Wu Hongjing, Pan Hao, Li Haixing
Nanchang University College of Science and Technology, Nanchang, China.
International Institute of Food Innovation Co., Ltd., Nanchang University, Nanchang, China.
Bio Protoc. 2025 Jan 20;15(2):e5161. doi: 10.21769/BioProtoc.5161.
PCR-based genome walking is one of the prevalent techniques implemented to acquire unknown flanking genomic DNAs. The worth of genome walking includes but is not limited to cloning full-length genes, mining new genes, and discovering regulatory regions of genes. Therefore, this technique has advanced molecular biology and related fields. However, the PCR amplification specificity of this technique needs to be further improved. Here, a practical protocol based on fork PCR is proposed for genome walking. This PCR uses a fork primer set of three arbitrary primers to execute walking amplification task, where the primary fork primer mediates walking by partially annealing to an unknown flank, and the fork-like structure formed between the three primers participates in inhibiting non-target amplification. In primary fork PCR, the low-annealing temperature (25 °C) cycle allows the primary fork primer to anneal to many sites of the genome, synthesizing a cluster of single-stranded DNAs; the subsequent 65 °C cycle processes the target single-strand into double-strand via the site-specific primer; then, the remaining 65 °C cycles selectively enrich this target DNA. However, any non-target single-stranded DNA formed in the 25 °C cycle cannot be further processed in the following 65 °C cycles because it lacks an exact binding site for any primer. Secondary, or even tertiary nested fork PCR further selectively enriches the target DNA. The practicability of fork PCR was validated by walking three genes in CD0817 and one gene in . The results indicated that the proposed protocol can serve as a supplement to the existing genome walking protocols. Key features • This protocol builds upon the method developed by Pan et al. [1], which is applicable to genome-walking for any species. • The developed protocol is a random priming PCR-based genome-walking scheme. • Two rounds of nested fork PCR amplifications suffice to release a positive walking result.
基于聚合酶链式反应（PCR）的基因组步移技术是获取未知侧翼基因组DNA的常用技术之一。基因组步移的价值包括但不限于克隆全长基因、挖掘新基因以及发现基因的调控区域。因此，该技术推动了分子生物学及相关领域的发展。然而，该技术的PCR扩增特异性仍需进一步提高。在此，提出了一种基于叉状PCR的实用基因组步移方案。这种PCR使用一组由三个任意引物组成的叉状引物来执行步移扩增任务，其中主叉状引物通过部分退火至未知侧翼来介导步移，三个引物之间形成的叉状结构参与抑制非靶标扩增。在初级叉状PCR中，低退火温度（25℃）循环使主叉状引物能够退火至基因组的多个位点，合成一簇单链DNA；随后的65℃循环通过位点特异性引物将靶标单链加工成双链；然后，剩余的65℃循环选择性地富集该靶标DNA。然而，在25℃循环中形成的任何非靶标单链DNA在随后的65℃循环中无法进一步加工，因为它缺乏与任何引物的精确结合位点。二级甚至三级巢式叉状PCR进一步选择性地富集靶标DNA。通过对CD0817中的三个基因和中的一个基因进行步移验证了叉状PCR的实用性。结果表明，所提出的方案可作为现有基因组步移方案的补充。关键特性•本方案基于Pan等人[1]开发的方法，适用于任何物种的基因组步移。•所开发的方案是一种基于随机引物PCR的基因组步移方案。•两轮巢式叉状PCR扩增足以获得阳性步移结果。
