College of Animal Science and Technology, Northwest A&F University, Yangling, 712100, Shaanxi, China.
Appl Microbiol Biotechnol. 2018 Dec;102(23):10119-10126. doi: 10.1007/s00253-018-9348-z. Epub 2018 Sep 12.
The full length of interested genes can be usually cloned by assembling exons or RACE products through overlap PCR. However, the procedure requires multiple PCR steps, which are prone to random mutagenesis. Here, we present a novel SSA-based method for gene cloning and seamless site-directed mutagenesis. We firstly cloned the full-length coding sequence of Cashmere goat (Capra hircus) Hoxc13 gene by assembling exons amplified from genomic DNA. Secondly, we created a Hoxc13 loss-function mutant seamlessly and further illustrated that direct repeat length of 25 bp is enough to trigger the SSA repair in routine E. coli strains including DH5α, Trans1t1, JM109, and Top10. Moreover, we cloned another full-length mutant of Foxn1 gene from Cashmere goat cDNA using further shortened direct repeats of 19 bp. In summary, our study provided an alternative method to overcome the difficulties during overlap PCR in some particular cases for gene cloning.
通过重叠 PCR 将外显子或 RACE 产物组装起来，通常可以克隆感兴趣基因的全长。然而，该过程需要多个 PCR 步骤，容易发生随机突变。在这里，我们提出了一种基于 SSA 的新型基因克隆和无缝定点诱变方法。我们首先通过从基因组 DNA 扩增的外显子组装克隆了绒山羊（Capra hircus）Hoxc13 基因的全长编码序列。其次，我们无缝地创建了一个 Hoxc13 失活突变体，并进一步表明，25 bp 的直接重复长度足以在包括 DH5α、Trans1t1、JM109 和 Top10 在内的常规大肠杆菌菌株中触发 SSA 修复。此外，我们使用进一步缩短的 19 bp 直接重复从绒山羊 cDNA 中克隆了另一个 Foxn1 全长突变体。总之，我们的研究为基因克隆中某些特定情况下重叠 PCR 中的困难提供了一种替代方法。
