Gene fragment swapping and site-directed mutagenesis are commonly required in dissecting functions of gene domains. While there are many approaches for seamless fusion of different gene fragments, new methods are yet to be developed to offer higher efficiency, better simplicity, and more affordability. In this study, we showed that in most cases overlap-PCR was highly effective in creating site-directed mutagenesis, gene fragment deletion, and substitutions using RUS1 and RUS2 as example. While for cases where the overlap-PCR approach is not feasible due to complex secondary structure of gene fragments, a unique restriction site can be generated at the overlapped region of the primers through synonymous mutations. Then different gene fragments can be seamlessly fused through traditional restriction digestion and subsequent ligation. In conclusion, while the classical overlap-PCR is not feasible, the modified overlap-PCR approaches can provide effective and alternative ways to seamlessly fuse different gene fragments.

在剖析基因域的功能时，通常需要基因片段交换和定点诱变。虽然有许多方法可以无缝融合不同的基因片段，但仍有待开发新方法以提供更高的效率、更好的简单性和更实惠的价格。在这项研究中，我们表明在大多数情况下，重叠 PCR 在使用RUS1和RUS2创建定点诱变、基因片段删除和替换方面非常有效作为例子。而对于由于基因片段二级结构复杂而导致重叠PCR方法不可行的情况，可以通过同义突变在引物的重叠区域产生独特的限制性位点。然后不同的基因片段可以通过传统的限制性消化和随后的连接进行无缝融合。总之，虽然经典的重叠 PCR 不可行，但改进的重叠 PCR 方法可以提供有效和替代的方法来无缝融合不同的基因片段。