分子生物学第九章分子生物学研究方法电子教案

第九章分子生物学研究方法

1 •课程教学内容

(1) 核酸技术1—基本操作

(2) 核酸技术2—克隆技术

(3) 核酸技术3—测序

(4) 基因表达和表达分析基因定点诱变

(5) 蛋白质与核酸的相互作用

(6) 其他(热点)技术

2 •课程重点、难点

基因克隆技术、杂交技术、测序技术、蛋白质与核酸的相互作用检测技术

3 •课程教学要求

掌握基因克隆技术、杂交技术、测序技术、蛋白质与核酸的相互作用检测等各种技术的原理。

本章内容

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? 核酸的凝胶电泳

DNA分子的酶切割

核酸的分子杂交

基因扩增

基因的克隆和表达

细菌的转化

DNA核苷酸序列分析

蛋白质的分离与纯化

研究DNA与蛋白质相互作用的方法

一、核酸的凝胶电泳

基本原理：当一种分子被放置在电场当中时，它们会以一定的速度移向适当的电极。电泳的迁移率：电泳分子在电场作用下的迁移速度，它同电场的强度和电泳分子本身所携带的净电荷成正比。

由于在电泳中使用了一种无反应活性的稳定的支持介质，如琼脂糖和丙烯酰胺，从而降低了对流运动，故电泳的迁移率又同分子的摩擦系数成反比。

在生理条件下，核酸分子的糖-磷酸骨架中的磷酸基团，是呈离子化状态的。从这个意义上讲，DNA和RNA勺多核苷酸链可叫做多聚阴离子，因此，当核酸分子放置在电场中时，它就会向正极移动。

在一定的电场强度下，DNA分子的这种迁移率，取决于核酸分子本身大小和构型。

分子量较小的DNA分子，比分子量较大的分子，具有较紧密的构型，所以其电泳迁移

率也就比同等分子量的松散型的开环DNA分子或线性DNA分子要快些。

Gel matrix ( 胶支持物) is an inserted, jello-like porous material that supports and allows macromolecules to move through.

Agarose ( 琼脂糖):

(1) a much less resolving power than polyacrylamide,

(2) but can separate DNA molecules of up to tens of kb

DNA can be visualized by staining the gel with fluorescent dyes, such as

ethidium bromide (EB 溴化乙锭)

Polyacrylamide ( 聚丙稀酰胺):

(1) has high resolving capability, and can resolve DNA that differ from each other as

little as a single base pair/nucleotide.

(2) but can only separate DNA over a narrow size range (1 to a few hundred bp). Pulsed-field gel electrophoresis ( 脉冲电泳)

(1) The electric field is applied in pulses that are oriented orthogonally ( 直角地) to each

other.

(2) Separate DNA molecules according to their molecule weight, as well

as to their shape and topological properties.

(3) Can effectively separate DNA molecules over 30-50 kb and up to several Mb in

length.

二、DNA分子的酶切割

Restriction endonucleases ( 限制性内切酶) cleave DNA molecules at particular sites Restriction endonucleases (RE) are the nucleases that cleave DNA at particular sites by the recognition of specific sequences.

RE used in molecular biology typically recognize ( 识别) short (4-8bp)

target sequences that are usually palindromic ( 回文结构), and cut ( 切割) at

a defined sequence within those sequences. e.g. EcoRI

The random occurrence of the hexameric ( 六核苷酸的) sequence: 1/4096 (4- 6=1/46)

(1) Restriction enzymes differ in the recognition specificity: target sites are different.

(2) Restriction enzymes differ in the length they recognized, and thus the frequencies differ.

(3) Restriction enzymes differ in the nature of the DNA ends they generate: blunt/flush ends ( 平末端), sticky/staggered ends ( 粘性末端).

(4) Restriction enzymes differ in the cleavage activity.