Trinh Quoclinh, Zhu Pengyu, Shi Hui, Xu Wentao, Hao Junran, Luo Yunbo, Huang Kunlun
Laboratory of Food Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
Laboratory of Food Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
Anal Biochem. 2014 Dec 1;466:24-6. doi: 10.1016/j.ab.2014.07.022. Epub 2014 Jul 30.
The polymerase chain reaction (PCR)-based genome walking method has been extensively used to isolate unknown flanking sequences, whereas nonspecific products are always inevitable. To resolve these problems, we developed a new strategy to isolate the unknown flanking sequences by combining A-T linker adapter PCR with inverse PCR (I-PCR) or thermal asymmetric interlaced PCR (TAIL-PCR). The result showed that this method can be efficiently achieved with the flanking sequence from the Arabidopsis mutant and papain gene. Our study provides researchers with an additional method for determining genomic DNA flanking sequences to identify the target band from bulk of bands and to eliminate the cloning step for sequencing.
基于聚合酶链反应（PCR）的基因组步移方法已被广泛用于分离未知的侧翼序列，然而非特异性产物总是不可避免的。为了解决这些问题，我们开发了一种新策略，通过将A-T接头衔接子PCR与反向PCR（I-PCR）或热不对称交错PCR（TAIL-PCR）相结合来分离未知的侧翼序列。结果表明，该方法可以有效地从拟南芥突变体和木瓜蛋白酶基因的侧翼序列中实现。我们的研究为研究人员提供了另一种确定基因组DNA侧翼序列的方法，以便从大量条带中识别目标条带并省去测序的克隆步骤。
