【正文】 s hypothesis with a purified bacterial restriction enzyme and showed that this enzyme cuts DNA in the middle of a specific symmetrical sequence. Other restriction enzymes have similar properties, but different enzymes recognize different sequences. ? Nathans pioneered the application of restriction enzymes to geics. He demonstrated their use for the construction of geic maps and developed and applied new methodology involving restriction enzymes to solve various problems in geics. ? This year39。s publication from 1971 no doubt served as a major source of inspiration for scientists who subsequently started to use restriction enzymes. Two years later he described the cleavage patterns of SV40 DNA obtained with two additional restriction enzymes. He could then piece together the fragments obtained from the three cleavages and construct the plete geic map of SV40 DNA, the first obtained by a chemical method. The general approach designed by Nathans for SV40 was later used by other scientists for mapping increasingly plex DNA structures. The map of SV40 DNA was further refined by other scientists. Today we know the plete nucleotide sequence of the molecule and thus can write the plete chemical formula for all the genes of an animal virus. Nathans himself continuously contributed new ideas and developed new methods for the application of restriction enzymes to geic problems and has continuously been a main source of inspiration in this field of research. Discovery of restriction endonucleases Phenomenon Hypothesis Experimental proof Mechanism practical use of restriction endonucleases 38 How does the bacteria prevent it39。1 Molecular Biology of the Gene, 6/E Watson et al. (2022) Part I: Chemistry and Geics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods 2 The revised central dogma Translation RNA processing DNA repair and rebination 基因組的保持 基因組的表達(dá) 3 Chapter 20 Techniques of Molecular Biology 4 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 5 The methods depend upon understanding of the properties of biological macromolecules themselves. ? Hybridization(分子雜交 ） the basepairing characteristics of DNA and RNA ? DNA cloning DNA polymerase, restriction endonucleases and DNA Ligase ? PCRThermophilic DNA polymerase 6 Outline ? Topic 1: Nucleic acids Techniques ? Topic 2: Protein Techniques ? Topic 3: Techniques for studying DNAprotein interaction ? Topic 4: Techniques for studying proteinprotein interaction Basic principle Basic procedure application 7 Topic 1: Nucleic acids Techniques by Electrophoresis (電泳分離 ) by Restriction endonuclease (限制性內(nèi)切酶切割 ) by Hybridization (雜交鑒定 ) Cloning and gene expression (基因克隆和表達(dá)技術(shù)） （聚合酶鏈?zhǔn)椒磻?yīng)） 8 Gel electrophoresis separates DNA and RNA molecules according to size, shape and topological properties 1. Gel Electrophoresis （ 凝膠電泳） 9 and RNA molecules are negatively charged, thus move in the gel matrix (膠支持物 ) toward the positive pole (正電極 ) DNA molecules are separated according to sizes. The large DNA molecules move slower than the small molecules. DNA gel mobility (electrophoretic mobility ， DNA在膠上的遷移率 ) 10 large moderate small Run gel(跑膠 ) 11 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 (線性 ) nicked or relaxed (缺刻或松散 ) 12 DNA can be visualized by staining the gel with fluorescent dyes, such as ethidium bromide (EB,溴化乙錠 ) DNA is separated by gel electrophoresis 1 kb kb 2 kb 3 kb 4 kb DNA marker 13 Gel matrix (膠支持物 ) is a jello(果凍狀 ） like porous material that supports and allows macromolecules to move through. Gel matrix (膠支持物 ) Polyacrylamide (聚丙烯酰胺 ) Agarose (瓊脂糖 ) 14 Polyacrylamide (聚丙烯酰胺 ): (1)has high resolving capability(分辨力高 ), and can resolve DNA/RNA that differ from each other as little as a single base pair. (2)but can separate DNA over a narrow size range (up to a few hundred bp, 幾百 bp). 15 Agarose (瓊脂糖 ): (1)a much less resolving power than polyacrylamide (2)but can separate DNA molecules of up to tens of kb 1 kb kb 2 kb 3 kb 4 kb 16 (1) RNA have a uniform negative charge as DNA does. (2) RNA is singlestranded and have extensive secondary and tertiary structure, which significantly influences their electrophoretic mobility. (3) RNA can be treated with reagent such as glyoxal (乙二醛 ) to prevent RNA base pairing, so that its mobility only correlates with the molecular weight Electrophoresis is also used to separate RNAs 17 2. Restriction endonucleases (限制性內(nèi)切酶 ) cleave DNA molecules at particular sites ? Why use endonucleases? ? To make large DNA molecules break into manageable fragments ? Why use restriction endonucleases? ? cleave DNA molecules at particular sites 18 ? 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 (識別 ) short (48bp) target sequences that are usually palindromic (回文結(jié)構(gòu) ), and cut (切割 ) at a defined sequence within those sequences. 19 5’… .GAATTC.… .3’ … .CTTAAG… . EcoRI 3’ 5’ 反向重復(fù)序列（回文結(jié)構(gòu)） 20 the 1st such enzyme