摘 要：分子克隆是一种利用体外重组技术实现在分子水平上扩增特定DNA片段的方法。最常用的基因克隆方法是双酶切构建载体方法，但该方法耗时耗力。无缝克隆技术是近年来发展的一种快速、简单且高效的DNA定向克隆方法，并经过不断的技术改进，实现了大分子DNA片段的克隆和多片段DNA无缝连接。但是目前的无缝克隆技术主要是双链DNA分子克隆，而且多需要先经过PCR扩增再与载体连接。而我们如今所研究的方法已经能实现单链DNA的克隆，只需要一步就能完成目的基因与载体的连接，外源基因并不需要经过PCR扩增，进一步简化了无缝克隆技术。并且对于载体需敲除或者突变一段基因时，该方法同样成功率高，并且操作更加简便。
Abstract
Molecular cloning is a method to amplify specific DNA fragments at molecular level by using in vitro recombination technology. The most commonly used gene cloning method is double enzyme vector digestion, but this method is time consuming and labor-intensive. The seamless cloning technology is a rapid, simple and efficient DNA directional cloning method developed in recent years, and through continuous technical improvements, the cloning of macromolecular DNA fragments and the seamless connection of multi-segment DNA are realized. However, the current seamless cloning technique is mainly the cloning of double-stranded DNA molecules, and it needs to be amplified by PCR before being connected to the vector. The method we are investigating now has been able to clone single-stranded DNA, which only takes one step to complete the connection between the target gene and the vector. The exogenous gene does not need to be amplified by PCR, further simplifying the seamless cloning technology. And when the vector needs to knock out or mutate a gene, the method has the same high success rate and is easier to operate.
关键词：无缝克隆； PCR；单引物克隆；基因敲除；基因突变
Keywords: Seamless cloning; PCR; Single primer cloning; Gene knockout; Gene mutation
目 录
1 引言 4
1.1 无缝克隆技术概述 4
1.2 无缝克隆技术的应用 4
1.3 无缝克隆技术研究进展 5
1.4 本研究的内容及意义 5
2 实验材料与方法 6
2.1 实验材料 6
2.2 实验方法 6
2.2.1 不同条件下T7核酸外切酶活性及外切速度比较 6
2.2.1.1 不同温度和时间下T7核酸外切酶活性及外切速度比较 6
2.2.1.2 不同的T7核酸外切酶活力下外切速度比较 6
2.2.2 单引物克隆到目的载体 7
2.2.3外源基因片段克隆到目的载体 10
3 结果与分析 14
3.1 不同条件下T7核酸外切酶活性及外切速度比较结果 14
3.1.1 不同温度和时间下T7核酸外切酶活性及外切速度比较 14
3.1.2 不同的T7核酸外切酶活力下外切速度比较 15
3.2 单引物克隆到目的载体与鉴定结果 15
3.3 外源基因克隆到目的载体与鉴定结果 16
3.3.1 目的基因的扩增 16
3.3.2 载体与目的片段重组连接及鉴定 引物桥接克隆方法研究:http://www.chuibin.com/shengwu/lunwen_206335.html