Gene cloning and seamless site-directed mutagenesis using single-strand annealing (SSA)
Gene cloning and seamless site-directed mutagenesis using single-strand annealing (SSA)
复制标题
使用单链退火 (SSA) 进行基因克隆和无缝定点诱变。
DOI：
10.1007/s00253-018-9348-z
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发表时间：
2018-12-01
影响因子：
5
通讯作者：
Wang, Xin
中科院分区：
文献类型：
--
作者：
Luo, Zhixin;Wang, Shanhe;Wang, Xin
通常通过重叠PCR组装外显子或RACE产物来克隆目的基因的全长。然而，该方法需要多个PCR步骤，这易于发生随机诱变。在这里，我们提出了一种新的基于SSA的基因克隆和无缝定点突变的方法。本研究首次克隆了绒山羊（Capra hircus）Hoxc13基因的全长编码序列。其次，我们成功地构建了Hoxc13基因的缺失突变体，并进一步证明了25bp的直接重复序列长度足以在常规大肠杆菌中引发SSA修复。大肠杆菌菌株包括DH5、Trans1t1、JM 109和Top10。此外，我们还进一步缩短了19bp的正向重复序列，从绒山羊cDNA中克隆了Foxn1基因的另一个全长突变体。总之，我们的研究提供了一种替代方法，以克服重叠PCR在某些特定情况下的基因克隆的困难。
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 25bp is enough to trigger the SSA repair in routine E. coli strains including DH5, Trans1t1, JM109, and Top10. Moreover, we cloned another full-length mutant of Foxn1 gene from Cashmere goat cDNA using further shortened direct repeats of 19bp. In summary, our study provided an alternative method to overcome the difficulties during overlap PCR in some particular cases for gene cloning.