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Cyclic Digestion and Ligation-Mediated PCR Used for Flanking Sequence Walking

文献类型： 外文期刊

作者： Yu, Dong 1 ; Zhou, Tianshun 2 ; Sun, Xuewu 2 ; Sun, Zhizhong 2 ; Sheng, Xiabing 1 ; Tan, Yanning 2 ; Liu, Ling 2 ; Ouyang 1 ;

作者机构： 1.Hunan Agr Univ, Coll Biosci & Biotechnol, 1 Nongda Rd, Changsha 410128, Peoples R China

2.Hunan Hybrid Rice Res Ctr, State Key Lab Hybrid Rice, 736 Yuanda Rd, Changsha 410125, Peoples R China

3.Hunan Agr Univ, Coll Agr, 1 Nongda Rd, Changsha 410128, Peoples R China

4.Hunan Univ, Grad Sch, Long Ping Branch, 892 Yuanda Rd, Changsha 410125, Peoples R China

期刊名称：SCIENTIFIC REPORTS （ 影响因子：4.379； 五年影响因子：5.133 ）

ISSN： 2045-2322

年卷期： 2020 年 10 卷 1 期

页码：

收录情况： SCI

摘要： Ligation-mediated PCR (LM-PCR) is a classical method for isolating flanking sequences; however, it has a common limitation of reduced success rate owing to the circularization or multimerization of target restriction fragments including the known sequence. To address this limitation, we developed a novel LM-PCR method, termed Cyclic Digestion and Ligation-Mediated PCR (CDL-PCR). The novelty of this approach involves the design of new adapters that cannot be digested after being ligated with the restriction fragment, and cyclic digestion and ligation may be manipulated to block the circularization or multimerization of the target restriction fragments. Moreover, to improve the generality and flexibility of CDL-PCR, an adapter precursor sequence was designed, which could be digested to prepare 12 different adapters at low cost. Using this method, the flanking sequences of T-DNA insertions were obtained from transgenic rice and Arabidopsis thaliana. The experimental results demonstrated that CDL-PCR is an efficient and flexible method for identifying the flanking sequences in transgenic rice and Arabidopsis thaliana.

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