Single restriction enzyme-assisted megaprimer polymerase chain reaction to fuse two DNA sequences on separate cloning vectors |
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Authors: | Umeki Nobuhisa Uyeda Taro Q P |
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Institution: | aBiomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Higashi, Tsukuba, Ibaraki 305-8562, Japan;bBiomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology, Aomi, Koto, Tokyo 135-0064, Japan |
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Abstract: | We describe a simple and versatile method to fuse two DNA sequences on separate cloning vectors in a single polymerase chain reaction (PCR). The method, termed restriction enzyme-assisted megaprimer PCR (REM–PCR), requires that the two cloning vectors share a common sequence and that the DNA sequences to be fused are cloned in the same orientation with respect to the common sequence. Fusion of the two sequences is achieved by mutual priming at the common sequence between two DNA fragments that were generated by restriction enzyme and linearly amplified by repetitive priming in the PCR reaction mixture. |
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