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Chapter 9
Techniques of
Molecular Biology
分子生物学技术
Yang Yang(杨洋教授)
Huazhong University of Sciences and Technology
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Techniques of Molecular Biology
Techniques of molecular biology have been one of the major driving forces in the research of molecular biology
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The methods depend upon understanding of the properties of biological macromolecules themselves.
DNA cloning--- DNA polymerase, restriction endonucleases and DNA Ligase
PCR---Thermophilic DNA polymerase
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Outline
Topic 1: Nucleic acids Techniques
Topic 2: Protein Techniques
Topic 3: Techniques for studying DNA-protein interaction
Topic 4: Techniques for studying protein-protein interaction
Basic principle
Basic procedure
application
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Topic 1: Nucleic acids Techniques
Separation by Electrophoresis (电泳分离)
Cut by Restriction endonuclease (限制性内切酶切割)
DNA Cloning and gene expression (基因克隆和表达技术)
PCR(聚合酶链式反应)
5. Hybridization (杂交技术): Southern hybridization, Northern hybridization, Western hybridization
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Gel electrophoresis separates DNA and RNA molecules according to size, shape and topological properties
1. Gel Electrophoresis (凝胶电泳)
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and RNA molecules are negatively charged, thus move in the gel matrix (胶支持物) toward the positive pole (正电极)
DNA molecules are separated according to sizes. The large DNA molecules move slower than the small molecules.
DNA gel mobility (electrophoretic mobility ,
DNA在胶上的迁移率)
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large
moderate
small
Run gel(跑胶)
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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)
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DNA can be visualized by staining the gel with fluorescent dyes, such as ethidium bromide (EB,溴化乙锭)
DNA is separated by gel electrophoresis
1 kb
kb
2 kb
3 kb
4 kb
DNA marker
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