Nelson Matthew D, Fitch David H A
Department of Biology, New York University, New York, NY, USA.
Methods Mol Biol. 2011;772:459-70. doi: 10.1007/978-1-61779-228-1_27.
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.
将基因或调控元件组合以构建杂交基因是整个生物科学领域广泛使用的方法。在此，我们描述了一种用于杂交基因构建的优化方法，称为重叠延伸PCR。在这种方法中，聚合酶链反应（PCR）被用于高效且可靠地构建杂交基因。基于PCR的方法不依赖于可用的限制性酶切位点或其他特定序列，这是相对于更传统的克隆或重组工程方法的一个优势。通过使用高保真DNA聚合酶，该方法可用于构建甚至非常大的构建体（>20 kb），同时产生最少的不必要突变。最后，重叠延伸PCR可作为一种定点诱变手段，将所需突变引入最终的杂交基因。
