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

Molecular biology of the Gene 5/E - Watson et al. ( 2004) Part l: Chemistry and Genetics Part Maintenance of the Genome Part l: Expression of the Genome Part IV: Regulation Part va methods 刻博来字 Wuhan Univ

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 hemselves bridization---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

CATER20: Techniques of Molecular iolo Topⅰc1: Nucleic acids 1. Separation by Electrophoresis(电泳分离) 2 Cut by Restriction endonuclease(限制性 内切酶切割) 3工 dentification by Hybridization(杂交鉴 定) 4 PCR 5. Genome sequence analysis 6. DNA Cloning and gene expression

6 Topic 1: Nucleic acids CHAPTER20: Techniques of Molecular Biology 1.Separation by Electrophoresis (电泳分离) 2.Cut by Restriction endonuclease (限制性 内切酶切割) 3.Identification by Hybridization (杂交鉴 定) 4.PCR 5.Genome sequence & analysis 6.DNA Cloning and gene expression

Gelelectrophoresis(凝胶电泳) 1. Gel electrophoresis separates DNA and RNA molecules according to size, shape and topological properties

7 1. Gel electrophoresis separates DNA and RNA molecules according to size, shape and topological properties Gel Electrophoresis （凝胶电泳）

DNA gel mobility(DNA在胶上的迁移性 1.DNA and RNA molecules are negatively charged, thus move in the gel matrix(胶支持物) toward the positive pole(正电极) 2. Linear DNA molecules are separated according to sizes. The large dna molecules move slower than the small molecules 3. 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(超螺旋)> linear(线性)> nicked or relaxed(缺刻或松散)

8 1.DNA and RNA molecules are negatively charged, thus move in the gel matrix (胶支持物) toward the positive pole (正电极). 2.Linear DNA molecules are separated according to sizes. The large DNA molecules move slower than the small molecules. 3.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 (超螺旋)> linear (线性) > nicked or relaxed (缺刻或松散) DNA gel mobility (DNA在胶上的迁移性)

DNA can be visualized by staining the gel with fluorescent dyes, such as ethidium bromide(EB溴化乙锭) electrophoresis chamber buffer solution DNA fragments agarose gel electrode electrode large moderate small Fig 20-1: DNA is separated by gel electrode electrode electrophoresis small dNA fragments move further through the gel than large fragments Copyright 2004 Pearson Education, Inc, publishing as Benjamin Cummings

9 Fig 20-1: DNA is separated by gel electrophoresis large moderate small DNA can be visualized by staining the gel with fluorescent dyes, such as ethidium bromide (EB 溴化乙锭)

Gel matrixκ(胶支持物) Gel matrix(胶支持物) is an inserted jello-like porous material that supports and allows macromolecules to move through 10

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

Polyacrylamide(聚丙稀酰 胺): (1)has high resolving capability and can MgCh(mM) resolve DNA/RNa that 0204060811215235710 differ from each other mana ERli- as little as a single base pair/nucleotide nissan ===== (2)but can separate DNA over a narrow size range〔 up to a few 三= hundred bp/nt)

11 Polyacrylamide (聚丙稀酰 胺): (1)has high resolving capability, and can resolve DNA/RNA that differ from each other as little as a single base pair/nucleotide. (2)but can separate DNA over a narrow size range (up to a few hundred bp/nt)