武汉大学生命科学学院：《分子生物学》英文版 CHAPTER 20 Techniques of Molecular Biology

Welcome each of You to My molecular Biology class @博来字 Wuhan University

Welcome Each of You to My Molecular Biology Class

Molecular Biology of the Gene 5/E --Watson et al. (2004) Part I: Chemistry and Genetics Part maintenance of the genome Part l: Expression of the Genome Part IV: Regulation Part v: methods Wuhan University

2 Molecular Biology of the Gene, 5/E --- Watson et al. (2004) Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods

Part v METHODs Ch 20: Techniques of Molecular Biology Ch 21: Model Organisms

3 Ch 20: Techniques of Molecular Biology Ch 21: Model Organisms Part V: METHODS

Molecular iology Course Chapter 20 Techniques of Molecular Biology Preparation, analysis and manipulation of nucleic acids and proteins

4 •Molecular Biology Course Chapter 20 Techniques of Molecular Biology Preparation, analysis and manipulation of nucleic acids and proteins

The methods depend upon, and were developed from, an understanding of the properties of biological macromolecules themselves Hybridization---the base-pairing characteristics of dna and rna DNA cloning DNA polymerase restriction endonucleases and dna ligase > PCR---Thermophilic DNA polymerase

5 The methods depend upon, and were developed from, an understanding of the properties of biological macromolecules themselves. ➢ Hybridization---the base-pairing characteristics of DNA and RNA ➢ DNA cloning--- DNA polymerase, restriction endonucleases and DNA ligase ➢ PCR---Thermophilic DNA polymerase

CHAPTER20: Techniques of Molecular Biology Topic1: Nucleic acids 1. Electrophoresis 2. Restriction 3. Hybridization 4. DNA Cloning and gene expression 5. PCR 6. Genome sequence analysis

6 Topic1: Nucleic acids CHAPTER20: Techniques of Molecular Biology 1.Electrophoresis 2.Restriction 3.Hybridization 4.DNA Cloning and gene expression 5.PCR 6.Genome sequence & analysis

Electrophoresis 1. Gel electrophoresis separates DNA and rna molecules according to size, shape and topological properties Gel matrix is an inserted, jello-like porous material that support and allows macromolecules to move througl h. Agarose and polyacrylamide are two different gel matrices

7 1. Gel electrophoresis separates DNA and RNA molecules according to size, shape and topological properties Gel matrix is an inserted, jello-like porous material that support and allows macromolecules to move through. Agarose and polyacrylamide are two different gel matrices Electrophoresis

Electrophoresis DNA and RNa molecules are negatively charged, thus move in the gel matrix toward the positive pole(+) Linear dNa molecules are separated according to size The mobility of circular dna molecules is affected by their topological structures. The mobility of the same molecular weight dna molecule with different shapes is supercoiled> Iinear> nicked or relaxe

8 ➢ DNA and RNA molecules are negatively charged, thus move in the gel matrix toward the positive pole (+) ➢ Linear DNA molecules are separated according to size ➢ The mobility of circular DNA molecules is affected by their topological structures. The mobility of the same molecular weight DNA molecule with different shapes is: supercoiled> linear> nicked or relaxed Electrophoresis

Fig 20-1: DNA separation by gel electrophoresis electrophoresis chamber buffer solution DNA fragments agarose gel electrode electrode large moderate small After electr electrode electrode small DNA fragments move further through the gel than large fragments Copyright C 2004 Pearson Education, Inc, publishing as Benjamin Cummings

9 Fig 20-1: DNA separation by gel electrophoresis large moderate small After electr

Electrophoresis To separate DNA of different SIze ranges a Narrow size range of DNA: use polyacrylamide Wide size range of DNA: use agarose gel Very large DNA(>30-50kb): use pulsed-field gel electrophoresis 10

10 To separate DNA of different size ranges ◼ Narrow size range of DNA: use polyacrylamide ◼ Wide size range of DNA: use agarose gel ◼ Very large DNA(>30-50kb): use pulsed-field gel electrophoresis Electrophoresis