[自然科学]Technique of molecular biology.ppt

3-18 chapter12:mechanisms of transcription Chapter 20 Techniques of Molecular Biology Techniques of Molecular Biology separating individual macromoleculars from the myriad mixtures in cell dissecting the genome into manageable sized segments for manipulation and analysis of specific DNA sequences Techniques of molecular biology have been one of the major driving forces in the research of molecular biology 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 Topic 1: Nucleic acids Techniques Gel electrophoresis separates DNA and RNA molecules according to size, shape and topological properties 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 can be visualized by staining the gel with fluorescent dyes, such as ethidium bromide (EB,溴化乙锭) 2. 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.(剪刀) 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. 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: Sau3AI, 5’-GATC-3’; NotI, 5’-GCGGCCGC-3’ (3) Restriction enzymes differ in the nature of the DNA ends they ge