Tian Bingkun, Wu Hongjing, Wang Rongrong, Chen Hong, Li Haixing
State Key Laboratory of Food Science and Resource, Nanchang University, Nanchang, 330047, China.
International Institute of Food Innovation Co., Ltd., Nanchang University, Nanchang, 330020, China.
Biochem Genet. 2024 Jul 30. doi: 10.1007/s10528-024-10896-1.
As a tool for acquiring uncharacterized genomic DNA adjacent to characterized DNA, genome-walking is integral to bioscience-related research works. Herein, a new genome-walking tool known as N-ended walker PCR (polymerase chain reaction) is presented. The key aspect for this method is the use of a degenerate walker primer in secondary/tertiary PCR. The 7 nt 5' tail of this primer completely degenerates to N relative to its corresponding primary walker primer. The degeneracy reduces the efficiency of annealing this primer to its predecessor site. Clearly, primary nontarget DNA defined by the primary walker primer prefers to form a hairpin structure via the inverted ends rather than hybridizing with the degenerate primer. As a result, N-ended walker PCR achieves genome-walking by selectively boosting the DNA of interest. The feasibility of the N-ended walker PCR method was proven by acquiring uncharacterized DNAs flanking several characterized DNAs.
作为一种获取与已知DNA相邻的未知基因组DNA的工具，基因组步移对于生物科学相关研究工作不可或缺。本文介绍了一种新的基因组步移工具，称为N端步移PCR（聚合酶链反应）。该方法的关键在于在二次/三次PCR中使用简并步移引物。相对于其相应的一次步移引物，该引物的7个核苷酸5'端完全退化为N。这种简并性降低了该引物与其前体位点退火的效率。显然，由一次步移引物定义的主要非靶DNA更倾向于通过反向末端形成发夹结构，而不是与简并引物杂交。因此，N端步移PCR通过选择性地扩增目标DNA实现基因组步移。通过获取几个已知DNA侧翼的未知DNA，证明了N端步移PCR方法的可行性。
