分子生物学杨洋第九章-分子生物学技术-techniquesofmolecularbiology.ppt

3-18 chapter12:mechanisms of transcription Outline Nucleic acids Techniques Protein Techniques Techniques for studying DNA-protein interaction Techniques for studying protein-protein interaction Nucleic acids Techniques （核酸技术） 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 generate: blunt ends (平末端), sticky ends (粘性末端). How does the bacteria prevent its own DNA from being cut by the restriction endonucleases? Modification One of the nucleotides in bacteria DNA is modified by an methylase enzyme (甲基化酶，modification enzyme) that attaches a methyl group to one of the bases The restriction enzyme doesnt bind to sites where one of the bases is modified by methylation 3.