第12章分子生物学实验技术(精)

第12章 分子生物学实验技术

1 本章主要内容

(1)基因操作:DNA克隆概述、质粒DNA的制备、限制酶与电泳、连接、转化与重组体分析

(2)克隆载体:载体设计、各种载体的特征 (3)工具酶的种类、特点 (4)PCR的基本原理及操作技术

2 教学目的和要求

(1) 掌握基因操作和PCR的基本原理和过程; (2) 熟悉常用克隆载体、工具酶的特性。

3 重点难点

(1) 克隆原理; (2) 质粒载体构建原理; (3) PCR原理及操作。

4 教学方法与手段

讲授与交流互动相结合,采用多媒体教学。

5 授课内容

第1节 DNA cloning (DNA 克隆)

一、DNA 克隆

克隆一词是英文单词clone的音译,作为名词,c1one通常被意译为无性繁殖系。同一克隆内所有成员的遗传构成是完全相同的,例外仅见于有突变发生时。

自然界早已存在天然植物、动物和微生物的克隆,例如:同卵双胞胎实际上就是一种克隆。

作为动词,cloning是指用人工方法产生无性繁殖系的一系列操作过程。 DNA cloning (DNA 克隆)的定义:将基因组中携有目的基因或其他相关序列的较小片段连接到一段可以自主复制的DNA即载体(vectors)上,形成通常可以在另一宿主中进行复制的重组DNA(recombinant DNA),这种复制是独立于原初基因组的。带有重组DNA的宿主(Hosts)细胞的增殖构成了一群具有遗传一致性的个体,或称为单克隆。这一系列操作过程被称为DNA克隆。

Blotting the DNA or RNA on a membrane

Hybridize the labeled probe with DNA membrane (Southern) or RNA (Northern) membrane

二、Hosts and vectors

1.Host organism/cell: where the plasmids get multiplied and propagated faithfully

忠实增殖, which is crucial for DNA cloning. 2. Hosts for DNA cloning vector:

Prokaryotic host : E. coli ( most cases)

Eukaryotic host : Yeast Saccharomyces cerevisiae 酿酒酵母 (large fragments of

human genome)

3. General features of a Vector

(1)autonomously replicating DNA independent of host?s genome. (2)Easily to be isolated from the host cell (3)Most are circular, some are linear

(4)Contains at least one selective marker, which allows host cells containing the vector to be selected amongst those which do not. (5)Contains a multiple cloning site (MCS) 4.Types of vectors (1)Cloning vectors

allowing the exogenous DNA to be inserted, stored, and manipulated at DNA level. E. coli cloning vector: plasmids, bacteriophages (l and M13), plasmid-bacteriophage l hybrids (cosmids).

Yeast cloning vector: yeast artificial chromosomes (YACs)

PCR直接产物载体

(2)Expression vectors

allowing the exogenous DNA to be inserted and expressed. Promoter and terminator for RNA transcription are required. ? bacterial expression vectors ? yeast expression vectors mammalian expression vectors

(3)Integration vectors DNA after a transformation.

A. allowing the exogenous DNA to be inserted and integrated into a chromosomal B. The integration is conducted by homologous recombination between the homologous sequence shared by the plasmid and the genome of the recipient cells.

C. bacterial integration vectors (Agrobacterium tumefaciens Ti plasmid is used to integrate DNA into plant genome) D. yeast integration vectors

E. Mammalian integration vector: virus based

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