chapter 4 dna and rna extraction. september 11, 2015september 11, 2015september 11, 2015 genetic...

Chapter 4

DNA and RNA

Extraction

April 21, 2023April 21, 2023 Genetic EngineeringGenetic Engineering 22

1.1 Introduction

Summarize

1.2 The purpose of DNA/RNA isolation

1.3 The procedures of DNA/RNA extraction

1.4 The methods of DNA/RNA extraction


本章主要内容

1.1 DNA 和 RNA 简介

1.2 分离 DNA/RNA 的目的

1.3 DNA/RNA 分离的流程

1.4 DNA/RNA 分离的具体方法


Objective目标

1.1. Understand the Understand the purposespurposes 目的目的 of DNA of DNA and RNA and RNA isolationisolation 分离分离 . .

2.2. Learn the procedures of DNA and RNA Learn the procedures of DNA and RNA extraction extraction 提取提取 ..

3.3. Learn the operations of DNA and RNA Learn the operations of DNA and RNA extraction extraction 提取提取 ..


目标1.1. 理解利用分离的理解利用分离的 DNA/RNADNA/RNA 能做些什么能做些什么 2.2. 学习提取学习提取 DNA/RNADNA/RNA 的流程的流程3.3. 学会学会 DNA/RNADNA/RNA 提取过程中的操作方法提取过程中的操作方法


1.1 Introduction 引言

核酸 nucleic acid

核苷酸 nucleotide

核苷 nucleoside 磷酸 phosphate

嘌呤碱 purine base 或嘧啶碱 pyrimidine base

（碱基 base ）

核糖 ribose 或脱氧核糖 deoxyribose

（戊糖 amyl sugar ）


1.1 Introduction

A gene is a sequence of DNA that produces another nucleic acid, RNA. The DNA has two strands of nucleic acid, and the RNA has only one strand. The sequence of

the RNA is determined by the sequence of the DNA, the RNA is in turn used to direct production of a protein.


The difference between prokaryotic gene and eucaryotic gene

exon1 intron1 exon2 intron2 exon3

geneprokaryotic gene

eucaryotic gene

mRNA

transcription

transcriptionexon1 intron1 exon2 intron2 exon3Pre-mRNA

exon1 exon2 exon3mRNAModified


Ribosomal RNAs (rRNA)

Ribosomal RNAs (rRNA) are components of the ribosome, a large ribonucleoprotein complex that contains many proteins as well as its RNA components, and which provides the apparatus for actually polymerizing amino acids into a polypeptide chain.


Transfer RNAs (tRNA)

Transfer RNAs (tRNA) are small RNAs that are used to provide amino acids corresponding to each particular codon in mRNA.


Messenger RNA (mRNA)

核苷酸

核苷酸

A A A A

The 5' end of eukaryotic mRNA is capped

The 3' terminus is polyadenylated


1.2 The purpose of DNA/RNA isolation

Get the gene of interest

Analyze the gene sequence of a certain organism

Analyze the polymorphism of a certain gene

Species identification for microorganism

Paternity testing, medical diagnosis


1.2 分离 DNA/RNA 的目的

得到感兴趣的目的基因

对特定物种的基因序列进行分析对一种基因的多态性进行分析

微生物的菌种鉴定

亲子鉴定、医学诊断


The polymorphism of a certain gene

Restriction Fragment Length Polymorphism-RFLP


1.3 The procedures of DNA extraction

1.Breaking the cells open, commonly referred to as cell disruption 破裂 or cell lysis 溶解 , to expose the DNA within. This is commonly achieved by chemical and physical methods-blending, grinding研磨 or sonicating 超声处理 the sample.

2.Removing membrane lipids by adding a detergent 去污剂 or surfactants 表面活性剂 .

3.Removing proteins by adding a protease (optional but almost always done).

4.Removing RNA by adding an RNase (often done).


1.3 The procedures of DNA extraction

5.Precipitating 沉淀 the DNA with an alcohol 酒精 — usually ice-cold ethanol 酒精 or isopropanol 异丙醇 . Since DNA is insoluble in these alcohols, it will aggregate 聚集 together, giving a pellet 沉淀 upon centrifugation 离心 . This step also removes alcohol-soluble salt.


1.3 The procedures of RNA extraction

The procedures of RNA extraction is complicated by the ubiquitous 普遍存在 presence of ribonuclease 核糖核酸酶 enzymes in cells and tissues, which can rapidly degrade RNA.

All the reagents and apparatus must be treated with RNAase inhibitors (DEPC), and the process should be carried out at low temperature.


下面列出了一些 DNA 提取时所需要的试剂请推测其作用

Lysozyme Lysozyme 溶菌酶溶菌酶 ;; 葡萄糖溶液葡萄糖溶液 ;EDTA;EDTA 0.2 mol/L NaOH0.2 mol/L NaOH －－ SDSSDS 液（液（ PH=12.6PH=12.6 ）） 3mol/L NaAC-HAC3mol/L NaAC-HAC 溶液（溶液（ PH=4.8PH=4.8 ）） CTAB;CTAB; 苯酚；氯仿；苯酚；氯仿； RNAase or DNAaseRNAase or DNAase SDS-KACSDS-KAC 溶液溶液 TRIzolTRIzol Tris-HCLTris-HCL 缓冲液缓冲液


The apparatus of DNA/RNA extraction

bead beater centrifuge gel box



There are a few things to know before isolating the DNA/RNA from the samples.

The species of the samples

Are the samples from cells or tissues?

What is our purpose?

What is your opinions?



Here are several videos about different methods of DNA/RNA extraction

DNA extraction

DNA Extraction by Phenol Chloroform 苯酚 - 氯仿Plasmid DNA Extraction

DNA Extraction from Tissue

DNA Extraction from Blood



Here are several videos about different methods of DNA/RNA extraction

RNA extraction

RNA Extraction by TRIzol

mRNA Extraction from Total RNA

RNA Extraction from Tissue

RNA Extraction from Cell Culture


Discussion in the class

What conclusions do you draw ?(from the methods you've watched)

What is the difference between DNA and RNA extraction?

Do you know other applications of DNA and RNA extraction? What are they?

chapter 4 dna and rna extraction. september 11, 2015september 11, 2015september 11, 2015 genetic...

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