例如，通过cDNA或基因组文库构建）或用于模拟PCR片段上的5'突出端粘性末端。
We have investigated the use of dU excision by uracil N-glycosylase (UDG) to create cohesive ends on PCR fragments "mimicking" those generated by restriction enzymes. The feasibility of this approach for directional and nondirectional cloning using cohesive ends mimicking SacI or PstI ends is demonstrated by the subcloning of a 383 to 388-bp fragment of bovine basic fibroblast growth factor into restriction enzyme-linearized pT7T318U. UDG-mediated cohesive ends imperfectly matched to PstI-generated vector ends gave reasonable cloning efficiency and accuracy, suggesting that the approach may be extended to mimicry of other restriction enzymes producing 3' overhangs. The rapid and specific excision of dU by UDG (within 30 min at 37 degrees C) has several potential advantages over the use of restriction site-modified primers, including the avoidance of restriction cleavage at internal sites within the PCR product. Also, following ligation, the approach described may be used to prevent subsequent cleavage of the joined DNA segments by the restriction enzyme, that is, by not recreating the restriction enzyme recognition sequence at the junction, which may find application in gene engineering. By adapting the approach to use dU-containing linkers or "vectorettes," the approach may be used for cloning unknown sequences (e.g., by cDNA or genomic library construction) or for mimicking 5' overhang cohesive ends on PCR fragments.