Various polymerase chain reaction (PCR)-based methods have been applied for the development of genome walking (GW) technique. These methods which could be based on the application of restriction enzymes or primers have various efficiencies to identify the unknown nucleotide sequences. The present study was conducted to design a new innovative double-strand adaptor using MAP30 gene sequence of Momordica charantia plant as a model to improve genome walking with convenient PCR. The adaptor was designed using multiple restriction sites of Hind III, BamH I, EcoR I, and Bgl II enzymes with no restriction site in a known sequence of the MAP30 gene. In addition, no modification was required to add phosphate, amine, or other groups to the adaptor, since restriction enzyme digestion of double-strand adaptor provided the 5′ phosphate group. Here, preparation of the phosphate group in the genomic DNA of the plant digestion with restriction enzymes was performed followed by ligation with digested adaptor containing 5′ phosphate group. PCR was done to amplify the unknown sequence using MAP30 gene-specific primer and adaptor primer. Results confirmed the ability of the technique for successful identification of the sequence. Consequently, a newly designed adaptor in the developed technique reduced the time and cost of the method compared to the conventional genome walking; also, cloning and culturing of bacterial steps could be eliminated.

中文翻译：

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简单创新的适配器，可通过方便的PCR改善基因组步移

各种基于聚合酶链反应（PCR）的方法已用于基因组步行（GW）技术的发展。这些可基于限制酶或引物的应用的方法具有鉴定未知核苷酸序列的各种效率。本研究进行了设计，以苦瓜属植物的MAP30基因序列为模型，设计了一种新型的创新双链衔接子，以通过方便的PCR改善基因组步移。使用Hind III，BamHI，EcoRI和Bgl II酶的多个限制性位点设计了衔接子，在MAP30基因的已知序列中没有限制性位点。另外，不需要修饰以将磷酸酯，胺或其他基团添加至衔接子，因为双链衔接子的限制性酶消化提供了5'磷酸酯基团。这里，在用限制酶消化的植物的基因组DNA中进行磷酸基的制备，然后与含有5'磷酸基的消化的衔接子连接。使用MAP30基因特异性引物和衔接子引物进行PCR扩增未知序列。结果证实了该技术成功鉴定序列的能力。因此，与常规的基因组步移相比，在开发的技术中新设计的适配器减少了该方法的时间和成本。同样，可以消除细菌步骤的克隆和培养。使用MAP30基因特异性引物和衔接子引物进行PCR扩增未知序列。结果证实了该技术成功鉴定序列的能力。因此，与常规的基因组步移相比，在开发的技术中新设计的适配器减少了该方法的时间和成本。同样，可以消除细菌步骤的克隆和培养。使用MAP30基因特异性引物和衔接子引物进行PCR扩增未知序列。结果证实了该技术成功鉴定序列的能力。因此，与常规的基因组步移相比，在开发的技术中新设计的适配器减少了该方法的时间和成本。同样，可以消除细菌步骤的克隆和培养。