Overlap Extension PCR: An Efficient Method for Transgene Construction
Overlap Extension PCR: An Efficient Method for Transgene Construction
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DOI：
10.1007/978-1-61779-228-1_27
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发表时间：
2011-01-01
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影响因子：
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通讯作者：
Fitch, David H. A.
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文献类型：
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作者：
Nelson, Matthew D.;Fitch, David H. A.
组合基因或调控元件以产生杂交基因是整个生物科学中广泛使用的方法。在这里，我们描述了一种优化的方法称为重叠延伸PCR杂交基因的建设。在该方法中，聚合酶链反应（PCR）用于高效和可靠的杂交基因的构建。基于PCR的方法不依赖于可用的限制性位点或其他特定序列，这是比更常规的克隆或重组方法更大的优势。通过使用高保真DNA聚合酶，该方法可以用于制备具有最小不需要突变的甚至非常大的构建体（>20 kb）。最后，重叠延伸PCR可以用作定点诱变的手段，将所需突变引入最终的杂交基因。
Combining genes or regulatory elements to make hybrid genes is a widely used methodology throughout the biological sciences. Here, we describe an optimized approach for hybrid gene construction called overlap extension PCR. In this method, the polymerase chain reaction (PCR) is employed for efficient and reliable construction of hybrid genes. A PCR-based approach does not rely on available restriction sites or other specific sequences, an advantage over more conventional cloning or recombineering methods. With the use of high-fidelity DNA polymerase, this method can be used for making even very large constructs (>20 kb) with minimal unwanted mutations Finally, overlap extension-PCR can be used as a means for site-directed mutagenesis, introducing desired mutations to the final hybrid gene.